Method for rapidly detecting molecular weight of hyaluronic acid in fermentation liquor

Abstract

The invention discloses a method for rapidly detecting the molecular weight of hyaluronic acid in fermentation liquor. The method specifically comprises the following steps: diluting and centrifuging hyaluronic acid fermentation liquor, precipitating with alcohol, redissolving, adsorbing with activated carbon, filtering with perlite to obtain a hyaluronic acid solution, and detecting the molecular weight of hyaluronic acid by adopting a gel permeation chromatography method. The method is simple, convenient and efficient, avoids loss of hyaluronic acid molecular weight caused by too complex purification process, reduces interference of a large number of impurities in fermentation liquor on molecular weight detection, has relatively high stability and reproducibility, provides timely and effective operation guidance for hyaluronic acid fermentation experiment and production, and has a wide application prospect. The method has great significance in improving the quality of hyaluronic acid products.

Description

Technical Field

[0001] The invention belongs to the technical field of fermentation analysis, and particularly relates to a method for rapidly detecting the molecular weight of hyaluronic acid in a fermentation liquid. Background Art

[0002] Hyaluronic acid (HA), also known as "hyaluronic acid", is an acidic macromolecular mucopolysaccharide. As a biopolymer, it has a unique molecular structure and physical and chemical properties that show a variety of important physiological functions in the body. Due to the different molecular weights of HA, its application fields are also different. For example, low molecular weight HA (Mr<1×10 6 Da) is generally used in cosmetics and food, while high molecular weight hyaluronic acid (Mr>2×10 6 Da) is generally used in the medical field, such as lubricating joints, regulating the permeability of blood vessel walls, regulating the diffusion and movement of water and electrolytes, promoting wound healing and soft tissue repair, and serving as a drug carrier. It has a high application value, especially in preventing and reducing tissue adhesion after surgical operations.

[0003] At present, the production and preparation methods of hyaluronic acid mainly include animal tissue extraction method and microbial fermentation method. Among them, animal tissue extraction method can obtain high molecular weight hyaluronic acid, but the purification process often needs to use some protease enzymolysis and organic solvent to remove impurities, which is easy to cause environmental pollution, and the raw material cost is high and the source is limited, and the yield is extremely low. Compared with the animal tissue extraction method, the microbial fermentation method has the advantages of low cost, no material restriction, and high yield, but it is difficult to obtain high molecular weight hyaluronic acid through microbial fermentation, and the hyaluronic acid produced by microbial fermentation is mixed with its own secondary metabolites and residual culture medium. It is difficult to quickly and accurately determine the molecular weight of hyaluronic acid after fermentation. If the molecular weight of hyaluronic acid after fermentation is to be detected, the fermentation liquid must be separated and purified, but in the purification process, due to the influence of external physical or chemical factors, it will inevitably cause the loss of part of the molecular weight of hyaluronic acid.

[0004] At present, the molecular weight of hyaluronic acid in the fermentation broth can be detected by multi-angle laser scattering instrument method (SEC-MALLS). This method does not require the hyaluronic acid fermentation broth to be complicatedly purified. Hyaluronic acid of different molecular weights in the fermentation broth is separated by the principle of gel exclusion chromatography, and the relevant parameters are detected by multi-angle laser scattering instrument to calculate its absolute molecular weight and molecular weight distribution. However, this equipment is expensive and requires professional people to operate. In addition, when measuring high molecular weight hyaluronic acid, the sample may be sheared and degraded in the porous medium, and the gel column may also be blocked. Therefore, this method is difficult to be widely used in production. At present, the intrinsic viscosity method is mostly used to detect the molecular weight of hyaluronic acid in the fermentation broth. It calculates the ratio of the outflow time of the test sample and the blank solution in the capillary at a constant temperature, and calculates the intrinsic viscosity η according to the formula η=(In T / T0) / C, where the relationship between the molecular weight (M) of sodium hyaluronate and the intrinsic viscosity η is: η=0.036M 0.78 , the molecular weight of hyaluronic acid is calculated. This method has high requirements on the purity of the sample, impurities have a great impact on the accuracy of the results, and the entire detection process is complicated and time-consuming, and the accuracy and stability are poor. Therefore, it is urgent to establish a simple, fast and accurate method for detecting the molecular weight of hyaluronic acid in fermentation broth. Summary of the invention

[0005] The purpose of the present invention is to provide a method for rapidly detecting the molecular weight of hyaluronic acid in fermentation liquid, which is different from the prior art in that the purification method of hyaluronic acid fermentation liquid is different. The separation and purification method adopted by the present invention is simple and fast, and can specifically remove impurities such as residual culture medium and secondary metabolites that have a large impact on the hyaluronic acid molecular weight detection result, such as macromolecular substances such as proteins and nucleic acids, and reduce the loss of hyaluronic acid molecular weight as much as possible, and separate and detect hyaluronic acid with different molecular weights using the principle of gel permeation chromatography. This method does not need to purify hyaluronic acid to dry powder, and does not need to accurately measure the concentration of hyaluronic acid in the liquid to be tested, and 15-30min can accurately detect the molecular weight of hyaluronic acid, and the detection cycle is greatly shortened, which provides a simple, fast, accurate and stable test method for hyaluronic acid fermentation molecular weight determination.

[0006] The above-mentioned object of the present invention is achieved through the following technical solutions:

[0007] In one aspect, the present invention provides a method for rapidly detecting the molecular weight of hyaluronic acid in a fermentation broth, comprising the following steps:

[0008] 1) diluting the hyaluronic acid fermentation liquid with water, centrifuging at high speed, taking the supernatant and adding ethanol to precipitate, taking the precipitate and dissolving it with sodium chloride solution, adding activated carbon for adsorption, and filtering with perlite as a pre-coating layer to obtain a hyaluronic acid test solution;

[0009] 2) Use gel permeation chromatography to determine the elution time of the hyaluronic acid test solution and several sodium polystyrene sulfonate reference substances with different molecular weights, record the chromatogram, draw a calibration curve, and obtain the molecular weight of sodium hyaluronate by the universal calibration method.

[0010] The gel permeation chromatography is referred to as GPC.

[0011] Preferably, in step 1), the hyaluronic acid fermentation broth is Escherichia coli hyaluronic acid fermentation broth, and the volume after dilution is 5-10 times of the original volume, preferably 5 times.

[0012] Preferably, in step 1), the amount of ethanol added is 2-3 times, preferably 3 times, the volume of the supernatant; the concentration of ethanol is 75%-95%, preferably 95%.

[0013] Preferably, in step 1), the concentration of the sodium chloride solution is 0.2 M to 0.5 M, preferably 0.2 M. The amount of sodium chloride solution added depends on the dissolution of the precipitate, and the precipitate can be just dissolved.

[0014] Preferably, in step 1), the activated carbon is 767# needle-shaped activated carbon, and the amount used is 0.2%-0.8% of the volume of the solution, preferably 0.2%. The solution here refers to the total volume of the solution after adding the sodium chloride solution.

[0015] Preferably, in step 1), the adsorption time is 10 min to 60 min, preferably 10 min.

[0016] Preferably, in step 1), the thickness of the perlite as a pre-coating layer is 3-5 cm, preferably 3 cm;.

[0017] Preferably, in step 2), both the hyaluronic acid test solution and the reference solution need to be filtered through a 0.22um-0.45um filter membrane, preferably 0.22um.

[0018] Preferably, in step 2), the concentration of the hyaluronic acid sample is below 0.1 g / L, preferably 0.03-0.09 g / L, more preferably 0.03 g / L.

[0019] Preferably, in step 2), the minimum molecular weight of hyaluronic acid is not less than 100 MDa.

[0020] Preferably, the specific method of step 2) gel permeation chromatography is:

[0021] Analysis conditions: Agilent 1260 Infinity high performance liquid chromatography system;

[0022] Chromatographic column: gel chromatography column, Shodex SB-806HQ 8.0mm×300mm;

[0023] Mobile phase: 0.2M sodium chloride solution

[0024] Flow rate: 0.3-0.7ml / min;

[0025] Injection volume: 50-100ul;

[0026] Detector: Refractive Index Detector (RID)

[0027] Analysis steps: Take sodium polystyrene sulfonate of known different molecular weights, prepare a 1 mg / ml reference solution with the mobile phase, inject 100 ul of each test sample and reference solution, record the chromatogram, and draw a calibration curve; using the universal calibration method, the K value of the reference is 0.00018, and the α value is 0.65; the K value of sodium hyaluronate is 0.00057, and the α value is 0.75; finally, the molecular weight of sodium hyaluronate is calculated.

[0028] The present invention has the following beneficial effects:

[0029] The present invention performs simple separation and purification on the hyaluronic acid fermentation broth, reducing the loss of molecular weight in the purification process. The molecular weight of hyaluronic acid is detected by gel permeation chromatography, reducing the influence of other impurities on the molecular weight, while also delaying the service life of the chromatographic column, and accurate and stable quality control data can be obtained. This method does not need to accurately measure the concentration of hyaluronic acid in the fermentation broth, avoiding the requirements of few impurities and high purity when detecting by the characteristic viscosity method, and the cumbersome steps of measuring the concentration of the solution in advance, and also avoiding the drawbacks of multi-angle laser scattering, low operability, high equipment cost, and the need for professional operation. The results of a series of methodological verifications show that this method has fast detection speed, strong stability and reproducibility, and small human operation error, which is of great significance to the quality control of hyaluronic acid raw material products. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 is the GPC calibration curve for the reference substances of sodium polystyrene sulfonate with different molecular weights of the present invention;

[0031] Figure 2 This is the GPC test chromatogram of the sample after the fermentation broth is purified in the present invention;

[0032] Figure 3 Glucuronic acid standard curve for HA content determination. DETAILED DESCRIPTION

[0033] The following will explain the implementation methods of the present application in detail through examples, so that the implementation process of how the present application uses technical means to solve technical problems and achieve technical effects can be fully understood and implemented accordingly.

[0034] The raw materials and equipment used in this application, unless otherwise specified, are all commonly used raw materials and equipment in this field, and are all commercially available products. The methods used in this application, unless otherwise specified, are all conventional methods in this field.

[0035] There are many other feasible technical solutions in this application, which are not listed here one by one. The technical solutions claimed in the claims of this application are all feasible.

[0036] "Comprising" or "including" is intended to indicate that compositions (e.g., media) and methods include the listed elements, but do not exclude other elements. When used to define compositions and methods, "consisting essentially of" means excluding other elements that have any significance for the combination for the stated purpose. Thus, a composition consisting essentially of the elements defined herein does not exclude other materials or steps that do not materially affect the basic and novel features of the claimed application. "Consisting of" means excluding trace elements and substantial method steps of other components. Embodiments defined by each of these transition terms are within the scope of the present application.

[0037] Example 1: Take 20ml of hyaluronic acid fermentation liquid fermented by Escherichia coli, dilute with 5 times the volume of pure water, centrifuge at 10000rpm for 30min, and detect the hyaluronic acid molecular weight of 2.17MDa by gel permeation chromatography-multi-angle laser scattering instrument. Take the supernatant and slowly add 3 times the volume (95%) of ethanol to precipitate the hyaluronic acid in the fermentation liquid. Collect the hyaluronic acid precipitate, dissolve the precipitate with 0.2M sodium chloride solution, add 0.2% 767# needle-shaped activated carbon for adsorption for 10min, and filter with perlite as a pre-coating layer with a thickness of 3cm to obtain a transparent and clear sodium hyaluronate test solution. The test solution is diluted with 0.2M sodium chloride solution to a hyaluronic acid concentration of 0.03g / L, and the hyaluronic acid molecular weight is determined by gel permeation chromatography. Analytical conditions: Instrument: Agilent 1260 Infinity HPLC system; Chromatographic column: gel chromatography column (Shodex SB-806HQ (8.0 mm × 300 mm); Mobile phase: 0.2 M sodium chloride solution; Flow rate: 0.7 ml / min; Injection volume: 100 ul; Detector: differential refractive index detector (RID); Analysis steps: Take sodium polystyrene sulfonate of known molecular weight, prepare 1 mg / ml reference solution with mobile phase, take 100 ul of each of the test solution and reference solution, record the chromatogram, and draw the calibration curve with GPC software, see Figure 1Using the universal calibration method, the K value of the reference substance is 0.00018, and the α value is 0.65; the K value of sodium hyaluronate is 0.00057, and the α value is 0.75. The calculation results give the molecular weight of sodium hyaluronate.

[0038] Figure 2 This is the GPC detection chromatogram of the sample in Example 1 after Primrose.

[0039] Example 2: Take 20ml of hyaluronic acid fermentation liquid fermented by Escherichia coli, dilute with 6 volumes of pure water, centrifuge at 10000rpm for 30min, and detect the hyaluronic acid molecular weight of 2.17MDa by gel permeation chromatography-multi-angle laser scattering instrument. Take the supernatant and slowly drip 3 volumes of ethanol (95%) to precipitate the hyaluronic acid in the fermentation liquid, collect the hyaluronic acid precipitate, dissolve the precipitate with 0.2M sodium chloride solution, add 0.2% 767# needle-shaped activated carbon for adsorption for 30min, filter with perlite as a pre-coating layer with a thickness of 3cm, and filter to obtain a transparent and clear sodium hyaluronate test solution. The test solution is diluted with 0.2M sodium chloride solution to a hyaluronic acid concentration of 0.06g / L, and the hyaluronic acid molecular weight is determined by gel permeation chromatography. Analysis conditions: Instrument: Agilent 1260 Infinity HPLC system; Chromatographic column: gel chromatography column (Shodex SB-806HQ (8.0mm×300mm); Mobile phase: 0.2M sodium chloride solution; Flow rate: 0.7ml / min; Injection volume: 100ul; Detector: Differential refractive index detector (RID); Analysis steps: Take sodium polystyrene sulfonate of known different molecular weights, prepare a 1mg / ml reference solution with the mobile phase, take 100ul of each of the test solution and the reference solution, record the chromatogram, and draw a calibration curve using GPC software. Using the universal calibration method, the K value of the reference is 0.00018, and the α value is 0.65; the K value of sodium hyaluronate is 0.00057, and the α value is 0.75. Calculate the results and obtain the molecular weight of sodium hyaluronate.

[0040] Example 3: Take 20ml of hyaluronic acid fermentation liquid fermented by Escherichia coli, dilute with 6 times the volume of pure water, centrifuge at 10000rpm for 30min, and detect the hyaluronic acid molecular weight of 2.17MDa by gel permeation chromatography-multi-angle laser scattering instrument. Take the supernatant and slowly drip 3 times the volume (95%) of ethanol to precipitate the hyaluronic acid in the fermentation liquid, collect the hyaluronic acid precipitate, dissolve the precipitate with 0.2M sodium chloride solution, add 0.2% 767# needle-shaped activated carbon for adsorption for 60min, filter with perlite as a pre-coating layer with a thickness of 3cm, and filter to obtain a transparent and clear sodium hyaluronate test solution. The test solution is diluted with 0.2M sodium chloride solution to a hyaluronic acid concentration of 0.09g / L, and the hyaluronic acid molecular weight is determined by gel permeation chromatography. Analysis conditions: Instrument: Agilent 1260 Infinity HPLC system; Chromatographic column: gel chromatography column (Shodex SB-806HQ (8.0mm×300mm); Mobile phase: 0.2M sodium chloride solution; Flow rate: 0.7ml / min; Injection volume: 100ul; Detector: Differential refractive index detector (RID); Analysis steps: Take sodium polystyrene sulfonate of known different molecular weights, prepare a 1mg / ml reference solution with the mobile phase, take 100ul of each of the test solution and the reference solution, record the chromatogram, and draw a calibration curve using GPC software. Using the universal calibration method, the K value of the reference is 0.00018, and the α value is 0.65; the K value of sodium hyaluronate is 0.00057, and the α value is 0.75. Calculate the results and obtain the molecular weight of sodium hyaluronate.

[0041] Example 4: Take 20ml of hyaluronic acid fermentation liquid fermented by Escherichia coli, dilute with 7 times volume of pure water, centrifuge at 10000rpm for 30min, and detect the hyaluronic acid molecular weight of 2.17MDa by gel permeation chromatography-multi-angle laser scattering instrument. Take the supernatant and slowly add 3 times volume (95%) of ethanol to precipitate the hyaluronic acid in the fermentation liquid, collect the hyaluronic acid precipitate, dissolve the precipitate with 0.2M sodium chloride solution, add 0.5% 767# needle-shaped activated carbon for adsorption for 10min, filter with perlite as a pre-coating layer, and filter to obtain a transparent and clear sodium hyaluronate test solution. Dilute the test solution with 0.2M sodium chloride solution to a hyaluronic acid concentration of 0.09g / L, and determine the hyaluronic acid molecular weight by gel permeation chromatography. Analysis conditions: Instrument: Agilent 1260 Infinity HPLC system; Chromatographic column: gel chromatography column (Shodex SB-806HQ (8.0mm×300mm); Mobile phase: 0.2M sodium chloride solution; Liquid flow rate: 0.7ml / min; Injection volume: 100ul; Detector: Differential refractive index detector (RID); Analysis steps: Take sodium polystyrene sulfonate of known different molecular weights, use the mobile phase to prepare a 1mg / ml reference solution, take 100ul of each of the test solution and the reference solution, record the chromatogram, and draw a calibration curve using GPC software. Using the universal calibration method, the K value of the reference is 0.00018, and the α value is 0.65; the K value of sodium hyaluronate is 0.00057, and the α value is 0.75. Calculate the results and obtain the molecular weight of sodium hyaluronate.

[0042] Example 5: Take 20ml of hyaluronic acid fermentation liquid fermented by Escherichia coli, dilute with 7 times volume of pure water, centrifuge at 10000rpm for 30min, and detect the hyaluronic acid molecular weight of 2.17MDa by gel permeation chromatography-multi-angle laser scattering instrument. Take the supernatant and slowly add 3 times volume (95%) of ethanol to precipitate the hyaluronic acid in the fermentation liquid. Collect the hyaluronic acid precipitate, dissolve the precipitate with 0.2M sodium chloride solution, add 0.5% 767# needle-shaped activated carbon for adsorption for 30min, filter with perlite as a pre-coating layer with a thickness of 3cm, and filter to obtain a transparent and clear sodium hyaluronate test solution. Dilute the test solution with 0.2M sodium chloride solution to a hyaluronic acid concentration of 0.03g / L, and determine the hyaluronic acid molecular weight by gel permeation chromatography. Analysis conditions: Instrument: Agilent 1260 Infinity HPLC system; Chromatographic column: gel chromatography column (Shodex SB-806HQ (8.0mm×300mm); Mobile phase: 0.2M sodium chloride solution; Flow rate: 0.7ml / min; Injection volume: 100ul; Detector: Differential refractive index detector (RID); Analysis steps: Take sodium polystyrene sulfonate of known different molecular weights, prepare a 1mg / ml reference solution with the mobile phase, take 100ul of each of the test solution and the reference solution, record the chromatogram, and draw a calibration curve using GPC software. Using the universal calibration method, the K value of the reference is 0.00018, and the α value is 0.65; the K value of sodium hyaluronate is 0.00057, and the α value is 0.75. Calculate the results and obtain the molecular weight of sodium hyaluronate.

[0043] Example 6: Take 20ml of hyaluronic acid fermentation liquid fermented by Escherichia coli, dilute with 8 times volume of pure water, centrifuge at 10000rpm for 30min, and detect the hyaluronic acid molecular weight of 2.17MDa by gel permeation chromatography-multi-angle laser scattering instrument. Take the supernatant and slowly drip 3 times volume (95%) of ethanol to precipitate the hyaluronic acid in the fermentation liquid. Collect the hyaluronic acid precipitate, dissolve the precipitate with 0.2M sodium chloride solution, add 0.5% 767# needle-shaped activated carbon for adsorption for 60min, filter with perlite as a pre-coating layer with a thickness of 3cm, and filter to obtain a transparent and clear sodium hyaluronate test solution. Dilute the test solution with 0.2M sodium chloride solution to a hyaluronic acid concentration of 0.06g / L, and determine the hyaluronic acid molecular weight by gel permeation chromatography. Analysis conditions: Instrument: Agilent 1260 Infinity HPLC system; Chromatographic column: gel chromatography column (Shodex SB-806HQ (8.0mm×300mm); Mobile phase: 0.2M sodium chloride solution; Flow rate: 0.7ml / min; Injection volume: 100ul; Detector: Differential refractive index detector (RID); Analysis steps: Take sodium polystyrene sulfonate of known different molecular weights, prepare a 1mg / ml reference solution with the mobile phase, take 100ul of each of the test solution and the reference solution, record the chromatogram, and draw a calibration curve using GPC software. Using the universal calibration method, the K value of the reference is 0.00018, and the α value is 0.65; the K value of sodium hyaluronate is 0.00057, and the α value is 0.75. Calculate the results and obtain the molecular weight of sodium hyaluronate.

[0044] Example 7: Take 20ml of hyaluronic acid fermentation liquid fermented by Escherichia coli, dilute with 8 times volume of pure water, centrifuge at 10000rpm for 30min, and detect the hyaluronic acid molecular weight of 2.17MDa by gel permeation chromatography-multi-angle laser scattering instrument. Take the supernatant and slowly add 3 times volume (95%) of ethanol to precipitate the hyaluronic acid in the fermentation liquid. Collect the hyaluronic acid precipitate, dissolve the precipitate with 0.2M sodium chloride solution, add 0.8% 767# needle-shaped activated carbon for adsorption for 10min, filter with perlite as a pre-coating layer with a thickness of 3cm, and filter to obtain a transparent and clear sodium hyaluronate test solution. Dilute the test solution with 0.2M sodium chloride solution to a hyaluronic acid concentration of 0.06g / L, and determine the hyaluronic acid molecular weight by gel permeation chromatography. Analysis conditions: Instrument: Agilent 1260 Infinity HPLC system; Chromatographic column: gel chromatography column (Shodex SB-806HQ (8.0mm×300mm); Mobile phase: 0.2M sodium chloride solution; Flow rate: 0.7ml / min; Injection volume: 100ul; Detector: Differential refractive index detector (RID); Analysis steps: Take sodium polystyrene sulfonate of known different molecular weights, prepare a 1mg / ml reference solution with the mobile phase, take 100ul of each of the test solution and the reference solution, record the chromatogram, and draw a calibration curve using GPC software. Using the universal calibration method, the K value of the reference is 0.00018, and the α value is 0.65; the K value of sodium hyaluronate is 0.00057, and the α value is 0.75. Calculate the results and obtain the molecular weight of sodium hyaluronate.

[0045] Example 8: Take 20ml of hyaluronic acid fermentation liquid fermented by Escherichia coli, dilute with 9 times volume of pure water, centrifuge at 10000rpm for 30min, and detect the hyaluronic acid molecular weight of 2.17MDa by gel permeation chromatography-multi-angle laser scattering instrument. Take the supernatant and slowly drip 3 times volume (95%) of ethanol to precipitate the hyaluronic acid in the fermentation liquid. Collect the hyaluronic acid precipitate, dissolve the precipitate with 0.2M sodium chloride solution, add 0.8% 767# needle-shaped activated carbon for adsorption for 30min, filter with perlite as a pre-coating layer with a thickness of 3cm, and filter to obtain a transparent and clear sodium hyaluronate test solution. Dilute the test solution with 0.2M sodium chloride solution to a hyaluronic acid concentration of 0.09g / L, and determine the hyaluronic acid molecular weight by gel permeation chromatography. Analysis conditions: Instrument: Agilent 1260 Infinity HPLC system; Chromatographic column: gel chromatography column (Shodex SB-806HQ (8.0mm×300mm); Mobile phase: 0.2M sodium chloride solution; Flow rate: 0.7ml / min; Injection volume: 100ul; Detector: Differential refractive index detector (RID); Analysis steps: Take sodium polystyrene sulfonate of known different molecular weights, prepare a 1mg / ml reference solution with the mobile phase, take 100ul of each of the test solution and the reference solution, record the chromatogram, and draw a calibration curve using GPC software. Using the universal calibration method, the K value of the reference is 0.00018, and the α value is 0.65; the K value of sodium hyaluronate is 0.00057, and the α value is 0.75. Calculate the results and obtain the molecular weight of sodium hyaluronate.

[0046] Example 9: Take 20ml of hyaluronic acid fermentation liquid fermented by Escherichia coli, dilute with 10 times the volume of pure water, centrifuge at 10000rpm for 30min, and detect the hyaluronic acid molecular weight of 2.17MDa by gel permeation chromatography-multi-angle laser scattering instrument. Take the supernatant and slowly add 3 times the volume (95%) of ethanol to precipitate the hyaluronic acid in the fermentation liquid. Collect the hyaluronic acid precipitate, dissolve the precipitate with 0.2M sodium chloride solution, add 0.8% 767# needle-shaped activated carbon for adsorption for 60min, filter with perlite as a pre-coating layer, and filter to obtain a transparent and clear sodium hyaluronate test solution. Dilute the test solution with 0.2M sodium chloride solution to a hyaluronic acid concentration of 0.03g / L, and determine the hyaluronic acid molecular weight by gel permeation chromatography. Analysis conditions: Instrument: Agilent 1260 Infinity HPLC system; Chromatographic column: gel chromatography column (Shodex SB-806HQ (8.0mm×300mm); Mobile phase: 0.2M sodium chloride solution; Flow rate: 0.7ml / min; Injection volume: 100ul; Detector: Differential refractive index detector (RID); Analysis steps: Take sodium polystyrene sulfonate of known different molecular weights, prepare a 1mg / ml reference solution with the mobile phase, take 100ul of each of the test solution and the reference solution, record the chromatogram, and draw a calibration curve using GPC software. Using the universal calibration method, the K value of the reference is 0.00018, and the α value is 0.65; the K value of sodium hyaluronate is 0.00057, and the α value is 0.75. Calculate the results and obtain the molecular weight of sodium hyaluronate.

[0047] Comparative Example 1, intrinsic viscosity method

[0048] Purification method: Take 20 ml of hyaluronic acid fermentation liquid fermented by Escherichia coli, dilute it with 5 times the volume of pure water, centrifuge at 10000rpm for 30 minutes, filter it with diatomaceous earth as a pre-coating layer and a thickness of 5 cm, slowly add 2 times the volume (95%) of ethanol to the filtrate for precipitation, dissolve the precipitate with 0.2M sodium chloride solution, add 0.5% 767# needle-shaped activated carbon for adsorption for 60 minutes, filter it with diatomaceous earth as a pre-coating layer and a thickness of 3 cm, add 1-3% of the volume of hexadecyl Trimethylammonium bromide (CTAB) was used for complexation for 30 minutes. After standing for 1 hour, the supernatant was poured out, and 5% sodium chloride solution (mass ratio) was added for dissociation for 30 minutes. After adding 0.3% 767# needle-shaped activated carbon for adsorption for 60 minutes, diatomaceous earth was used as a pre-coating layer, and the filter was filtered with a thickness of 3 cm. Two volumes (95%) of ethanol were added to the filtrate for precipitation. The precipitate was filtered with a sand core funnel, dehydrated with anhydrous ethanol twice, and placed in a vacuum drying oven (40°C) for drying for 12 hours to obtain hyaluronic acid powder.

[0049] Molecular weight determination method: Weigh the dried hyaluronic acid powder, dissolve it in 0.2M sodium chloride solution to obtain a sample solution, filter the solvent and the sample solution through a G-type vertical melting funnel, discard the primary filtrate, take 20ml of their subsequent filtrates, and use an Ubbelohde viscometer with a capillary inner diameter of 0.53mm in a constant temperature water bath at 25°C to measure the outflow time T0 of the blank solvent and the outflow time Ti of the sample solution, respectively. Measure three times each (the difference between the three values ​​shall not exceed 0.2 seconds), take the average value, and then calculate the intrinsic viscosity: η=Ti / T0, Ti / T0 should meet the following two conditions: 1) T0>100 seconds 2) Ti / T0=1.3~1.5

[0050] The intrinsic viscosity value η is calculated according to the following formula. The intrinsic viscosity is used to characterize the molecular weight (M) of sodium hyaluronate. The relationship between the two is: η = 0.036M 0.78

[0051]

[0052] Where: η—intrinsic viscosity (cm 3 / g)

[0053] Ti—average outflow time of the test solution (s)

[0054] T0—Blank reference substance (0.2M sodium chloride solution) elution time (s)

[0055] C—HA (hyaluronic acid) content (calculated on a dry basis)

[0056] HA content determination (carbazole sulfate method)

[0057] Preparation of standard reference solution: Accurately weigh about 0.1g of glucuronic acid standard reference substance that has been vacuum dried to constant weight at 105°C with phosphorus pentoxide as a desiccant, place it in a 100ml volumetric flask, add water to dissolve and dilute to scale, shake well, and use it as a stock solution. Accurately measure 5.0ml of the stock solution, place it in a 100ml volumetric flask, add water to dilute to scale, and shake well to make a solution containing about 50g of standard substance per 1ml.

[0058] Preparation of test solution: Accurately weigh about 0.10g of hyaluronic acid powder that has been vacuum dried at 40℃ for 12h, place it in a 100ml volumetric flask, add appropriate amount of water to swell the sample, add water to the scale after it is completely dissolved, and shake well. Then weigh about 4.0g of the solution (the density of the solution is calculated as 1.000g / ml, which is equivalent to 4.00ml) and place it in a 50ml volumetric flask, add water to dilute to the scale, shake well, and use it as the test solution.

[0059] Preparation of standard curve: Accurately measure 0.0, 0.2, 0.4, 0.6, 0.8 and 1.0 ml of standard reference solution and place them in stoppered test tubes respectively. Add water to each tube to 1.0 ml. Mix well and cool to below 4°C with ice water. Slowly add 5.0 ml of 0.025 mol / L borax sulfuric acid solution cooled to below 4°C under shaking. Mix well and heat in a boiling water bath for 10 minutes and cool in ice water. Accurately add 0.20 ml of carbazole test solution, shake well, heat in a boiling water bath for 15 minutes and cool to room temperature in ice water. Use UV-visible spectrophotometer to measure absorbance at a wavelength of 530 nm. Calculate the linear regression equation and correlation coefficient (r>0.990) by the concentration of glucuronic acid (g / m1) and absorbance. Figure 3 This is the glucuronic acid standard curve obtained by linear fitting based on the standard data.

[0060] Sample content determination: accurately weigh 1.0 ml of the test solution and place it in a stoppered test tube. Prepare two test tubes in parallel and operate in the same way as under the standard curve starting from "cool with ice water to below 4°C". After measuring the absorbance, the concentration of glucuronic acid in the test solution is obtained by the linear regression equation, and the content of hyaluronic acid in the sample is calculated according to the following formula.

[0061]

[0062] In the formula: ρ: the amount of glucuronic acid in the sample tube (μg / ml); m: the sample amount (mg).

[0063] Comparative Example 2: A hyaluronic acid solution was obtained by the purification method of Example 1, and 2 volumes of anhydrous ethanol were added for precipitation and dehydration, and the solution was placed in a vacuum drying oven (40° C.) for drying for 12 hours to obtain hyaluronic acid powder. The molecular weight of hyaluronic acid was determined by the intrinsic viscosity method of Comparative Example 1.

[0064] Table 1 Main process parameters and test results

[0065]

[0066]

[0067] Remark:

[0068] A260: is the wavelength of the absorption peak of nucleic acid molecules, which is used to estimate the content and concentration of nucleic acid in the solution. A280: is the wavelength of the absorption peak of protein molecules, which is used to estimate the content and concentration of protein in the solution.

[0069] As can be seen from Table 1 above, impurities (protein and nucleic acid content) and sample injection concentration have a great influence on the molecular weight test results. Because the product of hyaluronic acid fermentation broth is a high molecular polymer, the difference in dilution volume causes different degrees of separation of high molecular polysaccharides and other substances. At the same time, the injection concentration during detection will also cause differences in the degree of folding of its spatial conformation, resulting in different molecular weight detection structures. In addition, in addition to polysaccharides, macromolecular substances such as nucleic acids and proteins are also important factors that interfere with the test results. Activated carbon is an effective medium for adsorbing high molecular substances. Its degree of activation, sample adsorption time and amount of activated carbon will intercept high molecular substances to varying degrees, resulting in a small molecular weight test result. Therefore, in order to achieve relatively stable and accurate detection of the molecular weight of hyaluronic acid after fermentation, the type, amount and adsorption time of activated carbon are crucial.

[0070] Compared with Comparative Example 1, the purification and detection method of the present invention is simpler and faster, and the experimental error is small. Comparative Example 2 also reflects that the commonly used intrinsic viscosity method has high requirements for the purity of the measured sample, which is difficult to meet the needs of rapid detection in actual production.

[0071] Comparing the GPC test results of each embodiment with the test results of the multi-angle laser scattering instrument, the process of embodiment 1 is the preferred option. The description of the above embodiments is only used to help understand the method of the present invention and its core concept.

[0072] The contents not described in detail in the present application description belong to the common knowledge of those skilled in the art.

[0073] As mentioned in the entire specification and claims, "including" is an open term and should be interpreted as "including but not limited to". "Substantially" means that within an acceptable error range, those skilled in the art can solve the technical problem within a certain error range and basically achieve the technical effect.

[0074] It should also be noted that the terms "include", "comprises" or any other variations thereof are intended to cover non-exclusive inclusion, so that a product or system including a series of elements includes not only those elements, but also other elements not explicitly listed, or also includes elements inherent to such a product or system. In the absence of more restrictions, the elements defined by the sentence "comprises a ..." do not exclude the existence of other identical elements in the product or system including the elements.

[0075] The above description shows and describes several preferred embodiments of the present application, but as mentioned above, it should be understood that the present application is not limited to the form disclosed herein, and should not be regarded as excluding other embodiments, but can be used in various other combinations, modifications and environments, and can be modified within the scope of the invention concept described herein through the above teachings or the technology or knowledge of the relevant field. The changes and modifications made by those skilled in the art do not depart from the spirit and scope of the present application, and should be protected by the claims attached to the present application.

Claims

1. A method for rapidly detecting the molecular weight of hyaluronic acid in a fermentation broth, characterized in that: The steps are as follows: 1) diluting the hyaluronic acid fermentation liquid with water, centrifuging at high speed, taking the supernatant and adding ethanol to precipitate, taking the precipitate and dissolving it with sodium chloride solution, adding activated carbon for adsorption, and filtering with perlite as a pre-coating layer to obtain a hyaluronic acid test solution; 2) Use gel permeation chromatography to determine the elution time of the hyaluronic acid test solution and several sodium polystyrene sulfonate reference substances with different molecular weights, record the chromatogram, draw a calibration curve, and obtain the molecular weight of sodium hyaluronate by the universal calibration method.

2. The method according to claim 1, characterized in that In the step 1), the hyaluronic acid fermentation liquid is Escherichia coli hyaluronic acid fermentation liquid, and the volume after dilution is 5-10 times of the original volume, preferably 5 times.

3. The method according to claim 1, characterized in that In the step 1), the amount of ethanol added is 2-3 times, preferably 3 times, the volume of the supernatant; the concentration of ethanol is 75%-95%, preferably 95%.

4. The method according to claim 1, characterized in that: In the step 1), the concentration of the sodium chloride solution is 0.2M to 0.5M, preferably 0.2M.

5. The method according to claim 1, characterized in that In the step 1), the activated carbon is 767# needle-shaped activated carbon, and the amount used is 0.2%-0.8% of the volume of the solution, preferably 0.2%.

6. The method according to claim 1, characterized in that In the step 1), the adsorption time is 10 min to 60 min, preferably 10 min.

7. The method according to claim 1, characterized in that In the step 1), the thickness of the perlite pre-coating layer is 3-5 cm, preferably 3 cm; in the step 2), both the hyaluronic acid test solution and the reference solution need to be filtered through a 0.22um-0.45um filter membrane, preferably 0.22um.

8. The method according to claim 1, characterized in that In the step 2), the concentration of the hyaluronic acid sample is below 0.1 g / L, preferably 0.03-0.09 g / L, and more preferably 0.03 g / L.

9. The method according to claim 1, characterized in that: In the step 2), the minimum molecular weight of the hyaluronic acid is not less than 100 MDa.

10. The method according to claim 1, characterized in that The specific method of the step 2) gel permeation chromatography is: Analysis conditions: Agilent 1260 Infinity high performance liquid chromatography system; Chromatographic column: gel chromatography column, Shodex SB-806HQ 8.0mm×300mm; Mobile phase: 0.2M sodium chloride solution Flow rate: 0.3-0.7ml / min; Injection volume: 50-100ul; Detector: Refractive Index Detector (RID) Analysis steps: Take sodium polystyrene sulfonate of known different molecular weights, prepare a 1 mg / ml reference solution with the mobile phase, inject 100 ul of each test solution and reference solution, record the chromatogram, and draw a calibration curve; using the universal calibration method, the K value of the reference is 0.00018, and the α value is 0.65; the K value of sodium hyaluronate is 0.00057, and the α value is 0.75; finally, the molecular weight of sodium hyaluronate is calculated.

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