CN111562231B - Method for measuring molecular weight of hyaluronic acid - Google Patents
Method for measuring molecular weight of hyaluronic acid Download PDFInfo
- Publication number
- CN111562231B CN111562231B CN202010573896.8A CN202010573896A CN111562231B CN 111562231 B CN111562231 B CN 111562231B CN 202010573896 A CN202010573896 A CN 202010573896A CN 111562231 B CN111562231 B CN 111562231B
- Authority
- CN
- China
- Prior art keywords
- hyaluronic acid
- molecular weight
- enzymatic hydrolysate
- measuring
- hyaluronate
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
- KIUKXJAPPMFGSW-DNGZLQJQSA-N (2S,3S,4S,5R,6R)-6-[(2S,3R,4R,5S,6R)-3-Acetamido-2-[(2S,3S,4R,5R,6R)-6-[(2R,3R,4R,5S,6R)-3-acetamido-2,5-dihydroxy-6-(hydroxymethyl)oxan-4-yl]oxy-2-carboxy-4,5-dihydroxyoxan-3-yl]oxy-5-hydroxy-6-(hydroxymethyl)oxan-4-yl]oxy-3,4,5-trihydroxyoxane-2-carboxylic acid Chemical compound CC(=O)N[C@H]1[C@H](O)O[C@H](CO)[C@@H](O)[C@@H]1O[C@H]1[C@H](O)[C@@H](O)[C@H](O[C@H]2[C@@H]([C@@H](O[C@H]3[C@@H]([C@@H](O)[C@H](O)[C@H](O3)C(O)=O)O)[C@H](O)[C@@H](CO)O2)NC(C)=O)[C@@H](C(O)=O)O1 KIUKXJAPPMFGSW-DNGZLQJQSA-N 0.000 title claims abstract description 194
- 229920002674 hyaluronan Polymers 0.000 title claims abstract description 194
- 229960003160 hyaluronic acid Drugs 0.000 title claims abstract description 194
- 238000000034 method Methods 0.000 title claims abstract description 60
- 239000007788 liquid Substances 0.000 claims abstract description 36
- 238000002835 absorbance Methods 0.000 claims abstract description 33
- 238000000862 absorption spectrum Methods 0.000 claims abstract description 17
- 230000002255 enzymatic effect Effects 0.000 claims description 79
- 239000000413 hydrolysate Substances 0.000 claims description 50
- WCDDVEOXEIYWFB-VXORFPGASA-N (2s,3s,4r,5r,6r)-3-[(2s,3r,5s,6r)-3-acetamido-5-hydroxy-6-(hydroxymethyl)oxan-2-yl]oxy-4,5,6-trihydroxyoxane-2-carboxylic acid Chemical compound CC(=O)N[C@@H]1C[C@H](O)[C@@H](CO)O[C@H]1O[C@@H]1[C@@H](C(O)=O)O[C@@H](O)[C@H](O)[C@H]1O WCDDVEOXEIYWFB-VXORFPGASA-N 0.000 claims description 42
- 229940014041 hyaluronate Drugs 0.000 claims description 42
- 108010003272 Hyaluronate lyase Proteins 0.000 claims description 40
- 102000001974 Hyaluronidases Human genes 0.000 claims description 40
- 229960002773 hyaluronidase Drugs 0.000 claims description 40
- 239000000243 solution Substances 0.000 claims description 39
- 108010009736 Protein Hydrolysates Proteins 0.000 claims description 24
- 230000001580 bacterial effect Effects 0.000 claims description 23
- 102000004190 Enzymes Human genes 0.000 claims description 19
- 108090000790 Enzymes Proteins 0.000 claims description 19
- 229940088598 enzyme Drugs 0.000 claims description 19
- 241000894006 Bacteria Species 0.000 claims description 17
- 230000007071 enzymatic hydrolysis Effects 0.000 claims description 15
- 238000006047 enzymatic hydrolysis reaction Methods 0.000 claims description 15
- 239000002994 raw material Substances 0.000 claims description 13
- SQDAZGGFXASXDW-UHFFFAOYSA-N 5-bromo-2-(trifluoromethoxy)pyridine Chemical compound FC(F)(F)OC1=CC=C(Br)C=N1 SQDAZGGFXASXDW-UHFFFAOYSA-N 0.000 claims description 10
- 229920001287 Chondroitin sulfate Polymers 0.000 claims description 10
- 229940059329 chondroitin sulfate Drugs 0.000 claims description 10
- 238000010790 dilution Methods 0.000 claims description 10
- 239000012895 dilution Substances 0.000 claims description 10
- 238000003776 cleavage reaction Methods 0.000 claims description 7
- 230000007017 scission Effects 0.000 claims description 7
- 241000193830 Bacillus <bacterium> Species 0.000 claims description 6
- 238000007865 diluting Methods 0.000 claims description 4
- 238000000569 multi-angle light scattering Methods 0.000 claims description 4
- 239000003085 diluting agent Substances 0.000 claims description 3
- 230000000415 inactivating effect Effects 0.000 claims description 3
- 241000194110 Bacillus sp. (in: Bacteria) Species 0.000 claims description 2
- 238000000855 fermentation Methods 0.000 claims 1
- 230000004151 fermentation Effects 0.000 claims 1
- 150000003839 salts Chemical class 0.000 abstract description 26
- 238000001514 detection method Methods 0.000 abstract description 11
- 238000000691 measurement method Methods 0.000 abstract description 6
- 239000003814 drug Substances 0.000 abstract description 2
- 229920002385 Sodium hyaluronate Polymers 0.000 description 14
- 229940010747 sodium hyaluronate Drugs 0.000 description 14
- YWIVKILSMZOHHF-QJZPQSOGSA-N sodium;(2s,3s,4s,5r,6r)-6-[(2s,3r,4r,5s,6r)-3-acetamido-2-[(2s,3s,4r,5r,6r)-6-[(2r,3r,4r,5s,6r)-3-acetamido-2,5-dihydroxy-6-(hydroxymethyl)oxan-4-yl]oxy-2-carboxy-4,5-dihydroxyoxan-3-yl]oxy-5-hydroxy-6-(hydroxymethyl)oxan-4-yl]oxy-3,4,5-trihydroxyoxane-2- Chemical compound [Na+].CC(=O)N[C@H]1[C@H](O)O[C@H](CO)[C@@H](O)[C@@H]1O[C@H]1[C@H](O)[C@@H](O)[C@H](O[C@H]2[C@@H]([C@@H](O[C@H]3[C@@H]([C@@H](O)[C@H](O)[C@H](O3)C(O)=O)O)[C@H](O)[C@@H](CO)O2)NC(C)=O)[C@@H](C(O)=O)O1 YWIVKILSMZOHHF-QJZPQSOGSA-N 0.000 description 14
- 238000004519 manufacturing process Methods 0.000 description 9
- OYPRJOBELJOOCE-UHFFFAOYSA-N Calcium Chemical compound [Ca] OYPRJOBELJOOCE-UHFFFAOYSA-N 0.000 description 5
- FYYHWMGAXLPEAU-UHFFFAOYSA-N Magnesium Chemical compound [Mg] FYYHWMGAXLPEAU-UHFFFAOYSA-N 0.000 description 5
- ZLMJMSJWJFRBEC-UHFFFAOYSA-N Potassium Chemical compound [K] ZLMJMSJWJFRBEC-UHFFFAOYSA-N 0.000 description 5
- 239000011575 calcium Substances 0.000 description 5
- 229910052791 calcium Inorganic materials 0.000 description 5
- 239000011777 magnesium Substances 0.000 description 5
- 229910052749 magnesium Inorganic materials 0.000 description 5
- 239000011591 potassium Substances 0.000 description 5
- 229910052700 potassium Inorganic materials 0.000 description 5
- 238000005070 sampling Methods 0.000 description 5
- 238000003556 assay Methods 0.000 description 4
- 238000006243 chemical reaction Methods 0.000 description 3
- 238000005520 cutting process Methods 0.000 description 3
- 238000012544 monitoring process Methods 0.000 description 3
- 238000012360 testing method Methods 0.000 description 3
- 238000010521 absorption reaction Methods 0.000 description 2
- 230000015556 catabolic process Effects 0.000 description 2
- 238000006731 degradation reaction Methods 0.000 description 2
- 239000008213 purified water Substances 0.000 description 2
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 2
- 229920002683 Glycosaminoglycan Polymers 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- 238000009835 boiling Methods 0.000 description 1
- 238000005336 cracking Methods 0.000 description 1
- 238000013480 data collection Methods 0.000 description 1
- 238000009826 distribution Methods 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 238000002356 laser light scattering Methods 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 230000001105 regulatory effect Effects 0.000 description 1
- 239000012266 salt solution Substances 0.000 description 1
- 238000001542 size-exclusion chromatography Methods 0.000 description 1
- 210000003491 skin Anatomy 0.000 description 1
- 238000011895 specific detection Methods 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 210000003954 umbilical cord Anatomy 0.000 description 1
- 210000004127 vitreous body Anatomy 0.000 description 1
Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/17—Systems in which incident light is modified in accordance with the properties of the material investigated
- G01N21/25—Colour; Spectral properties, i.e. comparison of effect of material on the light at two or more different wavelengths or wavelength bands
- G01N21/31—Investigating relative effect of material at wavelengths characteristic of specific elements or molecules, e.g. atomic absorption spectrometry
- G01N21/33—Investigating relative effect of material at wavelengths characteristic of specific elements or molecules, e.g. atomic absorption spectrometry using ultraviolet light
Landscapes
- Physics & Mathematics (AREA)
- Spectroscopy & Molecular Physics (AREA)
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Immunology (AREA)
- Pathology (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The application discloses a method for measuring hyaluronic acid molecular weight, and relates to the technical field of biological medicine detection. The measurement method is to measure the molecular weight of hyaluronic acid in a hyaluronic acid solution based on the absorbance of the ultraviolet absorption spectrum of the hyaluronic acid solution. The method solves the problems that the prior method for measuring the molecular weight of the hyaluronic acid is complex and has limited detection range, and the molecular weight of the hyaluronic acid and the salt thereof in the hyaluronic acid enzymolysis liquid can not be rapidly detected.
Description
Technical Field
The application relates to the technical field of biological medicine detection, in particular to a method for measuring hyaluronic acid molecular weight.
Background
Hyaluronic Acid (HA) is also called Hyaluronic acid or Hyaluronic acid, and is a linear glycosaminoglycan with high molecular weight composed of (1-3) -2-acetamido-2-deoxy-D-glucose (1-4) -D-glucuronic acid disaccharide repeating units, widely existing in vitreous bodies, skin and umbilical cord of human and animals, and is an indispensable key functional substance in human body. Hyaluronate has many different functions depending on its molecular weight. Generally, we define hyaluronic acid with a molecular weight of ∈1000kDa as high molecular hyaluronic acid, hyaluronic acid with a molecular weight of 10kDa to 1000kDa as low molecular hyaluronic acid, and oligomeric hyaluronic acid with a molecular weight of ∈10 kDa.
The macromolecular hyaluronic acid can be used for preparing low-molecular and oligomeric hyaluronic acid and salts thereof by enzymolysis of hyaluronidase. In the process of producing low-molecular and oligomeric hyaluronic acid and salts thereof by adopting hyaluronidase enzymolysis, the hyaluronic acid and salts thereof in the enzymolysis liquid need to be monitored in real time so as to finally obtain the low-molecular and oligomeric hyaluronic acid and salts thereof with the required target molecular weight. However, currently, the molecular weight of hyaluronic acid and its salts is usually detected by using a multi-angle laser light scattering method (SEC-MALLS), i.e. HA in each molecular weight section in the solution is first separated by size exclusion chromatography, and then the related parameters are measured by a laser light scattering device and a differential detector, and the molecular weight and molecular weight distribution of HA are calculated by software. The Laurent method is a method for calculating the molecular weight of hyaluronic acid by detecting the viscosity of hyaluronic acid solution according to the conversion relation between the viscosity and the molecular weight of hyaluronic acid, but when the concentration of hyaluronic acid and its salt solution is low or the molecular weight is lower than 50kDa, the viscosity of hyaluronic acid solution is too low to detect the molecular weight. Therefore, the current determination method cannot monitor the molecular weight of hyaluronic acid and the salt thereof with the molecular weight smaller than 50kDa in the enzymolysis liquid in real time.
Disclosure of Invention
The embodiment of the application solves the problems that the conventional measuring method is complex and limited in detection range, and can not rapidly detect the molecular weight of hyaluronic acid and salt thereof in the hyaluronic acid enzymolysis liquid.
In order to achieve the above purpose, the embodiment of the present application mainly provides the following technical solutions:
the embodiment of the application provides a method for measuring the molecular weight of hyaluronic acid, which is used for measuring the molecular weight of hyaluronic acid in a hyaluronic acid solution based on the absorbance of an ultraviolet absorption spectrum of the hyaluronic acid solution.
Preferably, the absorbance is the absorbance of the hyaluronic acid solution at 232 nm.
Preferably, the hyaluronic acid solution is a hyaluronic acid enzymatic hydrolysate produced by enzymatic cleavage of hyaluronic acid.
Preferably, the method for measuring the molecular weight of hyaluronic acid comprises the following steps:
establishing a functional relation 'M-S function' between an S value of the hyaluronic acid enzymatic hydrolysate and the molecular weight M of hyaluronic acid in the hyaluronic acid enzymatic hydrolysate, wherein the S value is calculated by the following formula:
wherein A is absorbance of ultraviolet absorption spectrum of diluent of the hyaluronic acid enzymatic hydrolysate, V is total volume of the hyaluronic acid enzymatic hydrolysate, T is dilution multiple of the hyaluronic acid enzymatic hydrolysate, H is moisture content of raw material hyaluronate for enzymatic hydrolysis of the hyaluronic acid enzymatic hydrolysate, W is mass of raw material hyaluronate for enzymatic hydrolysis of the hyaluronic acid enzymatic hydrolysate, and R is molecular weight ratio of hyaluronic acid in hyaluronate;
inactivating and diluting the hyaluronic acid enzymatic hydrolysate sample to be detected, measuring the absorbance A of the ultraviolet absorption spectrum, calculating the S value of the hyaluronic acid enzymatic hydrolysate sample to be detected according to the formula, and calculating the molecular weight of hyaluronic acid in the hyaluronic acid enzymatic hydrolysate sample to be detected according to the S value and the M-S function of the hyaluronic acid enzymatic hydrolysate sample to be detected.
Preferably, the absorbance of the diluted solution at 232nm is in the range of 0.2 to 1.0 after the hyaluronic acid enzymatic hydrolysate is diluted.
Preferably, the raw material for enzymolysis of the hyaluronic acid enzymolysis liquid is hyaluronate with molecular weight more than or equal to 3kDa obtained by enzymolysis of bacterial hyaluronidase or bacterial chondroitin sulfate enzyme, or hyaluronate with molecular weight more than or equal to 200kDa obtained by enzymolysis of non-hyaluronidase or non-chondroitin sulfate enzyme.
Preferably, the molecular weight M of the hyaluronic acid in the "M-S function" is in the range of 3kDa to 50kDa.
Preferably, the mass fraction of the hyaluronic acid in the hyaluronic acid enzymatic hydrolysate is 1% -50%.
Preferably, when the "M-S function" is established, the molecular weight M of hyaluronic acid in the hyaluronic acid enzymatic hydrolysate is measured by the SEC-MALLS method or the Laurent method.
Preferably, the hyaluronic acid enzymatic hydrolysate is an enzymatic hydrolysate produced by the cleavage of hyaluronic acid by bacterial hyaluronidase or bacterial chondroitin sulfate enzyme.
Preferably, the bacterial hyaluronidase is a bacillus hyaluronidase.
Preferably, the bacterium produces hyaluronidase which is produced by fermenting Bacillus (Bacillus sp.) A50 CGMCC No. 5744.
One or more technical solutions provided in the embodiments of the present application at least have the following technical effects or advantages:
according to the determination method, the functional relation between the absorbance A of the ultraviolet absorption spectrum of the hyaluronic acid enzymatic hydrolysate and the molecular weight M of the hyaluronic acid is established, and the molecular weights of the hyaluronic acid and the salt thereof in the enzymatic hydrolysate can be calculated and obtained by determining the absorbance of the ultraviolet absorption spectrum of the enzymatic hydrolysate in the production process, and the detection process can obtain the results in a few minutes, so that the real-time monitoring of the molecular weights of the hyaluronic acid and the salt thereof in the enzymatic hydrolysate is realized; the measuring method has low equipment requirement, does not need to adopt HPLC-MALLS or viscosity measuring value for detection on the production site, and reduces the detection cost.
Drawings
FIG. 1 is a graph showing the "M-S function" between the molecular weight M of hyaluronic acid and the S value of the enzymatic hydrolysate of hyaluronic acid in the examples of the present application.
Detailed Description
In order to facilitate understanding of the present application by those skilled in the art, the following description will further illustrate the present application with reference to specific examples, which are to be construed as limiting the scope of the application.
The embodiment of the application solves the problems that the conventional measuring method is complex and limited in detection range, and can not rapidly detect the molecular weight of hyaluronic acid and salt thereof in the hyaluronic acid enzymolysis liquid.
In the process of producing low-molecular and oligomeric hyaluronic acid and salts thereof by enzymatic cleavage, real-time monitoring of the hyaluronic acid and salts thereof in the enzymatic hydrolysate is required in order to finally obtain the low-molecular and oligomeric hyaluronic acid and salts thereof of the desired target molecular weight. The bacterial hyaluronidase or bacterial chondroitin sulfate enzyme breaks the 1, 4-glycosidic bond in the (1-3) -2-acetamido-2-deoxy-D-glucose (1-4) -D-glucuronic acid disaccharide repeating unit to form unsaturated double bond, has specific ultraviolet absorption at 232nm, and can be measured by an ultraviolet spectrophotometer. In the process of preparing low-molecular and oligomeric hyaluronic acid and salt thereof by enzymolysis, along with the prolongation of the enzymolysis time of the hyaluronic acid and the salt thereof, the molecular weight of the hyaluronic acid and the salt thereof is gradually reduced, and the more unsaturated double bonds are generated in the hyaluronic acid solution, the higher the absorbance of the enzymolysis liquid at 232nm is.
Based on the principle, in order to conveniently and rapidly monitor the molecular weight of the hyaluronic acid and the salt thereof in the enzymolysis liquid, the embodiment of the application develops a method for calculating the molecular weight of the hyaluronic acid and the salt thereof in the enzymolysis liquid according to the absorbance of the ultraviolet absorption spectrum of the enzymolysis liquid by establishing the relation between the molecular weight of the hyaluronic acid in the hyaluronic acid enzymolysis liquid and the specific parameter of the enzymolysis liquid.
The technical scheme in the embodiment of the application aims to solve the problems, and the overall thought is as follows:
the embodiment of the application provides a method for measuring the molecular weight of hyaluronic acid, which is used for measuring the molecular weight of hyaluronic acid in a hyaluronic acid solution based on the absorbance of an ultraviolet absorption spectrum of the hyaluronic acid solution.
Since the hyaluronic acid obtained by enzymatic cleavage has a specific unsaturated double bond, has specific ultraviolet absorption, and as the molecular weight of the hyaluronic acid obtained by enzymatic cleavage is smaller, the more unsaturated double bonds are, the higher the absorbance is, and thus the molecular weight of the hyaluronic acid in the hyaluronic acid solution can be determined by measuring the absorbance thereof by an ultraviolet spectrophotometer. Because of the specific ultraviolet absorbance at 232nm of specific unsaturated double bonds in hyaluronic acid, the embodiment of the application preferably determines the absorbance of hyaluronic acid solution at 232 nm.
The determination method provided by the embodiment of the application is suitable for detecting the molecular weight of the hyaluronic acid obtained by enzymatic hydrolysis, and is more suitable for detecting the hyaluronic acid enzymatic hydrolysate generated by enzymatic hydrolysis so as to rapidly determine the molecular weight of the hyaluronic acid generated in the enzymatic hydrolysis process.
The measuring method provided by the embodiment of the application comprises the following steps:
establishing a functional relation 'M-S function' between an S value of the hyaluronic acid enzymatic hydrolysate and the molecular weight M of hyaluronic acid in the hyaluronic acid enzymatic hydrolysate, wherein the S value is calculated by the following formula:
wherein A is absorbance of ultraviolet absorption spectrum of diluent of the hyaluronic acid enzymatic hydrolysate, V is total volume of the hyaluronic acid enzymatic hydrolysate (L), T is dilution multiple of the hyaluronic acid enzymatic hydrolysate, H is moisture content (%) of raw material hyaluronate for enzymatic hydrolysis of the hyaluronic acid enzymatic hydrolysate, W is mass (kg) of raw material hyaluronate for enzymatic hydrolysis of the hyaluronic acid enzymatic hydrolysate, and R is molecular weight ratio of hyaluronic acid in hyaluronate, such as: the R value of sodium hyaluronate is 94.28%, the R value of potassium hyaluronate is 90.67%, the R value of magnesium hyaluronate is 96.93%, and the R value of calcium hyaluronate is 94.99%;
inactivating and diluting the hyaluronic acid enzymatic hydrolysate sample to be detected, measuring the absorbance A of the ultraviolet absorption spectrum, calculating the S value of the hyaluronic acid enzymatic hydrolysate sample to be detected according to the formula, and calculating the molecular weight of hyaluronic acid in the hyaluronic acid enzymatic hydrolysate sample to be detected according to the S value and the M-S function of the hyaluronic acid enzymatic hydrolysate sample to be detected.
According to the determination method provided by the embodiment of the application, the functional relation between the absorbance A of the ultraviolet absorption spectrum of the hyaluronic acid enzymatic hydrolysate and the molecular weight M of the hyaluronic acid is established, the molecular weight of the hyaluronic acid and the salt thereof in the enzymatic hydrolysate can be calculated and obtained by determining the absorbance of the ultraviolet absorption spectrum of the enzymatic hydrolysate in the production process, and the detection process can obtain the result in a few minutes, so that the real-time monitoring of the molecular weight of the hyaluronic acid and the salt thereof in the enzymatic hydrolysate is realized; the determination method has low requirements on equipment, does not need to adopt HPLC-MALLS to detect on a production site, and reduces the detection cost.
The measurement method provided by the embodiment of the application is suitable for the enzymolysis process of enzymes which can crack hyaluronic acid from various sources, preferably the hyaluronic acid enzymolysis liquid is the enzymolysis liquid generated after the hyaluronic acid is cracked by bacterial hyaluronidase or bacterial chondroitin sulfate enzyme, more preferably the hyaluronic acid enzymolysis liquid is the enzymolysis liquid generated after the hyaluronic acid is cracked by bacterial hyaluronidase. The bacterial hyaluronidase can be any kind of bacterial hyaluronidase, and the bacterial hyaluronidase is preferably Bacillus hyaluronidase, and more preferably the bacterial hyaluronidase is produced by fermenting Bacillus A50 CGMCC No. 5744.
The functional relation 'M-S function' between the S value of the hyaluronic acid enzymatic hydrolysate and the molecular weight M of the hyaluronic acid in the hyaluronic acid enzymatic hydrolysate is not influenced by the type and concentration of hyaluronate in the enzymatic hydrolysate, is not influenced by the enzymatic hydrolysis conditions and the enzymatic hydrolysis time, is influenced only by the enzymatic hydrolysis mode of the selected enzyme, namely, the mode of the selected enzyme for cracking the hyaluronic acid and the salt thereof is the influence of random degradation or terminal degradation, and adopts different enzymes to carry out enzymatic hydrolysis, so that the obtained 'M-S function' is different. After the types of enzymes selected for producing the enzyme-cleaved low-molecular and oligomeric hyaluronic acid and the salt thereof and the types of hyaluronate are determined, the M-S function applicable to the enzyme can be obtained through multiple data collection of the S value of the hyaluronate enzymatic hydrolysate and the molecular weight M of the hyaluronate, so that the molecular weight of the hyaluronate and the salt thereof in the production process of the enzyme-cleaved low-molecular and oligomeric hyaluronate and the salt thereof can be monitored.
In the enzymolysis process applicable to the measurement method of the embodiment of the application, the addition amount of the enzyme is any proper addition amount calculated according to the dosage, concentration, reaction condition, enzymolysis time and target hyaluronic acid molecular weight of the enzymolysis hyaluronic acid. The enzymolysis conditions are any conditions suitable for the reaction conditions of the enzyme used.
In the above measurement method provided by the embodiment of the application, the dilution factor of the hyaluronic acid enzymatic hydrolysate is calculated according to the initial concentration of hyaluronic acid, and the dilution factor can be 0-10000, and in the embodiment of the application, preferably, after the hyaluronic acid enzymatic hydrolysate is diluted, the absorbance of the dilution solution at 232nm is in the range of 0.2-1.0.
The embodiment of the application provides the measuring method which is suitable for the production process of obtaining low-molecular or oligomeric hyaluronic acid and salts thereof by enzymolysis of high-molecular hyaluronate. The preferred raw materials for enzymolysis of the hyaluronic acid enzymolysis liquid are hyaluronate with molecular weight more than or equal to 3kDa obtained by enzymolysis of bacterial hyaluronidase or bacterial chondroitin sulfate enzyme, or hyaluronate with molecular weight more than or equal to 200kDa obtained by enzymolysis of non-hyaluronidase or non-chondroitin sulfate enzyme.
The molecular weight range of the hyaluronic acid suitable for the M-S function in the determination method provided by the embodiment of the application is determined according to the factors such as the molecular weight of the hyaluronate raw material, the enzymolysis degree, the concentration of the enzymolysis liquid and the like in the actual enzymolysis process, so that the final detection result is within the required error range. In the embodiment of the application, the molecular weight M of the hyaluronic acid in the M-S function is preferably in the range of 3kDa to 50kDa.
The embodiment of the application provides the method for measuring the hyaluronic acid in the hyaluronic acid enzymatic hydrolysate, which is suitable for any concentration, and preferably the mass fraction of the hyaluronic acid in the hyaluronic acid enzymatic hydrolysate is 1% -50%.
In the measurement method provided by the embodiment of the application, when the 'M-S function' is established, any feasible method can be adopted to measure the molecular weight M of the hyaluronic acid in the hyaluronic acid enzymatic hydrolysate, and the SEC-MALLS method or the Laurent method is preferably adopted to measure the molecular weight M of the hyaluronic acid in the hyaluronic acid enzymatic hydrolysate.
In order to better understand the above technical solutions, the following detailed description will be given with reference to the accompanying drawings and specific embodiments, but not limiting the present application.
Example 1
The production process of the bacterial hyaluronidase production enzyme cutting low-molecular and oligomeric sodium hyaluronate is monitored, an M-S function is established, and the bacterial hyaluronidase adopted in the production process is the hyaluronidase produced by fermenting Bacillus A50 CGMCC No. 5744. The specific detection steps are as follows:
establishing an M-S function: purified water was used to prepare 5 different sodium hyaluronate solutions (moisture content of sodium hyaluronate raw material 7.8%) respectively: (1) 1L of 1% by mass sodium hyaluronate solution with molecular weight of 3000kDa was added with 5X 10 7 U bacteria produce hyaluronidase; (2) 1L of sodium hyaluronate solution with molecular weight of 2000kDa with mass fraction of 2%, 8×10 7 U bacteria produce hyaluronidase; (3) 1L of sodium hyaluronate solution with molecular weight of 1000kDa with mass fraction of 5%, 6×10 7 U bacteria produce hyaluronidase; (4) 1L of bacterial enzyme-cutting low molecular sodium hyaluronate solution with molecular weight of 300kDa with mass fraction of 10%, adding 7×10 7 U bacteria produce hyaluronidase; (5) 1L of bacterial enzyme-cutting low molecular sodium hyaluronate solution with molecular weight of 90kDa with mass fraction of 50%, adding 5×10 8 The bacterium of U produces hyaluronidase. The pH values of the 5 sodium hyaluronate solutions are respectively regulated to 4.0 to 9.0, and the temperature is 20 to the upper rangeCarrying out enzymolysis at 45 ℃ and sampling every 1h, sampling 10mL each time, boiling the enzymolysis liquid sample for 2min, diluting with purified water, ensuring that the absorbance of the ultraviolet absorption spectrum at 232nm after dilution is within the range of 0.2-1.0, and measuring the absorbance A of the dilution liquid of each enzymolysis liquid sample at 232nm 232 The S value is calculated according to the following formula (1), and the molecular weight M of the hyaluronic acid in each enzymolysis liquid sample is measured by adopting a SEC-MALLS method, and an 'M-S function' is obtained by computer fitting, as shown in figure 1.
Wherein A is 232 The method is characterized in that the method comprises the steps of (1) the absorbance of a diluted solution of a hyaluronic acid enzymatic hydrolysate sample at 232nm, V is the total volume (L) of the hyaluronic acid enzymatic hydrolysate, T is the dilution multiple of the hyaluronic acid enzymatic hydrolysate, H is the moisture content (%) of a raw material hyaluronate for enzymatic hydrolysis of the hyaluronic acid enzymatic hydrolysate, W is the mass (kg) of the raw material hyaluronate for enzymatic hydrolysis of the hyaluronic acid enzymatic hydrolysate, and R is the molecular weight ratio of the hyaluronic acid in the hyaluronate.
And (3) detecting enzymolysis liquid: 1000L of sodium hyaluronate solution containing 10kg of 2000kDa sodium hyaluronate (sodium hyaluronate moisture content 7.8%) was added to 2.0X10% sodium hyaluronate solution 7 U bacteria produce hyaluronidase, after enzymolysis for 4 hours at 40deg.C and pH of 5.5, 10mL is sampled every 1 hour, total sampling is 4 times, each sample is boiled and inactivated, and after dilution for 100 times, absorbance A at 232nm is measured 232 Calculating the S value according to the formula (1), and calculating the molecular weight M of hyaluronic acid in the enzymolysis liquid sample according to the M-S function in figure 1 1 As shown in table 1. In addition, the molecular weight M of hyaluronic acid in each enzymolysis liquid sample is measured by adopting a SEC-MALLS method 2 By comparison, the error of the molecular weight obtained by the measuring method is within +/-5.5 percent.
TABLE 1 example 1 enzymatic hydrolysate sampling assay A 232 And calculating the molecular weight
Example 2
This example uses the same bacteria as in example 1 to produce hyaluronidase and uses the "M-S function" obtained in example 1 during the test.
And (3) detecting enzymolysis liquid: 200L of potassium hyaluronate solution containing 60kg of 400kDa potassium hyaluronate (the moisture content of potassium hyaluronate is 8.5%) was added to 3.0X10% potassium hyaluronate solution 8 U bacteria produce hyaluronidase, after enzymolysis for 5h at 35deg.C and pH of 6.0, 10mL is sampled every 1h, total sampling is 4 times, each sample is boiled and inactivated, and diluted 1000 times, and ultraviolet absorption spectrum absorbance A at 232nm is measured 232 Calculating the S value according to the formula (1), and calculating the molecular weight M of hyaluronic acid in the enzymolysis liquid sample according to the M-S function in figure 1 1 As shown in table 2. In addition, the molecular weight M of hyaluronic acid in each enzymolysis liquid sample is measured by adopting a SEC-MALLS method 2 By comparison, the error of the molecular weight obtained by the measuring method is within +/-6 percent.
TABLE 2 sample assay A for enzymatic hydrolysate 232 And calculating the molecular weight
Example 3
This example uses the same bacteria as in example 1 to produce hyaluronidase and uses the "M-S function" obtained in example 1 during the test.
And (3) detecting enzymolysis liquid: 500L of magnesium hyaluronate solution containing 25kg of 1000kDa magnesium hyaluronate (moisture content of magnesium hyaluronate 8.8%) was added to 1.0X10% magnesium hyaluronate solution 7 U bacteria produce hyaluronidase, after enzymolysis for 7h at 30deg.C and pH of 5.0, 10mL is sampled every 1h for 4 times, each sample is boiled and inactivated, diluted again for 50 times, and its absorbance A at 232nm is measured 232 Calculating the S value according to the above formula (1) and then according to the "M-S function" in FIG. 1Calculating the molecular weight M of hyaluronic acid in the enzymolysis liquid sample 1 As shown in table 3. In addition, the molecular weight M of hyaluronic acid in each enzymolysis liquid sample is measured by adopting a SEC-MALLS method 2 By comparison, the error of the molecular weight obtained by the measuring method is within +/-3 percent.
TABLE 3 sample assay A for enzymatic hydrolysate 232 And calculating the molecular weight
Example 4
This example uses the same bacteria as in example 1 to produce hyaluronidase and uses the "M-S function" obtained in example 1 during the test.
And (3) detecting enzymolysis liquid: 2000L of calcium hyaluronate solution containing 1000kg of 50kDa bacterial enzyme-digested low molecular weight calcium hyaluronate (calcium hyaluronate moisture content 5.4%) was added to 8.0X10% calcium hyaluronate solution 8 U bacteria produce hyaluronidase, after enzymolysis for 3h at 38deg.C and pH of 7.0, 10mL is sampled every 1h for 4 times, each sample is boiled and inactivated, diluted 10000 times, and its absorbance A at 232nm is measured 232 Calculating the S value according to the formula (1), and calculating the molecular weight M of hyaluronic acid in the enzymolysis liquid sample according to the M-S function in figure 1 1 As shown in table 4. In addition, the molecular weight M of hyaluronic acid in each enzymolysis liquid sample is measured by adopting a SEC-MALLS method 2 By comparison, when the molecular weight is more than or equal to 3kDa, the error of the molecular weight obtained by the determination method is within +/-3 percent. However, when the molecular weight is less than or equal to 3kDa, the error in the molecular weight obtained by the measurement method of the present application increases, and the smaller the molecular weight, the larger the error, and the method is no longer suitable for measuring the molecular weight by the method.
TABLE 4 sample assay A for enzymatic hydrolysate 232 And calculating the molecular weight
Finally, it is noted that the above embodiments are only for illustrating the technical solution of the present application and not for limiting the same, and although the present application has been described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that modifications and equivalents may be made thereto without departing from the spirit and scope of the technical solution of the present application, which is intended to be covered by the scope of the claims of the present application.
Claims (6)
1. A method for measuring the molecular weight of hyaluronic acid is characterized by measuring the molecular weight of hyaluronic acid in the hyaluronic acid solution based on the absorbance of an ultraviolet absorption spectrum of the hyaluronic acid solution, wherein the absorbance is the absorbance of the hyaluronic acid solution at 232nm, the hyaluronic acid solution is a hyaluronic acid enzymatic hydrolysate generated after the hyaluronic acid is subjected to enzymatic cleavage,
the method comprises the following steps:
establishing a functional relation 'M-S function' between an S value of the hyaluronic acid enzymatic hydrolysate and the molecular weight M of hyaluronic acid in the hyaluronic acid enzymatic hydrolysate, wherein the S value is calculated by the following formula:
wherein A is absorbance of ultraviolet absorption spectrum of diluent of the hyaluronic acid enzymatic hydrolysate, V is total volume of the hyaluronic acid enzymatic hydrolysate, T is dilution multiple of the hyaluronic acid enzymatic hydrolysate, H is moisture content of raw material hyaluronate for enzymatic hydrolysis of the hyaluronic acid enzymatic hydrolysate, W is mass of raw material hyaluronate for enzymatic hydrolysis of the hyaluronic acid enzymatic hydrolysate, and R is molecular weight ratio of hyaluronic acid in hyaluronate;
inactivating and diluting a hyaluronic acid enzymatic hydrolysate sample to be detected, measuring the absorbance A of an ultraviolet absorption spectrum of the hyaluronic acid enzymatic hydrolysate sample to be detected, calculating the S value of the hyaluronic acid enzymatic hydrolysate sample to be detected according to the formula, and calculating the molecular weight of hyaluronic acid in the hyaluronic acid enzymatic hydrolysate sample to be detected according to the S value and the M-S function of the hyaluronic acid enzymatic hydrolysate sample to be detected; the molecular weight M of hyaluronic acid in each enzymolysis liquid sample is measured by adopting a SEC-MALLS method, and an M-S function is obtained by computer fitting;
the molecular weight M of the hyaluronic acid in the M-S function is in the range of 3kDa to 50kDa.
2. The method for measuring the molecular weight of hyaluronic acid according to claim 1, wherein the mass fraction of hyaluronic acid in the hyaluronic acid enzymatic hydrolysate is 1% -50%.
3. The method for measuring the molecular weight of hyaluronic acid according to claim 1 or 2, wherein the raw material for enzymolysis of the hyaluronic acid enzymolysis liquid is hyaluronate with molecular weight not less than 3kDa obtained by enzymolysis of bacterium producing hyaluronidase or bacterium producing chondroitin sulfate enzyme, or hyaluronate with molecular weight not less than 200kDa obtained by enzymolysis of non-hyaluronidase or non-chondroitin sulfate enzyme.
4. The method for measuring the molecular weight of hyaluronic acid according to claim 3, wherein the enzymatic hydrolysate of hyaluronic acid is an enzymatic hydrolysate produced by cleavage of hyaluronic acid by bacterial hyaluronidase or bacterial chondroitin sulfate enzyme.
5. The method for measuring the molecular weight of hyaluronic acid according to claim 4, wherein the hyaluronidase produced by the bacterium is a bacillus hyaluronidase.
6. The method for measuring the molecular weight of hyaluronic acid according to claim 5, wherein the hyaluronidase produced by the bacterium is a hyaluronidase produced by fermentation of Bacillus sp. A50 CGMCC No. 5744.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202010573896.8A CN111562231B (en) | 2020-06-22 | 2020-06-22 | Method for measuring molecular weight of hyaluronic acid |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202010573896.8A CN111562231B (en) | 2020-06-22 | 2020-06-22 | Method for measuring molecular weight of hyaluronic acid |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN111562231A CN111562231A (en) | 2020-08-21 |
| CN111562231B true CN111562231B (en) | 2023-09-15 |
Family
ID=72073904
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202010573896.8A Active CN111562231B (en) | 2020-06-22 | 2020-06-22 | Method for measuring molecular weight of hyaluronic acid |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN111562231B (en) |
Citations (11)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP0070559A2 (en) * | 1981-07-20 | 1983-01-26 | The Coca-Cola Company | Process for treating caramel colors |
| JP2008267941A (en) * | 2007-04-19 | 2008-11-06 | Toshitsu Kagaku Kenkyusho:Kk | Method for measuring molecular weight of hyaluronan, method for diagnosing disease, and kit for the same |
| WO2012032150A2 (en) * | 2010-09-09 | 2012-03-15 | Altergon S.A. | Process for obtaining hyaluronic acid of defined molecular weight |
| CN102876748A (en) * | 2012-04-13 | 2013-01-16 | 华熙福瑞达生物医药有限公司 | Method for preparing oligomeric hyalurate by digestion method, and oligomeric hyalurate and application thereof |
| CN103255187A (en) * | 2012-02-21 | 2013-08-21 | 华熙福瑞达生物医药有限公司 | Low molecular hyaluronate, preparation method and purpose thereof |
| CN105254779A (en) * | 2015-10-13 | 2016-01-20 | 青海大学 | Extraction and purification method of hyaluronic acid with large molecular weight |
| CN105699578A (en) * | 2016-04-22 | 2016-06-22 | 广东东阳光药业有限公司 | Analysis method of glycoform fingerprint atlas formed by sodium hyaluronate |
| CN109836511A (en) * | 2019-04-03 | 2019-06-04 | 上海应用技术大学 | A method of extracting tremella polysaccharides from fermentation liquid |
| CN110331178A (en) * | 2019-06-27 | 2019-10-15 | 青岛海洋生物医药研究院股份有限公司 | A kind of enzyme cutting method prepares the method for micromolecule hyaluronic acid and gained micromolecule hyaluronic acid is applied with it |
| CN110642963A (en) * | 2019-10-18 | 2020-01-03 | 福州大学 | Method for extracting chondroitin sulfate/dermatan sulfate/hyaluronic acid from donkey-hide gelatin |
| CN110982862A (en) * | 2019-12-27 | 2020-04-10 | 华熙生物科技股份有限公司 | Method for large-scale preparation of high-purity unsaturated hyaluronic acid disaccharide |
-
2020
- 2020-06-22 CN CN202010573896.8A patent/CN111562231B/en active Active
Patent Citations (12)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP0070559A2 (en) * | 1981-07-20 | 1983-01-26 | The Coca-Cola Company | Process for treating caramel colors |
| JP2008267941A (en) * | 2007-04-19 | 2008-11-06 | Toshitsu Kagaku Kenkyusho:Kk | Method for measuring molecular weight of hyaluronan, method for diagnosing disease, and kit for the same |
| WO2012032150A2 (en) * | 2010-09-09 | 2012-03-15 | Altergon S.A. | Process for obtaining hyaluronic acid of defined molecular weight |
| CN103255187A (en) * | 2012-02-21 | 2013-08-21 | 华熙福瑞达生物医药有限公司 | Low molecular hyaluronate, preparation method and purpose thereof |
| CN103255076A (en) * | 2012-02-21 | 2013-08-21 | 华熙福瑞达生物医药有限公司 | Bacillus spp and hyaluronidase and preparation method and purpose thereof |
| CN102876748A (en) * | 2012-04-13 | 2013-01-16 | 华熙福瑞达生物医药有限公司 | Method for preparing oligomeric hyalurate by digestion method, and oligomeric hyalurate and application thereof |
| CN105254779A (en) * | 2015-10-13 | 2016-01-20 | 青海大学 | Extraction and purification method of hyaluronic acid with large molecular weight |
| CN105699578A (en) * | 2016-04-22 | 2016-06-22 | 广东东阳光药业有限公司 | Analysis method of glycoform fingerprint atlas formed by sodium hyaluronate |
| CN109836511A (en) * | 2019-04-03 | 2019-06-04 | 上海应用技术大学 | A method of extracting tremella polysaccharides from fermentation liquid |
| CN110331178A (en) * | 2019-06-27 | 2019-10-15 | 青岛海洋生物医药研究院股份有限公司 | A kind of enzyme cutting method prepares the method for micromolecule hyaluronic acid and gained micromolecule hyaluronic acid is applied with it |
| CN110642963A (en) * | 2019-10-18 | 2020-01-03 | 福州大学 | Method for extracting chondroitin sulfate/dermatan sulfate/hyaluronic acid from donkey-hide gelatin |
| CN110982862A (en) * | 2019-12-27 | 2020-04-10 | 华熙生物科技股份有限公司 | Method for large-scale preparation of high-purity unsaturated hyaluronic acid disaccharide |
Non-Patent Citations (4)
| Title |
|---|
| 《Compared molecular characterization of hyaluronan using multiple-detectiontechniques》;Tu Luan 等;《Polymer》;20111015;全文 * |
| 《Instability of recombinant pUB110 plasmids in Bacillus subtilis: Plasmid-encoded stability function and effects of DNA inserts》;S Bron 等;《Plasmid》;全文 * |
| 姜旭淦 等.《分光光度计的使用》.《全国高等医药院校医学检验技术专业第四轮规划教材 临床生物化学检验实验指导 供医学检验技术专业使用 第4版》.中国医药科技出版社,2020,第12-14页. * |
| 谢江川 等.《载门冬酰胺酶的自组装聚乙二醇- 透明质酸/ 二甲基-- 环糊精纳米粒 体外稳定性的初步考察》.《中国医药工业杂志》.2015,第46卷(第7期),全文. * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN111562231A (en) | 2020-08-21 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Wang et al. | Structural characterization of the symbiotically important low-molecular-weight succinoglycan of Sinorhizobium meliloti | |
| Campa et al. | Biochemical analysis of the processive mechanism for epimerization of alginate by mannuronan C-5 epimerase AlgE4 | |
| Holtan et al. | Characterization of the hydrolysis mechanism of polyalternating alginate in weak acid and assignment of the resulting MG-oligosaccharides by NMR spectroscopy and ESI− mass spectrometry | |
| Jay et al. | Analysis of structure and function of gellans with different substitution patterns | |
| US10704068B2 (en) | Method of producing heparan sulfate having anticoagulant activity | |
| CN105087456A (en) | Construction method of recombinant bacillus subtilis capable of generating hyaluronic acid with specific molecular weight | |
| CN110331178B (en) | Method for preparing micromolecular hyaluronic acid by enzyme cleavage method, obtained micromolecular hyaluronic acid and application thereof | |
| HU214986B (en) | Process for producing preparation containing n,o-sulfated heparosanes | |
| CN106906161B (en) | Microbacterium, broad-spectrum glycosaminoglycan lyase expressed by same, and coding gene and application thereof | |
| CN109929859B (en) | Kappa-carrageenan enzyme coding gene and preparation and application thereof | |
| Candia et al. | Effect of the nitrogen source on pyruvate content and rheological properties of xanthan | |
| Borges et al. | Influence of agitation and aeration in xanthan production by Xanthomonas campestris pv pruni strain 101 | |
| CN111562231B (en) | Method for measuring molecular weight of hyaluronic acid | |
| Favorov et al. | A study of the reaction catalysed by alginate lyase VI from the sea mollusc, Littorina sp. | |
| Jouanneau et al. | Hybridity of carrageenans water-and alkali-extracted from Chondracanthus chamissoi, Mazzaella laminarioides, Sarcothalia crispata, and Sarcothalia radula | |
| CN110951805B (en) | Enzymolysis preparation method of low molecular weight laver polysaccharide and laver oligosaccharide | |
| Petersen et al. | Mannuronate C-5 epimerases and their use in alginate modification | |
| Tait et al. | Synthesis and properties of a mutant type of xanthan | |
| US10301658B2 (en) | Method for in vivo production of glycosaminoglycans | |
| Heyraud et al. | An enzymatic method for preparation of homopolymannuronate blocks and strictly alternating sequences of mannuronic and guluronic units | |
| Bobkova et al. | Discovery and characteristic of hyaluronidases from filamentous fungi | |
| Ding et al. | Physicochemical studies of extracellular polysaccharides of Erwinia chrysanthemi spp | |
| Manzoni et al. | Production of K5 polysaccharides of different molecular weight by Escherichia coli | |
| Kool | Enzymatic modification and characterization of xanthan | |
| JP2008295450A (en) | Quick assay method of acidic polysaccharide |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| TA01 | Transfer of patent application right | ||
| TA01 | Transfer of patent application right |
Effective date of registration: 20230613 Address after: Tianchen Avenue, Ji'nan hi tech Development Zone of Shandong Province, No. 678 250101 Applicant after: BLOOMAGE BIOTECH Co.,Ltd. Address before: Tianchen Avenue, Ji'nan hi tech Development Zone of Shandong Province, No. 678 250101 Applicant before: BLOOMAGE BIOTECH Co.,Ltd. Applicant before: SHANDONG BLOOMAGE HYINC BIOPHARM Corp.,Ltd. |
|
| GR01 | Patent grant | ||
| GR01 | Patent grant |