阅读说明：本技术 一种低分子量透明质酸的制备方法 (Preparation method of low-molecular-weight hyaluronic acid ) 是由 不公告发明人 于 2020-11-13 设计创作，主要内容包括：本发明属于透明质酸技术领域,具体涉及一种低分子量透明质酸的制备方法。本发明首先将高分子量透明质酸进行预处理降解,然后经中性盐分级沉淀去除蛋白质和小分子杂质,然后经超滤纳滤过滤分离得到目标分子量的透明质酸。中性盐分级沉淀可以去除透明质酸中的蛋白质和小分子在杂质,得到纯度较高的透明质酸,并减少膜分离过程中的污染问题；膜过滤可以通过控制膜的孔径将透明质酸按照不同分子量分离,以得到特定分子量的透明质酸；该方法简单高效,无杂质残留,制得的透明质酸具有高度的皮肤亲和性和渗透性,能促进毛细血管合成、促进创伤愈合、促进胶原蛋白合成。(The invention belongs to the technical field of hyaluronic acid, and particularly relates to a preparation method of low-molecular-weight hyaluronic acid. The method comprises the steps of firstly, pretreating and degrading high molecular weight hyaluronic acid, then removing proteins and small molecular impurities through neutral salt fractional precipitation, and then, filtering and separating through ultrafiltration and nanofiltration to obtain the hyaluronic acid with the target molecular weight. The neutral salt fractional precipitation can remove the protein and micromolecule impurities in the hyaluronic acid, obtain the hyaluronic acid with higher purity and reduce the pollution problem in the membrane separation process; membrane filtration hyaluronic acid can be separated according to different molecular weights by controlling the pore size of the membrane to obtain hyaluronic acid with a specific molecular weight; the method is simple and high-efficiency, has no impurity residue, and the prepared hyaluronic acid has high skin affinity and permeability, and can promote capillary synthesis, wound healing and collagen synthesis.)

1. A method for preparing low molecular weight hyaluronic acid, which is characterized by comprising the following steps:

1) pretreatment: dissolving high molecular weight hyaluronic acid with the molecular weight of 50-1000 KDa in 15-30 times of water by weight, adjusting the pH of the solution to 2.8-9.3 by using dilute acid or dilute alkali solution, fully stirring and swelling at room temperature, slowly heating to 80-100 ℃, and hydrolyzing at constant temperature for 2-4 hours; then leaching, filtering residues, repeating the leaching operation, and combining the two filtrates for later use;

2) two-stage precipitation of neutral salt: heating the solution obtained by pretreatment to 30-40 ℃, then adding neutral salt solid or solution, stirring to completely dissolve, and then carrying out ultrafiltration; adding neutral salt solid or solution into the filtrate again, stirring until the neutral salt solid or solution is completely dissolved, standing for precipitation, then filtering, ultrafiltering, drying, adding purified water of 4-6 times into the precipitate for redissolving, and then filtering and separating;

3) filtering and separating: performing ultrafiltration pretreatment on the solution by using a microfiltration membrane with the diameter of 0.03 mu m under the transmembrane pressure of 0.3 MPa; vacuum freeze drying the ultrafiltration retentate to obtain hyaluronic acid with molecular weight of 5-200 KDa and high molecular weight; and (3) performing nanofiltration concentration on the percolate through a nanofiltration membrane with the molecular weight cutoff of 5KDa, and performing vacuum freeze drying on the finally obtained concentrated solution to obtain hyaluronic acid with the molecular weight lower than 5 KDa.

2. The method according to claim 1, wherein the hyaluronic acid with a molecular weight of less than 5kDa obtained by vacuum freeze-drying has the following general structural formula:

in the formula: n is 0-12, and X is H, K or Na.

3. The method as claimed in claim 1, wherein the diluted acid solution in step 1) is one of citric acid, maleic acid and tartaric acid, and the concentration of the diluted acid solution is 0.05-1 mol/L.

4. The method according to claim 1, wherein the dilute alkali solution in step 1) is at least one of sodium methoxide solution, potassium ethoxide solution and potassium tert-butoxide solution, and the concentration of the dilute alkali solution is 0.01-2 mol/L.

5. The method of claim 1, wherein during the precipitation of the neutral salt fraction, the neutral salt is one of sodium sulfate, magnesium sulfate, and sodium phosphate.

6. The method according to claim 1, wherein during the fractional precipitation of the neutral salt, the first addition amount of the neutral salt is 20-30% of the weight of the solution, and the second addition amount is 40-45% of the weight of the system.

7. The method as claimed in claim 1, wherein the nanofiltration membrane and the ultrafiltration membrane are hollow fiber membranes or flat sheet membranes, and the membrane material is polyvinyl alcohol, sulfonated polysulfone or polyamide.

8. A low molecular weight hyaluronic acid, which is produced by the method according to any one of claims 1 to 7.

9. The use of the low molecular weight hyaluronic acid according to any of claims 1-8 in the fields of food, medicine and cosmetics, wherein the low molecular weight hyaluronic acid prepared by the method according to any of claims 1-8 is used as a main active ingredient in the fields of food, medicine and cosmetics.

10. A composition, which is characterized by consisting of the low molecular weight hyaluronic acid and a pharmaceutically or dietetically acceptable carrier thereof, is used in the fields of food, medicine and cosmetics.

Technical Field

The invention belongs to the technical field of hyaluronic acid, and particularly relates to a preparation method of low-molecular-weight hyaluronic acid.

Background

Hyaluronic Acid (HA), also known as Hyaluronic acid, is an important component in the human body. Hyaluronic acid is a polymeric acidic mucopolysaccharide with an unbranched structure consisting of repeating units of N-acetylglucosamine (GlcNAc) and D-glucuronic acid (GlcA) disaccharide via β - (1 → 4) and β - (1 → 3) glycosidic linkages, and is widely distributed in the intercellular substance of animal tissues and the capsule of some bacteria. With the continuous and deep research on the distribution, chemical structure, physicochemical properties and other aspects of HA at home and abroad, it is found that HA HAs various biological functions, such as participating in signal conduction of lung and vascular diseases and embryogenesis, wound healing, skin moisturizing and the like, so that HA HAs wide and unique application values in the fields of food, medicine, cosmetics and the like. It HAs also been found that the physiological role of HA is closely related to the molecular weight, with smaller molecular weight being more bioactive. When the molecular weight of HA is about 1000Da, the composition can promote angiogenesis, promote wound healing and resist tumors; when the molecular weight is less than 1000Da, the skin shows excellent skin affinity and rapid permeability, and can improve skin nutrition metabolism, make skin tender and smooth, remove wrinkles, increase elasticity, prevent aging, etc.

The methods for preparing low molecular weight hyaluronic acid disclosed so far mainly include enzymatic, physical and chemical methods. Wherein; the enzymolysis method has mild reaction conditions, easy control, uniform product and good product quality, but needs subsequent steps of enzyme deactivation and enzyme removal, does not have industrialized hyaluronidase, and has extremely high enzyme cost in laboratory; the physical degradation force is limited, so that nano hyaluronic acid with small molecular weight is difficult to obtain; the chemical method comprises acid hydrolysis, alkali hydrolysis and oxidative degradation, the relative molecular mass of the degradation product of the chemical degradation method can be controlled by changing the addition amount and reaction time of acid and alkali or an oxidant, the degradation cost is low, the large-scale production is easy, but the oxidative degradation may have oxidant residues.

As a novel separation method, the membrane separation process has the advantages of mild operation conditions, no phase or chemical change in the separation process, reusability, environmental friendliness, low energy consumption, high efficiency, no secondary pollution and the like, is widely applied to separation and purification of natural polysaccharides and oligosaccharides, and preliminarily realizes integrated and large-scale production. The research of molecular weight grading of sodium hyaluronate by using a PVDF membrane and extraction of sodium hyaluronate from hyaluronic acid fermentation liquor is facilitated, but the PVDF membrane is seriously polluted in the separation and purification process, the separation efficiency and the repeated use times of the membrane are seriously reduced, the membrane is difficult to clean, and the service life is greatly shortened. Therefore, aiming at the existing separation and purification process, a new preparation method of the low molecular weight hyaluronic acid is developed, and the method has practical significance for realizing scale and breaking through the bottleneck of the existing production process.

Disclosure of Invention

The invention aims to provide a preparation method of low-molecular-weight hyaluronic acid aiming at the defects of the prior art, which has the advantages of simple process, low cost, repeated utilization for many times and high-efficiency and large-scale production of the low-molecular-weight hyaluronic acid. The hyaluronic acid prepared by the method has controllable molecular weight and no impurity residue, can obtain hyaluronic acid with molecular weight of 5-200 KDa, can also obtain hyaluronic acid oligosaccharide monomer with molecular weight lower than 5KDa, and can be applied to the fields of food, medicine, cosmetics and the like.

To achieve the above object, the present invention adopts a technical means including the following items [1] to [4 ].

[1] A method for preparing low molecular weight hyaluronic acid comprises the following steps:

1) pretreatment: dissolving high molecular weight hyaluronic acid with the molecular weight of 50-1000 KDa in 15-30 times of water by weight, adjusting the pH of the solution to 2.8-9.3 by using dilute acid or dilute alkali solution, fully stirring and swelling at room temperature, slowly heating to 80-100 ℃, and hydrolyzing at constant temperature for 2-4 hours; then leaching, filtering residues, repeating the leaching operation, and combining the two filtrates for later use;

2) two-stage precipitation of neutral salt: heating the solution obtained by pretreatment to 30-40 ℃, then adding neutral salt solid or solution, stirring to completely dissolve, and then carrying out ultrafiltration; and adding the neutral salt solid or solution into the filtrate again, stirring until the neutral salt solid or solution is completely dissolved, standing for precipitation, then filtering, ultrafiltering, drying, adding 4-6 times of purified water into the precipitate for redissolving, and then filtering and separating.

3) Filtering and separating: carrying out ultrafiltration pretreatment on the solution by using a microfiltration membrane with the diameter of 0.03 mu m under the transmembrane pressure of 0.3 MPa; vacuum freeze drying the ultrafiltration retentate to obtain hyaluronic acid with molecular weight of 5-200 KDa and high molecular weight; and (2) performing nanofiltration concentration on the percolate through a nanofiltration membrane with the molecular weight cutoff of 5KDa, and performing vacuum freeze-drying on the finally obtained concentrated solution to obtain hyaluronic acid with the molecular weight lower than 5KDa, wherein the structural general formula of the hyaluronic acid with the molecular weight lower than 5KDa is shown as the following formula (1):

in the formula: n is 0-12, and X is H, K or Na.

The hyaluronic acid with the specific molecular weight is efficiently and quickly separated by a membrane separation technology, the method is simple to operate and can be repeatedly utilized, and the prepared hyaluronic acid with the specific molecular weight can be used in the fields of food, medicines and cosmetics.

Further, in the step 1), the dilute acid is one of citric acid, maleic acid and tartaric acid, and the concentration of the dilute acid solution is 0.05-1 mol/L.

Further, the dilute alkali solution in the step 1) is at least one of a sodium methoxide solution, a potassium ethoxide solution and a potassium tert-butoxide solution, and the concentration of the dilute alkali solution is 0.05-2 mol/L.

Further, the neutral salt in the step 2) is one of sodium sulfate, magnesium sulfate and sodium phosphate.

Further, the neutral salt in step 2) may be added in the form of a solid or a solution, and

the first addition amount is 20-30% of the solution weight,

the second addition amount is 40-45% of the solution weight. Different amounts of neutral salt is added in sequence and a small amount of neutral salt is added for fractional precipitation, so that partial impurities such as protein, nucleic acid and the like in the solution can be precipitated and separated out, the protein residue of the prepared hyaluronic acid finished product is greatly reduced, the problem of membrane pollution can be effectively improved, the membrane separation efficiency is greatly improved, the repeated use times are multiple, and the service life is prolonged.

Further, the membrane in the filtration and separation in the step 3) is a hollow fiber membrane or a flat membrane.

Further, the material of the membrane in the filtration separation in the step 3) is one of polyvinyl alcohol, sulfonated polysulfone and polyamide. The microfiltration, ultrafiltration and nanofiltration membrane materials selected by the application are hydrophilic materials, so that the membrane has good anti-pollution capacity, and the problems of membrane separation efficiency reduction and service life shortening caused by protein adsorption can be effectively reduced in the membrane separation process; in addition, the membrane material can resist chemical reagents and high temperature and can be repeatedly used.

The inventor unexpectedly finds that the pretreated hyaluronic acid solution is precipitated by neutral salt twice, so that partial impurities such as protein, nucleic acid and the like in the solution can be precipitated, and the protein residue of the prepared hyaluronic acid finished product is greatly reduced; in addition, in the membrane separation process, the membrane cleaning difficulty is increased due to the fact that double images are severely influenced by protein adsorption on the surface of the membrane, and the membrane separation efficiency and the service life are prolonged, and the problem of membrane pollution can be effectively solved by removing the protein through twice neutral salt precipitation, so that the membrane separation efficiency is greatly improved, the number of times of repeated use is large, and the service life is prolonged.

According to the method, firstly, high molecular weight hyaluronic acid is subjected to pretreatment degradation, then protein and small molecular impurities are removed through neutral salt fractional precipitation, then hyaluronic acid with a target molecular weight is obtained through ultrafiltration, nanofiltration, filtration and separation, and the hyaluronic acid with a specific molecular weight can be obtained through controlling degradation conditions and the aperture of a nanofiltration membrane ultrafiltration membrane; the neutral salt fractional precipitation can remove the protein and micromolecule impurities in the hyaluronic acid, obtain the hyaluronic acid with higher purity, and reduce the pollution problem in the membrane separation process; membrane filtration hyaluronic acid can be separated according to different molecular weights by controlling the pore size of the membrane to obtain hyaluronic acid with a specific molecular weight; the method is simple and high-efficiency, has no impurity residue, and the prepared hyaluronic acid has high skin affinity and permeability, and can promote capillary synthesis, wound healing and collagen synthesis.

[2] A low molecular weight hyaluronic acid produced by the method of item [1 ].

[3] Use of the low-molecular-weight hyaluronic acid according to any one of items [1] to [2] in the fields of foods, medicines and cosmetics.

[4] A composition comprising the low-molecular-weight hyaluronic acid according to any one of items [1] to [2] as a main active ingredient, together with a pharmaceutically or dietetically acceptable carrier or adjuvant.

The composition of the invention can be used in the cosmetic field, such as cosmetics for moisturizing, resisting aging and wrinkles, shampoo and hair conditioner, and the like; the nano-particle is used in the field of medicines, can be used as an excellent medicine carrier, and can achieve the purposes of medicine thickening, medicine slow release, medicine penetration capacity promotion and targeting; it is used in food field, and is an oral health product with the functions of raising skin water holding capacity, raising skin elasticity and reducing wrinkles.

The hyaluronic acid with specific molecular weight is obtained by pretreatment degradation, and then by neutral salt fractional precipitation and membrane filtration separation, so that the hyaluronic acid has the following beneficial effects:

1) the low molecular weight hyaluronic acid is prepared by the prior art and the prior equipment without additionally arranging a production line, so that the process operation is greatly simplified, and the preparation efficiency of the hyaluronic acid is improved;

2) the low molecular hyaluronic acid has high skin affinity and permeability, can promote capillary synthesis, promote wound healing and promote collagen synthesis, and can be used in the fields of food, medicine, cosmetics and the like;

3) by utilizing two-stage precipitation of neutral salt, the problem of residual protein and small molecular impurities is effectively solved, the purity of the obtained hyaluronic acid is greatly improved, the problem of membrane pollution is effectively improved, the membrane separation efficiency is greatly improved, the repeated use times are multiple, and the service life is prolonged;

4) the hyaluronic acid is filtered and purified by using the microfiltration membrane and the nanofiltration membrane, so that hyaluronic acid with different molecular weights can be prepared by controlling the pore diameter of the membrane, a hyaluronic acid oligosaccharide monomer with a low polymer can be obtained, the molecular weight of the hyaluronic acid is controllable, and no impurity residue exists;

5) the low molecular hyaluronic acid provided by the invention has the characteristics of simple preparation process, low cost, high yield, harmlessness, no pollution and large-scale industrialization;

the invention adopts the technical scheme to make up the defects of the prior art, and has reasonable design and convenient operation.

Drawings

In order to make the aforementioned and other objects, features, and advantages of the invention, as well as others which will become apparent, reference is made to the following description taken in conjunction with the accompanying drawings in which:

FIG. 1 is a schematic representation of an infrared spectrum of a low molecular weight hyaluronic acid obtained in example 1 of the present invention;

FIG. 2 is a schematic view showing the contamination of the membrane.

Detailed Description

Unless defined otherwise, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The materials, methods, and examples described herein are illustrative only and are not intended to be limiting. All publications, patent applications, patents, provisional applications, database entries, and other references mentioned herein, and the like, are incorporated by reference herein in their entirety. In case of conflict, the present specification, including definitions, will control.

The technical solution of the present invention will be described in further detail below with reference to the following detailed description and accompanying drawings.

Example 1: low molecular weight hyaluronic acid

The present example provides a method for preparing low molecular weight hyaluronic acid, comprising the steps of:

1) pretreatment: dissolving 50g of high molecular weight hyaluronic acid with the molecular weight of 1000kDa in 30 times of weight parts of water, adjusting the pH of the solution to 8.5 by using 0.1mol/L sodium methoxide solution, fully stirring and swelling at room temperature, slowly heating to 90 ℃, hydrolyzing at constant temperature for 4 hours, then leaching and filtering, repeating the leaching operation on filter residues, and combining the two filtrates for later use;

2) and (3) fractional precipitation of neutral salts: heating the solution obtained in the step 1) to 40 ℃, adding 25% of magnesium sulfate solid of the solution weight, stirring to completely dissolve the solution, then carrying out ultrafiltration by using a 0.1 mu m sulfonated polysulfone ultrafiltration membrane, then adding 43% of magnesium sulfate solid of the solution weight into the filtrate again, stirring to completely dissolve the solution, standing for precipitation, then carrying out filtration, ultrafiltration and drying, and then adding 6 times of purified water into the dried precipitate for redissolving;

3) and (3) filtering and purifying: carrying out ultrafiltration pretreatment on the solution obtained in the step 2) by using a sulfonated polysulfone microfiltration membrane with the thickness of 0.03 mu m under the membrane permeation pressure of 0.3 MPa; vacuum freeze drying the ultrafiltered retentate to obtain high molecular weight hyaluronic acid; and (3) performing nanofiltration concentration on the percolate through a sulfonated polysulfone nanofiltration membrane with the intercepted molecular weight of 5KDa, and finally performing vacuum freeze drying on the obtained concentrated solution to obtain hyaluronic acid with the target molecular weight.

Without limitation, the sodium methoxide solution in this example can be replaced by potassium ethoxide solution or potassium tert-butoxide; the sulfonated polysulfone membrane can be changed into a polyvinyl alcohol or polyamide membrane.

Example 2: another low molecular weight hyaluronic acid

Example 2 provides another low molecular weight hyaluronic acid, which is prepared substantially in the same manner as in example 1, except that in example 2, in the pretreatment of step 1), the pH of the solution is adjusted to 8.5 with 0.1mol/L sodium hydroxide solution.

Example 3: another low molecular weight hyaluronic acid

Example 3 provides another low molecular weight hyaluronic acid, which is prepared substantially in the same manner as in example 1, except that in example 3, the pH of the solution is adjusted to 4 using 0.1mol/L citric acid in the pretreatment of step 1).

Example 4: another low molecular weight hyaluronic acid

Example 4 provides another low molecular weight hyaluronic acid, which is prepared substantially in the same manner as in example 1, except that in example 4, the pH of the solution is adjusted to 7 in the pretreatment of step 1).

Example 5: another low molecular weight hyaluronic acid

Example 5 provides another low molecular weight hyaluronic acid, which is prepared substantially in the same manner as in example 1, except that in example 5, the degradation time is 1.5 hours in the pretreatment of step 1).

Example 6: another low molecular weight hyaluronic acid

Example 6 provides another low molecular weight hyaluronic acid, which is prepared substantially in the same manner as in example 1, except that in example 6, the degradation time is 5 hours in the step 1) pretreatment.

Example 7: another low molecular weight hyaluronic acid

Example 7 provides another low molecular weight hyaluronic acid, which is prepared substantially in the same manner as in example 1, except that magnesium sulfate is added in an amount of 15% by weight and 35% by weight of the solution in the first time and the second time during the purification of the neutral salt fraction in step 2) of example 7.

Example 8: another low molecular weight hyaluronic acid

Example 8 provides another low molecular weight hyaluronic acid, which is prepared substantially in the same manner as in example 1, except that in example 8, the neutral salt fraction purification process of step 2) is: heating the solution obtained in the step 1) to 40 ℃, adding 25% magnesium sulfate solid of the solution weight, stirring to completely dissolve the solution, then performing ultrafiltration by using a 0.1 mu m sulfonated polysulfone ultrafiltration membrane, then drying the precipitate, and adding 6 times of purified water into the dried precipitate for redissolving.

Example 9: another low molecular weight hyaluronic acid

Example 9 provides another low molecular weight hyaluronic acid prepared substantially in the same manner as in example 1, except that in example 9, sodium sulfate is used as a neutral salt in the step 2) of the neutral salt fractionation.

Example 10: another low molecular weight hyaluronic acid

Example 10 provides another low molecular weight hyaluronic acid prepared substantially in the same manner as in example 1, except that in example 10, the neutral salt is sodium phosphate during the step 2) of fractional purification of the neutral salt.

Example 11: another low molecular weight hyaluronic acid

Example 11 provides another low molecular weight hyaluronic acid, which is prepared substantially in the same manner as in example 1, except that in example 11, the preparation steps are alkaline degradation and filtration purification, and there is no fractional precipitation of neutral salt.

Example 12: another low molecular weight hyaluronic acid

Example 12 provides another low molecular weight hyaluronic acid, which was prepared substantially as in example 1, except that in example 11, the membrane material used in the filtration purification process was a polytetrafluoroethylene membrane.

Experimental example 1: detection of hyaluronic acid degradation

The molecular weight and degradation weight ratio of the products degraded in examples 1 to 12 were measured.

As can be seen from Table 1, the present invention degrades high molecular weight hyaluronic acid by using acidic or alkaline conditions, with a higher degradation weight ratio, wherein the alkaline conditions are more favorable for degradation of hyaluronic acid; as can be seen from examples 1 and 2, the organic base degrades hyaluronic acid more thoroughly, low molecular weight hyaluronic acid and specific gravity are higher; as can be seen from examples 1, 3 and 4, the pH of the solution has great influence on the degradation of hyaluronic acid, and the hyaluronic acid with smaller molecular weight can be degraded with higher pH; from examples 5 to 7, it can be seen that the degradation time has a great influence on both the degraded molecular weight and the degradation weight ratio, wherein the longer the degradation time, the easier the hyaluronic acid with smaller molecular weight can be obtained, and the higher the degradation rate.

Experimental example 2: measuring content, recovery rate and impurity residue of hyaluronic acid in product

The content, recovery rate, protein residue and small molecular impurity residue of hyaluronic acid in the product obtained by the neutral salt fractional precipitation in the embodiment 1-12 are measured;

the hyaluronic acid oligosaccharide monomer or the low molecular hyaluronic acid and the common hyaluronic acid are both composed of N-acetylglucosamine and D-glucuronic acid disaccharide repeating units, so that the content of the hyaluronic acid oligosaccharide monomer or the low molecular hyaluronic acid and the common hyaluronic acid is equal to the content of disaccharide, and the content of disaccharide can be measured by an HPLC method;

high performance liquid chromatography: firstly, weighing a certain amount of standard reference substances (Hyaluronic acid diaschisde Delta DiHA sodium salt, H9649, Sigam), adding into a 50mL volumetric flask, adding ultrapure water for diluting to a scale, and then adding ultrapure water for diluting to obtain a standard reference solution with a certain concentration gradient; adopting a sugar molecular column to carry out high performance liquid chromatography determination, wherein the mobile phase is 0.4mol/L NaH₂PO₄A solution; the flow rate is 0.6ml/min, the column temperature is 35 ℃, the detection wavelength is 232nm, the sample injection amount is 20 mu L, and a standard curve is drawn; weighing 300mg of the product obtained after fractional precipitation of the neutral salt in the embodiment 1-12, adding 3.5mL of 72% sulfuric acid for dissolving, adding 100mL of distilled water, heating in a water bath at 100 ℃ for 2.5h, cooling to room temperature, adding barium carbonate, fully reacting until the pH of the solution is about 7, centrifuging at 3000r/min, washing the precipitate with distilled water for three times, mixing the supernatant and the washing solution together, carrying out vacuum rotary concentration at 45 ℃, fixing the volume to 10mL, and carrying out HPCL determination by injecting 10 mu L of the sample; the content of low molecular weight hyaluronic acid was calculated by peak area according to an external standard method according to the following formula (2):

ax: peak area of hyaluronic acid disaccharide of the sample to be detected;

A_R: peak area of hyaluronic acid disaccharide of the standard control;

W_X: weighing a sample to be detected in mg;

C_R: the concentration of the standard reference substance solution is mg/mL;

h (%): and (5) the sample to be tested loses weight after drying.

TABLE 2 measurement results of hyaluronic acid content, recovery rate, protein residue and small molecular impurity residue in the product

As can be seen from Table 2, the hyaluronic acid prepared by the method has high recovery rate and no residual protein and small molecular impurities. It can also be seen that the kind or amount of the neutral salt has a great influence on the removal of proteins and small molecular impurities and the recovery of hyaluronic acid; compared with inorganic salt degradation, the content of hyaluronic acid degraded by organic base is higher, and the recovery rate of hyaluronic acid is higher; wherein, the protein is removed more thoroughly as the addition amount of the neutral salt is higher, and the removal rate of the neutral salt to the protein is ranked as follows: magnesium sulfate > sodium phosphate > sodium sulfate; as can be seen from comparative example 11, the hyaluronic acid which has not undergone the neutral salt fractionation precipitation contains proteins and small molecular impurities, and the hyaluronic acid prepared by the present invention has a high purity.

Experimental example 3: membrane fouling assay

Firstly, measuring the pure water flux of the membrane, then cleaning the nanofiltration membrane subjected to filtering separation in the embodiments 1-12 for 30min by using an ultrasonic instrument, then washing the nanofiltration membrane by using deionized water, testing the water flux of the cleaned membrane, and calculating the flux recovery rate FRR of the membrane according to the following formula (3):

FRR＝(J_W2/J_W1)×100％ (3)

in the formula: jw₂Pure water flux of the washed membranes, Jw₁The results of the test are shown in fig. 2 for pure water flux of the clean membrane.

By comparing the pollution conditions of the membranes obtained in the examples 1 to 12 after filtration and separation, the membranes can be obviously polluted to different degrees; comparing examples 1-10 and 11, it can be seen that the recovery rate of pure flux of the membrane is higher after the two-stage precipitation of the neutral salt, which indicates that the two-stage precipitation of the neutral salt can effectively remove impurities such as protein and the like, can effectively alleviate the problem of membrane pollution, and prolong the service life of the membrane; as can be seen from comparative example 12, the membrane utilized by the present invention has a high anti-contamination capability ratio and a high recycling rate; it can also be seen that the recovery rate of membrane flux is high up to 95.7% after the membrane is washed by deionized water. Therefore, the method for preparing the low molecular weight hyaluronic acid by using the membrane separation technology makes up for the defects of the prior art, and has great practical significance.

Conventional techniques in the above embodiments are known to those skilled in the art, and therefore, detailed descriptions thereof are omitted here. While the above detailed description has shown, described, and pointed out novel features as applied to various embodiments, it will be understood that various omissions, substitutions, and changes in the form and details of the device or method illustrated may be made without departing from the spirit of the disclosure. In addition, the various features and methods described above may be used independently of one another, or may be combined in various ways. All possible combinations and sub-combinations are intended to fall within the scope of the present disclosure. Many of the embodiments described above include similar components, and thus, these similar components are interchangeable in different embodiments. Although the present invention has been disclosed in the context of certain embodiments and examples, it will be understood by those skilled in the art that the present invention extends beyond the specifically disclosed embodiments to other alternative embodiments and/or uses and obvious modifications and equivalents thereof. Accordingly, the invention is not intended to be limited by the specific disclosure of preferred embodiments herein.