A protein adsorbent for the separation and purification of hyaluronic acid, and its preparation and application methods.

Abstract

This invention belongs to the technical field of hyaluronic acid separation and purification, specifically relating to a protein adsorbent for hyaluronic acid separation and purification, its preparation method, and its usage method. The preparation method of the protein adsorbent includes (1) preparing a sol: first, mixing and stirring the raw material sodium silicate solution with sulfuric acid solution to carry out the reaction; when the reaction has proceeded to 4%-40% of the total reaction time, adding a hydroxyl reversible shielding agent, continuing the reaction, and obtaining a sol after the reaction is completed; the hydroxyl reversible shielding agent is composed of an esterification reaction promoter, a carboxyl activator, and a carboxylic acid compound; (2) preparing a gel: aging, soaking in alkali, and washing with water to obtain a gel; (3) preparing the protein adsorbent: drying and activating the obtained gel to obtain the protein adsorbent. By using the method described, a protein adsorbent with small pore size and abundant surface hydroxyl groups is prepared, achieving high selective adsorption of proteins in hyaluronic acid fermentation broth and achieving the purpose of specific adsorption.

Description

Technical Field

[0001] This invention belongs to the technical field of hyaluronic acid separation and purification, specifically relating to a protein adsorbent for hyaluronic acid separation and purification, its preparation method, and its usage method. Background Technology

[0002] Hyaluronic acid is an essential substance for living organisms, playing a vital role in maintaining eye and skin health, lubricating joints, maintaining cell space, and regulating cell osmotic pressure. It is an important raw material for cosmetics, pharmaceuticals, food, and health products. Therefore, research on the preparation, purification, and separation of hyaluronic acid is of great significance to human health and related industries. Hyaluronic acid is usually prepared by fermentation using specific enzymes. The final fermentation product is a complex colloidal solution rich in enzymes and other proteins. These enzymes and proteins are impurities that must be removed from the hyaluronic acid product. If not completely removed, they will continue to undergo complex biochemical reactions with the hyaluronic acid, seriously affecting product quality. Therefore, it is essential to promptly separate the unwanted enzymes and proteins from the hyaluronic acid product after fermentation.

[0003] Currently, commonly used methods for treating hyaluronic acid fermentation broth mainly include the following categories: ① Adding adsorbents to adsorb and separate unwanted enzymes and proteins from the reaction system after fermentation; ② Adding adsorbents to adsorb and separate hyaluronic acid, the product of fermentation, from the reaction system: both of these methods selectively bind and remove certain substances in the fermentation broth; ③ Adding proteases to indiscriminately and completely decompose all proteins in the fermentation broth, etc. Although the above methods can achieve the purpose of separating hyaluronic acid and proteins to a certain extent, they often have drawbacks such as poor adsorption selectivity, high cost, and introduction of subsequent impurities. However, as a product of the fermentation broth, the content of hyaluronic acid far exceeds that of the enzymes and other proteins used. Therefore, a better approach is to retain the hyaluronic acid product in the fermentation broth as much as possible, while selectively adsorbing and removing enzymes and other proteins, which has greater economic and practical significance.

[0004] Silica gel is a material with a large specific surface area and strong adsorption capacity, often used for impurity adsorption. More importantly, its surface is rich in hydroxyl groups, which have a strong binding force to proteins, making it extremely suitable for the adsorption treatment of protein-rich solutions. In addition, silica gel adsorbents have several other significant advantages: no leaching substances are introduced, preventing the introduction of impurities into the fermentation broth; it does not cause significant loss of the fermentation broth; it is easy to handle after use; and it is non-toxic and odorless, making it one of the safest adsorbents currently available. After protein removal via silica gel adsorption, the shelf life of hyaluronic acid can be greatly extended while maintaining high clarity. Furthermore, compared to other adsorbents, silica gel has a significant cost advantage, thus being considered a very ideal adsorbent.

[0005] However, silica gel adsorbents still face some unresolved challenges in practical applications. On the one hand, existing silica gels typically adsorb all types of substances indiscriminately, exhibiting low selectivity for proteins in fermentation broths. They cannot effectively distinguish between proteins that need to be removed and hyaluronic acid that needs to be retained in the fermentation broth. This makes it difficult to completely separate proteins and also easily leads to the loss of hyaluronic acid, resulting in a reduced hyaluronic acid yield and severely impacting economic value.

[0006] Therefore, there is an urgent need to develop a new type of silica gel with strong selectivity for protein adsorption and weak adsorption for hyaluronic acid, so as to achieve the purpose of specifically adsorbing enzymes and other proteins in fermentation broth. Summary of the Invention

[0007] The purpose of this invention is to address the problem of poor selectivity in the adsorption of enzymes and other proteins by existing silica gels, and to provide a method for preparing a protein adsorbent for the separation and purification of hyaluronic acid. This method allows for precise control of the pore size of the adsorbent material, which not only reduces the pore size but also maximizes the protection of the silanol groups. The resulting protein adsorbent has small pore size and abundant surface hydroxyl groups, achieving highly selective adsorption of proteins in hyaluronic acid fermentation broth, thus achieving specific adsorption.

[0008] During their research, the inventors discovered that enzymes and other proteins, which have the most significant impact on the quality of hyaluronic acid, differ considerably in molecular weight and particle size from hyaluronic acid. Hyaluronic acid has a molecular weight of 1×10⁻⁶. 7 -5×10 7 Between Da, the molecular size is greater than 15 nm; while the molecular weight of enzyme proteins is between 1 × 10⁻⁶. 4 Up to 1×10 5 The particle size is approximately 5-10 nm. However, existing silica gel adsorbents have relatively large pore sizes, which not only reduces the specific surface area but also allows larger particles of hyaluronic acid to diffuse into the large pores, resulting in indiscriminate adsorption of hyaluronic acid in the fermentation broth and reduced selectivity for protein adsorption. If the particle size difference could be utilized for selective separation, by precisely controlling the pore size of the silica gel adsorbent to fall between that of hyaluronic acid and protein, it would be possible to block the diffusion of larger hyaluronic acid particles into the pores through a smaller pore size, reducing unwanted adsorption; simultaneously, it would not affect the diffusion of smaller protein particles into the pores, allowing them to adsorb onto the inner surface of the silica gel, thereby achieving the selective removal of enzymes and other proteins.

[0009] Therefore, achieving precise control over the pore size of adsorbent materials has become crucial to solving the technical problem. In traditional preparation processes, the most critical step in forming the silica gel microstructure is acidification by adding dilute sulfuric acid to a sodium silicate solution. This causes silicate ions to combine with hydrogen ions to form orthosilicic acid. However, orthosilicic acid is unstable and spontaneously undergoes dehydration condensation to form polysilicic acid. The formation of polysilicic acid in this stage has a vital impact on the pore structure and the number of surface hydroxyl groups in the final silica gel product. The degree of dehydration condensation of orthosilicic acid is usually difficult to control effectively; the reaction rate can often only be adjusted by temperature or the pH of the solution. This can easily lead to excessive condensation of orthosilicic acid, resulting in pore closure. Closed or fewer pores are detrimental to the selective diffusion adsorption between hyaluronic acid and proteins.

[0010] To address this issue, the inventors further researched and, for the first time, proposed an inventive concept of acidification-aging with reversible shielding of silanol groups. By reversibly shielding the hydroxyl groups on orthosilicic acid, excessive polymerization of orthosilicic acid is inhibited, making the degree of polymerization highly controllable. This achieves the inhibition of orthosilicic acid dehydration condensation and precise control of the degree of condensation, allowing for precise adjustment of the pore size of the silica gel adsorbent within a certain range. This effectively distinguishes between proteins that need to be removed and hyaluronic acid that needs to be retained in the fermentation broth, improving the purity of the hyaluronic acid product and enhancing product quality. Furthermore, it simultaneously retains a certain amount of hydroxyl groups on the silica gel surface, with a hydroxyl group retention rate of 59-75%. This avoids excessive loss of silanol groups due to their participation in the excessive dehydration condensation reaction of orthosilicic acid, which would affect the binding capacity of silica gel to proteins and consequently the separation and purification effect of hyaluronic acid.

[0011] The specific technical solution is as follows:

[0012] A method for preparing a protein adsorbent for the separation and purification of hyaluronic acid includes the following steps:

[0013] (1) Preparation of sol:

[0014] First, the raw material sodium silicate solution and sulfuric acid solution are mixed and stirred to carry out the reaction, and the pH value is adjusted to 2-4.

[0015] Then, when the reaction has proceeded to 4%-40% of the total reaction time, add the hydroxyl reversible shielding agent and mix thoroughly.

[0016] Continue the reaction until it is complete, then maintain the pH at 3-4 to prepare the sol.

[0017] The hydroxyl reversible shielding agent is composed of an esterification reaction promoter, a carboxyl activator, and a carboxylic acid compound.

[0018] (2) Preparation of gel: The sol obtained in step (1) is aged and soaked in alkali, and then washed with water using a washing solution to obtain a gel; wherein the washing solution is composed of ethanol and an alkaline aqueous solution with a pH of 8-14.

[0019] The purpose of using a water washing solution here is to hydrolyze the ester formed by the combination of carboxylic acid and silanol groups under appropriate conditions. This hydrolysis reduces the silanol groups on the silica gel surface to free carboxylic acid, ultimately restoring a certain number of hydroxyl groups to the silica gel surface. This allows for precise control of the silica gel pore structure while maintaining a certain number of surface silanol groups, preventing excessive loss due to excessive dehydration and condensation of orthosilicic acid, which could affect the adsorption of subsequent enzymes and proteins. Simultaneously, water washing removes impurities such as the hydrolyzed carboxylic acid.

[0020] (3) Preparation of protein adsorbent: The protein adsorbent is obtained by drying and activating the gel obtained in step (2).

[0021] In the above preparation method, when the reaction has proceeded to 4%-40% of the total reaction time, a hydroxyl reversible shielding agent composed of an esterification reaction promoter, a carboxyl activator, and a carboxylic acid is added. At this time, the added hydroxyl reversible shielding agent can play a role in inhibiting the excessively rapid increase in the concentration of hydroxyl groups on orthosilicic acid in the reaction system, reducing the reaction potential and Gibbs free energy of the spontaneous dehydration polymerization of orthosilicic acid to form polysilicic acid, thereby achieving controllable reaction rate of the dehydration polymerization process from orthosilicic acid to polysilicic acid.

[0022] The specific mechanism of action is as follows: The three components in the hydroxyl reversible shielding agent work together synergistically. In the presence of the esterification reaction promoter and the carboxyl activator, the carboxylic acid becomes a temporary shielding or protective agent for the silanol group. In other words, under the catalytic action of the esterification reaction promoter and the carboxyl activator, the carboxylic acid reacts with the hydroxyl group on the orthosilicic acid to form a silicic acid-carboxylic acid ester.

[0023] The so-called dehydration condensation of orthosilicic acid is essentially the dehydration condensation between the four hydroxyl groups contained in orthosilicic acid. Two adjacent hydroxyl groups of orthosilicic acid will condense and lose a water molecule to form polysilicic acid (i.e., silica gel). Therefore, by controlling the number of hydroxyl groups in the orthosilicic acid in the reaction system, the polymerization rate and degree of orthosilicic acid can be controlled, thus achieving precise control over the formation and condensation of orthosilicic acid. However, if the number of hydroxyl groups used for mutual polymerization is reduced too much, it will lead to incomplete polymerization of orthosilicic acid, resulting in a non-dense silica gel with many pores and gaps. Therefore, the timing of adding a hydroxyl reversible shielding agent has a significant impact on the precise and quantitative shielding of the surface hydroxyl groups of orthosilicic acid.

[0024] In this invention, the mass fraction of sodium silicate solution in step (1) of the protein adsorbent preparation method can be 2wt%-50wt%, and the mass fraction of sulfuric acid can be 2wt%-70wt%.

[0025] In this invention, the molar ratio of carboxylic acid: esterification reaction promoter: carboxylic acid group activator in step (1) of the protein adsorbent preparation method is 0.15-6:1:0.5-2; the molar ratio of carboxylic acid: sodium silicate is 0.5-10:1.

[0026] Preferably, the molar ratio of carboxylic acid: esterification reaction promoter: carboxylic acid group activator in the hydroxyl reversible shielding agent is 2:1:1.

[0027] In this invention, the carboxylic acid in step (1) of the protein adsorbent preparation method is one or more of propionic acid, butyric acid, valeric acid, hexanoic acid, benzoic acid, phenylacetic acid, succinic acid, adipic acid, phthalic acid, isophthalic acid, terephthalic acid, fumaric acid, or maleic acid.

[0028] Preferably, the carboxylic acid is one or more of butyric acid, hexanoic acid, phenylacetic acid, adipic acid, phthalic acid, or maleic acid.

[0029] The carboxyl activator in the hydroxyl reversible shielding agent is one or more of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCI), dicyclohexylcarbodiimide (DCC), diisopropylcarbodiimide (DIC), or 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDC).

[0030] The esterification reaction promoter in the hydroxyl reversible shielding agent is one or more of N-hydroxysuccinimide, N-hydroxythiosuccinimide, or N-hydroxysulfosuccinimide.

[0031] In this invention, the alkaline aqueous solution in step (2) of the protein adsorbent preparation method can be a K2CO3 aqueous solution or a Na2CO3 aqueous solution with a mass fraction of 2wt%-90wt%.

[0032] In this invention, the volume ratio of ethanol to alkaline aqueous solution in the washing solution of step (2) of the protein adsorbent preparation method is 0.5-10:1.

[0033] In this invention, the water washing temperature in step (2) of the protein adsorbent preparation method is 10-80℃; preferably, the water washing temperature is 20-25℃.

[0034] In this invention, the drying temperature in step (3) of the protein adsorbent preparation method is 60-180℃, and the drying time is 2-48h.

[0035] In this invention, the activation in step (3) of the protein adsorbent preparation method is carried out by high-temperature calcination, with a calcination temperature of 400-500℃ and a calcination time of 2-96h.

[0036] A protein adsorbent for the separation and purification of hyaluronic acid is prepared using the above-described method; the protein adsorbent has a pore size of 7-10 nm and a specific surface area of ​​520-700 m². 2 / g, with a silanol retention rate of 59-75%.

[0037] The method of using the above-mentioned protein adsorbent includes the following steps: First, at 5-25℃, add the protein adsorbent to the fermentation broth to be treated and stir and react for 1-2.5 hours; wherein, the mass ratio of protein adsorbent to fermentation broth is 1:5-10; then, filter out the protein adsorbent.

[0038] The beneficial effects of this invention are as follows: Based on the reversible shielding of silanol groups on orthosilicic acid, this invention proposes for the first time the inventive concept of acidification-aging with reversible shielding of silanol groups. That is, when the reaction has proceeded to 4%-40% of the total reaction time, a hydroxyl reversible shielding agent composed of an esterification reaction promoter, a carboxyl activator, and a carboxylic acid compound is added. By precisely controlling the timing of the addition of the hydroxyl reversible shielding agent, the surface hydroxyl groups of orthosilicic acid can be reversibly shielded precisely and quantitatively. This enables highly controllable polymerization of orthosilicic acid and controllable adjustment of the silica gel pore structure, allowing the pore size of the silica gel adsorbent to be precisely adjusted within a certain range. By obtaining smaller pores, the enzymes and other proteins that need to be removed from the fermentation broth and the hyaluronic acid that needs to be retained can be effectively distinguished, thus improving the purity of the product and enhancing product quality.

[0039] The protein adsorbent described in this invention is silica gel. Hydroxyl groups are the structural basis for silica gel's adsorption properties; the ability of silica gel to adsorb enzymes and other proteins in fermentation broth relies on the binding of a large number of hydroxyl groups on its surface. Silica gel adsorbs proteins effectively due to its abundant hydroxyl groups. Therefore, to ensure the superior adsorption capacity of silica gel, a certain number of hydroxyl groups must be retained. The ester groups formed by the esterification reaction promoter described above have reversible reaction properties and can undergo hydrolysis with water under appropriate conditions, releasing the shielded silanol groups. Washing can restore the silanol groups, greatly maintaining the number of hydroxyl groups on the silica gel surface and preventing their reduction due to excessive dehydration and condensation, which would decrease adsorption performance. Thus, during the polymerization of silicic acid, a reversible hydroxyl shielding agent can be added at an appropriate time to protect the hydroxyl groups, preventing them from participating in the polymerization of silicic acid into silica gel. Releasing the silanol groups after silica gel has solidified allows for the retention of a large number of hydroxyl groups for the adsorption of enzymes and proteins in the fermentation broth; the appropriate incomplete polymerization of orthosilicic acid also helps protect the porous structure of the silica gel.

[0040] As can be seen, the preparation method described in this invention can combine with hydroxyl groups to form esters when temporary protection of hydroxyl groups is required, and can reversibly remove the hydroxyl-blocking agent at any time to reduce the hydroxyl groups when silanol groups are needed. This allows for the simultaneous control of the silica gel pore structure and the maintenance of the number of surface silanol groups, avoiding the loss of silanol groups due to excessive dehydration and condensation reactions of orthosilicic acid.

[0041] The preparation method described in this invention improves the controllability of silicone channel size to a new level and can simultaneously protect the number of silanol groups. Attached Figure Description

[0042] Figure 1 This is a SEM image of the protein adsorbent described in this invention. Detailed Implementation

[0043] The technical solution of the present invention will be described in detail below.

[0044] Example 1

[0045] The specific steps for preparing the protein adsorbent used for the separation and purification of hyaluronic acid are as follows:

[0046] (1) Preparation of sol:

[0047] First, a sodium silicate solution with a mass fraction of 25 wt% and a sulfuric acid solution with a mass fraction of 35 wt% were mixed and stirred at 30°C for 10 min to carry out the reaction, and the pH value of the mixture was adjusted to 3.

[0048] Then, when the reaction has proceeded for 18 minutes (which is 15% of the total reaction time), add the hydroxyl reversible shielding agent and mix well.

[0049] Continue the reaction until the total reaction time is 2 hours. Once the reaction is complete, maintain the pH at 4 to prepare the sol.

[0050] The hydroxyl reversible shielding agent is composed of N-hydroxythiosuccinimide, an esterification reaction promoter, EDC, a carboxyl activator, and adipic acid; wherein the molar ratio of adipic acid:N-hydroxythiosuccinimide:EDC is 2:1:1; and the molar ratio of adipic acid:sodium silicate is 3:1.

[0051] (2) Preparation of gel: First, the sol obtained in step (1) is aged at 70°C for 2 hours; then, it is soaked in a 30wt% NaOH solution for 15 minutes and then washed with a water washing solution at 25°C to obtain the gel; wherein, the water washing solution is composed of ethanol and K2CO3 aqueous solution with pH value of 10 in a volume ratio of 2:1.

[0052] (3) Preparation of protein adsorbent: After drying the gel obtained in step (2) at 100°C for 12 hours, it is placed in a muffle furnace at 500°C for high-temperature activation for 2 hours to obtain the protein adsorbent.

[0053] At 25°C, the prepared protein adsorbent is mixed with the fermentation broth at a mass ratio of 1:6. After reacting for 2 hours, the protein adsorbent is removed by diatomaceous earth filtration, thus completing the fermentation broth treatment.

[0054] Example 2

[0055] The specific steps for preparing the protein adsorbent used for the separation and purification of hyaluronic acid are as follows:

[0056] (1) Preparation of sol:

[0057] First, a sodium silicate solution with a mass fraction of 25 wt% and a sulfuric acid solution with a mass fraction of 35 wt% were mixed and stirred at 40°C for 10 min to carry out the reaction, and the pH value of the mixture was adjusted to 2.

[0058] Then, when the reaction has proceeded for 5 minutes (which is 4% of the total reaction time), add the hydroxyl reversible shielding agent and mix well.

[0059] Continue the reaction until the total reaction time is 2 hours. Once the reaction is complete, maintain the pH value at 3 to prepare the sol.

[0060] The hydroxyl reversible shielding agent is composed of esterification reaction promoter N-hydroxysulfosuccinimide, carboxyl activator DCC and hexanoic acid; wherein the molar ratio of hexanoic acid:N-hydroxysulfosuccinimide:DCC is 0.2:1:0.5; and the molar ratio of hexanoic acid:sodium silicate is 10:1.

[0061] (2) Preparation of gel: First, the sol obtained in step (1) is aged at 60°C for 2 hours; then, it is soaked in a 30wt% NaOH solution for 15 minutes and then washed with a water washing solution at 20°C to obtain the gel; wherein, the water washing solution is composed of ethanol and a Na2CO3 aqueous solution with a pH of 12 in a volume ratio of 7:1.

[0062] (3) Preparation of protein adsorbent: The gel obtained in step (2) is dried at 180°C for 2 hours and then placed in a muffle furnace at 400°C for high-temperature activation for 96 hours to obtain the protein adsorbent.

[0063] At 5℃, the prepared protein adsorbent is mixed with the fermentation broth at a mass ratio of 1:10. After reacting for 2.5 hours, the protein adsorbent is removed by diatomaceous earth filtration, thus completing the fermentation broth treatment.

[0064] Example 3

[0065] The specific steps for preparing the protein adsorbent used for the separation and purification of hyaluronic acid are as follows:

[0066] (1) Preparation of sol:

[0067] First, a sodium silicate solution with a mass fraction of 25 wt% and a sulfuric acid solution with a mass fraction of 35 wt% were mixed and stirred at 25°C for 10 min to carry out the reaction, and the pH value of the mixture was adjusted to 3.

[0068] Then, when the reaction has proceeded for 48 minutes (which is 40% of the total reaction time), add the hydroxyl reversible shielding agent and mix well.

[0069] Continue the reaction until the total reaction time is 2 hours. Once the reaction is complete, maintain the pH at 4 to prepare the sol.

[0070] The hydroxyl reversible shielding agent is composed of N-hydroxysuccinimide, an esterification reaction promoter, DIC, a carboxyl activator, and phenylacetic acid; wherein the molar ratio of phenylacetic acid:N-hydroxysuccinimide:DIC is 6:1:2; and the molar ratio of phenylacetic acid:sodium silicate is 0.5:1.

[0071] (2) Preparation of gel: First, the sol obtained in step (1) is aged at 60°C for 2 hours; then, it is soaked in a 30wt% NaOH solution for 15 minutes and then washed with a water washing solution at 25°C to obtain the gel; wherein, the water washing solution is composed of ethanol and K2CO3 aqueous solution with pH 9 in a volume ratio of 5:1.

[0072] (3) Preparation of protein adsorbent: After drying the gel obtained in step (2) at 100°C for 24 hours, it is placed in a muffle furnace at 450°C for high-temperature activation for 48 hours to obtain the protein adsorbent.

[0073] At 15℃, the prepared protein adsorbent is mixed with the fermentation broth at a mass ratio of 1:5. After reacting for 2 hours, the protein adsorbent is removed by diatomaceous earth filtration, thus completing the fermentation broth treatment.

[0074] Example 4

[0075] The difference from Example 1 is that the hydroxyl reversible shielding agent in step (1) is composed of N-hydroxysulfosuccinimide, an esterification reaction promoter, EDCI, a carboxyl activator, and maleic acid.

[0076] The others are the same as in Example 1.

[0077] Example 5

[0078] The difference from Example 1 is that the hydroxyl reversible shielding agent in step (1) is composed of N-hydroxysuccinimide, an esterification reaction promoter, EDC, a carboxyl activator, and phthalic acid.

[0079] The others are the same as in Example 1.

[0080] Example 6

[0081] The difference from Example 1 is that the hydroxyl reversible shielding agent in step (1) is composed of N-hydroxythiosuccinimide, an esterification reaction promoter, EDC, a carboxyl activator, and butyric acid.

[0082] The others are the same as in Example 1.

[0083] Example 7

[0084] The difference from Example 1 is that the hydroxyl reversible shielding agent in step (1) is composed of N-hydroxysuccinimide, an esterification reaction promoter, EDC, a carboxyl activator, and maleic acid.

[0085] The others are the same as in Example 1.

[0086] Example 8

[0087] The difference from Example 1 is that the hydroxyl reversible shielding agent in step (1) is composed of N-hydroxythiosuccinimide, an esterification reaction promoter, EDC, a carboxyl activator, and propionic acid.

[0088] The others are the same as in Example 1.

[0089] Example 9

[0090] The difference from Example 1 is that the hydroxyl reversible shielding agent in step (1) is composed of N-hydroxythiosuccinimide, an esterification reaction promoter, EDC, a carboxyl activator, and adipic acid; wherein the molar ratio of adipic acid:N-hydroxythiosuccinimide:EDC is 2:1:2.

[0091] The others are the same as in Example 1.

[0092] Example 10

[0093] The difference from Example 1 is that the hydroxyl reversible shielding agent in step (1) is composed of N-hydroxythiosuccinimide, an esterification reaction promoter, EDC, a carboxyl activator, and adipic acid; wherein the molar ratio of adipic acid:N-hydroxythiosuccinimide:EDC is 2:1:0.5.

[0094] The others are the same as in Example 1.

[0095] Comparative Example 1

[0096] The difference from Example 1 is that the hydroxyl reversible shielding agent in step (1) is composed of N-hydroxythiosuccinimide, an esterification reaction promoter, EDC, a carboxyl activator, and formic acid.

[0097] The others are the same as in Example 1.

[0098] Comparative Example 2

[0099] The difference from Example 1 is that the hydroxyl reversible shielding agent in step (1) is composed of N-hydroxythiosuccinimide, an esterification reaction promoter, EDC, a carboxyl activator, and acetic acid.

[0100] The others are the same as in Example 1.

[0101] Comparative Example 3

[0102] The difference from Example 1 is that the hydroxyl reversible shielding agent in step (1) is composed of N-hydroxythiosuccinimide, an esterification reaction promoter, EDC, a carboxyl activator, and oxalic acid.

[0103] The others are the same as in Example 1.

[0104] Comparative Example 4

[0105] The difference from Example 1 is that the hydroxyl reversible shielding agent in step (1) is composed of N-hydroxythiosuccinimide, an esterification reaction promoter, EDC, a carboxyl activator, and citric acid.

[0106] The others are the same as in Example 1.

[0107] Comparative Example 5

[0108] The difference from Example 1 is that the hydroxyl reversible shielding agent in step (1) is composed of N-hydroxythiosuccinimide, an esterification reaction promoter, EDC, a carboxyl activator, and lactic acid.

[0109] The others are the same as in Example 1.

[0110] Comparative Example 6

[0111] The difference from Example 1 is that the hydroxyl reversible shielding agent in step (1) is composed of N-hydroxythiosuccinimide, an esterification reaction promoter, EDC, a carboxyl activator, and acrylic acid.

[0112] The others are the same as in Example 1.

[0113] The relevant indicators of the protein adsorbents prepared in each embodiment and comparative example are detailed in Table 1.

[0114] Table 1. Relevant Indicators of Various Protein Adsorbents

[0115]

[0116]

[0117] Experimental Example 1

[0118] I. Experimental Objective: To investigate the effect of the timing of the addition of the hydroxyl reversible shielding agent on various indicators of the protein adsorbent.

[0119] II. Experimental Methods: Following the preparation method steps described in Example 1, the single variable was set as the percentage of the total reaction time when the hydroxyl reversible shielding agent was added in step (1), which were 2%, 4%, 8%, 15%, 25%, 35%, 40%, and 45%, respectively. Other steps and conditions were the same as in Example 1.

[0120] III. Experimental Results: See Table 2 for details.

[0121] Table 2. Relevant Indicators of Various Protein Adsorbents

[0122]

[0123] Experiment Example 2

[0124] I. Experimental Objective: To investigate the effect of carboxylic acid dosage on various indicators of protein adsorbent.

[0125] II. Experimental Methods: Following the preparation method steps described in Example 1, the single variable was set as the molar ratio of adipic acid to sodium silicate in step (1) as 0.2:1, 0.5:1, 1:1, 3:1, 5:1, 7:1, 9:1, 10:1, and 11:1, respectively. All other steps and conditions were the same as in Example 1.

[0126] III. Experimental Results: See Table 3 for details.

[0127] Table 3. Relevant Indicators of Various Protein Adsorbents

[0128]

Claims

1. A method for preparing a protein adsorbent for the separation and purification of hyaluronic acid, characterized in that, Includes the following steps: (1) Preparation of sol: First, the raw material sodium silicate solution and sulfuric acid solution are mixed and stirred to carry out the reaction, and the pH value is adjusted to 2-4. Then, when the reaction has proceeded to 4%-40% of the total reaction time, add the hydroxyl reversible shielding agent and mix thoroughly. Continue the reaction until it is complete, then maintain the pH at 3-4 to prepare the sol. The hydroxyl reversible shielding agent is composed of an esterification reaction promoter, a carboxyl activator, and a carboxylic acid compound. (2) Preparation of gel: First, the sol obtained in step (1) is aged and soaked in alkali, and then washed with water solution to obtain gel; The washing solution consists of ethanol and an alkaline aqueous solution with a pH of 8-14. (3) Preparation of protein adsorbent: The protein adsorbent is obtained by drying and activating the gel obtained in step (2).

2. The method for preparing the protein adsorbent according to claim 1, characterized in that, In step (1), the molar ratio of carboxylic acid: esterification reaction promoter: carboxylic acid group activator in the hydroxyl reversible shielding agent is 0.15-6:1:0.5-2; and the molar ratio of carboxylic acid: sodium silicate is 0.5-10:

1.

3. The method for preparing the protein adsorbent according to claim 2, characterized in that, In step (1), the molar ratio of carboxylic acid: esterification reaction promoter: carboxylic acid group activator in the hydroxyl reversible shielding agent is 2:1:

1.

4. The method for preparing the protein adsorbent according to claim 1, characterized in that, The carboxylic acid in the hydroxyl reversible shielding agent in step (1) is one or more of the following: propionic acid, butyric acid, valeric acid, hexanoic acid, benzoic acid, phenylacetic acid, succinic acid, adipic acid, phthalic acid, isophthalic acid, terephthalic acid, fumaric acid, or maleic acid. The carboxyl activator is one or more of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, dicyclohexylcarbodiimide, diisopropylcarbodiimide or 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide; The esterification reaction promoter is one or more of N-hydroxysuccinimide, N-hydroxythiosuccinimide, or N-hydroxysulfosuccinimide.

5. The method for preparing the protein adsorbent according to claim 4, characterized in that, The carboxylic acid in the hydroxyl reversible shielding agent in step (1) is one or more of butyric acid, hexanoic acid, phenylacetic acid, adipic acid, phthalic acid, or maleic acid.

6. The method for preparing the protein adsorbent according to claim 1, characterized in that, The alkaline aqueous solution in step (2) is a K2CO3 aqueous solution or a Na2CO3 aqueous solution.

7. The method for preparing a protein adsorbent according to claim 1, characterized in that, The volume ratio of ethanol to alkaline aqueous solution in the washing solution of step (2) is 0.5-10:

1.

8. The method for preparing a protein adsorbent according to claim 1, characterized in that, The water washing temperature in step (2) is 20-25℃.

9. The method for preparing a protein adsorbent according to claim 1, characterized in that, The drying temperature in step (3) is 60-180℃, and the drying time is 2-48h.

10. The method for preparing a protein adsorbent according to claim 1, characterized in that, The activation in step (3) is carried out by calcination, with a calcination temperature of 400-500℃ and a calcination time of 2-96h.

11. A protein adsorbent for the separation and purification of hyaluronic acid, characterized in that, The protein adsorbent is prepared by the method according to any one of claims 1-10; the pore size is 7-10 nm and the specific surface area is 520-700 m². 2 / g, with a silanol retention rate of 59-75%.

12. The method of using the protein adsorbent as described in claim 11, characterized in that, Includes the following steps: First, the protein adsorbent is added to the fermentation broth to be treated at 5-25℃ and stirred and reacted for 1-2.5 hours; wherein the mass ratio of protein adsorbent to fermentation broth is 1:5-10. Then, filter out the protein adsorbent.

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