A zeolite pearlite powder gel chitosan resin adsorbent and preparation method thereof and purification and production method of hyaluronic acid

Abstract

The present invention belongs to the technical field of microbial extraction, and relates to a zeolite perlite powder gel chitosan resin adsorbent and a preparation method thereof, and a purification and production method of hyaluronic acid. The zeolite perlite powder gel chitosan resin adsorbent includes a gel chitosan resin and zeolite powder and perlite powder loaded thereon; the preparation method of the gel chitosan resin includes: dissolving chitosan in a dilute acid solution to obtain a chitosan dilute acid solution; mixing a metal hydroxide solution with a cross-linking agent to obtain a solidified solution; under stirring, slowly dropping the chitosan dilute acid solution into the solidified solution, filtering after the dropping is completed, washing the wet resin obtained after filtration with water until it is neutral, decolorizing and drying. The above zeolite perlite powder gel chitosan resin adsorbent is used to remove impurities such as bacteria, proteins, pigments, metal ions, etc. in hyaluronic acid fermentation broth, which has the advantages of simple operation, safety, low pollution, low cost, and high impurity removal rate.

Description

Technical Field

[0001] The invention belongs to the technical field of microbial extraction, and specifically relates to a zeolite pearlite powder gel chitosan resin adsorbent and a preparation method thereof, and a purification and production method of hyaluronic acid. Background Art

[0002] Hyaluronic acid (HA), also known as hyaluronic acid or hyaluronic acid, has a molecular structure composed of D-glucuronic acid and N-acetylamino-D-glucose repeatedly connected by β-1,4 and β-1,3 glycosidic bonds. With its unique molecular structure and physical and chemical properties, hyaluronic acid shows a variety of important physiological functions in the body, such as lubricating joints, regulating the permeability of blood vessel walls, regulating the diffusion and operation of proteins, water and electrolytes, and promoting wound healing. Hyaluronic acid is also an inherent component in the human body. It is a glucuronic acid with no species specificity. It is widely present in tissues such as the placenta, amniotic fluid, lens, articular cartilage, and skin dermis. It has excellent water retention and penetration-assisting properties. More importantly, hyaluronic acid has a special water retention effect and is the best moisturizing substance found in nature. It is called an ideal natural moisturizing factor. Hyaluronic acid is widely used in clinical medicine as a binder and in the cosmetics industry as a high-grade water retention factor to replace glycerol. Its demand is increasing day by day, and its application field is gradually expanding. It has been valued by countries around the world.

[0003] At present, hyaluronic acid usually exists in the form of sodium salt. Sodium hyaluronate is a more stable hyaluronic acid with the chemical formula (C 14 H 20 NO 11 Na) n The specific structure is shown in formula (1). Sodium hyaluronate is a white fibrous or paste-like powder, odorless and tasteless, with strong hygroscopicity, soluble in water, and insoluble in organic solvents. In sodium chloride solution, the -COOH group in glucuronic acid dissociates to produce H + This makes hyaluronic acid appear in an acidic polyanion state, giving it the characteristics of an acidic mucopolysaccharide.

[0004]

[0005] Currently, the production process of sodium hyaluronate is divided into two categories: animal tissue extraction method and microbial fermentation method.

[0006] The characteristics of the animal tissue extraction method are simple process flow, large molecular weight of obtained hyaluronic acid, high viscosity and good moisture retention. However, due to the limited constraints of raw material sources, and low hyaluronic acid content in raw materials, hyaluronic acid also coexists with mucopolysaccharides such as chondroitin sulfate in biological tissues, so this method has low output, poor quality and high cost, is difficult to be applied to large-scale production, and is more suitable for small-scale production with dispersed raw material sources. In addition, animal-derived products are easy to become carriers of virus transmission, and there is a risk of causing cross infection, including many countries including China, expressly restricting the sale and use of cosmetics, oral health products and medicines containing these raw materials. This has brought good opportunities for the development of microbial fermentation production.

[0007] The microbial fermentation method utilizes some streptococci in the process of growth and reproduction, secretes metabolites in the capsule outside the cell and forms hyaluronic acid. Compared with the animal tissue extraction method, the microbial fermentation method has the characteristics of production scale not limited by raw materials, infection of pathogenic viruses without animal sources, low cost, high yield, high molecular weight, simple extraction, separation and purification process, easy batch production, etc., and has become the main development direction of hyaluronic acid production. However, the hyaluronic acid fermentation liquid produced by the microbial fermentation method belongs to a typical non-Newtonian Kaisson fluid, and the viscosity coefficient is relatively large, which brings a lot of technical difficulties to subsequent extraction and purification work, and the hyaluronic acid fermentation liquid produced by fermentation contains impurities such as thalline, protein, pigment, metal ions, etc., and the presence of these impurities can cause the extraction rate of hyaluronic acid to reduce, making hyaluronic acid difficult to effectively separate, hindering the industrial production of hyaluronic acid. Therefore, the hyaluronic acid fermentation liquid must be pre-treated to reduce the impact of impurities such as foreign proteins and thalline on the extraction of hyaluronic acid sodium salt in the later stage, so that high-quality sodium hyaluronate products can be obtained.

[0008] In addition, the presence of bacteria and proteins in hyaluronic acid fermentation broth can cause allergies or inflammation when hyaluronic acid is used in cosmetics or pharmaceutical products, so the bacteria and proteins contained therein must be removed as much as possible. It can be seen from the records of existing patent documents and non-patent documents that the current existing technology for removing bacteria and proteins is mainly achieved by adopting the following methods: (1) high-speed centrifugation, which can effectively remove bacteria, but because the solid particles such as bacteria in the fermentation broth are small, a very high speed is required for solid-liquid separation. Therefore, this method has very high requirements for equipment; (2) using chloroform and trichloroacetic acid precipitation method to denature protein and effectively separate protein, but the bacterial precipitate carries a lot of hyaluronic acid, resulting in a low recovery rate of hyaluronic acid, and chloroform has moderate toxicity and is not environmentally friendly; (3) using ultrafiltration membrane technology Separation method can effectively remove protein and bacteria, the process has no phase change, it is easy to operate continuously and scale up, but it is affected by the characteristics of the membrane itself, it is easy to pollute, it is easy to cause membrane pore blockage, the flux decreases quickly, and because the viscosity of hyaluronic acid increases after concentration, the material is easy to remain in the membrane and is difficult to remove, resulting in a decrease in yield; (4) quaternary ammonium salt precipitation method such as CPC and CTAB is used to directly precipitate the fermentation broth, with high purification efficiency, but this method has a large demand for quaternary ammonium salts, is expensive, and has high cost; (5) enzymatic hydrolysis method, mild conditions, can effectively remove protein, but this method may introduce new proteins, resulting in a slower separation speed. The above-mentioned methods for removing bacteria and proteins all have many disadvantages and are not suitable for industrial production of hyaluronic acid.

[0009] At the same time, hyaluronic acid prepared by microbial fermentation usually contains some multivalent metal ions (such as Fe 3+ Mg 2 + , Cu 2+ , Ca 2+ 、Zn 2+ 、As 2+ 、Cd 2+ , Hg 2+ , Pb 2+ Etc.) and some colored impurities, if they cannot be completely removed, will seriously affect the application and sensory quality of hyaluronic acid. However, from the records of existing patent documents and non-patent literature, it is known that the removal methods of metal ions and colored substances are mostly removed by using ethanol multiple fractionation and ultrafiltration processes, but when using ethanol multi-stage separation process to remove metal ions and colored substances, the process is too cumbersome, and the processing capacity is very large when used for large-scale production, resulting in high costs, which is not suitable for industrial production; when using ultrafiltration process to remove metal ions, although the metal ion removal effect is very good, due to the hyaluronic acid viscosity will increase significantly with the reduction of filtered concentrate, which will cause membrane clogging, difficult to handle, this is not only costly, and the maintenance of the membrane and the requirements for processing feed liquid are also increased.

[0010] At the same time, the existing public technology shows that the removal of colored substances in hyaluronic acid can be completed by using some adsorbents, such as activated carbon, diatomaceous earth, perlite, alumina, silica gel, clay, etc. Among them, activated carbon is a porous substance with a developed pore structure and a huge specific surface area. It has a certain adsorption capacity for color, smell, taste, disinfection byproducts, trace organic pollutants, etc. At the same time, it is also the most ideal decolorizer in the market with a significant decolorization effect. However, since activated carbon contains a small amount of oxygen, hydrogen, sulfur, nitrogen, chlorine and other elements, it will also produce new solid salts in the solution on the basis of decolorization, thereby affecting the quality of hyaluronic acid. In addition, the market price of activated carbon is expensive and the cost is high, which is not suitable for industrial use. Perlite and diatomaceous earth are combined with activated carbon to adsorb and decolorize hyaluronic acid fermentation liquid. Perlite and diatomaceous earth have basically the same properties. Because of their porous properties and light specific surface density, they have strong adsorption, low price, easy regeneration, and easy activity control. However, perlite and diatomaceous earth have the highest adsorption for polyvalent metal ions and proteins under slightly acidic conditions, so they are selective for removing impurities in hyaluronic acid and have a low removal rate. Perlite and diatomaceous earth are more used as filter aids and are the best and preferred materials for filter aids on the market so far. Alumina and silica gel are also commonly used strong polar adsorbents, but due to their super strong adsorption, they will cause the loss of nutrients while adsorbing impurities, so they are not easy to choose. White clay is a kind of decolorizing agent, but because white clay has a light specific surface density and high viscosity, it is not suitable for adsorbing impurities in hyaluronic acid with greater viscosity, which will cause difficulty in filtration after adsorption and difficult to regenerate.

[0011] In summary, when using microbial fermentation engineering technology to mass produce hyaluronic acid, it is currently necessary to find a method that is simple to operate and can quickly remove bacteria, proteins, metal ions and colored substances to reduce production costs and make it suitable for large-scale industrial production. Summary of the invention

[0012] The purpose of the present invention is to overcome the defects of complicated process and low impurity removal rate when using existing methods to remove impurities such as bacteria, proteins, pigments, metal ions in hyaluronic acid fermentation broth, and to provide a new zeolite perlite powder gel chitosan resin adsorbent and a preparation method thereof and a purification and production method of hyaluronic acid. When the zeolite perlite powder gel chitosan resin adsorbent is used to remove impurities such as bacteria, proteins, pigments, metal ions in hyaluronic acid fermentation broth, it has the advantages of simple operation, safety, low pollution, low cost and high impurity removal rate.

[0013] Specifically, the present invention provides a zeolite perlite powder gel chitosan resin adsorbent, wherein the zeolite perlite powder gel chitosan resin adsorbent comprises a gel chitosan resin and zeolite powder and perlite powder loaded on the gel chitosan resin;

[0014] The preparation method of the gel chitosan resin comprises the following steps:

[0015] S11, dissolving chitosan in a dilute acid solution to obtain a chitosan dilute acid solution;

[0016] S12, mixing the metal hydroxide solution and the cross-linking agent uniformly to obtain a solidified solution;

[0017] S13, under stirring, slowly drop the chitosan dilute acid solution into the curing solution, filter after the dropwise addition is complete, wash the wet resin obtained after filtration with water until it is neutral and then decolorize, and dry the decolorized resin to obtain a gel chitosan resin.

[0018] In a preferred embodiment, in step S11, the dilute acid solution is selected from at least one of hydrochloric acid solution, acetic acid solution, oxalic acid solution and phosphoric acid solution.

[0019] In a preferred embodiment, in step S11, the concentration of the dilute acid solution is 0.5-5% v / v.

[0020] In a preferred embodiment, in step S11, the chitosan and the dilute acid solution are used in such amounts that the concentration of the obtained chitosan dilute acid solution is 0.01-5%.

[0021] In a preferred embodiment, in step S12, the metal hydroxide in the metal hydroxide solution is potassium hydroxide and / or sodium hydroxide.

[0022] In a preferred embodiment, in step S12, the concentration of the metal hydroxide solution is 1-30% w / v.

[0023] In a preferred embodiment, in step S12, the cross-linking agent is selected from at least one of formaldehyde, glutaraldehyde and epichlorohydrin.

[0024] In a preferred embodiment, in step S12, the amount of the cross-linking agent added is 0.1 to 0.5 times the total volume of the curing solution.

[0025] In a preferred embodiment, in step S13, the decolorization method is to immerse the neutral resin obtained by washing with water in acetone.

[0026] In a preferred embodiment, in step S13, the amount of acetone used is 1-10% v / v of the total volume of the neutral resin.

[0027] In a preferred embodiment, in step S13, the immersion conditions include room temperature and a time of 1 to 5 hours.

[0028] The present invention also provides a method for preparing the zeolite perlite powder gel chitosan resin adsorbent, wherein the method comprises loading zeolite powder and perlite powder on the gel chitosan resin.

[0029] In a preferred embodiment, the method of loading zeolite powder and perlite powder on the gel chitosan resin comprises the following steps:

[0030] S21, placing zeolite powder and perlite powder with a particle size of less than 100 meshes in a dilute acid solution A for soaking, washing with water until neutral, and drying to obtain a zeolite powder-perlite powder composite;

[0031] S22, stirring and mixing the zeolite powder-perlite powder composite and the gel chitosan resin in a dilute acid solution B, and then dropping the obtained mixture into an alkaline solution, and then washing the obtained loaded gel chitosan zeolite perlite powder pellets with water until neutral, drying and sieving, to obtain a zeolite perlite powder gel chitosan resin adsorbent.

[0032] In a preferred embodiment, in step S21, the mass ratio of the zeolite powder to the perlite powder is 1:(0.5-5).

[0033] In a preferred embodiment, in step S21, the soaking conditions include room temperature and a soaking time of 2 to 10 hours.

[0034] In a preferred embodiment, in step S21, the concentration of the dilute acid solution A is 0.1 to 10 mol·L -1 .

[0035] In a preferred embodiment, in step S22, the usage ratio of the zeolite powder-perlite powder composite, the gel chitosan resin and the dilute acid solution B is (50-300) g: 1 g: (100-3000) mL.

[0036] In a preferred embodiment, in step S22, the concentration of the dilute acid solution B is 0.1 to 10 mol·L -1 .

[0037] In a preferred embodiment, in step S22, the stirring and mixing conditions include a temperature of 10 to 60° C. and a time of 1 to 10 hours.

[0038] In a preferred embodiment, in step S22, the alkaline solution is a sodium hydroxide solution and / or a potassium hydroxide solution.

[0039] In a preferred embodiment, in step S22, the concentration of the alkaline solution is 0.1 to 10 mol·L -1 .

[0040] The present invention also provides application of the zeolite pearlite powder gel chitosan resin adsorbent in purifying hyaluronic acid.

[0041] The present invention also provides a method for purifying hyaluronic acid, wherein the method comprises the following steps:

[0042] S31, hyaluronic acid dissociation: dissociating the hyaluronic acid fermentation liquid with a viscosity of 0.1-5 Pa.s with sodium chloride to obtain a sodium hyaluronate dissociation liquid;

[0043] S32, impurity removal: adding composite adsorbent A to the sodium hyaluronate dissociation solution obtained in step S31 to perform stirring and adsorption for initial impurity removal, and performing a first filtration after the initial impurity removal is completed, wherein the composite adsorbent B is used as a filtration medium for the first filtration to obtain an impurity-removed solution;

[0044] S33. The pH value of the impurity removal liquid is adjusted to alkaline for degradation. After the degradation is completed, the composite adsorbent C is added to the obtained degradation liquid for stirring and adsorption to remove impurities in depth. After the deep impurity removal is completed, a second filtration is performed, and the pH value of the obtained filtrate is adjusted to 6-8 to obtain a hyaluronic acid filtrate; the composite adsorbent A, composite adsorbent B and composite adsorbent C are all the above-mentioned zeolite perlite powder gel chitosan resin adsorbents.

[0045] In a preferred embodiment, in step S31, the ratio of the amount of sodium chloride to the hyaluronic acid fermentation broth is (0.01-5.0) g:1 mL.

[0046] In a preferred embodiment, in step S31, the dissociation conditions include a temperature of 30 to 60°C and a time of 0.5 to 2 hours.

[0047] In a preferred embodiment, in step S32, the amount of the composite adsorbent A is 0.01-10% of the volume of the sodium hyaluronate dissociation solution.

[0048] In a preferred embodiment, in step S32, the conditions for the initial impurity removal by stirring and adsorption include a temperature of 30 to 60° C. and a time of 0.5 to 5 h.

[0049] In a preferred embodiment, in step S32, the conditions for the first filtration include a filter medium coating amount of 0.5-5% of the total volume of the reaction solution, a filtration rate of 20-30 L / h, and a filtration pressure of 0.1-0.3 MPa.

[0050] In a preferred embodiment, in step S33, the reagent used for adjusting the pH value is alkaline solution.

[0051] In a preferred embodiment, in step S33, the alkali solution is a sodium hydroxide solution.

[0052] In a preferred embodiment, in step S33, the concentration of the alkali solution is 0.1 to 20 mol·L -1 .

[0053] In a preferred embodiment, in step S33, the degradation conditions are such that the number average molecular weight of the obtained degradation solution is 1.0×10 6 ~2.0×10 6 Da.

[0054] In a preferred embodiment, in step S33, the amount of the composite adsorbent C is 0.1-10% of the volume of the degradation liquid.

[0055] In a preferred embodiment, in step S33, the pore size of the filter membrane used for the second filtration is 0.1-10 μm.

[0056] In addition, the present invention also provides a method for producing hyaluronic acid, wherein the method comprises the following steps:

[0057] S41, adjusting the viscosity of the hyaluronic acid fermentation liquid obtained by the microbial fermentation method to 0.1-5 Pa.s;

[0058] S42, purifying the hyaluronic acid fermentation liquid with a viscosity of 0.1 to 5 Pa.s by the above method to obtain a hyaluronic acid refined filtrate;

[0059] S43, concentrating the hyaluronic acid filtrate, filtering the obtained concentrated solution after alcohol precipitation and crystallization, and drying the obtained filter cake to obtain sodium hyaluronate crystals.

[0060] In a preferred embodiment, in step S43, the concentration is carried out in a jacketed coupled MVR stirred multi-effect steam kettle, which comprises a heat exchanger, a low-pressure steam kettle, a high-pressure steam kettle, a flash tank-1, a flash tank-2 and a compressor, wherein the outlet of the heat exchanger is connected to the top of the low-pressure steam kettle, the bottom of the low-pressure steam kettle is connected to the top of the high-pressure steam kettle, the jacket outlet of the low-pressure steam kettle is connected to the inlet of the flash tank-2, the outlet of the flash tank-2 is connected to the jacket inlet of the high-pressure steam kettle via the compressor, the jacket outlet of the high-pressure steam kettle is connected to the inlet of the flash tank-1, and the flash tank-1 is connected to the jacket inlet of the high-pressure steam kettle. The outlet is connected to the jacket inlet of the low-pressure steam kettle; the hyaluronic acid refined filtrate is heated to 45-55°C by a heat exchanger and then sent to a low-pressure steam kettle and a high-pressure steam kettle in sequence for concentration, the pressure in the low-pressure steam kettle is controlled at 50-60kPa, and the pressure in the high-pressure steam kettle is controlled at 70-80kPa, and the concentrated liquid finally obtained is discharged from the bottom of the high-pressure steam kettle for alcohol precipitation and crystallization; the secondary steam produced by the low-pressure evaporator is first recovered part of the steam through the flash tank-2 and then pressurized and heated by the compressor as a heat source in the high-pressure steam kettle, and the secondary steam produced by the high-pressure evaporator is recovered part of the steam through the flash tank-1 and used as a heat source for the low-pressure evaporator.

[0061] In a preferred embodiment, in step S43, the alcohol precipitation and crystallization method is to add 1 to 2 times the volume of ethanol with a concentration of more than 90% to the concentrated solution, stir and precipitate, and then let it stand. When the supernatant is clear, the upper layer is separated. When the supernatant is clear, 1 to 1.5 times the volume of ethanol with a concentration of 70% to 80% is added to the remaining base material, stir and precipitate, and then let it stand. When the supernatant is clear, the upper layer is separated, and 1 to 1.5 times the volume of ethanol with a concentration of more than 90% is added to the remaining base material again, stir and precipitate.

[0062] In a preferred embodiment, the drying conditions include a temperature of 30° C. to 60° C. and a time of 8 h to 24 h.

[0063] The beneficial effects of the present invention are as follows:

[0064] (1) The key to the present invention is to use a composite adsorbent formed by compounding gel chitosan resin, zeolite powder and perlite powder as an adsorbent for purifying hyaluronic acid fermentation broth. The composite adsorbent can effectively remove pigments and polyvalent metal ions in the hyaluronic acid fermentation broth, and significantly remove bacteria and proteins in the hyaluronic acid fermentation broth. Compared with similar technical solutions, the advantages of using this composite adsorbent are: (a) it still has a strong adsorption and impurity removal ability for bacteria and proteins in non-inactivated high-viscosity fermentation broth, and has a wide range of applicability; (b) based on the synergistic characteristics of the three adsorbents chitosan, zeolite powder and perlite powder, the special structure formed by the combination of the three enables its adsorption of proteins, pigments and heavy metals to far exceed the technical effects brought by the three or the simple mixture of the three, while still retaining the advantages of no adsorption of nutrients and minimal loss rate; (c) the use of composite adsorbents has a high treatment efficiency, which can greatly shorten the process flow, reduce the requirements for process equipment, and save costs; (d) the raw materials of the above composite adsorbents are cheap, have a long life, and can be regenerated for use; (e) it is suitable for industrial production, with low cost and high efficiency.

[0065] (2) The method provided by the present invention is used to purify the hyaluronic acid fermentation liquid, which can quickly and effectively remove the protein, bacteria, some heavy metals, polyvalent metal ions and colored impurities in the hyaluronic acid fermentation liquid at the same time, avoiding the use of trichloroacetic acid and chloroform to remove the protein after denaturation and causing unfriendly to people and the environment; there is no need to use enzymatic hydrolysis to cause the introduction of new proteins and the problem of slowing down the separation speed; at the same time, it is also possible to avoid the high cost and low yield problems when using enzymatic hydrolysis, ethanol precipitation, quaternary ammonium salt precipitation and membrane separation to remove proteins and metal ions. In addition, the entire purification and production process of the present invention is simple to operate, low in cost, and suitable for large-scale industrial production.

[0066] (3) The method for extracting sodium hyaluronate from microbial fermentation broth provided by the present invention has a very simple entire extraction and purification process. No toxic or harmful substances are added during the entire process, and no environmental pollution is caused. The material and energy consumption is low. At the same time, the equipment requirements used in the entire process are simpler than those of the existing process, and the amount of solvent used is significantly reduced. The prepared sodium hyaluronate product has a content of more than 95% and a purity of >99.0%. It contains no heavy metals or proteins, is very suitable for large-scale industrial production, and has broad application prospects.

[0067] In a preferred embodiment, the concentration of the hyaluronic acid filtrate is carried out in a jacketed coupled MVR stirred multi-effect steam kettle. The advantages of the jacketed coupled MVR stirred multi-effect steam kettle are: (a) It has strong practicality for the hyaluronic acid to be extracted. Because hyaluronic acid is a heat-sensitive substance, the equipment can achieve evaporation at 50°C without freezing, and continuous circulation is carried out by using relatively little energy. In this case, primary steam is not required as a heating medium, the unit energy consumption is low, and the operating cost is low. At the same time, the designed stirring device can prevent the material from scaling and the decrease of the heat transfer coefficient during the concentration process, which makes it difficult to continue the concentration of the raw material liquid; (b) Due to the low temperature difference, the evaporation of the product is gentle, the hyaluronic acid is highly practical, the operation is stable, the degree of automation is high, and the energy saving is high. The advantages of the present invention are as follows: (a) high evaporation efficiency, simple operation, high equipment anti-corrosion performance, convenient descaling, and low overall system operating costs; (b) the system can be operated under normal pressure or negative pressure conditions; and (c) the negative pressure conditions not only reduce the evaporation temperature of the raw material liquid, but also reduce the material requirements for system-related equipment and pipelines, while ensuring the continuous and stable production of the system; and (d) compared with the single-stage MVR equipment, the equipment evaporates part of the feed water at a lower concentration, thereby greatly reducing the steam flow rate processed by the compressor of the multi-effect evaporation system coupled with MVR. In addition, the flash tank is used to recover part of the steam energy, which greatly reduces the power consumption of the compressor; and (e) the volume of the hyaluronic acid filtrate is reduced without consuming a lot of energy, thereby saving the consumption of ethanol and the high cost caused by the recovery of a large amount of ethanol. BRIEF DESCRIPTION OF THE DRAWINGS

[0068] Figure 1 A schematic diagram of the structure of a jacketed coupled MVR stirring multi-effect steam kettle provided by the present invention;

[0069] Figure 2 The decolorization results of hyaluronic acid fermentation liquid using pearlite powder, zeolite powder, the gel chitosan resin obtained in Preparation Example 1, and the zeolite pearlite powder gel chitosan resin adsorbent obtained in Preparation Example 1;

[0070] Figure 3 The infrared spectra of the gel chitosan resin obtained in Preparation Example 1 and the zeolite perlite powder gel chitosan resin adsorbent obtained in Preparation Example 1, wherein a is the infrared absorption spectrum corresponding to the gel chitosan resin, and b is the infrared absorption spectrum corresponding to the zeolite perlite powder gel chitosan resin adsorbent;

[0071] Figure 4 The infrared spectra of hyaluronic acid before and after filtering using the zeolite pearlite powder gel chitosan resin adsorbent obtained in Preparation Example 1;

[0072] Figure 5It is a graph showing the adsorption results of hyaluronic acid by pearlite powder, zeolite powder, the gel chitosan resin obtained in Preparation Example 1, and the zeolite pearlite powder gel chitosan resin adsorbent obtained in Preparation Example 1;

[0073] Figure 6 It is a void distribution curve diagram of pearlite powder, zeolite powder, the gel chitosan resin obtained in Preparation Example 1, and the zeolite pearlite powder gel chitosan resin adsorbent obtained in Preparation Example 1;

[0074] Figure 7 The surface morphology of the chitosan gel particles obtained in Preparation Example 1 and the zeolite perlite powder gel chitosan resin adsorbent obtained in Preparation Example 1, wherein A is the surface morphology of the chitosan gel particles, and B is the surface morphology of the zeolite perlite powder gel chitosan resin adsorbent;

[0075] Figure 8 The antibacterial effect diagrams of the chitosan gel particles obtained in Preparation Example 1 and the zeolite perlite powder gel chitosan resin adsorbent obtained in Preparation Example 1 on Staphylococcus aureus ATCC 6538 (a) and Escherichia coli ATCC 8739 (b), wherein A is the antibacterial effect diagram of the chitosan gel particles, and B is the antibacterial effect diagram of the zeolite perlite powder gel chitosan resin adsorbent. DETAILED DESCRIPTION

[0076] The zeolite perlite powder gel chitosan resin adsorbent (composite adsorbent) provided by the present invention comprises a gel chitosan resin and zeolite powder and perlite powder loaded on the gel chitosan resin. This specific composite adsorbent can improve the adsorption capacity of proteins, metal ions and colored substances in a non-Newtonian Chisson fluid such as hyaluronic acid, thereby effectively removing the above substances, and has the advantages of low price, easy regeneration, small loss of hyaluronic acid nutrients, antibacterial, etc.

[0077] From the molecular structure, we can see that chitosan contains abundant amino and hydroxyl groups, which can effectively inhibit Pb 2+ , Cu 2+ Cr 2+ 、Cd 2+ 、As 2+ , Hg 2+ , Pb 2+ , Mn 2+ It has coordination and chelation effects on heavy metal ions such as ions. Due to the presence of free amino groups in chitosan molecules, they are protonated in dilute solutions, making the molecular chain carry a large amount of positive charge and become a typical cationic flocculant. Therefore, it has the dual effects of charge neutralization and adsorption flocculation.

[0078] Chitosan has a strong effect on protein adsorption, because the protein molecule chain carries basic groups (such as amino groups) and acidic groups (such as carboxyl groups), which can both accept and release protons in the solution, and is an amphoteric compound. The solubility of protein is the lowest at the isoelectric point. When the value of the solution is higher than the isoelectric point of the protein, the protein is negatively charged. Conversely, when the value of the solution is lower than the isoelectric point of the protein, the protein is positively charged. The adsorption of chitosan on protein is mainly electrical neutralization, that is, the positively charged amino groups in the chitosan molecules and the negatively charged groups in the protein molecules attract each other, effectively neutralizing the surface charge of the protein and causing it to condense and sink. Therefore, chitosan has a strong ability to aggregate proteins. The mechanism of chitosan adsorption of protein is shown in the following formulas (2) and (3).

[0079]

[0080] However, chitosan still has some shortcomings: it is not very convenient to use chitosan directly as a material, the scope of application is narrow, it is non-porous and easy to lose, so the present invention needs to make chitosan into a gel chitosan resin. Specifically, the preparation method of the gel chitosan resin includes the following steps: S11, dissolving chitosan in a dilute acid solution to obtain a chitosan dilute acid solution; S12, mixing a metal hydroxide solution and a cross-linking agent evenly to obtain a solidifying solution; S13, under stirring, slowly adding the chitosan dilute acid solution to the solidifying solution, filtering after the addition is complete, washing the wet resin obtained after filtration with water to neutrality and then decolorizing, and the decolorized resin obtained is dried to obtain a gel chitosan resin.

[0081] In step S11, specific examples of the dilute acid solution include, but are not limited to, at least one of hydrochloric acid solution, acetic acid solution, oxalic acid solution and phosphoric acid solution, preferably hydrochloric acid solution. The concentration of the dilute acid solution is preferably 0.5-5% v / v, more preferably 0.5-2% v / v. The amount of chitosan and the dilute acid solution is preferably such that the concentration of the obtained chitosan dilute acid solution is 0.01-5% (w / v, g / mL), more preferably 1-3%.

[0082] In step S12, the metal hydroxide in the metal hydroxide solution may be potassium hydroxide and / or sodium hydroxide. The concentration of the metal hydroxide solution is preferably 1 to 30% w / v, more preferably 2 to 8% w / v. Step S12 requires compounding the alkaline metal hydroxide solution with a cross-linking agent, wherein specific examples of the cross-linking agent include, but are not limited to, at least one of formaldehyde, glutaraldehyde, and epichlorohydrin. Among them, selecting methanol as a cross-linking agent can make the prepared gel chitosan resin scaffold pore size more regular, the porosity, water absorption rate and swelling rate higher, the pore connections more compact, and the size more uniform. Therefore, the cross-linking agent is preferably methanol. In addition, the amount of the cross-linking agent added is preferably 0.1 to 0.5 times the total volume of the curing solution, more preferably 0.1 to 0.3 times.

[0083] In step S13, the reason why it is necessary to carry out the process under stirring is to prevent the generated chitosan wet resin from sticking. The decolorization method is preferably to impregnate the neutral resin obtained by washing with acetone. Among them, the amount of acetone is preferably 1-10% v / v of the total volume of the neutral resin, and more preferably 2-8% v / v. In addition, the impregnation conditions generally include a temperature of room temperature (20-40°C) and a time of 1-5h. The wet gel chitosan resin (wet resin) obtained in step S13 is dark yellow, and some fat-soluble and water-insoluble substances adhere to the surface of the gel chitosan resin. In order to avoid affecting the subsequent hyaluronic acid extraction and further improve the adsorption of the gel chitosan resin, the present invention uses acetone to decolorize the wet resin. Acetone is the simplest ketone in the saturated fatty ketone series and is an important organic solvent. It can completely dissolve the water-insoluble lipid substances on the surface of the gel chitosan resin, thereby widening the pores of the gel chitosan resin and playing a decolorizing role. In addition, the drying temperature can be 50-80°C. The drying is preferably vacuum drying. In order to be more conducive to improving the adsorption performance, preferably, the drying further includes a grinding or crushing and screening step.

[0084] Zeolite is a porous aluminosilicate mineral with a skeleton structure. The most basic structure of its skeleton is silicon-oxygen tetrahedron and aluminum-oxygen tetrahedron. It is a highly polar adsorbent with a strong affinity for polar molecules and unsaturated molecules. It also has a high selective adsorption advantage for molecules with large polarization rates among non-polar molecules. The cations in the pores and channels of zeolite also have strong selective ion exchange properties, which can remove heavy metal ions and cyanide that are harmful to animals. In addition, zeolite also has unique acid resistance, thermal stability, antibacterial properties and good decolorization effects. In combination with perlite powder, it can have a good adsorption and filtration effect on hyaluronic acid, an acidic high-molecular mucopolysaccharide. At the same time, it can produce strong adsorption and local catalysis for the pigments in the hyaluronic acid material, and has a very strong adsorption and treatment capacity for proteins and metal ions. The experimental results show that the zeolite perlite powder gel chitosan resin adsorbent provided by the present invention has a certain antibacterial ability, and has a certain antibacterial ability against Staphylococcus aureus and Escherichia coli. Moreover, the adsorption can still reach the same level after multiple regenerations, and has a good application prospect.

[0085] The present invention does not particularly limit the manner in which zeolite powder and perlite powder are loaded onto the gel chitosan resin, and preferably includes the following steps: S21, placing zeolite powder and perlite powder with a particle size of less than 100 meshes together in a dilute acid solution A for immersion treatment, washing with water to neutrality after immersion, and drying to obtain a zeolite powder-perlite powder complex; S22, stirring and mixing the zeolite powder-perlite powder complex and the gel chitosan resin in a dilute acid solution B, and then dropping the obtained mixture into an alkaline solution, and then washing the obtained loaded gel chitosan zeolite perlite powder pellets with water to neutrality, drying and sieving to obtain a zeolite perlite powder gel chitosan resin adsorbent.

[0086] In step S21, since the particle size of zeolite powder and perlite powder is generally large, it is usually necessary to perform screening treatment before soaking to control the particle size below 100 mesh. The particle size of the zeolite powder and perlite powder used initially can be 20 to 600 meshes independently, preferably 100 to 200 meshes independently. The mass ratio of the zeolite powder and perlite powder is preferably 1: (0.5 to 5), such as 1: 0.5, 1: 1, 1: 1.5, 1: 2, 1: 2.5, 1: 3, 1: 3.5, 1: 4, 1: 4.5, 1: 5, etc. The soaking conditions preferably include a temperature of room temperature (20 to 40 ° C); a time of 2 to 10 hours.

[0087] In step S22, the usage ratio of the zeolite powder-perlite powder composite, the gel chitosan resin and the dilute acid solution B is preferably (50-300) g: 1 g: (100-3000) mL, more preferably (80-120) g: 1 g: (800-1200) mL. Specifically, based on the amount of gel chitosan resin as 1g, the amount of the zeolite powder-perlite powder composite is preferably 50-300g, such as 50, 60, 70, 80, 90, 100, 120, 150, 180, 200, 220, 250, 280, 300g; the amount of the dilute acid solution B is preferably 100-3000mL, such as 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1200, 1500, 1800, 2000, 2200, 2500, 2800, 3000, etc. The conditions for stirring and mixing uniformly generally include a temperature of 10-60°C, preferably 35-45°C; and a time of 1-10h, preferably 2-6h. In addition, the alkaline solution may be a sodium hydroxide solution and / or a potassium hydroxide solution. The concentration of the alkaline solution is preferably 0.1 to 10 mol·L -1 , more preferably 0.5 to 1 mol·L -1 .

[0088] For ease of description, the dilute acid solution used in step S21 is referred to as "dilute acid solution A", and the dilute acid solution used in step S22 is referred to as "dilute acid solution B". The dilute acid solution A and the dilute acid solution B can be independently selected from at least one of hydrochloric acid solution, acetic acid solution, oxalic acid solution and phosphoric acid solution. In addition, the concentrations of the dilute acid solution A and the dilute acid solution B can be independently 0.1 to 10 mol·L -1 , preferably each independently 0.1 to 1 mol·L -1 .

[0089] After in-depth research, the inventors of the present invention found that the composite adsorbent has the best adsorption effect under weakly acidic conditions with a pH value of 4.5 to 5.5.

[0090] The present invention also provides application of the zeolite pearlite powder gel chitosan resin adsorbent in purifying hyaluronic acid.

[0091] The purification method of hyaluronic acid provided by the present invention comprises the following steps: S31, hyaluronic acid dissociation: dissociating hyaluronic acid fermentation liquid with a viscosity of 0.1 to 5 Pa.s with sodium chloride to obtain sodium hyaluronate dissociation liquid; S32, impurity removal: adding composite adsorbent A to the sodium hyaluronate dissociation liquid obtained in step S31 to carry out stirring and adsorption for initial impurity removal, and performing a first filtration after the initial impurity removal, wherein the first filtration uses composite adsorbent B as a filter medium to obtain impurity-removed liquid; S33, adjusting the pH value of the impurity-removed liquid to alkaline for degradation, and after the degradation is completed, adding composite adsorbent C to the obtained degradation liquid for stirring and adsorption for deep impurity removal, and performing a second filtration after the deep impurity removal is completed, and adjusting the pH value of the obtained filtrate to 6 to 8 to obtain hyaluronic acid refined filtrate.

[0092] Since hyaluronic acid is an acidic mucopolysaccharide, a special non-Newtonian Chisson fluid, the viscosity of the hyaluronic acid fermentation broth can generally reach 1 to 10 Pa.s. High viscosity not only affects the adsorption effect of the adsorbent, but also causes troubles in transportation. Therefore, the fermentation broth should be diluted to 0.1 to 5 Pa.s before purification.

[0093] The hyaluronic acid is usually present in the form of sodium salt. Sodium hyaluronate is a more stable hyaluronic acid. Therefore, before preparing the hyaluronic acid crystalline powder, it is necessary to use a certain amount of sodium chloride to dissociate it. Because in the sodium chloride solution, the -COOH in the glucuronic acid dissociates to produce H + Make hyaluronic acid into acidic polyanion state, dissociated -COO and Na + Recombines into desired sodium hyaluronate product.

[0094] In step S31, the mass of NaCl required for the dissociation is quantified according to the concentration of salt in the total solution. After extensive and in-depth research, the inventors of the present invention found that hyaluronic acid is precipitated in ethanol, and the salt concentration needs to be >1.5% (w / v, g / mL). At the same time, the composite adsorbent provided by the present invention has the best adsorption effect in the range of pH 4.5 to 5.5, so that the mass volume concentration of salt can be determined to be preferably 1.5% to 5.0% (w / v, g / mL), and more preferably 2% to 3% (w / v, g / mL). That is, the ratio of the amount of sodium chloride to the hyaluronic acid fermentation broth is preferably (0.01 to 5.0) g:1mL, and more preferably (0.01 to 3) g:1mL. In addition, the conditions for the dissociation generally include a temperature of 30 to 60°C, preferably 45 to 55°C; a time of 0.5 to 2h, preferably 0.5 to 1h.

[0095] In step S32, composite adsorbent A is added to the sodium hyaluronate dissociation solution for adsorption and impurity removal. The amount of the composite adsorbent A is 0.01-10% (w / v, g / mL) of the volume of the sodium hyaluronate dissociation solution, and more preferably 0.01-3% (w / v, g / mL). The conditions for the initial impurity removal by stirring and adsorbing generally include a temperature of 30-60°C, preferably 45-55°C; a time of 0.5-5h, preferably 0.5-2h. In addition, the first filtration adopts a modern deep filtration process, with composite adsorbent B as the filter medium, so that a small amount of protein, heavy metals and bacteria remaining in the solution can continue to be adsorbed for a second time while assisting the filtration. The amount of the filter medium plated is preferably 0.5-5% of the total volume of the reaction solution, and more preferably 1-3% of the total volume of the reaction solution. The "reaction solution" described here refers to the solution treated with composite adsorbent A. In addition, the filtration rate is preferably 20-30L / h, and the filtration pressure is preferably 0.1-0.3MPa.

[0096] Since sodium hyaluronate is a high molecular chain product, in order to obtain the target product of the required molecular weight, it is necessary to adjust the impurity removal liquid obtained in step S32 to alkaline with a certain concentration of alkali solution such as sodium hydroxide solution for degradation. After the degradation is completed, the composite adsorbent C is added again to further remove some alkaline protein impurities. After a period of adsorption reaction, the filtrate is filtered. The pH value of the filtrate is adjusted to 6 to 8 with a certain concentration of acid solution such as dilute hydrochloric acid solution, and then preferably passed through a filter membrane to obtain the hyaluronic acid filtrate. The alkali solution can be, for example, potassium hydroxide solution and / or sodium hydroxide solution. The concentration of the alkali solution is preferably 0.1 to 20 mol·L -1 , more preferably 0.5 to 5 mol·L -1 The degradation time and degradation pH value depend on the molecular weight. The longer the time, the higher the pH value, and the smaller the molecular weight of the obtained sodium hyaluronate. If you want to obtain a number average molecular weight of 1.0×10 6 ~2.0×10 6 Da of high molecular weight sodium hyaluronate, the degradation pH is preferably 10.5, and the degradation temperature is preferably 50° C. Furthermore, the amount of the composite adsorbent C is 0.1-10% (w / v, g / mL) of the volume of the degradation solution, more preferably 0.1-1% (w / v, g / mL). In addition, the pore size of the filter membrane used in the second filtration is preferably 0.1-10 μm, more preferably 0.2-0.6 μm.

[0097] For the convenience of description, the adsorbent used in the initial impurity removal by stirring adsorption is called "composite adsorbent A", the filter medium used in the first filtration is called "composite adsorbent B", and the adsorbent used in the deep impurity removal by stirring adsorption is called "composite adsorbent C". The composite adsorbent A, composite adsorbent B and composite adsorbent C are all the above-mentioned zeolite perlite powder gel chitosan resin adsorbent. Furthermore, in order to obtain a better impurity removal effect, the filter medium used in the second filtration is also preferably the above-mentioned zeolite perlite powder gel chitosan resin adsorbent.

[0098] In addition, in order to make the obtained hyaluronic acid filtrate have a higher purity, the purification method of hyaluronic acid provided by the present invention further includes a filtration step after the pH value is finally adjusted to 6 to 8. The pore size of the filter membrane used for filtration is preferably 0.1 to 10 μm, more preferably 0.2 to 0.6 μm.

[0099] In addition, the production method of hyaluronic acid provided by the present invention comprises the following steps: S41, adjusting the viscosity of the hyaluronic acid fermentation liquid obtained by the microbial fermentation method to 0.1-5 Pa.s; S42, purifying the hyaluronic acid fermentation liquid with a viscosity of 0.1-5 Pa.s by the above method to obtain a hyaluronic acid filtrate; S43, concentrating the hyaluronic acid filtrate, filtering the obtained concentrated liquid after alcohol precipitation and crystallization, and drying the obtained filter cake to obtain sodium hyaluronate crystals.

[0100] The production method of hyaluronic acid provided by the present invention preferably uses a jacketed coupling MVR stirring multi-effect steam kettle to concentrate the hyaluronic acid fine filtrate, which can reduce the volume of the hyaluronic acid solution without losing too much energy consumption, thereby greatly reducing the consumption of the later purification solvent, and then significantly reducing the high energy consumption caused by solvent recovery. The jacketed coupling MVR stirring multi-effect steam kettle does not require heat supplementation during the entire concentration process, which fully meets the application of industrial evaporation and concentration, can achieve a good energy-saving effect, and hyaluronic acid is a high-viscosity thermosensitive substance, and the jacketed coupling MVR stirring multi-effect steam kettle can be evaporated at 40°C without freezing, by using relatively little energy, that is, the mechanical energy of the compressor impeller in the case of a compression heat pump, the energy is added to the process heating medium and enters a continuous cycle, in this case, no steam is required as a heating medium, the unit energy consumption is low, and the operating cost is low. The whole operation process is simple in process, strong in practicality, stable in operation, high in automation, and saves personnel consumption.

[0101] Since the production method of hyaluronic acid provided by the present invention requires the use of ethanol for alcohol precipitation and crystallization to realize the extraction of sodium hyaluronate, the volume of the hyaluronic acid filtrate obtained in the extraction process determines the amount of ethanol used. At the same time, sodium hyaluronate is a high-viscosity heat-sensitive substance and is easily degraded at high temperatures. If existing traditional concentration equipment (such as single-effect vacuum concentration, double-effect vacuum concentration, multi-effect vacuum concentration, etc.) is used, it is necessary to choose direct alcohol precipitation without concentration due to its large volume, complex system and operation, and high energy consumption and time consumption at low temperature. This will result in the use of a large amount of ethanol, which will cause great trouble and production cost for the subsequent ethanol recovery, and is not suitable. Industrial production, therefore, the present invention designs a jacketed coupled MVR multi-effect evaporator to concentrate the hyaluronic acid refined filtrate. This technology saves the use of ethanol solvent to a great extent without increasing the difficulty of purification process operation and solves the high energy consumption and high cost problems caused by large-scale solvent recovery. At the same time, because hyaluronic acid is a highly viscous material, direct concentration may cause the material to stick to the wall and agglomerate, which is difficult to remove. Therefore, a stirring device is designed, and the MVR thin film evaporator is replaced by a jacketed coupled MVR stirring multi-effect evaporator, which can prevent the material from scaling and a decrease in heat transfer coefficient during the concentration process, which makes it difficult to continue the concentration of the raw material liquid.

[0102] Compared with traditional concentration methods, MVR technology operates on the principle of reverse Carnot cycle, and the latent heat of secondary steam can be fully recovered and reused. Compared with the multiple-effect evaporation system, the steam from the compressor outlet of the MVR system will introduce a small amount of condensed water before entering the evaporator to eliminate the steam superheat, thereby eliminating the need for a secondary steam cooling water system, which has more significant energy-saving effects and better economic benefits.

[0103] At present, most domestic and foreign scholars have conducted relevant research on the falling film MVR evaporation system, but there are few reports on the jacketed coupled MVR stirring multi-effect evaporation system.

[0104] For the MVR evaporation and concentration process, if the boiling point increase is too large, the effective heat transfer temperature difference of the heat exchanger will be reduced, so that the compressor needs to increase the temperature to a higher level to overcome the impact of the boiling point increase, resulting in excessive energy consumption of the system. According to relevant literature: For the MVR evaporation system, when the boiling point of the solution increases by more than 15°C, the secondary steam temperature provided by the single-stage steam compressor cannot maintain the effective heat transfer temperature difference during the evaporation process, and a two-stage MVR or graded compression system is required. If it is lower than 15°C, a single-stage MVR evaporation system can be used. Based on the variation law of the boiling point increase of the material solution extracted by the present invention at different evaporation temperatures, it can be seen that the boiling point increase in the evaporation and concentration stage is less than 10°C, so a single-stage MVR system can be used. However, for the single-stage MVR system, the amount of steam processed by the compressor is all the secondary steam that needs to be produced in the concentration stage, and the boiling point increase of the wastewater is large, so the power consumption will be large.

[0105] In this regard, in order to reduce the compressor power of the single-stage MVR system, the present invention designs a coupled MVR multi-effect evaporation system, pre-concentrates the feed liquid at a lower concentration, and then further evaporates the water to a set concentration through the MVR system. Considering the thermal integration of the system, two flash tanks are used in combination with a feed preheater to reduce the energy consumption of the system. At the same time, because the product has a large viscosity and is a heat-sensitive raw material liquid, scaling and a decrease in heat transfer coefficient may occur during the concentration process, which will make it difficult to continue the concentration of the raw material liquid. The present invention applies the coupled MVR multi-effect evaporation system to a jacketed evaporator with a stirring device, and uses a single-screw compressor as the driving source of the entire system, thereby obtaining a jacketed coupled MVR stirring multi-effect steam kettle.

[0106] At the same time, there are many types of compressors used as power sources. In recent years, there are three main types that are widely used in China: Roots steam compressors, centrifugal steam compressors, and fan steam compressors, while single-screw compressors are rarely used. Among them, Roots steam compressors are widely used in small and medium-low boiling point material evaporation, but due to their structural characteristics, they have a short service life, poor heat exchange, small displacement, bulky body, complex structure, poor continuous operation performance, loud vibration and noise, and high maintenance costs. Centrifugal steam compressors are widely used in large and medium-sized and high boiling point material evaporation units, but because the rotor speed is high, in order to ensure a certain width of the impeller, centrifugal steam compressors must be used in large and medium flow occasions, limiting their application in small evaporation units. Fan steam compressors are widely used in large and medium-sized evaporation units and low boiling point material evaporation. Medium and high boiling point materials are used in two or three stages in series. Because the single-stage compression ratio of fan steam compressors is relatively small, the cascade equipment is complex, there are many control points, and the area occupied is large, there are also restrictions on their application.

[0107] The equipment of the present invention uses a single screw compressor to provide a power source for the jacket-type coupled MVR multi-effect evaporator. Compared with twin screw, Roots, centrifugal, and fan compressors, the air flow artery is small and the exhaust volume is large during operation. It can operate under high pressure, and the first-stage compression can reach 6.16MPa, while other compressors do not exceed 3MPa at most. Because its screw force is completely balanced, it can work under very small gaps, with high speed and light load, few parts, small leakage, high solvent efficiency, and good energy-saving special effects. Thereby greatly reducing energy consumption and saving costs, it is very suitable for industrial production of sodium hyaluronate.

[0108] This design takes a 600MW coal-fired unit as an example. The processing capacity of hyaluronic acid fine filtrate is 10t / h. The process simulation software Aspen plus is used to simulate the different thermal evaporation and crystallization systems of the fine filtrate after pretreatment, and the material and energy balance of thermal concentration is obtained. The performance of each system is calculated according to the three commonly used energy statistics and conversion methods at home and abroad (heat value equivalent method, equivalent value method or power generation coal consumption method, equivalent electricity method). The simulation calculation results of different concentration processes are shown in Table 1. The performance calculation results of each scheme are simulated under the same feed flow, components, the same evaporation scale (9481kg / h) and the same crystallization process.

[0109] According to calculation, for the traditional three-effect evaporation system, the required steam consumption is 4551.7 kg / h, that is, the unit steam consumption (the amount of steam consumed per unit evaporation) is 0.48. The secondary steam produced by the last effect of the three-effect evaporation system needs to be condensed by the condenser, and 47904 kg / h of condensed water is required (assuming that the condensed water temperature rises to 10 ° C). For the single-stage MVR system, since the compressor is used to pressurize and improve the quality of the secondary steam produced during the evaporation process, it is not necessary to consume raw steam, and the condenser is also cancelled. Compared with the three-effect evaporation system, its unit energy consumption is greatly reduced; when different energy consumption conversion methods are used, the system energy saving rate is 67.7% to 94.9%. Compared with the single-stage MVR system, due to the evaporation of part of the feed water (about 40% of the total evaporation) at a lower concentration, the steam flow rate handled by the compressor of the multi-effect evaporation system coupled with MVR is greatly reduced; in addition, the use of flash tanks to recover part of the steam energy reduces the compressor power consumption of the multi-effect evaporation system coupled with MVR by about 30% compared with the single-stage MVR system, and the system energy efficiency coefficient is increased by 24.5%.

[0110] Table 1 Performance comparison of different systems

[0111]

[0112]

[0113] The schematic diagram of the jacketed coupled MVR stirring multi-effect steam kettle provided by the present invention is as follows Figure 1 Specifically, see Figure 1The jacket-coupled MVR stirring multi-effect steam kettle comprises a heat exchanger 10, a low-pressure steam kettle 20, a high-pressure steam kettle 30, a flash tank-1 60, a flash tank-2 40 and a compressor 50, wherein the outlet of the heat exchanger 10 is connected to the tower top of the low-pressure steam kettle 20, the tower bottom of the low-pressure steam kettle 20 is connected to the tower top of the high-pressure steam kettle 30, the jacket outlet of the low-pressure steam kettle 20 is connected to the inlet of the flash tank-2 40, the outlet of the flash tank-2 40 is connected to the jacket inlet of the high-pressure steam kettle 30 via the compressor 50, the jacket outlet of the high-pressure steam kettle 30 is connected to the inlet of the flash tank-1 60, and the outlet of the flash tank-1 60 is connected to the jacket inlet of the low-pressure steam kettle 20. The compressor 50 is a single-screw compressor. Both the low-pressure steam kettle 20 and the high-pressure steam kettle 30 are provided with a stirring device. In addition, the jacket-type coupled MVR stirred multi-effect steam kettle further comprises a water tank 70, the inlet of which is connected to the jacket outlet of the heat exchanger 10 to recover the heat exchange medium (usually water) in the jacket of the heat exchanger.

[0114] Correspondingly, the working principle of the jacket-coupled MVR stirring multi-effect steam kettle is as follows: the refined filtrate obtained by extracting the fermented liquid after adsorption and impurity removal is heated to 45-55°C through a heat exchanger, and then sent to a low-pressure evaporator (50-60kPa), and the concentrated liquid produced is sent to a high-pressure evaporator (70-80kPa), and the concentrated liquid is finally discharged from the bottom of the kettle and sent to the finished alcohol precipitation unit. Among them, the jacket-coupled MVR stirring multi-effect steam kettle is provided with two flash tanks (i.e., flash tank-1 and flash tank-2) for recovering part of the steam heat energy, wherein the secondary steam produced by the low-pressure evaporator is recovered by flash tank-2, and then pressurized and heated by the compressor as the evaporation heat source of the high-pressure steam kettle; the secondary steam generated by the high-pressure evaporator is recovered by flash tank-1 as the heat source of the low-pressure evaporator. In addition, preferably, the heat source of the heat exchanger is the condensed water at the outlet of flash tank-2. Compared with the single-stage MVR system, the steam flow rate handled by the compressor is greatly reduced, and the power consumption of the system compressor is greatly reduced. In the present invention, the pressure refers to absolute pressure.

[0115] The jacketed coupled MVR stirring multi-effect steam kettle provided by the present invention can accelerate the heat transfer of the raw material liquid and prevent the occurrence of scaling of heat-sensitive materials and the like. It has the advantages of high evaporation efficiency, simple operation, high equipment corrosion resistance, convenient descaling, low overall system operating cost, etc. It can be operated under normal pressure or negative pressure conditions. Under negative pressure conditions, not only the evaporation temperature of the raw material liquid can be reduced, but also the material requirements for system-related equipment and pipelines can be reduced, while ensuring continuous and stable production of the system.

[0116] In the present invention, the single screw compressor adopts a variable frequency speed regulating three-phase asynchronous motor to provide a power source for the jacketed coupled MVR with stirring multi-effect evaporator. The single screw compressor achieves different speeds and torques under the drive of the frequency converter, which can adapt to the needs of different workloads. In the present invention, the system energy efficiency coefficient is the ratio of the heat absorbed by the evaporation and crystallization process of the raw material liquid to the system energy consumption.

[0117] The inventor of the present invention has conducted several operation tests on the jacketed coupled MVR with stirring multi-effect evaporator, and the results show that the evaporation rate is 1m 3 The power consumption of water does not exceed 25kW·h. The balanced output of condensate is about 92% to 95% of the total concentrated liquid, usually at a temperature of 20℃ to 40℃, and no cooling or heat exchange is required, and it can directly enter the next process.

[0118] The evaporation temperature of the jacketed coupled MVR with stirring multi-effect evaporator is usually controlled at 45-55°C. Specifically, the refined filtrate obtained after the fermentation liquid is adsorbed and impurities are removed is heated to the required temperature of 45-55°C by a heat exchanger, and then enters the above-mentioned jacketed coupled MVR with stirring multi-effect evaporator for concentration. The concentrated liquid is finally discharged into the alcohol precipitation tank through the bottom of the kettle to wait for alcohol precipitation and crystallization. The temperature of the discharged material is usually 20-30°C. The secondary steam is transported to the single-screw compressor, and the pressurized secondary steam is returned to the evaporation system according to the steam temperature and pressure required by the heating chamber of the evaporator (the filtrate from the pre-filtration process corresponds to the boiling point required for its evaporation and concentration) and recycled as a heat source (after the system operates normally, the primary steam is saved). In addition, the concentration multiple is preferably 1-2 times the volume of the fermentation liquid.

[0119] In step S43, the concentrated solution obtained by concentration is filtered after alcohol precipitation and crystallization, and the obtained filter cake is dried to obtain sodium hyaluronate crystals. Among them, the quality of the alcohol precipitation and crystallization effect directly affects the final effect of the next step of drying. When the dehydration effect is not good, the finished product is usually in a block or coarse fiber shape after drying; when the dehydration effect is good, the finished product is usually in a fine fiber or powder shape after drying, and this state meets the commercialization standard. In order to ensure a better dehydration effect, preferably, the alcohol precipitation and crystallization process is segmented using ethanol of different concentrations. In the most preferred embodiment, the alcohol precipitation and crystallization method is to add 1 to 2 times the volume of ethanol with a concentration of more than 90% to the concentrated solution, stir the alcohol precipitation and let it stand, and when the supernatant is clear, separate the upper layer, add 1 to 1.5 times the volume of ethanol with a concentration of 70% to 80% to the remaining base material, stir the alcohol precipitation and let it stand, and when the supernatant is clear, separate the upper layer, and add 1 to 1.5 times the volume of ethanol with a concentration of more than 90% to the remaining base material again. Stir the alcohol precipitation. The purpose of the alcohol precipitation and crystallization in the above segmented manner is to continue to effectively remove water-soluble impurities such as starch, mucilage, protein, sulphur, pigment, inorganic salt, etc. in the concentrated solution. Furthermore, the alcohol precipitation and crystallization are generally carried out under stirring conditions, wherein the stirring speed is preferably 10 to 1000 r / h. In addition, the drying can be carried out in a constant temperature vacuum oven. The drying conditions generally include a temperature of 30°C to 60°C and a time of 8h to 24h.

[0120] The finished sodium hyaluronate product obtained by the method of the present invention is a white powder crystal with a purity of ≥99%, a sodium hyaluronate content of >95%, an average extraction yield of more than 95%, no protein or heavy metal detected, and a light transmittance of more than 99%.

[0121] The present invention will be described in detail below through examples.

[0122] Preparation Example 1

[0123] This preparation example is used to illustrate the zeolite perlite powder gel chitosan resin adsorbent provided by the present invention and its preparation method.

[0124] (1) Preparation of gel chitosan resin:

[0125] S11. Prepare 1.5 L of a dilute hydrochloric acid solution with a concentration of 1% v / v, add 30 g of chitosan into the dilute hydrochloric acid solution and stir until dissolved to obtain a dilute acid solution of chitosan.

[0126] S12, add 200g NaOH solid into 500mL pure water and stir until dissolved to obtain sodium hydroxide aqueous solution, add 200mL methanol solution after cooling and stir evenly to obtain a solidified solution for standby use.

[0127] S13. The chitosan dilute acid solution obtained in step S11 is slowly dripped into the curing solution obtained in step S12 using a syringe needle under slow stirring. After the dropwise addition is completed, the solution is stirred for 10 minutes and filtered. The wet resin obtained is washed with water until it is neutral. The neutral resin obtained is then decolorized by soaking it in acetone at room temperature for 2 hours. After the decolorization is completed, it is filtered. The solid product obtained is then vacuum dried at 60°C. The dried product is a gel chitosan resin, which is recorded as NZ-1.

[0128] (2) Preparation of zeolite pearlite powder gel chitosan resin adsorbent:

[0129] S21, sieve zeolite powder and pearlite powder through 100 mesh, take 100g zeolite powder and 150g pearlite powder respectively, add them into 2L of 1mol·L -1 The mixture was immersed in a dilute hydrochloric acid solution at room temperature for 4 hours. After immersion, it was washed with distilled water until neutral and filtered. The obtained solid product was placed in a vacuum oven at 60°C and dried to obtain a zeolite powder-perlite powder composite.

[0130] S22, add 100g of zeolite powder-perlite powder composite and 1g of gel chitosan resin to 1L of 0.2mol·L -1 The mixture was stirred at 40 °C for 3 h, and then a 0.5 mol·L -1 The zeolite perlite powder gel chitosan resin adsorbent D1 was obtained by adding the zeolite perlite powder gel chitosan resin adsorbent D1 to a NaOH solution with a loading mass of 1%. The pellets were washed with distilled water until neutral and then dried. The zeolite perlite powder gel chitosan resin adsorbent D1 was obtained by passing through a 120-mesh sieve.

[0131] Preparation Example 2

[0132] This preparation example is used to illustrate the zeolite perlite powder gel chitosan resin adsorbent provided by the present invention and its preparation method.

[0133] (1) Preparation of gel chitosan resin:

[0134] S11. Prepare 2 L of a dilute acetic acid solution with a concentration of 1% v / v, add 50 g of chitosan into the dilute acetic acid solution and stir until dissolved to obtain a dilute acid solution of chitosan.

[0135] S12, add 250g NaOH solid into 1000mL pure water and stir until dissolved to obtain sodium hydroxide aqueous solution, add 300mL methanol solution after cooling and stir evenly to obtain a solidified solution for standby use.

[0136] S13. The chitosan dilute acid solution obtained in step S11 is slowly dripped into the curing solution obtained in step S12 using a syringe needle under slow stirring. After the dropwise addition is completed, the solution is stirred for 10 minutes and filtered. The wet resin obtained is washed with water until it is neutral. The neutral resin obtained is then decolorized by soaking it in acetone at room temperature for 1 hour. After the decolorization is completed, it is filtered. The solid product obtained is then vacuum dried at 60°C. The dried product is a gel chitosan resin, which is recorded as NZ-2.

[0137] (2) Preparation of zeolite pearlite powder gel chitosan resin adsorbent:

[0138] S21, sieve zeolite powder and pearlite powder through 100 mesh, take 100g zeolite powder and 150g pearlite powder respectively, add them into 2L of 1mol·L -1 The mixture was immersed in a dilute acetic acid solution at room temperature for 4 hours. After immersion, it was washed with distilled water until neutral and filtered. The obtained solid product was placed in a vacuum oven at 60°C and dried to obtain a zeolite powder-perlite powder composite.

[0139] S22, add 100g of zeolite powder-perlite powder composite and 2g of gel chitosan resin to 1L of 0.2mol·L -1 acetic acid solution at 40 °C, stirred for 3 h, and then the resulting mixture was slowly dripped with a 0.2 mol·L -1 The zeolite perlite powder gel chitosan resin adsorbent D2 was obtained by adding the zeolite perlite powder gel chitosan resin adsorbent D2 to a NaOH solution with a loading mass of 2%. The pellets were washed with distilled water until neutral and then dried. The zeolite perlite powder gel chitosan resin adsorbent D2 was obtained by passing through a 150-mesh sieve.

[0140] Preparation Example 3

[0141] This preparation example is used to illustrate the zeolite perlite powder gel chitosan resin adsorbent provided by the present invention and its preparation method.

[0142] (1) Preparation of gel chitosan resin:

[0143] S11. Prepare 2 L of a dilute oxalic acid solution with a concentration of 0.5% v / v, add 80 g of chitosan into the dilute oxalic acid solution and stir until dissolved to obtain a dilute acid solution of chitosan.

[0144] S12, add 300g NaOH solid into 1000mL pure water and stir until dissolved to obtain sodium hydroxide aqueous solution, add 300mL methanol solution after cooling and stir evenly to obtain a solidified solution for standby use.

[0145] S13. The chitosan dilute acid solution obtained in step S11 is slowly dripped into the curing solution obtained in step S12 using a syringe needle under slow stirring. After the dropwise addition is completed, the solution is stirred for 10 minutes and filtered. The wet resin obtained is washed with water until it is neutral. The neutral resin obtained is then decolorized by soaking it in acetone at room temperature for 2 hours. After the decolorization is completed, it is filtered. The solid product obtained is then vacuum dried at 60° C. The dried product is a gel chitosan resin, which is recorded as NZ-3.

[0146] (2) Preparation of zeolite pearlite powder gel chitosan resin adsorbent:

[0147] S21, sieve the zeolite powder and pearlite powder through a 120-mesh sieve, take 100g of zeolite powder and 200g of pearlite powder respectively, and add them to 2L of 1mol·L -1 The mixture was immersed in oxalic acid solution at room temperature for 5 hours, washed with distilled water until neutral, filtered, and the obtained solid product was placed in a vacuum oven at 60°C and dried to obtain a zeolite powder-perlite powder composite.

[0148] S22, add 200g zeolite powder-perlite powder composite and 3g gel chitosan resin into 1L of 0.3mol·L -1 The mixture was stirred at 50°C for 3 h, and then a 0.5 mol·L oxalic acid solution was slowly added with a 5# syringe. -1 The pellets were washed with distilled water until neutral and then dried. The zeolite perlite powder gel chitosan resin adsorbent D3 was obtained after passing through a 120-mesh sieve.

[0149] Test Example 1

[0150] Perlite powder, zeolite powder, gel chitosan resin NZ-1 obtained in Preparation Example 1, and zeolite perlite powder gel chitosan resin adsorbent (composite adsorbent D1) obtained in Preparation Example 1 were used as adsorbents in the hyaluronic acid fermentation broth extraction process, and the decolorization of each adsorbent was characterized. After adding equal amounts of adsorbent, the hyaluronic acid fermentation broth was stirred and reacted under the same conditions, and samples were taken at regular intervals, filtered with a 0.45 μm filter membrane, and the absorbance at each time was measured at 265 nm with a spectrophotometer. Each experiment was measured in parallel 3 times and the average value was taken as the result. Deionized water was used as a blank, and the absorbance at each time was measured at 265 nm with a spectrophotometer to calculate the decolorization rate. Decolorization rate = A0-A / A0*100%, where A0 is the absorbance of the undecolorized hyaluronic acid fermentation broth, and A is the absorbance of the hyaluronic acid fermentation broth at a certain time of decolorization. The results are shown in Figure 2 .

[0151] Depend on Figure 2It can be seen that within the first 25 minutes, the decolorization rates of the four decolorizers increased rapidly. As time went on, the decolorization rates of pearlite powder, zeolite powder and gel chitosan resin basically stopped increasing, indicating that adsorption equilibrium was reached. The highest decolorization rates were 23.75%, 32.15% and 11.34%, respectively; while the decolorization rate of the composite adsorbent D1 provided by the present invention continued to increase slowly within 25 to 125 minutes. When the decolorization time was 125 minutes, the removal rate of the composite adsorbent D1 was the highest, reaching 78.76%, and the decolorization rate basically stopped rising thereafter, indicating that the adsorption of the composite adsorbent D1 on the pigment was saturated, that is, the decolorization reached equilibrium.

[0152] Test Example 2

[0153] The gel chitosan resin NZ-1 obtained in Preparation Example 1 and the zeolite pearlite powder gel chitosan resin adsorbent (composite adsorbent D1) obtained in Preparation Example 1 were analyzed by infrared spectroscopy (IR) using a 8400S Fourier transform infrared spectrometer. The results corresponding to the gel chitosan resin NZ-1 are shown in FIG. Figure 3 In a, the results corresponding to composite adsorbent D1 are shown in Figure 3 b. By Figure 3 The infrared absorption spectrum of the chitosan resin NZ-1 shows that at 3442.7 cm -1 The superimposed stretching vibration peak of -OH and -NH2, which is unique to chitosan, appears at 2923.9cm -1 The stretching vibration absorption peak of the -CH2- group appeared at 1604.4 cm -1 The absorption peak at 1384.8 cm is the out-of-plane vibration absorption peak of the -C=C bond. -1 The bending vibration peak of -CH bond is 1089.7cm -1 The characteristic peak of β-D-pyranoglucoside is 553.5 cm -1 Out-of-plane bending vibration of the =CH bond occurs at Figure 3 From the infrared absorption spectrum of the composite adsorbent D1, it can be seen that the main characteristic peaks of chitosan have not changed, indicating that the chemical structure of the gel chitosan resin has not changed after loading zeolite powder and perlite powder.

[0154] The zeolite pearlite powder gel chitosan resin adsorbent (composite adsorbent D1) obtained in Preparation Example 1 was used to filter the hyaluronic acid, and the infrared spectra (IR) of the hyaluronic acid before and after filtration were characterized. Figure 4 .from Figure 4 It can be seen that 3500~3000cm -1 The stronger broad peak is the -OH stretching vibration absorption peak; 2923cm -1 The left and right sides are the saturated CH stretching vibration absorption peaks; 1604cm-1 The left and right sides are -C=C stretching vibration absorption; 1243cm -1 The bending vibration peak of -OH is at 1040~1070cm -1 These characteristic peaks are basically consistent with the results of hyaluronic acid standard products and are consistent with the structure of hyaluronic acid, indicating that the adsorption effect of the composite adsorbent D1 provided by the present invention on the hyaluronic acid extraction process does not affect the structure of hyaluronic acid.

[0155] Test Example 3

[0156] Since they do not have selective specificity, perlite powder, zeolite powder, gel chitosan resin and the composite adsorbent provided by the present invention will have a certain adsorption on hyaluronic acid, resulting in product loss. Perlite powder, zeolite powder, gel chitosan resin obtained in Preparation Example 1 and composite adsorbent D1 obtained in Preparation Example 1 were applied to the hyaluronic acid fermentation broth extraction process, and the adsorption of each adsorbent was characterized. After adding equal amounts of adsorbents, the hyaluronic acid fermentation broth was stirred and reacted under the same conditions, and filtered under a certain pressure using a flat plate filter. After filtration, the filtrate was taken and the hyaluronic acid content in the filtrate was detected using an ultraviolet spectrophotometer and the yield was calculated, so that the adsorption of hyaluronic acid by each adsorbent can be known. Each experiment was measured twice in parallel, and the results are shown in Table 1. Figure 5 and Table 2. The specific surface area and pore structure of each adsorbent were characterized, and the pore distribution curve results are shown in Figure 6 .

[0157] Table 2 Adsorption of hyaluronic acid by various adsorbents

[0158] Serial number Adsorbent Addition amount (g / L) Hyaluronic acid loss (%) 1 Zeolite powder 10 20.3 2 Perlite powder 10 7.9 3 Gel Chitosan Resin 10 5.8 4 <![CDATA[Composite adsorbent D1]]> 10 1.3

[0159] Depend on Figure 5 It can be seen that the hysteresis phenomenon at a relative pressure of 0.4-1.0 indicates that the tissue porosity of the sample is relatively high, and the four adsorbents all conform to the type IV adsorption isotherm, indicating that they are all mesoporous materials; at a relative pressure of 0.9-1.0, the composite adsorbent D1 produces an H3 type hysteresis loop, indicating that it is a slit pore formed by the extrusion of flaky particles; at a relative pressure of 0.5-1.0, the gel chitosan resin, perlite powder and zeolite powder produce an H4 type hysteresis loop, indicating that it is a slit pore formed by the extrusion of layered particles; as shown in Table 2, the composite adsorbent D1 provided by the present invention has the least loss of 1.3%, followed by gel chitosan resin 5.8%, perlite 7.9%, and zeolite powder 20.3%.

[0160] The pore distribution curves of the four adsorbents were obtained from the desorption branch of the isotherm using the BJH method. Figure 6 As shown. Figure 6It can be seen that by comparing the specific surface areas of perlite powder, zeolite powder and composite adsorbent D1, the composite adsorbent D1 provided by the present invention has a relatively wide pore size distribution, mainly composed of mesopores (peak at 5-6nm), and a small amount of macropores (peak distribution at 126-148nm), and the proportion of micropores is relatively small, the average pore size is larger, and the specific surface area is relatively small; while chitosan gel particles, perlite powder and zeolite powder are mainly composed of micropores (peak at 1-2nm), the pore size distribution is relatively narrow, and the specific surface area is large, but due to the small pore size, the adsorption of slightly larger pigment impurities is poor.

[0161] Test Example 4

[0162] The surface morphology of the chitosan gel particles obtained in Preparation Example 1 and the composite adsorbent D1 obtained in Preparation Example 1 was observed using a S-530 transmission electron microscope. Figure 7 , Figure 7 A in the figure is the surface morphology of chitosan gel particles. Figure 7 B is the surface morphology of composite adsorbent D1. Figure 7 It can be seen that the surface pores of the composite adsorbent D1 are significantly larger than those of the chitosan gel particles. The pore structure parameters of zeolite powder, perlite powder, chitosan gel particles obtained in Preparation Example 1, and the composite adsorbent D1 obtained in Preparation Example 1 were tested. Figure 7 The results of A and B and Table 3 further confirm the above conclusions. The new adsorbent after compounding has larger pore size and wider pore size distribution.

[0163] Table 3

[0164]

[0165] Test Example 5

[0166] The antibacterial effects of the chitosan gel particles obtained in Preparation Example 1 and the composite adsorbent D1 obtained in Preparation Example 1 on Staphylococcus aureus ATCC 6538 (a) and Escherichia coli ATCC 8739 (b) were tested by the Oxford cup method, and the results were observed by using a S-530 transmission electron microscope. Figure 8 .in, Figure 8 A in the middle is the antibacterial ability of chitosan gel particles; Figure 8 B is the antibacterial ability of composite adsorbent D1.

[0167] Depend on Figure 8It can be seen that the chitosan gel composite adsorbent D1 loaded with zeolite powder and perlite powder has a larger antibacterial zone, indicating that the composite adsorbent D1 provided by the present invention has a better antibacterial effect on Escherichia coli and Staphylococcus aureus than single chitosan gel particles. In addition, the composite adsorbent has a better inhibitory effect on Staphylococcus aureus than Escherichia coli. The minimum diameter of the antibacterial ring formed is 9mm and the maximum can reach 14mm, which is much larger than the antibacterial performance certification standard of the diameter of the formed antibacterial ring ≥7mm. Studies have shown that chitosan gel itself has certain antibacterial properties. After being compounded with zeolite powder, it has a synergistic antibacterial effect and its antibacterial ability is significantly enhanced.

[0168] In summary, the characterization results of the research show that, after a series of loading modifications, the chemical structure of chitosan itself does not change in the composite adsorbent provided by the present invention, and the structure of hyaluronic acid is not affected; compared with other adsorbents (chitosan gel, perlite powder, zeolite powder), the composite adsorbent provided by the present invention has a significant change in pore structure while having the functions of the three, and the pore size becomes larger and wider. When used in hyaluronic acid extraction, the loss rate is very small. After being compounded with zeolite powder and perlite powder, it has a synergistic antibacterial effect, and the antibacterial ability is significantly enhanced.

[0169] Example 1 (using the composite adsorbent D1 obtained in Preparation Example 1 of the present invention)

[0170] A method for extracting and preparing sodium hyaluronate from a microbial fermentation broth, the specific steps are as follows:

[0171] (1) Add 4 times the volume of deionized water to 1000 mL of hyaluronic acid fermentation liquid and stir for 30 min to control the viscosity of the hyaluronic acid fermentation liquid at 2.5 Pa.s, add 102.04 g of NaCl solid with a total salt concentration of 2.0%, heat to 40°C, stir for 2 h, the solution pH = 5.0, add 51.02 g of composite adsorbent D1 with a total volume of 1.0% (w / v), stir at 60°C for 0.5 h and then filter, the filtration uses composite adsorbent D1 as the filtration medium, the coating amount of composite adsorbent D1 is 4% of the volume of the reaction liquid, and the filtration is carried out at a pressure of 0.2 MPa at a rate of 20 L / h, the volume of the obtained filtrate is 5010 mL, protein is not detected, Gu 2+ 、Zn 2+ 、As 2+ , Hg 2+ Mg 2+ , Ca 2+ , Fe 3+ Not detected, Cd 2+ :0.002ppm, Pb 2+ : 0.005ppm, the filtrate is milky white.

[0172] (2) The filtrate obtained in step (1) was adjusted to pH 10.5 using a 2.5 mol / L NaOH solution, and the mixture was stirred and degraded at 50°C for 15 min. 25.05 g of composite adsorbent D1 (0.5% by volume of the filtrate) was added, and the mixture was stirred at 50°C for 1 h and filtered. The filtrate was adjusted to pH 6.5 using a 5% (v / v) hydrochloric acid solution, and filtered again using a 0.22 μm filter membrane to obtain 4995 mL of refined filtrate. 2 + 、Zn 2+ 、As 2+ 、Cd 2+ , Hg 2+ Mg 2+ , Ca 2+ , Fe 3+ Not detected, Pb 2+ : 0.001ppm, no protein was detected, the filtrate was clear and bright, and the transmittance was 99.9%.

[0173] (3) The refined filtrate obtained in step (2) was concentrated using a laboratory jacketed MVR evaporator to a volume that was 1 times the volume of the original fermentation liquid. 1.5 times the volume of 95% ethanol was added to the concentrate, stirred for 30 minutes, and then allowed to stand for alcohol precipitation. The upper layer was separated until the supernatant was clear. 1.2 times the volume of 75% ethanol was added to the base material, stirred for 30 minutes, and then allowed to stand for alcohol precipitation. The supernatant was discarded, and 1.3 times the volume of 93% ethanol was added to the base material again, stirred for 30 minutes, and allowed to stand for alcohol precipitation. After dehydration, the filter cake was placed in a constant temperature vacuum oven at 50°C for 18 hours. The obtained finished product had a dry content of 95.8%, a transmittance of 99.5%, a yield of 95.5%, a moisture content of 7.8%, and a molecular weight of sodium hyaluronate: 1.5×10 6 DA, protein and heavy metals were not detected, the volume of the refined filtrate was reduced by 5 times compared with that before concentration, and the amount of ethanol used in the alcohol precipitation step was reduced by 80%.

[0174] Example 2 (using the composite adsorbent D2 obtained in Preparation Example 2 of the present invention)

[0175] The specific steps of the method for extracting and preparing sodium hyaluronate from a microbial fermentation broth are as follows:

[0176] (1) Add 3 times the volume of deionized water to 1000 mL of hyaluronic acid fermentation liquid and stir for 30 min to control the viscosity of the hyaluronic acid fermentation liquid at 3.3 Pa.s, add 45.92 g of NaCl solid with a total salt concentration of 1.5%, heat to 60°C, stir for 0.5 h, the solution pH = 5.13, add 45.69 g of the new composite adsorbent D2 with a total volume of 1.5% (w / v), stir at 50°C for 1 h and then filter, the filtration uses the composite adsorbent D2 as the filtration medium, the coating amount of the composite adsorbent D2 is 2% of the volume of the reaction liquid, and the filtration is carried out at a pressure of 0.25 MPa at a rate of 30 L / h, the volume of the obtained filtrate is 3050 mL, no protein is detected, Gu 2+ 、As 2+ , Hg 2+ Mg 2+ , Ca 2+ , Fe 3+ Not detected, Cd 2+ :0.003ppm, Pb 2+ :0.002ppm, Zn 2+ : 0.001ppm, the filtrate is milky white.

[0177] (2) The filtrate obtained in step (1) was adjusted to pH 10.3 using a 2.5 mol / L NaOH solution, and the mixture was stirred and degraded at 50°C for 30 min. 30.5 g of composite adsorbent D2 (1%) was added, and the mixture was stirred at 50°C for 1 h and filtered. The filtrate was adjusted to pH 6.45 using a 5% (v / v) hydrochloric acid solution, and filtered again using a 0.22 μm filter membrane to obtain 3000 mL of refined filtrate. 2+ 、Zn 2 + 、As 2+ 、Cd 2+ , Hg 2+ Mg 2+ , Ca 2+ , Fe 3+ Not detected, Pb 2+ : 0.001ppm, no protein was detected, the filtrate was clear and bright, and the transmittance was 99.8%.

[0178] (3) The refined filtrate obtained in step (2) was concentrated using a laboratory jacketed MVR evaporator to a volume that was 1 times the volume of the original fermentation liquid. 1.5 times the volume of 95% ethanol was added to the concentrate, stirred for 30 minutes, and then allowed to stand for alcohol precipitation. The upper layer was separated until the supernatant was clear. 1.2 times the volume of 80% ethanol was added to the base material, stirred for 30 minutes, and then allowed to stand for alcohol precipitation. The supernatant was discarded, and 1.2 times the volume of 95% ethanol was added to the base material again, stirred for 30 minutes, and allowed to stand for alcohol precipitation. After dehydration, the filter cake was placed in a constant temperature vacuum oven at 50°C and dried for 18 hours. The obtained finished product had a dry content of 95.2%, a transmittance of 99.7%, a yield of 94%, a moisture content of 7.5%, and a molecular weight of sodium hyaluronate: 1.2×10 6 DA, protein and heavy metals were not detected, the volume of the refined filtrate was reduced by 3 times compared with that before concentration, and the amount of ethanol used in the alcohol precipitation step was reduced by 67%.

[0179] Example 3 (Using the novel composite adsorbent D3 obtained in Preparation Example 3 of the present invention)

[0180] The specific steps of the method for extracting and preparing sodium hyaluronate from a microbial fermentation broth are as follows:

[0181] (1) Add 3 times the volume of deionized water to 5 L of hyaluronic acid fermentation liquid and stir for 1 h to control the viscosity of the hyaluronic acid fermentation liquid at 3.3 Pa.s, add 306 g of NaCl solid with a total salt concentration of 2.0%, heat to 50° C., stir for 1 h, the solution pH = 5.13, add 306 g of composite adsorbent D3 with a total volume of 2.0% (w / v), stir at 30° C. for 5 h and then filter, the filtration uses composite adsorbent D3 as the filtration medium, the coating amount of composite adsorbent D3 is 4% of the volume of the reaction liquid, and the filtration is carried out at a pressure of 0.25 MPa at a rate of 25 L / h, the volume of the obtained filtrate is 15.5 L, protein is not detected, Gu 2+ 、As 2+ 、Cd 2+ , Hg 2+ Mg 2+ , Ca 2 + , Fe 3+ Not detected, Pb 2+ :0.005ppm, Zn 2+ : 0.007ppm, the filtrate is milky white.

[0182] (2) The filtrate obtained in step (1) was adjusted to pH 10.8 using a 2.5 mol / L NaOH solution, and the mixture was degraded at 50°C for 30 min by stirring. 155 g of composite adsorbent D3 (1%) was added, and the mixture was stirred at 50°C for 1.5 h before filtration. The filtrate was adjusted to pH 6.5 using a 5% (v / v) hydrochloric acid solution, and filtered again using a 0.22 μm filter membrane to obtain 15 L of refined filtrate. 2+ 、Zn 2+ , Pb 2+ 、Cd 2+ , Hg 2+ Mg 2+ , Fe 3+ , Ca 2+ 、As 2+ No protein was detected, the filtrate was clear and bright, with a transmittance of 100%.

[0183] (3) The refined filtrate obtained in step (2) was concentrated using a laboratory jacketed MVR evaporator to a volume that was 1 times the volume of the original fermentation liquid. 1.5 times the volume of 95% ethanol was added to the concentrate, stirred for 30 minutes, and then allowed to stand for alcohol precipitation. The upper layer was separated until the supernatant was clear. 1.2 times the volume of 75% ethanol was added to the base material, stirred for 30 minutes, and then allowed to stand for alcohol precipitation. The supernatant was discarded, and 1.2 times the volume of 95% ethanol was added to the base material again, stirred for 30 minutes, and allowed to stand for alcohol precipitation. After dehydration, the filter cake was placed in a constant temperature vacuum oven at 50°C for 18 hours. The obtained finished product had a dry content of 96.1%, a transmittance of 99.6%, a yield of 93.5%, a moisture content of 7.0%, and a molecular weight of sodium hyaluronate: 1.1×10 6 DA, protein and heavy metals were not detected, the volume of the filtrate was reduced by 3 times compared with that before concentration, and the amount of ethanol used in the alcohol precipitation step was reduced by 67%.

[0184] Example 4 (using the composite adsorbent D1 obtained in Preparation Example 1 of the present invention)

[0185] A method for extracting and preparing sodium hyaluronate from a microbial fermentation broth, the specific steps are as follows:

[0186] (1) Add 4 times the volume of deionized water to 1000 mL of hyaluronic acid fermentation liquid and stir for 30 min to control the viscosity of the hyaluronic acid fermentation liquid at 2.5 Pa.s, add 102.04 g of NaCl solid with a total salt concentration of 2.0%, heat to 40°C, stir for 2 h, the solution pH = 5.01, adjust the pH of the dissolved liquid to 4.5 with 10% dilute hydrochloric acid, add 1.02 g of composite adsorbent D15 with a total volume of 1.0% (w / v), stir at 60°C for 0.5 h and filter, the filtration uses composite adsorbent D1 as the filtration medium, the coating amount of composite adsorbent D1 is 4% of the volume of the reaction liquid, and the filtration is carried out at a pressure of 0.2 MPa at a rate of 20 L / h. The volume of the obtained filtrate is 5010 mL, and no protein is detected. Gu 2+ 、Zn 2+ 、As 2+ 、Cd 2+ , Hg 2+ Mg 2+ , Ca 2+ , Fe 3+ Not detected, Pb 2+ : 0.002ppm, the filtrate is milky white.

[0187] (2) The filtrate obtained in step (1) was adjusted to pH 10.5 using a 2.5 mol / L NaOH solution, and the mixture was stirred and degraded at 50°C for 15 min. 25.05 g of composite adsorbent D1 (0.5% by volume of the filtrate) was added, and the mixture was stirred at 50°C for 1 h and filtered. The filtrate was adjusted to pH 6.5 using a 5% (v / v) hydrochloric acid solution, and filtered again using a 0.22 μm filter membrane to obtain 4995 mL of refined filtrate. 2 + 、Zn 2+ 、As 2+ 、Cd 2+ , Hg 2+ Mg 2+ , Ca 2+ , Fe 3+ Not detected, Pb 2+ : 0.001ppm, no protein was detected, the filtrate was clear and bright, and the transmittance was 100%.

[0188] (3) The refined filtrate obtained in step (2) was concentrated using a laboratory jacketed MVR evaporator to a volume that was 1 times the volume of the original fermentation liquid. 2 times the volume of 90% ethanol was added to the concentrate, stirred for 30 minutes, and then allowed to stand for alcohol precipitation. The upper layer was separated until the supernatant was clear. 1.5 times the volume of 70% ethanol was added to the base material, stirred for 30 minutes, and then allowed to stand for alcohol precipitation. The supernatant was discarded, and 1.5 times the volume of 90% ethanol was added to the base material again, stirred for 30 minutes, and allowed to stand for alcohol precipitation. After dehydration, the filter cake was placed in a constant temperature vacuum oven at 50°C for 18 hours. The obtained finished product had a dry content of 96.2%, a transmittance of 99.7%, a yield of 95.8%, a moisture content of 7.5%, and a molecular weight of sodium hyaluronate: 1.35×10 6 DA, protein and heavy metals were not detected, the volume of the refined filtrate was reduced by 5 times compared with that before concentration, and the amount of ethanol used in the alcohol precipitation step was reduced by 80%.

[0189] Example 5 (using regenerated composite adsorbent D1)

[0190] The new composite adsorbent D1 used in Example 4 was regenerated and reused. The regeneration method was as follows: the used composite adsorbent D1 was placed in a dry pan, heated at low temperature until the steam was completely removed, then placed in a muffle furnace at 750°C for 5 hours, taken out and placed in a dryer to cool before use. The regeneration was repeated for 10 batches, and the experimental conditions were the same as in Example 4. The results are shown in Table 4.

[0191] Table 4

[0192]

[0193] Comparative Example 1 (using common diatomaceous earth for adsorption and removal of impurities, CATB complexation and resin for removal of metal ions)

[0194] The method for extracting and preparing sodium hyaluronate product from hyaluronic acid fermentation liquid by using common diatomaceous earth for adsorption and impurity removal, CATB complexation, and resin separation and purification is as follows:

[0195] (1) Add 3 times the volume of deionized water to 500 mL of hyaluronic acid fermentation liquid and stir evenly to control the viscosity of the hyaluronic acid fermentation liquid at 3.3 Pa.s, add 3 g / L of diatomaceous earth and 2 g / L of activated carbon to the dissolved liquid, stir at 50° C. for two hours, filter to obtain a filtrate, add 10% CTAB to the filtrate for complexation, the added amount is HA:CTAB=1:2.5 (g / g), stir for complexation, and filter to obtain a complex.

[0196] (2) Add 0.8 M sodium chloride solution to the original volume of the fermentation liquid, stir at 200 rpm for 24 h, pass the dissociation liquid through the chromatography column at 2.0 BV / h, collect the column liquid, and the detection results of the column liquid are: protein 0.03 mg / mL, Hg2+ ,Gu 2+ , Fe 3+ Mg 2+ Not detected, Zn 2+ :0.97mg / mL,As 2+ :3.8mg / mL,Cd 2+ :1.6mg / mL,Pb 2+ :0.24mg / mL,Ca 2+ : 6.62ppm, transmittance 96.23%.

[0197] (3) 1500 mL of 95% ethanol was added to the column liquid in an amount of HA solution: ethanol = 1:3 (v / v), and stirred at 150 rpm. 15-20% (mass percentage) sodium chloride solution was added until small white flocs appeared. After stirring for 30 minutes, centrifugal drying was performed to obtain a sodium hyaluronate product. The solid was hard. The obtained product had a dry content of 92.3%, a transmittance of 98.1%, a yield of 84.2%, a moisture content of 7.8%, a protein content of 0.08%, and a heavy metal Zn 2+ :5ppm,As 2+ : 2.5ppm, Cd 2+ :2ppm, Pb 2+ :3.9ppm,Ca 2+ : 2.21ppm, others were not detected, molecular weight of sodium hyaluronate: 1.1×10 6 DA.

[0198] Comparative Example 2 (using diatomaceous earth to adsorb bacterial proteins and ultrafiltration membrane to remove metal ions)

[0199] The method for extracting and preparing sodium hyaluronate product from hyaluronic acid fermentation liquid by using diatomaceous earth to adsorb and remove bacterial proteins and ultrafiltration membrane to filter and remove metal ions is as follows:

[0200] 1000mL of hyaluronic acid fermentation liquid was measured, 2L of 95% ethanol was slowly added, and it was stirred and allowed to stand for precipitation for 2-3h; the precipitated hyaluronic acid was taken out, and ethanol was added to fully dehydrate it, the amount of ethanol was 2L, and the obtained crude product was added with 6 times distilled water to dissolve into 0.1% concentration, and the volume after dissolution was 6.6L. After the precipitate was completely dissolved, 1% activated carbon was added, and it was stirred at 50°C for 2h, and then 1% diatomaceous earth was added and stirred evenly, and it was filtered through a plate and frame pre-coated with diatomaceous earth until it was clarified, and ultrafiltration was performed at 0.2MPa using a PVDF ultrafiltration membrane with a molecular weight cutoff of 30,000nm, and the concentrated liquid after ultrafiltration was discharged, and the concentrated liquid volume was 5.05L. The detection results of the concentrated liquid were: protein 0.05mg / mL, Hg 2+ Mg 2+ , Ca 2+ , Fe 3+Not detected, As 2+ :3.8mg / mL,Cu 2+ :1.8mg / mL,Cd 2+ :1.6ppm, Pb 2+ :0.24ppm,Zn 2+ : 0.8 mg / mL, transmittance 97.08%.

[0201] 0.8 mol of sodium chloride was added to the concentrate and stirred until dissolved, then 1.5 times the volume of ethanol was added to precipitate to an alcohol content of 52%, and the supernatant was separated by standing. The base material was washed twice with 80% ethanol by volume of the base material, and then dehydrated twice with anhydrous ethanol and dried to obtain sodium hyaluronate. The obtained finished product had a dry content of 93.8%, a transmittance of 97.9%, a yield of 70.8%, a moisture content of 7.4%, a protein of 0.02%, and heavy metal Zn 2+ :10ppm,Gu 2+ : 11ppm, molecular weight of sodium hyaluronate: 1.4×10 6 DA, hyaluronic acid products are dark in color and in the form of hard particles.

[0202] Comparative Example 3 (using perlite powder)

[0203] The composite adsorbent D1 in Example 1 was replaced with pearlite powder of the same mass, and the other conditions were the same as in Example 1. The volume of the filtrate obtained in step (1) was 4850 mL, protein: 0.57 mg / mL, Gu 2+ 、Zn 2+ 、As 2+ Not detected, Hg 2+ :0.98mg / mL,Mg 2+ :0.52mg / mL,Ca 2+ :0.72mg / mL,Cd 2+ :0.01mg / mL,Pb 2+ :0.3mg / mL,Fe 3+ : 0.73mg / mL, transmittance 95.08%, filtrate is dark yellow. Step (2) obtains a refined filtrate volume of 4680mL, protein: 0.48mg / mL, Gu 2+ 、Zn 2+ 、As 2+ 、Cd 2+ Not detected, Hg 2+ :0.88mg / mL,Mg 2+ :0.45mg / mL,Ca 2+ :0.66mg / mL,Pb 2+ :0.25mg / mL,Fe 3+: 0.63 mg / mL, transmittance 96.38%, the filtrate is yellow. The finished product obtained in step (3) has a dry content of 80.02%, a transmittance of 95.5%, a yield of 78%, a moisture content of 7.5%, and a molecular weight of sodium hyaluronate: 1.45×10 6 DA, protein: 1.2mg / g, heavy metals: Gu 2+ 、Zn 2+ 、As 2+ 、Cd 2+ Not detected, Hg 2+ :20.5ppm,Mg 2+ :23.3ppm,Ca 2+ :6ppm, Pb 2+ :1.51ppm,Fe 3+ : 4.8ppm, the color of sodium hyaluronate crystals is light yellow, and the particles are hard.

[0204] Comparative Example 4 (using zeolite powder)

[0205] The composite adsorbent D1 in Example 1 was replaced with zeolite powder of the same mass, and the other conditions were the same as in Example 1. The volume of the filtrate obtained in step (1) was 4700 mL, protein: 0.66 mg / mL, Gu 2+ 、Zn 2+ 、As 2+ Not detected, Hg 2+ :0.68mg / mL、Mg 2 + :0.48mg / mL,Ca 2+ :0.59mg / mL,Cd 2+ :0.05mg / mL,Pb 2+ :0.22mg / mL,Fe 3+ : 0.55mg / mL, transmittance 96.13%, filtrate is yellow, filtration rate is slow. Step (2) The volume of the refined filtrate is 4530mL, protein: 0.44mg / mL, Gu 2+ 、Zn 2+ 、As 2+ 、Cd 2+ Not detected, Hg 2+ :0.32mg / mL,Mg 2+ :0.25mg / mL,Ca 2+ :0.55mg / mL,Pb 2+ :0.18mg / mL,Fe 3+: 0.14 mg / mL, transmittance 97.33%, the filtrate is yellow, the filtration rate is slow. The finished product obtained in step (3) has a dry content of 84.22%, a transmittance of 96.25%, a yield of 65%, and a moisture content of 7.3%. The molecular weight of sodium hyaluronate is 1.43×10 6 DA, protein: 1.15mg / g, heavy metals: Gu 2+ 、Zn 2+ 、As 2+ 、Cd 2+ , Pb 2+ Not detected, Hg 2+ :18.3ppm,Mg 2+ :21.5ppm,Ca 2+ :15.3ppm, Pb 2+ :5.60ppm,Fe 3+ : 4.52ppm, sodium hyaluronate crystals are slightly yellow.

[0206] Comparative Example 5 (using the gel chitosan resin obtained in step (2) of Preparation Example 1)

[0207] The composite adsorbent D1 in Example 1 was replaced with the gel chitosan resin prepared in step (2) of Preparation Example 1 of the same mass, and the other conditions were the same as in Example 1. The volume of the filtrate obtained in step (1) was 4900 mL, protein: 0.36 mg / mL, Gu 2+ 、Zn 2+ 、As 2+ 、Cd 2+ Not detected, Hg 2+ :0.49mg / mL,Mg 2+ :0.48mg / mL,Ca 2+ :0.50mg / mL,Pb 2+ :0.21mg / mL,Fe 3+ : 0.35mg / mL, transmittance 96.43%, filtrate yellow. Step (2) The volume of the refined filtrate was 4850mL, protein: 0.40mg / mL, Gu 2+ 、Zn 2+ 、As 2+ 、Cd 2+ Not detected, Hg 2+ :0.32mg / mL,Mg 2+ :0.32mg / mL,Ca 2+ :0.31mg / mL,Pb 2+ :0.10mg / mL,Fe 3+: 0.24 mg / mL, transmittance 97.93%, the filtrate is yellow. The finished product obtained in step (3) has a dry content of 89.92%, a transmittance of 97.95%, a yield of 80.23%, a moisture content of 7.3%, and a molecular weight of sodium hyaluronate: 1.42×10 6 DA, protein: 1.15mg / g, heavy metals: Gu 2+ 、Zn 2+ 、As 2+ 、Cd 2+ Not detected, Hg 2+ :12.8ppm,Mg 2+ :16.5ppm,Ca 2+ :3.3ppm, Pb 2+ :0.52ppm,Fe 3+ : 0.31ppm, sodium hyaluronate crystals are slightly yellow.

[0208] Comparative Example 6 (using three adsorbents, namely, uncompounded zeolite powder, perlite powder, and the gel chitosan resin obtained in step (2) of Preparation Example 1)

[0209] The composite adsorbent D1 in Example 1 was replaced with a mixed adsorbent of equal mass, wherein the mixed adsorbent was obtained by simply dry mixing zeolite powder, perlite powder and the gel chitosan resin obtained in step (2) of Preparation Example 1 at a mass ratio of 40:60:1. The experimental conditions were the same as those in Example 1. The volume of the filtrate obtained in step (1) was 4780 mL, protein: 1.58 mg / mL, Zn 2+ 、As 2+ 、Cd 2+ Not detected, Cu 2+ :0.32mg / mL,Hg 2+ :0.49mg / mL,Mg 2+ :0.88mg / mL,Ca 2+ :0.90mg / mL,Pb 2+ :0.91mg / mL,Fe 3+ : 1.22 mg / mL, transmittance 93.5%, filtrate dark yellow, filtration rate slow. Step (2) The volume of the refined filtrate was 4650 mL, protein: 0.91 mg / mL, Zn 2+ 、As 2+ 、Cd 2+ Not detected, Gu 2+ :0.11mg / mL,Hg 2+ :0.38mg / mL,Mg 2+ :0.82mg / mL,Ca 2+ :0.71mg / mL,Pb 2+ :0.02mg / mL,Fe 3+: 0.44 mg / mL, transmittance 94.54%, the filtrate is yellow, and the filtration rate is slow. The finished product obtained in step (3) has a dry content of 77.92%, a transmittance of 96.85%, a yield of 81.23%, a moisture content of 7.9%, and a molecular weight of sodium hyaluronate: 1.45×10 6 DA, protein: 1.02mg / g, heavy metals: Gu 2+ 、Zn 2+ 、As 2+ 、Cd 2+ , Pb 2+ Not detected, Hg 2+ :22.8ppm,Mg 2+ :26.5ppm,Ca 2+ :15.3ppm,Fe 3+ : 2.31ppm, sodium hyaluronate crystals are slightly yellow, and the particles are hard.

[0210] Although the embodiments of the present invention have been shown and described above, it is to be understood that the above embodiments are exemplary and are not to be construed as limitations on the present invention. A person skilled in the art may change, modify, replace and modify the above embodiments within the scope of the present invention without departing from the principles and intent of the present invention.

Claims

1. A zeolite pearlite powder gel chitosan resin adsorbent, characterized in that: The zeolite perlite powder gel chitosan resin adsorbent comprises a gel chitosan resin and zeolite powder and perlite powder loaded on the gel chitosan resin; The preparation method of the gel chitosan resin comprises the following steps: S11, dissolving chitosan in a dilute acid solution to obtain a chitosan dilute acid solution; S12, mixing the metal hydroxide solution and methanol uniformly to obtain a solidified solution; S13, under stirring, slowly adding the chitosan dilute acid solution to the curing solution, filtering after the addition is complete, washing the wet resin obtained after filtration to neutrality and then decolorizing, and drying the decolorized resin to obtain a gel chitosan resin; The method of loading zeolite powder and pearlite powder on gel chitosan resin includes the following steps: S21, placing zeolite powder and perlite powder with a particle size of less than 100 meshes in a dilute acid solution A for soaking treatment, wherein the mass ratio of the zeolite powder to the perlite powder is 1:(0.5-5), washing with water to neutrality after soaking, and drying to obtain a zeolite powder-perlite powder composite; S22, stirring and mixing the zeolite powder-perlite powder composite and the gel chitosan resin in a dilute acid solution B, wherein the amount ratio of the zeolite powder-perlite powder composite, the gel chitosan resin and the dilute acid solution B is (50-300) g: 1 g: (100-3000) mL, and then dropping the obtained mixture into an alkaline solution, and then washing the obtained loaded gel chitosan zeolite perlite powder pellets with water until neutral, drying and sieving, to obtain a zeolite perlite powder gel chitosan resin adsorbent.

2. The zeolite pearlite powder gel chitosan resin adsorbent according to claim 1, characterized in that: In step S11, the dilute acid solution is selected from at least one of hydrochloric acid solution, acetic acid solution, oxalic acid solution and phosphoric acid solution.

3. The zeolite pearlite powder gel chitosan resin adsorbent according to claim 1, characterized in that: In step S11, the concentration of the dilute acid solution is 0.5-5% v / v.

4. The zeolite pearlite powder gel chitosan resin adsorbent according to claim 1, characterized in that: In step S11, the chitosan and the dilute acid solution are used in such amounts that the concentration of the obtained chitosan dilute acid solution is 0.01-5%.

5. The zeolite pearlite powder gel chitosan resin adsorbent according to claim 1, characterized in that: In step S12, the metal hydroxide in the metal hydroxide solution is potassium hydroxide and / or sodium hydroxide.

6. The zeolite pearlite powder gel chitosan resin adsorbent according to claim 1, characterized in that: In step S12, the concentration of the metal hydroxide solution is 1-30% w / v.

7. The zeolite pearlite powder gel chitosan resin adsorbent according to claim 1, characterized in that: In step S12, the amount of methanol added is 0.1 to 0.5 times the total volume of the solidification solution.

8. The zeolite pearlite powder gel chitosan resin adsorbent according to claim 1, characterized in that: In step S13, the decolorization method is to immerse the neutral resin obtained by washing with water in acetone.

9. The zeolite pearlite powder gel chitosan resin adsorbent according to claim 8, characterized in that: In step S13, the amount of acetone used is 1-10% v / v of the total volume of the neutral resin.

10. The zeolite pearlite powder gel chitosan resin adsorbent according to claim 8, characterized in that: In step S13, the immersion conditions include room temperature and a time of 1 to 5 hours.

11. The method for preparing the zeolite pearlite powder gel chitosan resin adsorbent according to any one of claims 1 to 10, characterized in that: The method comprises the following steps: S21, placing zeolite powder and perlite powder with a particle size of less than 100 meshes in a dilute acid solution A for soaking treatment, wherein the mass ratio of the zeolite powder to the perlite powder is 1:(0.5-5), washing with water to neutrality after soaking, and drying to obtain a zeolite powder-perlite powder composite; S22, stirring and mixing the zeolite powder-perlite powder composite and the gel chitosan resin in a dilute acid solution B, wherein the amount ratio of the zeolite powder-perlite powder composite, the gel chitosan resin and the dilute acid solution B is (50-300) g: 1 g: (100-3000) mL, and then dropping the obtained mixture into an alkaline solution, and then washing the obtained loaded gel chitosan zeolite perlite powder pellets with water until neutral, drying and sieving, to obtain a zeolite perlite powder gel chitosan resin adsorbent.

12. The method for preparing the zeolite pearlite powder gel chitosan resin adsorbent according to claim 11, characterized in that: In step S21, the soaking conditions include room temperature and a soaking time of 2 to 10 hours.

13. The method for preparing the zeolite pearlite powder gel chitosan resin adsorbent according to claim 11, characterized in that: In step S21, the concentration of the dilute acid solution A is 0.1-10 molˑL -1 .

14. The method for preparing the zeolite pearlite powder gel chitosan resin adsorbent according to claim 11, characterized in that: In step S22, the concentration of the dilute acid solution B is 0.1-10 molˑL -1 .

15. The method for preparing the zeolite pearlite powder gel chitosan resin adsorbent according to claim 11, characterized in that: In step S22, the stirring and mixing conditions include a temperature of 10-60°C and a time of 1-10 hours.

16. The method for preparing the zeolite pearlite powder gel chitosan resin adsorbent according to claim 11, characterized in that: In step S22, the alkaline solution is a sodium hydroxide solution and / or a potassium hydroxide solution.

17. The method for preparing the zeolite pearlite powder gel chitosan resin adsorbent according to claim 11, characterized in that: In step S22, the concentration of the alkaline solution is 0.1-10 molˑL -1 .

18. Use of the zeolite pearlite powder gel chitosan resin adsorbent according to any one of claims 1 to 10 in the purification of hyaluronic acid.

19. A method for purifying hyaluronic acid, characterized in that: The method comprises the following steps: S31, hyaluronic acid dissociation: dissociating the hyaluronic acid fermentation liquid with a viscosity of 0.1-5 Pa.s with sodium chloride to obtain a sodium hyaluronate dissociation liquid; S32, impurity removal: adding composite adsorbent A to the sodium hyaluronate dissociation solution obtained in step S31 to perform stirring and adsorption for initial impurity removal, and performing a first filtration after the initial impurity removal is completed, wherein the composite adsorbent B is used as a filtration medium for the first filtration to obtain an impurity-removed solution; S33. The pH value of the impurity removal liquid is adjusted to alkaline for degradation. After the degradation is completed, the composite adsorbent C is added to the obtained degradation liquid for stirring and adsorption to remove impurities in depth. After the deep impurity removal is completed, a second filtration is performed, and the pH value of the obtained filtrate is adjusted to 6-8 to obtain a hyaluronic acid filtrate; the composite adsorbent A, composite adsorbent B and composite adsorbent C are all the zeolite perlite powder gel chitosan resin adsorbents described in any one of claims 1 to 10.

20. The method for purifying hyaluronic acid according to claim 19, characterized in that: In step S31, the ratio of the amount of sodium chloride to the hyaluronic acid fermentation liquid is (0.01~5.0)g:1mL.

21. The method for purifying hyaluronic acid according to claim 19, characterized in that: In step S31, the dissociation conditions include a temperature of 30-60°C and a time of 0.5-2h.

22. The method for purifying hyaluronic acid according to claim 19, characterized in that: In step S32, the amount of the composite adsorbent A is 0.01-10% of the volume of the sodium hyaluronate dissociation solution; the conditions for the initial impurity removal by stirring and adsorption include a temperature of 30-60° C. and a time of 0.5-5 h.

23. The method for purifying hyaluronic acid according to claim 19, characterized in that: In step S32, the conditions for the first filtration include a filter medium coating amount of 0.5-5% of the total volume of the reaction solution, a filtration rate of 20-30 L / h, and a filtration pressure of 0.1-0.3 MPa.

24. The method for purifying hyaluronic acid according to claim 19, characterized in that: In step S33, the reagent used for adjusting the pH value is alkaline solution.

25. The method for purifying hyaluronic acid according to claim 24, characterized in that: In step S33, the alkaline solution is a sodium hydroxide solution.

26. The method for purifying hyaluronic acid according to claim 24, characterized in that: In step S33, the concentration of the alkali solution is 0.1~20molˑL -1 .

27. The method for purifying hyaluronic acid according to claim 19, characterized in that: In step S33, the degradation conditions are such that the number average molecular weight of the obtained degradation solution is 1.0×10 6 ~2.0×10 6 Da.

28. The method for purifying hyaluronic acid according to claim 19, characterized in that: In step S33, the amount of the composite adsorbent C used is 0.1-10% of the volume of the degradation liquid.

29. The method for purifying hyaluronic acid according to claim 19, characterized in that: In step S33, the pore size of the filter membrane used for the second filtration is 0.1-10 μm.

30. A method for producing hyaluronic acid, characterized in that: The method comprises the following steps: S41, adjusting the viscosity of the hyaluronic acid fermentation liquid obtained by the microbial fermentation method to 0.1-5 Pa.s; S42, purifying the hyaluronic acid fermentation liquid with a viscosity of 0.1-5 Pa.s by the method described in any one of claims 19-29 to obtain a hyaluronic acid refined filtrate; S43, concentrating the hyaluronic acid filtrate, filtering the obtained concentrated solution after alcohol precipitation and crystallization, and drying the obtained filter cake to obtain sodium hyaluronate crystals.

31. The method for producing hyaluronic acid according to claim 30, characterized in that: In step S43, the concentration is carried out in a jacketed coupled MVR stirred multi-effect steam kettle, which comprises a heat exchanger, a low-pressure steam kettle, a high-pressure steam kettle, a flash tank-1, a flash tank-2 and a compressor, wherein the outlet of the heat exchanger is connected to the top of the low-pressure steam kettle, the bottom of the low-pressure steam kettle is connected to the top of the high-pressure steam kettle, the jacket outlet of the low-pressure steam kettle is connected to the inlet of the flash tank-2, the outlet of the flash tank-2 is connected to the jacket inlet of the high-pressure steam kettle via the compressor, the jacket outlet of the high-pressure steam kettle is connected to the inlet of the flash tank-1, and the outlet of the flash tank-1 is connected to the low-pressure steam kettle. The jacket inlet of the high-pressure steam kettle is connected; the hyaluronic acid refined filtrate is heated to 45-55°C by a heat exchanger and then sent to a low-pressure steam kettle and a high-pressure steam kettle in sequence for concentration, the pressure in the low-pressure steam kettle is controlled at 50-60kPa, and the pressure in the high-pressure steam kettle is controlled at 70-80kPa, and the concentrated liquid finally obtained is discharged from the bottom of the high-pressure steam kettle for alcohol precipitation and crystallization; the secondary steam produced by the low-pressure steam kettle is first recovered through a flash tank-2 and then pressurized and heated by a compressor as a heat source in the high-pressure steam kettle, and the secondary steam produced by the high-pressure steam kettle is recovered through a flash tank-1 and then used as a heat source for the low-pressure steam kettle; In step S43, the alcohol precipitation and crystallization method is to add 1 to 2 times the volume of ethanol with a concentration of 90% or more to the concentrated solution, stir and precipitate, and then let it stand. When the supernatant is clear, the upper layer is separated. When the supernatant is clear, 1 to 1.5 times the volume of ethanol with a concentration of 70% to 80% is added to the remaining base material, stir and precipitate, and then let it stand. When the supernatant is clear, the upper layer is separated, and 1 to 1.5 times the volume of ethanol with a concentration of 90% or more is added to the remaining base material again, stir and precipitate.

32. The method for producing hyaluronic acid according to claim 30, characterized in that: In step S43, the drying conditions include a temperature of 30°C to 60°C and a time of 8h to 24h.

Images