CN110922504A - Method for extracting hyaluronic acid from squid eyes - Google Patents
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- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
- C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
- C08B37/0003—General processes for their isolation or fractionation, e.g. purification or extraction from biomass
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- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
- C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
- C08B37/006—Heteroglycans, i.e. polysaccharides having more than one sugar residue in the main chain in either alternating or less regular sequence; Gellans; Succinoglycans; Arabinogalactans; Tragacanth or gum tragacanth or traganth from Astragalus; Gum Karaya from Sterculia urens; Gum Ghatti from Anogeissus latifolia; Derivatives thereof
- C08B37/0063—Glycosaminoglycans or mucopolysaccharides, e.g. keratan sulfate; Derivatives thereof, e.g. fucoidan
- C08B37/0072—Hyaluronic acid, i.e. HA or hyaluronan; Derivatives thereof, e.g. crosslinked hyaluronic acid (hylan) or hyaluronates
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- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
- Y02P20/00—Technologies relating to chemical industry
- Y02P20/50—Improvements relating to the production of bulk chemicals
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Abstract
The invention discloses a method for extracting hyaluronic acid from squid eyes, which solves the problems of complex preparation process, high cost and low extraction purity of the conventional hyaluronic acid. Which comprises the following steps: 1) pretreating raw materials; 2) extracting hyaluronic acid; 3) removing impurities; 4) dialyzing and drying. The invention adopts acetone to pretreat squid eyes to denature and aggregate proteins, then extracts hyaluronic acid by a flotation technology, removes impurities such as fat, protein and the like by supercritical carbon dioxide fluid and enzymolysis, and finally prepares the hyaluronic acid with a certain molecular weight range by dialysis. The method has the advantages of simple operation process, low cost, high safety, high product purity and stable molecular structure, and ensures the biological activity of the product.
Description
Technical Field
The invention relates to preparation of hyaluronic acid, in particular to a method for extracting hyaluronic acid from squid eyes.
Background
Hyaluronic Acid (HA), also known as hyaluronic acid, is a sulfur-free natural large polysaccharide, an anionic acidic mucopolysaccharide consisting of the disaccharide units D-glucuronic acid and N-acetylglucosamine. Due to its ultra-strong water-holding capacity, viscoelasticity, permeability and biological function, it is widely used in the fields of health products, cosmetics and medicines.
Currently, animal tissue extraction and bacterial fermentation methods are mainly used for preparing HA, but in the HA purification process, impurities such as fat, protein and zymophyte reduce the purity of HA, so that the application of HA is limited. The purification methods reported in the prior art include quaternary ammonium salt method, chloroform precipitation method, chromatography, alcohol precipitation method, membrane filtration method, centrifugation method and flocculation method. However, the HA prepared by the quaternary ammonium salt method contains protein, the HA purity is low, and the quaternary ammonium salt dosage is large; the chloroform precipitation method has complex process and causes harm to human bodies and environment; the high viscosity of polysaccharides makes it difficult to remove impurities by centrifugation; chromatography, alcohol precipitation, membrane filtration and flocculation have high production cost and incomplete impurity removal. The development of a method for obtaining high-purity hyaluronic acid is significant.
The biological activity and the using effect of the HA are directly related to the relative molecular weight of the HA, and researches show that the molecular weight of the HA with better functions of viscoelasticity, moisture retention, lubricity, bacteriostasis and the like is more than 2000 KDa; the HA molecular weight with good lubricating property, moisture retention property, drug slow release and other effects is in the range of 1000-2000 KDa; the HA molecular weight is less than 1000KD, and HAs the functions of resisting tumor, regulating immunity, promoting wound healing, promoting angiogenesis and osteogenesis, etc. Therefore, it becomes critical to produce HA of different molecular weights as desired. In the prior art, HA is directly degraded through enzyme degradation, acid-base treatment, physical treatment and the like, such as HA prepared in patents CN102876748A, CN102648214A, CN104059166A, CN105777937A and the like, but the research processes are complex and the cost is high.
The squid is an important aquatic product processing raw material in China, generates 30 percent of processing byproducts, lacks an effective utilization approach and is generally used as a raw material for processing fish meal. The squid eyes in the processing leftovers account for about 5 percent, contain vitreous bodies, can be used as HA extraction resources, and are not fully utilized at present.
Disclosure of Invention
In order to overcome the defects of complex preparation process, high cost and low extraction purity of the conventional hyaluronic acid, the invention aims to provide a method for extracting hyaluronic acid from squid eyes. The method comprises the steps of treating crushed squid with acetone for eye, denaturing the crushed squid, extracting HA through an ion flotation technology, removing impurities such as fat, protein and the like through supercritical carbon dioxide fluid and enzymolysis, and finally preparing high-purity HA with a certain molecular weight range by utilizing a dialysis and alcohol precipitation combined method. The method has the advantages of simple operation process, low cost and high safety.
The technical scheme adopted by the invention for solving the technical problems is as follows:
a method for extracting hyaluronic acid from squid eyes is characterized by comprising the following steps: which comprises the following steps:
(1) pretreatment of raw materials: pulverizing fresh squid eyes, adding acetone 2-5 times of the fresh squid eyes, standing for 10-15min, and centrifuging to remove acetone to obtain squid eye raw materials;
(2) and (3) hyaluronic acid extraction: weighing the squid eye raw material obtained in the step (1), adding 5-10 times of the aqueous solution of lauryl polyether sulfate ammonium with the mass fraction of 1-15%, continuously stirring, and collecting generated bubbles; adding ethanol with the mass concentration of 95% 2-5 times of the weight of the bubbles into the collected bubbles, adjusting the pH value to 5-6 with an acid solution, fully stirring, centrifuging, collecting precipitate, and repeatedly washing with acetone to obtain crude hyaluronic acid;
(3) removing impurities: treating the crude hyaluronic acid obtained in the step (2) with supercritical carbon dioxide; selecting the residual materials after treatment, adding distilled water with equal mass, simultaneously adding protease with 2-6% of the weight of the distilled water, carrying out enzymolysis, inactivating after the enzymolysis is finished, centrifuging, and taking supernate;
(4) and (3) dialysis and drying: and (4) transferring the supernatant obtained in the step (3) into a dialysis bag, dialyzing for 20-24h at room temperature through two steps, adding ethanol with the mass concentration of 95% and the volume of 1-3 times that of the deionized water obtained in the second step into the deionized water obtained in the second step, standing, centrifuging, taking the precipitate, and freeze-drying to obtain the hyaluronic acid.
In a preferred aspect of the present invention, in the step (2), the acid solution is one or more of a hydrochloric acid solution, a citric acid solution, a sulfuric acid solution, and an acetic acid solution.
In a preferred aspect of the present invention, in the step (3), the supercritical carbon dioxide treatment conditions are: the pressure is controlled to be 20-25MPa, the temperature is controlled to be 20-30 ℃, the flow rate of carbon dioxide is 15-20 kg.h < -1 >, and the treatment time is 20-25 min.
In a preferred aspect of the present invention, in the step (3), the protease is composed of two enzymes selected from trypsin, pepsin, neutral protease and papain, and the ratio of the two enzymes is 1: 1.
In a preferred aspect of the present invention, in step (3), the conditions of the enzymatic hydrolysis are: the enzymolysis temperature is 40-50 deg.C, the enzymolysis time is 60-180min, and the pH value is 6-8.
In a preferred aspect of the present invention, in the step (4), the two-step dialysis is performed, wherein hyaluronic acid with a molecular weight of 400000Da is cut off by a dialysis bag in the first step of dialysis, and the dialysis time is 10-12 h; the second step of dialysis is to transfer the dialysate into a dialysis bag with the molecular weight of 2200000Da of the intercepted hyaluronic acid, put the dialysis bag into deionized water, perform dialysis for 10 to 12 hours, and finally collect the deionized water.
In the method, the squid eyes are pretreated by acetone, so that the protein is denatured and aggregated, the impurities such as protein and the like are reduced for subsequent treatment, and the purification of HA is facilitated; the squid eyes are treated by adopting a flotation technology, so that impurities are reduced to the maximum extent, the purity of the initial HA is improved, the HA is extracted to the maximum extent to form a minimum amount of extracting solution, and the use amount of ethanol in subsequent treatment is reduced; the method of removing impurities by combining supercritical carbon dioxide with enzymolysis is adopted, so that fat and protein are thoroughly removed; and finally, removing impurities such as salt and the like and simultaneously obtaining HA with different molecular weight ranges by adopting a dialysis combined organic reagent method. The method has the advantages of simple operation process, low cost, high safety, high product purity and stable molecular structure, and ensures the biological activity of the product.
Detailed Description
The invention is described in detail below with reference to specific embodiments, which are intended to facilitate the understanding and implementation of the invention and are not intended to limit the invention.
Example 1
A method for extracting hyaluronic acid from squid eyes comprises the following steps:
(1) pretreatment of raw materials: crushing fresh squid eyes, adding acetone with the weight 3 times of that of the fresh squid eyes, standing for 15min, and centrifuging to remove the acetone to obtain a squid eye raw material;
(2) and (3) hyaluronic acid extraction: weighing the squid eye raw material obtained in the step (1), adding 7 times of laureth sulfate ammonium aqueous solution with the weight percentage of 5%, continuously stirring, and collecting generated bubbles; adding ethanol with the mass concentration of 95% which is 3 times of the weight of the bubbles into the collected bubbles, adjusting the pH value to 6 by using a hydrochloric acid solution, fully stirring, centrifuging, collecting precipitates, and repeatedly washing by using acetone to obtain crude hyaluronic acid;
(3) removing impurities: treating the crude hyaluronic acid obtained in the step (2) with supercritical carbon dioxide, wherein the conditions of the supercritical carbon dioxide treatment are as follows: controlling the pressure at 20MPa, the temperature at 25 ℃, the flow rate of carbon dioxide at 18 kg.h < -1 >, and the treatment time at 20 min; selecting the remaining materials after treatment, adding distilled water with equal mass, and simultaneously adding protease with 5% of the weight of the distilled water for enzymolysis, wherein the protease consists of trypsin and neutral protease, and the adding ratio is 1: 1; the enzymolysis conditions are as follows: the enzymolysis temperature is 45 ℃, the enzymolysis time is 90min, and the pH value is 7; inactivating after enzymolysis, centrifuging, and taking supernatant;
(4) and (3) dialysis and drying: transferring the supernatant obtained in the step (3) into a dialysis bag, dialyzing at room temperature for 22h through two steps of dialysis, wherein the hyaluronic acid with the molecular weight cutoff of 400000Da is cut off by the dialysis bag in the first step of dialysis, and the dialysis time is 10 h; the second step of dialysis is to transfer the dialysate into a dialysis bag with the molecular weight of 2200000Da of the intercepted hyaluronic acid, put the dialysis bag into deionized water, perform dialysis for 12 hours, and finally collect the deionized water; adding deionized water into ethanol with 3 times of volume and mass concentration of 95%, standing, centrifuging, collecting precipitate, and freeze drying to obtain hyaluronic acid.
Example 2
A method for extracting hyaluronic acid from squid eyes comprises the following steps:
(1) pretreatment of raw materials: crushing fresh squid eyes, adding acetone 2 times of the fresh squid eyes, standing for 15min, and centrifuging to remove acetone to obtain squid eye raw materials;
(2) and (3) hyaluronic acid extraction: weighing the squid eye raw material obtained in the step (1), adding 5 times of laureth sulfate ammonium aqueous solution with the weight of 2 percent, continuously stirring, and collecting generated bubbles; adding ethanol with the mass concentration of 95% which is 5 times of the weight of the bubbles into the collected bubbles, adjusting the pH value to 5 by using a citric acid solution, fully stirring, centrifuging, collecting precipitates, and repeatedly washing by using acetone to obtain crude hyaluronic acid;
(3) removing impurities: treating the crude hyaluronic acid obtained in the step (2) with supercritical carbon dioxide, wherein the conditions of the supercritical carbon dioxide treatment are as follows: controlling the pressure at 25MPa, the temperature at 20 ℃, the flow rate of carbon dioxide at 15 kg.h < -1 >, and the treatment time at 25 min; selecting the remaining materials after treatment, adding distilled water with equal mass, and simultaneously adding protease with the weight of 2% of the distilled water for enzymolysis, wherein the protease consists of pepsin and papain, and the adding ratio is 1: 1; the enzymolysis conditions are as follows: the enzymolysis temperature is 50 ℃, the enzymolysis time is 60min, and the pH value is 6; inactivating after enzymolysis, centrifuging, and taking supernatant;
(4) and (3) dialysis and drying: transferring the supernatant obtained in the step (3) into a dialysis bag, and dialyzing at room temperature for 20h through two-step dialysis, wherein hyaluronic acid with the molecular weight cutoff of 400000Da is cut off by the dialysis bag in the first-step dialysis, and the dialysis time is 10 h; the second step of dialysis is to transfer the dialysate into a dialysis bag with the molecular weight of 2200000Da of the intercepted hyaluronic acid, put the dialysis bag into deionized water, perform dialysis for 10 hours, and finally collect the deionized water; adding deionized water into ethanol with the volume of 1 time and the mass concentration of 95%, standing, centrifuging, taking precipitate, and freeze-drying to obtain hyaluronic acid.
Example 3
A method for extracting hyaluronic acid from squid eyes comprises the following steps:
(1) pretreatment of raw materials: crushing fresh squid eyes, adding acetone which is 5 times of the fresh squid eyes, standing for 12min, and centrifuging to remove acetone to obtain a squid eye raw material;
(2) and (3) hyaluronic acid extraction: weighing the squid eye raw material obtained in the step (1), adding 10 times of laureth sulfate ammonium aqueous solution with the weight of 10 percent, continuously stirring, and collecting generated bubbles; adding ethanol with the mass concentration of 95% and the weight 5 times of that of the bubbles into the collected bubbles, adjusting the pH value to 6 by using a sulfuric acid solution, fully stirring, centrifuging, collecting precipitates, and repeatedly washing by using acetone to obtain crude hyaluronic acid;
(3) removing impurities: treating the crude hyaluronic acid obtained in the step (2) with supercritical carbon dioxide, wherein the conditions of the supercritical carbon dioxide treatment are as follows: controlling the pressure at 25MPa, the temperature at 30 ℃, the flow rate of carbon dioxide at 20 kg.h < -1 >, and the treatment time at 25 min; selecting the remaining materials after treatment, adding distilled water with equal mass, and simultaneously adding protease with the weight of 6% of the distilled water for enzymolysis, wherein the protease consists of trypsin and pepsin, and the adding ratio is 1: 1; the enzymolysis conditions are as follows: the enzymolysis temperature is 45 ℃, the enzymolysis time is 180min, and the pH value is 8; inactivating after enzymolysis, centrifuging, and taking supernatant;
(4) and (3) dialysis and drying: transferring the supernatant obtained in the step (3) into a dialysis bag, and dialyzing at room temperature for 24h through two-step dialysis, wherein hyaluronic acid with the molecular weight cutoff of 400000Da is cut off by the dialysis bag in the first-step dialysis, and the dialysis time is 12 h; the second step of dialysis is to transfer the dialysate into a dialysis bag with the molecular weight of 2200000Da of the intercepted hyaluronic acid, put the dialysis bag into deionized water, perform dialysis for 12 hours, and finally collect the deionized water; adding deionized water into ethanol with 3 times of volume and mass concentration of 95%, standing, centrifuging, collecting precipitate, and freeze drying to obtain hyaluronic acid.
Example 4
A method for extracting hyaluronic acid from squid eyes comprises the following steps:
(1) pretreatment of raw materials: crushing fresh squid eyes, adding acetone with the weight 3 times of that of the fresh squid eyes, standing for 10min, and centrifuging to remove the acetone to obtain a squid eye raw material;
(2) and (3) hyaluronic acid extraction: weighing the squid eye raw material obtained in the step (1), adding 8 times of laureth sulfate ammonium aqueous solution with the weight percentage of 15%, continuously stirring, and collecting generated bubbles; adding ethanol with the mass concentration of 95% 2 times of the weight of the bubbles into the collected bubbles, adjusting the pH value to 6 by using an acetic acid solution, fully stirring, centrifuging, collecting precipitates, and repeatedly washing by using acetone to obtain crude hyaluronic acid;
(3) removing impurities: treating the crude hyaluronic acid obtained in the step (2) with supercritical carbon dioxide, wherein the conditions of the supercritical carbon dioxide treatment are as follows: controlling the pressure at 22MPa, the temperature at 25 ℃, the flow rate of carbon dioxide at 18 kg.h < -1 >, and the treatment time at 20 min; selecting the remaining materials after treatment, adding distilled water with equal mass, and simultaneously adding protease with 5% of the weight of the distilled water for enzymolysis, wherein the protease consists of neutral protease and papain, and the adding ratio is 1: 1; the enzymolysis conditions are as follows: the enzymolysis temperature is 40 ℃, the enzymolysis time is 90min, and the pH value is 7; inactivating after enzymolysis, centrifuging, and taking supernatant;
(4) and (3) dialysis and drying: transferring the supernatant obtained in the step (3) into a dialysis bag, dialyzing at room temperature for 22h through two steps of dialysis, wherein the hyaluronic acid with the molecular weight cutoff of 400000Da is cut off by the dialysis bag in the first step of dialysis, and the dialysis time is 12 h; the second step of dialysis is to transfer the dialysate into a dialysis bag with the molecular weight of 2200000Da of the intercepted hyaluronic acid, put the dialysis bag into deionized water, perform dialysis for 10 hours, and finally collect the deionized water; and adding deionized water into ethanol with 2 times of volume and mass concentration of 95%, standing, centrifuging, collecting precipitate, and freeze-drying to obtain hyaluronic acid.
Comparative example 1
In this comparative example, the process is essentially the same as in example 1, except that: step 1) processing the squid eye raw material without using acetone.
Comparative example 2
In this comparative example, the process is essentially the same as in example 1, except that: and 2) replacing the ammonium laureth sulfate aqueous solution with deionized water.
Comparative example 3
In this comparative example, the process is essentially the same as in example 1, except that: step 1) squid eyes are not treated by using acetone, and step 2) the laureth sulfate ammonium aqueous solution is replaced by deionized water.
Comparative example 4
In this comparative example, the process was substantially the same as in comparative example 3, except that: the supercritical carbon dioxide impurity removal treatment of the step 3) is not carried out.
Comparative example 5
In this comparative example, the process was substantially the same as in comparative example 3, except that: the enzymolysis impurity removal treatment of the step 3) is not carried out.
Comparative example 6
In this comparative example, the process was substantially the same as in comparative example 5, except that: the supercritical carbon dioxide impurity removal treatment of the step 3) is not carried out.
Comparative example 7
In this comparative example, the process was substantially the same as in comparative example 6, except that: the dialysis treatment of step 4) was not performed.
Quality evaluation of hyaluronic acid extract: the hyaluronic acid extracted in the above examples and comparative examples was compared in quality with the glucuronic acid content, protein content, fat content, ash content, hyaluronic acid extraction rate as indicators, and the results are shown in table 1.
TABLE 1 comparison of hyaluronic acid quality indicators extracted in different examples and comparative examples
As can be seen from table 1, the purity and extraction rate of hyaluronic acid obtained by the present invention both have good effects, and the results of comparative examples 1 to 7 show that the purity and extraction rate of hyaluronic acid are significantly affected by the above extraction operation by changing the operations of acetone treatment, surfactant extraction, supercritical carbon dioxide treatment, enzymatic hydrolysis, dialysis, etc. and that good hyaluronic acid extraction effect can be obtained only by the synergistic effect of the above operations, as also demonstrated in comparative examples 1 to 4. The results of example 4 show that the invention can obtain hyaluronic acid with a certain molecular weight range according to needs, and the quality of hyaluronic acid is not influenced.
Evaluation of water retention of hyaluronic acid extract: 0.5g of hyaluronic acid prepared in each example and comparative example was taken, and the water-holding property of hyaluronic acid was observed by spraying water under a constant humidity environment, and the water content of hyaluronic acid was determined by weighing with no occurrence of significant water exudation as an end point, and the results are shown in table 2.
TABLE 2 comparison of water holding Properties of hyaluronic acids extracted in various examples and comparative examples
As can be seen from table 2, the hyaluronic acid obtained by the present invention has good water retention property and can carry 486 times of water, which indicates that the molecular structure of hyaluronic acid is not damaged during the extraction process, and the biological activity of the extracted hyaluronic acid is ensured.
Claims (6)
1. A method for extracting hyaluronic acid from squid eyes is characterized by comprising the following steps: which comprises the following steps:
(1) pretreatment of raw materials: pulverizing fresh squid eyes, adding acetone 2-5 times of the fresh squid eyes, standing for 10-15min, and centrifuging to remove acetone to obtain squid eye raw materials;
(2) and (3) hyaluronic acid extraction: weighing the squid eye raw material obtained in the step (1), adding 5-10 times of the aqueous solution of lauryl polyether sulfate ammonium with the mass fraction of 1-15%, continuously stirring, and collecting generated bubbles; adding ethanol with the mass concentration of 95% 2-5 times of the weight of the bubbles into the collected bubbles, adjusting the pH value to 5-6 with an acid solution, fully stirring, centrifuging, collecting precipitate, and repeatedly washing with acetone to obtain crude hyaluronic acid;
(3) removing impurities: treating the crude hyaluronic acid obtained in the step (2) by using supercritical carbon dioxide; selecting the residual materials after treatment, adding distilled water with equal mass, simultaneously adding protease with 2-6% of the weight of the distilled water, carrying out enzymolysis, inactivating after the enzymolysis is finished, centrifuging, and taking supernate;
(4) and (3) dialysis and drying: and (4) transferring the supernatant obtained in the step (3) into a dialysis bag, dialyzing for 20-24h at room temperature through two steps, adding ethanol with the mass concentration of 95% and the volume of 1-3 times that of the deionized water obtained in the second step into the deionized water obtained in the second step, standing, centrifuging, taking the precipitate, and freeze-drying to obtain the hyaluronic acid.
2. The method for extracting hyaluronic acid from squid eyes as claimed in claim 1, wherein the method comprises the following steps: in the step (2), the acid solution is one or more than two of hydrochloric acid solution, citric acid solution, sulfuric acid solution and acetic acid solution.
3. The method for extracting hyaluronic acid from squid eyes as claimed in claim 1, wherein the method comprises the following steps: in the step (3), the supercritical carbon dioxide treatment conditions are as follows: the pressure is controlled to be 20-25MPa, the temperature is controlled to be 20-30 ℃, the flow rate of carbon dioxide is 15-20 kg.h < -1 >, and the treatment time is 20-25 min.
4. The method for extracting hyaluronic acid from squid eyes as claimed in claim 1, wherein the method comprises the following steps: in the step (3), the protease is composed of two enzymes of trypsin, pepsin, neutral protease and papain, and the adding ratio of the two enzymes is 1: 1.
5. The method for extracting hyaluronic acid from squid eyes as claimed in claim 1 or 4, wherein: in the step (3), the conditions of the enzymolysis are as follows: the enzymolysis temperature is 40-50 deg.C, the enzymolysis time is 60-180min, and the pH value is 6-8.
6. The method for extracting hyaluronic acid from squid eyes as claimed in claim 1, wherein the method comprises the following steps: in the step (4), the two-step dialysis is carried out, wherein hyaluronic acid with the molecular weight of 400000Da is cut off by a dialysis bag in the first step of dialysis, and the dialysis time is 10-12 h; the second step of dialysis is to transfer the dialysate into a dialysis bag with the molecular weight of 2200000Da of the intercepted hyaluronic acid, put the dialysis bag into deionized water, perform dialysis for 10 to 12 hours, and finally collect the deionized water.
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| CN105907820A (en) * | 2016-06-26 | 2016-08-31 | 吴迪 | Method for extracting hyaluronic acid in squid eyes with fermentation method |
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| CN110863024A (en) * | 2019-12-13 | 2020-03-06 | 山东人和集团有限公司 | Method for preparing micromolecular hyaluronic acid by utilizing squid eyes |
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|---|---|---|---|---|
| CN105907820A (en) * | 2016-06-26 | 2016-08-31 | 吴迪 | Method for extracting hyaluronic acid in squid eyes with fermentation method |
| CN108250322A (en) * | 2017-12-31 | 2018-07-06 | 厦门大学 | the foam separation method of micro hyaluronic acid in aqueous solution |
| CN110863024A (en) * | 2019-12-13 | 2020-03-06 | 山东人和集团有限公司 | Method for preparing micromolecular hyaluronic acid by utilizing squid eyes |
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