CN110372810B - Method for extracting hyaluronic acid from eggshell membrane - Google Patents
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- CN110372810B CN110372810B CN201910756887.XA CN201910756887A CN110372810B CN 110372810 B CN110372810 B CN 110372810B CN 201910756887 A CN201910756887 A CN 201910756887A CN 110372810 B CN110372810 B CN 110372810B
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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
- C08B37/0003—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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Abstract
The inventionDiscloses a method for extracting hyaluronic acid by using eggshell membrane, which comprises the following steps: (1) pretreating raw materials to obtain eggshell membrane powder; adding deionized water into the eggshell membrane powder, and uniformly stirring; adding trypsin, performing water bath, performing enzymolysis, centrifuging, collecting supernatant, and concentrating; adding chloroform, shaking, separating water phase, adding 95% ethanol, collecting precipitate, and vacuum drying; dissolving, adjusting pH with hydrochloric acid, adding papain and cystine, maintaining at 65-75 deg.C for 14-16 hr, adding cellulose suspension containing cetylpyridinium chloride, treating for 3-5 hr, and adding NaCl and MgCl2Washing the solution, and drying in vacuum to obtain the product. The method is simple, and the obtained hyaluronic acid has high purity, stable molecular weight, safety and no toxic or side effect.
Description
Technical Field
The present invention relates to the field of biological extraction. More particularly, relates to a method for extracting hyaluronic acid by using eggshell membrane.
Background
China is the first major egg-laying country in the world, the yield of eggs in China in 2013 is 2876 ten thousand tons, and 316 ten thousand tons of eggshells and 14 ten thousand tons of eggshell membranes are produced in China every year according to the fact that the weight of the eggshells accounts for 11 percent of the weight of the eggs. The processing and utilization degree of the eggshells in China is low at present, and the eggshells are mainly discarded as waste, so that the environment is polluted, and the waste of the eggshell and eggshell membrane resources is also caused.
The eggshell membrane (commonly known as eggshell membrane) is a fibrous thin film located between the eggshell and the egg white, accounting for about 4-5% of the eggshell weight, and contains about 90% protein, 3% liposomes, and 2% carbohydrates. The proteins of eggshell membrane include keratin, collagen, sialoglycoprotein, ovalbumin, lysozyme, and porphyrin. The eggshell membrane also contains N-acetylglucosamine galactose, glucuronic acid, hyaluronic acid, chondroitin sulfate and other bioactive substances.
Hyaluronic acid is a linear macromolecular polysaccharide rich in anions, and can be combined with various cell growth factors, enzymes, cytokines and receptors in an animal body to induce signal transduction, so that cell growth, differentiation and apoptosis are regulated and controlled. Therefore, hyaluronic acid is widely used in the fields of medicine, food health, and cosmetics. When the hyaluronic acid is used as a medicine, the hyaluronic acid can be used for ophthalmic therapeutic operations such as crystal implantation, removal, corneal transplantation, anti-glaucoma operation and the like, and also can be used for treating arthritis, tendon and soft tissue injuries, accelerating wound healing, serving as a cancer nano-drug adjuvant and the like; when hyaluronic acid is used for producing cosmetics, it can keep skin moist, smooth, fine, tender and elastic, and has the functions of preventing wrinkle, removing wrinkle, beautifying, protecting health and restoring skin physiological function.
Therefore, it is important to provide a method for effectively increasing the extraction amount of hyaluronic acid.
Disclosure of Invention
The invention aims to provide a method for extracting hyaluronic acid from eggshell membrane, which effectively improves the yield and quality of hyaluronic acid.
In order to achieve the purpose, the invention adopts the following technical scheme:
a method for extracting hyaluronic acid from eggshell membrane comprises the following steps:
(1) pretreatment of raw materials: cleaning egg shell, separating egg shell from egg shell membrane, oven drying egg shell membrane at 30-40 deg.C, pulverizing, and sieving to obtain egg shell membrane powder;
(2) adding 30-50 times of deionized water into 10g of eggshell membrane powder, and uniformly stirring;
(3) adding trypsin into the step (2), performing enzymolysis extraction for 5-10h in water bath at 40-50 ℃, centrifuging, collecting supernate, and concentrating to obtain a crude extract;
(4) the volume ratio of the crude extract is 1: (2-3) adding chloroform, shaking up, separating out an aqueous phase, and mixing the aqueous phase and the aqueous phase according to a volume ratio of 1: (3-4) adding 95% ethanol, collecting precipitate, and vacuum drying;
(5) dissolving the dried powder obtained in the step (4), adjusting pH to 6-7 with hydrochloric acid, adding papain and cystine, keeping the temperature at 65-75 deg.C for 14-16 hr, adding 2-3 times volume of cellulose suspension containing 1% cetylpyridinium chloride, treating for 3-5 hr, and adding 1mol/L NaCl and 1mol/L MgCl2Washing for 2-3 times, and vacuum drying.
Further, the adding amount of the trypsin added in the step (3) is 0.6-0.8mg, and the enzyme activity of the trypsin is 5000-;
further, 0.3-0.4mg of papain and 30-50umol of cystine are added in the step (5), and the enzyme activity of the papain is 3000-4000U/g.
The invention has the following beneficial effects:
compared with the prior art, the method for extracting hyaluronic acid from the poultry eggshell membrane can separate high-purity hyaluronic acid from the poultry eggshell membrane, has wide sources and stable molecular weight, and does not contain endotoxin, so that the requirements of the market on hyaluronic acid are met. The hyaluronic acid prepared by the method is improved in quality and yield.
Detailed Description
In order to more clearly illustrate the invention, the invention is further described below in connection with preferred embodiments. It is to be understood by persons skilled in the art that the following detailed description is illustrative and not restrictive, and is not to be taken as limiting the scope of the invention.
Example 1A method for extracting hyaluronic acid from eggshell membrane
A method for extracting hyaluronic acid from eggshell membrane comprises the following steps:
(1) pretreatment of raw materials: cleaning egg shell, separating egg shell from egg shell membrane, oven drying egg shell membrane at 30 deg.C, pulverizing, and sieving to obtain egg shell membrane powder;
(2) adding 30 times of deionized water into 10g of eggshell membrane powder, and uniformly stirring;
(3) adding 0.6mg of trypsin into the step (2), performing water bath at 40 ℃, performing enzymolysis extraction for 5 hours, centrifuging, collecting supernate, and concentrating to obtain a crude extract;
(4) the volume ratio of the crude extract is 1: 2, adding chloroform, shaking up, separating out an aqueous phase, and mixing the aqueous phase and the aqueous phase according to a volume ratio of 1: 3, adding 95% ethanol, collecting the precipitate, and drying in vacuum;
(5) dissolving the dried powder obtained in step (4), adjusting pH to 6 with hydrochloric acid, adding 0.3mg of papain and 30umol cystine, maintaining at 65 deg.C for 14 hr, adding 2 times volume of 1% cetylpyridinium chloride suspension, treating for 3 hr, adding 1mol/L NaCl and 1mol/L MgCl2Washing for 2 times, and vacuum drying.
Example 2 method for extracting hyaluronic acid from eggshell membrane
A method for extracting hyaluronic acid from eggshell membrane comprises the following steps:
(1) pretreatment of raw materials: cleaning egg shell, separating egg shell from egg shell membrane, oven drying egg shell membrane at 40 deg.C, pulverizing, and sieving to obtain egg shell membrane powder;
(2) adding deionized water 50 times the mass of 10g of eggshell membrane powder, and uniformly stirring;
(3) adding 0.8mg of trypsin into the step (2), performing enzymolysis extraction for 10 hours in a water bath at 50 ℃, centrifuging, collecting supernate, and concentrating to obtain a crude extract;
(4) the volume ratio of the crude extract is 1: 3, adding chloroform, shaking uniformly, separating out an aqueous phase, and mixing the aqueous phase and the aqueous phase according to a volume ratio of 1: 4, adding 95% ethanol, collecting the precipitate, and drying in vacuum;
(5) dissolving the dried powder obtained in step (4), adjusting pH to 7 with hydrochloric acid, adding 0.4mg of papain and 50umol of cystine, maintaining at 75 deg.C for 16 hr, adding 3 times volume of 1% cetylpyridinium chloride suspension, treating for 5 hr, adding 1mol/L NaCl and 1mol/L MgCl2Washing for 3 times, and vacuum drying.
Example 3 method for extracting hyaluronic acid from eggshell membrane
A method for extracting hyaluronic acid from eggshell membrane comprises the following steps:
(1) pretreatment of raw materials: cleaning egg shell, separating egg shell from egg shell membrane, oven drying egg shell membrane at 35 deg.C, pulverizing, and sieving to obtain egg shell membrane powder;
(2) adding deionized water 40 times the mass of 10g of eggshell membrane powder, and uniformly stirring;
(3) adding 0.7mg of trypsin into the step (2), performing water bath at 45 ℃, performing enzymolysis extraction for 7 hours, centrifuging, collecting supernate, and concentrating to obtain a crude extract;
(4) the volume ratio of the crude extract is 1: 2.5 adding chloroform, shaking up, separating out the water phase, and mixing the water phase and the water phase according to the volume ratio of 1: 3.5 adding 95% ethanol, collecting precipitate, and vacuum drying;
(5) dissolving the dried powder obtained in step (4), adjusting pH to 6.5 with hydrochloric acid, adding papain 0.35mg and papain 40 mgThe umol cystine is incubated at 70 ℃ for 15 hours, then treated with 2.5 volumes of a 1% cetylpyridinium chloride suspension in cellulose for 4 hours, followed by 1mol/L NaCl and 1mol/L MgCl2Washing for 2 times, and vacuum drying.
Comparative example 1 method for extracting hyaluronic acid from eggshell membrane
A method for extracting hyaluronic acid from eggshell membrane comprises the following steps:
(1) pretreatment of raw materials: cleaning egg shell, separating egg shell from egg shell membrane, oven drying egg shell membrane at 35 deg.C, pulverizing, and sieving to obtain egg shell membrane powder;
(2) adding deionized water 40 times the mass of 10g of eggshell membrane powder, and uniformly stirring;
(3) adding 0.7mg of trypsin into the step (2), performing water bath at 45 ℃, performing enzymolysis extraction for 7 hours, centrifuging, collecting supernate, and concentrating to obtain a crude extract;
(4) the volume ratio of the crude extract is 1: 2.5 adding chloroform, shaking up, separating out the water phase, and mixing the water phase and the water phase according to the volume ratio of 1: 3.5 adding 95% ethanol, collecting precipitate, and vacuum drying;
(5) dissolving the dried powder obtained in step (4), adjusting pH to 6.5 with hydrochloric acid, adding 0.35mg of trypsin and 40umol of cystine, keeping at 70 deg.C for 15 hr, adding 2.5 times volume of 1% cetylpyridinium chloride suspension, treating for 4 hr, and adding 1mol/L NaCl and 1mol/L MgCl2Washing for 2 times, and vacuum drying.
Comparative example 2 method for extracting hyaluronic acid from eggshell membrane
A method for extracting hyaluronic acid from eggshell membrane comprises the following steps:
(1) pretreatment of raw materials: cleaning egg shell, separating egg shell from egg shell membrane, oven drying egg shell membrane at 35 deg.C, pulverizing, and sieving to obtain egg shell membrane powder;
(2) adding deionized water 40 times the mass of 10g of eggshell membrane powder, and uniformly stirring;
(3) adding 0.7mg of trypsin into the step (2), performing water bath at 45 ℃, performing enzymolysis extraction for 7 hours, centrifuging, collecting supernate, and concentrating to obtain a crude extract;
(4) the volume ratio of the crude extract is 1: 2.5 adding chloroform, shaking up, separating out the water phase, and mixing the water phase and the water phase according to the volume ratio of 1: 3.5 adding 95% ethanol, collecting precipitate, and vacuum drying;
(5) dissolving the dried powder obtained in step (4), adjusting pH to 6.5 with hydrochloric acid, adding 0.35mg pronase and 40umol cystine, keeping at 70 deg.C for 15 hr, adding 2.5 times volume of 1% cetyl pyridinium chloride suspension, treating for 4 hr, and adding 1mol/L NaCl and 1mol/L MgCl2Washing for 2 times, and vacuum drying.
Comparative example 3 method for extracting hyaluronic acid from eggshell membrane
A method for extracting hyaluronic acid from eggshell membrane comprises the following steps:
(1) pretreatment of raw materials: cleaning egg shell, separating egg shell from egg shell membrane, oven drying egg shell membrane at 35 deg.C, pulverizing, and sieving to obtain egg shell membrane powder;
(2) adding deionized water 40 times the mass of 10g of eggshell membrane powder, and uniformly stirring;
(3) adding 0.7mg of trypsin into the step (2), performing water bath at 45 ℃, performing enzymolysis extraction for 7 hours, centrifuging, collecting supernate, and concentrating to obtain a crude extract;
(4) the volume ratio of the crude extract is 1: 2.5 adding chloroform, shaking up, separating out the water phase, and mixing the water phase and the water phase according to the volume ratio of 1: 3.5 adding 95% ethanol, collecting precipitate, and vacuum drying;
(5) dissolving the dried powder obtained in step (4), adjusting pH to 6.5 with hydrochloric acid, adding 0.35mg neutral protease and 40umol cystine, keeping at 70 deg.C for 15 hr, adding 2.5 times volume of 1% cetyl pyridinium chloride suspension, treating for 4 hr, and adding 1mol/L NaCl and 1mol/L MgCl2Washing for 2 times, and vacuum drying.
The purity and yield of hyaluronic acid obtained in examples 1 to 3 and comparative examples 1 to 3 were compared, and the results are shown in Table 1. As can be seen from the tables, the purity and yield of hyaluronic acid obtained in examples 1 to 3 of the present invention are significantly higher than those of comparative examples 1 to 3.
TABLE 1 hyaluronic acid purity and yield from eggshell membrane
It should be understood that the above-mentioned embodiments of the present invention are only examples for clearly illustrating the present invention, and are not intended to limit the embodiments of the present invention, and it will be obvious to those skilled in the art that other variations or modifications may be made on the basis of the above description, and all embodiments may not be exhaustive, and all obvious variations or modifications may be included within the scope of the present invention.
Claims (1)
1. The method for extracting hyaluronic acid from eggshell membrane is characterized by comprising the following steps:
(1) pretreatment of raw materials: cleaning egg shell, separating egg shell from egg shell membrane, oven drying egg shell membrane at 30-40 deg.C, pulverizing, and sieving to obtain egg shell membrane powder;
(2) adding 30-50 times of deionized water into 10g of eggshell membrane powder, and uniformly stirring;
(3) adding trypsin into the step (2), performing enzymolysis extraction for 5-10h in water bath at 40-50 ℃, centrifuging, collecting supernate, and concentrating to obtain a crude extract;
(4) the volume ratio of the crude extract is 1: (2-3) adding chloroform, shaking up, separating out an aqueous phase, and mixing the aqueous phase and the aqueous phase according to a volume ratio of 1: (3-4) adding 95% ethanol, collecting precipitate, and vacuum drying;
(5) dissolving the dried powder obtained in the step (4), adjusting the pH value to 6-7 by using hydrochloric acid, adding papain and cystine, preserving the temperature for 14-16 hours at 65-75 ℃, then adding 2-3 times of cellulose suspension of 1% cetylpyridinium chloride with the volume concentration for treating for 3-5 hours, and then using 1mol/L NaCl and 1mol/L MgCl2Washing for 2-3 times, and vacuum drying;
the adding amount of the trypsin added in the step (3) is 0.6-0.8mg, and the enzyme activity of the trypsin is 5000-8000U/g;
0.3-0.4mg of papain and 30-50umol of cystine are added in the step (5), and the enzyme activity of the papain is 3000-4000U/g.
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| CN111808215B (en) * | 2020-07-10 | 2022-05-27 | 上海林清轩生物科技有限公司 | Method for preparing bioactive substances from eggshell membrane by complex enzyme hydrolysis method |
| CN115852548A (en) * | 2022-12-07 | 2023-03-28 | 南通郝旺新材料科技有限公司 | Preparation method and application of biocompatible biomass yarn |
| CN115976739A (en) * | 2022-12-07 | 2023-04-18 | 江南大学 | Method for improving piezoelectric performance of regenerated eggshell membrane |
| CN116084089A (en) * | 2022-12-07 | 2023-05-09 | 江南大学 | Preparation method of nanofiber membrane by taking waste eggshell inner membrane as raw material |
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