CN103601814A - Method for extracting hyaluronic acid from pigskin - Google Patents

Method for extracting hyaluronic acid from pigskin Download PDF

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Publication number
CN103601814A
CN103601814A CN201310338352.3A CN201310338352A CN103601814A CN 103601814 A CN103601814 A CN 103601814A CN 201310338352 A CN201310338352 A CN 201310338352A CN 103601814 A CN103601814 A CN 103601814A
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Prior art keywords
pigskin
extract
hyaluronic acid
hyaluronic
water
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CN201310338352.3A
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CN103601814B (en
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刘乃山
宋超龙
刘翠珍
刘振海
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QINGDAO JIULONG BIO-PHARMACEUTICAL Co Ltd
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QINGDAO JIULONG BIO-PHARMACEUTICAL Co Ltd
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Priority to CN201511002148.XA priority patent/CN105504091A/en
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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
    • 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

Abstract

The method discloses a method for extracting hyaluronic acid from pigskin. The method takes pigskin as the main raw material, and then subjects the raw material to processes of crude extraction, protein elimination, deposition, enzyme hydrolysis, complexation, dissociation, deposition, and drying so as to obtain the hyaluronic acid. The method has the advantages of short production period, low cost, high yield, no generation of any toxic or harmful substance, and high product safety, and is capable of being applied to the medicine field.

Description

From pigskin, extract hyaluronic method
Technical field
The inventive method relates to pigskin deep process technology field, is specifically related to a kind of hyaluronic extracting method, a kind ofly take pigskin as raw material, by biological enzymolysis technology, thereby extracts hyaluronic method.
Background technology
Hyaluronic acid (Haluronic acid, HA) has another name called Hyaluronic Acid, is a kind of acidic mucopolysaccharide, and first U.S. Meyer in 1934 etc. isolate this material from bovine vitreous body.The linear polysaccharide structures that hyaluronic acid is comprised of the repeating structure of N-amido glucose and D-Glucose aldehydic acid.
Hyaluronic acid has very large viscosity, and osteoarthrosis is had to lubrication.Can also promote diffusivity, adjusting cell surface and the pericellular Ca of material in skin 2+, Mg 2+, K +, Na +ion motion.Powerful water retention in tissue is one of its most important physiological function, therefore be called as desirable natural moisturizing factor, its theoretical water-retention value is up to 800ml/g, and the actual water-retention value in reticular tissue is 80ml/g.In addition, hyaluronic acid promotes fibroplasia, accelerated wound healing effect in addition.
Hyaluronic acid is mainly used in ophtalmic treatments operation as medicine, as crystal is implanted, is extractd, and corneal transplantation, resisting glaucoma operations etc., are also used for the treatment of osteoarthritis, traumatic arthritis and bursitis and accelerating wound healing.The application of hyaluronic acid in makeup is more extensive, and it can keep skin wet smooth, fine and smooth tender, and high resilience has the effect of wrinkle resistant, crease-resistant, beauty and health care and recovery skin physiology function.
Hyaluronic production technique is mainly divided into two large classes: take extraction method and the fermentation using bacteria method that animal tissues is raw material.Domesticly at present severally take cockscomb as the hyaluronic producer of raw material production, limited because of raw material, cannot form scale.The domestic enterprise that has at present 2 research on producing hyaluronic acid by fermentation methods.2000 kilograms of annual output less thaies can not meet domestic demand at all, and domestic main name-brand cosmetics is as Ao Lisi, virtue is all used hyaluronic acid forever, but because output in domestic is limited, therefore used for cosmetic hyaluronic acid mainly relies on import at present.
Lu Wenxiong etc. once delivered the paper of the one piece of hyaluronic preparation research > of < < pigskin by name > in 1999, the more detailed introduction in the inside a kind of method of preparing phaneroplasm acid with pigskin, its transparent hyaluronic acid obtaining meets correlated quality standard, hyaluronic acid quality prepared by employing this patent method is slightly more advanced than the preparations such as Lu Wenxiong, and yield promotes greatly.
Summary of the invention
For the deficiencies in the prior art; the object of the present invention is to provide a kind of hyaluronic method of extracting from pigskin; a kind of to expand raw material sources; form large-scale production chain; simplify again technique simultaneously; by the modification to parameter, improve yield, and energy-and time-economizing is the novel method that object develops, the method comprises the steps:
(1) slightly carry: after freezing pigskin thaws, with acetone dehydration is rear, pulverize, add water and wear into pasty state with colloidal mill, by every 1kg pigskin, add water 6～8L, add sodium-chlor 80～100g, be heated with stirring to 80～90 ℃, be incubated 10～20min, be cooled to 40～50 ℃, with filter cloth, add diatomite pressure filtration, obtain clear filtrate.
(2) except albumen, precipitation: crude extract is 1 by volume: (0.5～1.5) ratio and chloroform mix and blend 3h, separate water, be 1 by volume: (1.5～3.0) ratio adds 95% ethanol, collecting precipitation, acetone dehydration, vacuum-drying, obtains crude product hyaluronic acid.
(3) enzymolysis: crude product hyaluronic acid is dissolved in 0.1mol/L sodium-chlor, concentration of ordinary dissolution is 0.5%～1.5%, with 1mol/L hydrochloric acid, adjust pH4.0～5.0, be 1 by volume: (0.5～1.5) ratio adds chloroform to stir, divide water-yielding stratum, with dilute sodium hydroxide, adjust pH7.0～8.0, add proteolytic ferment, in 30～40 ℃, enzymolysis 20～30h.
(4) complexing, dissociate, precipitate: in enzymolysis solution, is first 1 by volume: (0.5～1.5) ratio adds chloroform to remove foreign protein, then be 1 by volume: (0.5～1.5) ratio adds 1% cetylpyridinium chloride (CPC), after placement, collecting precipitation, with 0.4mol/L NaCl, dissociate, centrifugal, take out clear liquid, be 1 by volume: (2～4) ratio adds 95% ethanol, collecting precipitation, acetone dehydration, vacuum-drying, obtains fine work hyaluronic acid.
Compared with prior art, advantage of the present invention and positively effect are:
It is that raw material extracts hyaluronic acid that the present invention adopts pigskin; broken market element and take cockscomb as the hyaluronic present situation of raw material production, and pigskin source is wide, wherein hyaluronic acid rich content again; can form the production chain of mass-producing, to meet market to hyaluronic demand.Compare with the method for Lu Wenxiong etc., the prepared hyaluronic acid yield of the inventive method promotes greatly, and quality also slightly rises.
Embodiment
Below in conjunction with specific embodiment, the invention will be further described.
Embodiment 1
(1) slightly carry: get freezing pigskin 20kg, after thawing, with acetone dehydration is rear, pulverize, adding purified water wears into after pasty state with colloidal mill, add purified water 120L, add sodium-chlor 1600g, be heated with stirring to 80 ℃, insulation 10min, be cooled to 40 ℃, with filter cloth, add diatomite pressure filtration, obtain clear filtrate 117.6L.
(2) except albumen, precipitation: crude extract and chloroform 58.8L mix and blend 3h, separate water 117.3L, add 95% ethanol 176L, collecting precipitation, acetone dehydration, vacuum-drying, obtains crude product hyaluronic acid 216g.
(3) enzymolysis: crude product hyaluronic acid is dissolved in 43.2L0.1mol/L sodium chloride solution, adjusts pH4.0 with 1mol/L hydrochloric acid, adds chloroform 21.6L to stir, and minute water-yielding stratum 43.1L, adjusts pH7.0 with dilute sodium hydroxide, adds PRONASE A 8.62g, in 30 ℃, and enzymolysis 20h.
(4) complexing, dissociate, precipitate: in enzymolysis solution, first add chloroform 21.6L except foreign protein, then add 1% cetylpyridinium chloride (CPC) 21.6L, after placement, collecting precipitation, adding 0.4mol/LNaC11.2L dissociates, centrifugal, take out clear liquid, add 95% ethanol 2.4L, collecting precipitation, acetone dehydration, vacuum-drying, obtains fine work hyaluronic acid 164g.
Embodiment 2
(1) slightly carry: get freezing pigskin 20kg, after thawing, with acetone dehydration is rear, pulverize, adding purified water wears into after pasty state with colloidal mill, add purified water 160L, add sodium-chlor 2000g, be heated with stirring to 90 ℃, insulation 20min, be cooled to 50 ℃, with filter cloth, add diatomite pressure filtration, obtain clear filtrate 158.2L.
(2) except albumen, precipitation: crude extract and chloroform 237L mix and blend 3h, separate water 236.7L, add 95% ethanol 710.1L, collecting precipitation, acetone dehydration, vacuum-drying, obtains crude product hyaluronic acid 221g.
(3) enzymolysis: crude product hyaluronic acid is dissolved in 14.7L0.1mol/L sodium chloride solution, adjusts pH5.0 with 1mol/L hydrochloric acid, adds chloroform 22.0L to stir, and minute water-yielding stratum 14.6L, adjusts pH8.0 with sodium hydroxide, adds PRONASE A 3.6g, in 40 ℃, and enzymolysis 30h.
(4) complexing, dissociate, precipitate: in enzymolysis solution, first add chloroform 21.9L except foreign protein, then add 1% cetylpyridinium chloride (CPC) 21.9L, after placement, collecting precipitation, with the NaC11.2L of 0.4mol/L, dissociate, centrifugal, take out clear liquid, add 95% ethanol 2.4L, collecting precipitation, acetone dehydration, vacuum-drying, obtains fine work hyaluronic acid 166g.
Adopt the detection method of Lu Wen hero etc., the detected result of the comparative examples such as embodiment detected result and Lu Wenxiong is as following table:
The detected result contrast of table 1: embodiment and comparative example
Figure 1
as can be seen from Table 1, adopt the hyaluronic acid yield that method of the present invention is produced greatly to promote, quality also slightly rises.
The above, be only preferred embodiment of the present invention, is not the present invention to be done to the restriction of other form, and any those skilled in the art may utilize the technology contents of above-mentioned announcement to be changed or be modified as the equivalent embodiment of equivalent variations.But every technical solution of the present invention content that do not depart from, any simple modification, equivalent variations and the remodeling above embodiment done according to technical spirit of the present invention, still belong to the protection domain of technical solution of the present invention.

Claims (9)

1. from pigskin, extract hyaluronic method, it is characterized in that, comprise the following steps: (1) is slightly carried; (2) except albumen, precipitation; (3) enzymolysis; (4) complexing, dissociate, precipitate.
2. method claimed in claim 1, is characterized in that, comprises the following steps:
(1) slightly carry: by pulverizing after pigskin dehydration, soak extraction, filter, obtain crude extract;
(2) except albumen, precipitation: crude extract and chloroform mix and blend, separate water, water adds 95% ethanol, collecting precipitation, acetone dehydration, vacuum-drying, obtains crude product hyaluronic acid;
(3) enzymolysis: crude product hyaluronic acid is dissolved in 0.1mol/L sodium-chlor, adjusts pH, adds chloroform to stir, and separates water, and water is adjusted pH, adds proteolytic ferment, enzymolysis;
(4) complexing, dissociate, precipitate: enzymolysis solution with chloroform except foreign protein, then adding concentration is 1% cetylpyridinium chloride (CPC), after placement, and collecting precipitation, dissociate, centrifugal, take out clear liquid, clear liquid adds 95% ethanol, collecting precipitation, acetone dehydration, vacuum-drying, obtains fine work hyaluronic acid.
3. according to claim 2ly from pigskin, extract hyaluronic method, it is characterized in that, raw materials used is the freezing pigskin after thawing.
4. according to claim 2ly from pigskin, extract hyaluronic method, it is characterized in that, in described step (1), " soaking extraction " is specially " add water, and grind the pigskin after dehydration is pulverized with colloidal mill; by every 1kg pigskin, add water 6～8L; add sodium-chlor 80～100g, be heated with stirring to 80-90 ℃, be incubated 10～20min; be cooled to 40～50 ℃ ", and " filtration " is specially " with filter cloth, adding diatomite pressure filtration ".
5. according to claim 2ly from pigskin, extract hyaluronic method, it is characterized in that, the volume ratio that crude extract or enzymolysis solution mix with chloroform is 1: (0.5～1.5).
6. according to claim 2ly from pigskin, extract hyaluronic method, it is characterized in that, in described step (2), the volume ratio of crude extract and 95% ethanol is 1: (1.5～3.0).
7. according to claim 2ly from pigskin, extract hyaluronic method, it is characterized in that, described step (3) is specially " crude product hyaluronic acid is dissolved in 0.1mol/L sodium-chlor, and dissolving mass concentration is 0.5%～1.5%, with 1mol/L hydrochloric acid adjust pH to 4.0～5.0; be 1 by volume: (0.5～1.5) ratio adds chloroform to stir; separate water, and water, with dilute sodium hydroxide adjust pH to 7.0～8.0, adds proteolytic ferment; in 30～40 ℃, enzymolysis 20～30h ".
8. according to claim 2ly from pigskin, extract hyaluronic method, it is characterized in that, in described step (3), proteolytic ferment used is: pancreatin, papoid, PRONASE A, neutral protease, Sumizyme MP etc.
9. according to claim 2ly from pigskin, extract hyaluronic method, it is characterized in that, in described step (4), enzymolysis solution and concentration are that the volume ratio of 1% cetylpyridinium chloride (CPC) is 1: (0.5～1.5), the volume ratio of clear liquid and 95% ethanol is 1: (2～4).
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CN105254779A (en) * 2015-10-13 2016-01-20 青海大学 Extraction and purification method of hyaluronic acid with large molecular weight
CN108101998A (en) * 2018-01-22 2018-06-01 南京农业大学 A kind of method that starch is extracted from flour

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CN106188338B (en) * 2016-07-13 2019-09-20 浙江景嘉医疗科技有限公司 A method of reducing foreign protein content in hyaluronic acid sodium raw materials
CN107522796A (en) * 2017-08-31 2017-12-29 浙江工业大学 A kind of method that hyaluronic acid is extracted in the eye from tuna

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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103947819A (en) * 2014-04-30 2014-07-30 桂林军供生化技术开发有限公司 Method for extracting protein from pigskin
CN105254779A (en) * 2015-10-13 2016-01-20 青海大学 Extraction and purification method of hyaluronic acid with large molecular weight
CN105254779B (en) * 2015-10-13 2017-11-21 青海大学 A kind of method for extraction and purification of macromolecule hyaluronic acid
CN108101998A (en) * 2018-01-22 2018-06-01 南京农业大学 A kind of method that starch is extracted from flour

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