CN106279465B - The method of sodium hyaluronate is extracted from giant salamander skin or mucus - Google Patents
The method of sodium hyaluronate is extracted from giant salamander skin or mucus Download PDFInfo
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- CN106279465B CN106279465B CN201610667457.7A CN201610667457A CN106279465B CN 106279465 B CN106279465 B CN 106279465B CN 201610667457 A CN201610667457 A CN 201610667457A CN 106279465 B CN106279465 B CN 106279465B
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- giant salamander
- sodium hyaluronate
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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 present invention discloses a kind of method that sodium hyaluronate is extracted from giant salamander skin or mucus, it is characterised in that successively carries out in accordance with the following steps: giant salamander skin or/and giant salamander mucus are homogenized at 0 ~ 10 DEG C；The complex enzyme that 0.01 ~ 0.1M phosphate buffer of 5 ~ 10 times of volume pH 7 ~ 8 is added into homogenate and is 0.1 ~ 1% with homogenate mass ratio, digests 12 ~ 48 h；The complex enzyme be the pronase of 3500 u/mg, the papain of 6000u/mg and 6000 u/mg trypsase in unit of activity ratio be 1:5:5 ratio mixing；Enzymolysis liquid pH after enzymatic hydrolysis is adjusted to 4 ~ 5, stands 12h, 6000rpm is centrifuged 20 min, collects supernatant；Supernatant is separated by the ultrafiltration membrane that molecular cut off is 50000 Da, the liquid for taking ultrafiltration membrane to retain；The ratio of 1:1.5 ~ 3 adds 95% ethyl alcohol of mass concentration by volume, collects precipitating, and freeze-drying obtains sodium hyaluronate.
Description
Technical field
The present invention relates to a kind of preparation method of sodium hyaluronate, especially a kind of preparation process is simple, yield and purity is high, matter
The measured method that sodium hyaluronate is extracted from giant salamander skin or mucus.
Technical background
Sodium hyaluronate, that is, hyaluronic acid (Hyaluronic acid, HA), is a kind of macromolecule straight-chain polysaccharide, by N- acetyl ammonia
Base glucose and D-Glucose aldehydic acid disaccharide unit repetitive structure composition, are distributed widely in human body and the connective tissue of animal,
The sodium hyaluronate structure of separate sources is identical, only has differences in relative molecular weight size.Studies have shown that sodium hyaluronate has
High flexibility, plasticity, permeability and good biocompatibility, as one kind, absorbable, degradable biological material, exists
The fields such as drug, cosmetics have a wide range of applications.If sodium hyaluronate theory water retention value may be up to 800ml/g, can be used as natural
Moisturizing factor；Sodium hyaluronate is as medicinal application in ophthalmology Lens implantation, corneal transplantation, resisting glaucoma operation and treatment of arthritis
In.
Currently, there are two types of the main sources of sodium hyaluronate: one is from animals such as cockscomb, people's umbilical cord, animal eyeball and pigskins
It is extracted in tissue；Another kind is obtained from streptococcus microorganism in the method for fermentation." pig has been delivered within 1999 as Lu Wen is male
The preparation research of skin hyaluronic acid ", it is to be digested pigskin to obtain sodium hyaluronate with neutral proteinase, although yield etc. is opposite
Other technologies increase, but also can only achieve 0.14%.Due to being limited by raw material and yield, annual output less than 2000kg,
It is far from satisfying the market demand.
Since the nineties in last century, Hunan, Hubei, Shaanxi and Guangxi all establish a large amount of giant salamander chartering cost enterprise,
Whole nation giant salamander culture scale has 500 ~ 6,000,000 tails at present.Giant salamander body surface constantly divides during the growth process without squama, more glandula integumentarias
Secrete the mucus for including various active substance.But so far, there are no about the extraction sodium hyaluronate from giant salamander skin or mucus
Relevant report.
Summary of the invention
The present invention be in order to solve above-mentioned technical problem present in the prior art, provide a kind of preparation process it is simple,
Rate and purity is high, the high-quality method that sodium hyaluronate is extracted from giant salamander skin or mucus.
The technical solution of the invention is as follows: a method of extracting sodium hyaluronate from giant salamander skin or mucus, feature exists
In successively progress in accordance with the following steps:
A. giant salamander skin or/and giant salamander mucus are homogenized at 0 ~ 10 DEG C；
B. 0.01 ~ 0.1M phosphate buffer of 5 ~ 10 times of volume pH 7 ~ 8 is added into homogenate and is with homogenate mass ratio
0.1 ~ 1% complex enzyme digests 12 ~ 48 h；The complex enzyme is the pawpaw egg of the pronase of 3500 u/mg, 6000u/mg
The trypsase of white enzyme and 6000 u/mg are the mixing of 1:5:5 ratio in unit of activity ratio；Enzymolysis liquid pH after enzymatic hydrolysis is adjusted to 4 ~
5,12h is stood, 6000rpm is centrifuged 20 min, collects supernatant；
C. supernatant is separated by the ultrafiltration membrane that molecular cut off is 50000 Da, the liquid for taking ultrafiltration membrane to retain
Body；The ratio of 1:1.5 ~ 3 adds 95% ethyl alcohol of mass concentration by volume, collects precipitating, and freeze-drying obtains sodium hyaluronate.
The present invention is to be configured to compound protease in proportion using three kinds of enzymes to carry out enzyme to giant salamander skin or the homogenate of giant salamander mucus
Solution, various enzyme interactings, sodium hyaluronate can be made with protein pilotaxitic texture, and (20 ~ 30 DEG C) enzymatic hydrolysis separate at normal temperature, make to collect
Supernatant in contain the higher sodium hyaluronate of concentration, by molecular cut off 50kDa ultrafiltration membrance filter removal enzymolysis liquid in
Enzyme and enzymolysis product, and with ethanol precipitation, can be obtained purity >=96%(mass percent) sodium hyaluronate, yield is up to 1%(matter
Measure percentage), preparation process is simple, and at low cost, prepared sodium hyaluronate relative molecular weight is 1.2 × 106Da, it can be applied to
The fields such as cosmetics, drug.
Specific embodiment
Embodiment 1:
A. giant salamander skin 5kg is homogenized at 0 ~ 10 DEG C；
B. the 0.05M phosphate buffer of 20 L pH 7 ~ 8 and the complex enzyme of 50g are added into homogenate, digests 30 h；Institute
Stating complex enzyme is by pronase (3500 u/mg), papain (6000 u/mg) and trypsase (6000 u/mg)
It is the mixing of 1:5:5 ratio in unit of activity ratio；The enzymolysis liquid pH after enzymatic hydrolysis is adjusted to 5 with food grade hydrochloric acid, stands 12h,
6000rpm is centrifuged 20 min, collects supernatant 13.5L；
C. supernatant is separated by the ultrafiltration membrane that molecular cut off is 50000 Da, the liquid for taking ultrafiltration membrane to retain
The total 5L of body；Add 95% ethanol precipitation of mass concentration, collect precipitating, freeze-drying obtains 51g desciccate.
The Bitter-Muir carbazole method and Elson-Morgan method reported with Lu Wen hero are respectively to the D- in desciccate
Glucuronic acid and N-acetylglucosamine content are detected, it is found that D-Glucose aldehydic acid and N-acetylglucosamine contain
Amount molar ratio is 1:1, and respective content reaches the 43% and 49% of gross mass, wherein the protein only containing mass percent 0.07%,
And it is higher using Intrinsic Viscosity Method to measure prepared sodium hyaluronate (desciccate) relative molecular weight, is 1.2 × 106Da。
By the inspection in the testing result of embodiment 1 and male " preparation research of pigskin hyaluronic acid " delivered for 1999 of Lu Wen
It surveys Comparative result and is shown in Table 1, it can be seen that be much higher than pair using sodium hyaluronate yield, purity and the quality that method of the invention produces
Ratio.
Table 1: 1 testing result of embodiment and comparative example compare
| Protein content (%) | D-Glucose aldehydic acid (%) | Molecular weight | Yield (%) | |
| Comparative example | <0.11 | 40.2 | 9.5×105Da | 0.14 |
| Embodiment 1 | 0.07 | 43.0 | 1.2×106Da | 1.00 |
Embodiment 2:
A. giant salamander mucus 5kg is homogenized at 0 ~ 10 DEG C；
B. the 0.1M phosphate buffer of 30L pH 7 ~ 8 and the complex enzyme of 50g are added into homogenate, digests 40 h；It is described
Complex enzyme is to press pronase (3500 u/mg), papain (6000 u/mg) and trypsase (6000 u/mg)
Unit of activity ratio is the mixing of 1:5:5 ratio；The enzymolysis liquid pH after enzymatic hydrolysis is adjusted to 4 with food grade hydrochloric acid, stands 12h, 6000rpm
20 min are centrifuged, supernatant 14.5L is collected；
C. supernatant is separated by the ultrafiltration membrane that molecular cut off is 50000 Da, the liquid for taking ultrafiltration membrane to retain
The total 5.1L of body；Add 95% ethanol precipitation of mass concentration, collect precipitating, freeze-drying obtains 52g desciccate.
The Bitter-Muir carbazole method and Elson-Morgan method reported with Lu Wen hero are respectively to the D- in desciccate
Glucuronic acid and N-acetylglucosamine content are detected, it is found that D-Glucose aldehydic acid and N-acetylglucosamine contain
Amount molar ratio is 1:1, and respective content reaches the 42.8% and 48.7% of gross mass, wherein the egg only containing mass percent 0.06%
White matter, and it is higher using Intrinsic Viscosity Method to measure prepared sodium hyaluronate (desciccate) relative molecular weight, is 1.2 × 106Da。
By the inspection in the testing result of embodiment 2 and male " preparation research of pigskin hyaluronic acid " delivered for 1999 of Lu Wen
It surveys Comparative result and is shown in Table 2, it can be seen that be much higher than pair using sodium hyaluronate yield, purity and the quality that method of the invention produces
Ratio.
Table 2: 2 testing result of embodiment and comparative example compare
| Protein content (%) | D-Glucose aldehydic acid (%) | Molecular weight | Yield (%) | |
| Comparative example | <0.11 | 40.2 | 9.5×105Da | 0.14 |
| Embodiment 1 | 0.06 | 42.8 | 1.2×106Da | 1.00 |
It can extract giant salamander mucus 500g every year according to an adult giant salamander to calculate, sodium hyaluronate 5g can be produced, with 500 ~ 6,000,000
Tail adult giant salamander calculates, and can prepare sodium hyaluronate 25000kg, greatly alleviate the demand and supply contraction.
Claims (1)
1. a kind of method for extracting sodium hyaluronate from giant salamander skin or mucus, it is characterised in that successively carry out in accordance with the following steps:
A. giant salamander skin or giant salamander mucus are homogenized at 0 ~ 10 DEG C；
B. be added into homogenate 5 ~ 10 times of volume pH 7 ~ 8 0.01 ~ 0.1M phosphate buffer and with homogenate mass ratio be 0.1 ~
1% complex enzyme digests 12 ~ 48 h；The complex enzyme is the papain of the pronase of 3500 u/mg, 6000u/mg
It in unit of activity ratio is that 1:5:5 ratio mixes with the trypsase of 6000 u/mg；Enzymolysis liquid pH after enzymatic hydrolysis is adjusted to 4 ~ 5, it is quiet
12h is set, 6000rpm is centrifuged 20 min, collects supernatant；
C. supernatant is separated by the ultrafiltration membrane that molecular cut off is 50000 Da, the liquid for taking ultrafiltration membrane to retain；It presses
The ratio of volume ratio 1:1.5 ~ 3 adds 95% ethyl alcohol of mass concentration, collects precipitating, and freeze-drying obtains sodium hyaluronate.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1063692A (en) * | 1991-01-31 | 1992-08-19 | 无锡市肉类厂 | From pork skin, extract hyaluronic manufacture method |
| CN105285202A (en) * | 2015-11-14 | 2016-02-03 | 普定县瑞鸿生产力促进有限责任公司 | Health tea and processing method thereof |
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2016
- 2016-08-15 CN CN201610667457.7A patent/CN106279465B/en active Active
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1063692A (en) * | 1991-01-31 | 1992-08-19 | 无锡市肉类厂 | From pork skin, extract hyaluronic manufacture method |
| CN105285202A (en) * | 2015-11-14 | 2016-02-03 | 普定县瑞鸿生产力促进有限责任公司 | Health tea and processing method thereof |
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