CN108997446A - The extraction process of small molecule fucoidin and the application in cosmetics - Google Patents
The extraction process of small molecule fucoidin and the application in cosmetics Download PDFInfo
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
- C07H3/00—Compounds containing only hydrogen atoms and saccharide radicals having only carbon, hydrogen, and oxygen atoms
- C07H3/04—Disaccharides
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K8/00—Cosmetics or similar toiletry preparations
- A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
- A61K8/30—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds
- A61K8/60—Sugars; Derivatives thereof
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
- A61Q19/00—Preparations for care of the skin
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
- A61Q19/00—Preparations for care of the skin
- A61Q19/08—Anti-ageing preparations
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
- C07H1/00—Processes for the preparation of sugar derivatives
- C07H1/06—Separation; Purification
- C07H1/08—Separation; Purification from natural products
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
- C07H3/00—Compounds containing only hydrogen atoms and saccharide radicals having only carbon, hydrogen, and oxygen atoms
- C07H3/06—Oligosaccharides, i.e. having three to five saccharide radicals attached to each other by glycosidic linkages
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Abstract
Application the present invention provides the extraction process of small molecule fucoidin and in cosmetics.After kelp broken wall slurries are made in kelp by the present invention, first with chitosan-acetic acid solution and it is aided with sodium perborate sedimentation polysaccharide, after distillation water dispersion, lysozyme combination trifluoroacetic acid is recycled to degrade, the separation for realizing small molecule fucoidin is handled finally by multigelation, products obtained therefrom molecular weight is low (200~300Da), purity is high, and there is appropriate good mobility, can be used for preparing cosmetics.
Description
Technical field
The present invention relates to technical field of natural product extraction, extraction process more particularly to small molecule fucoidin and
Application in cosmetics.
Background technique
1913, Sweden scientist Kylin had found the viscous ingredients in brown alga in University of Uppsala laboratory ---
Fucoidin.With the progress of science and technology, it was recognized that fucoidin is that " iuntercellular is more for one of brown algae plant
Sugar ", this polysaccharide is primarily present in the cell wall in brown algae plant, and content is seldom.
Fucoidin is a kind of natural complex polysaccharide, and natural sulfate is contained in molecule chain end, shows it
Excellent, apparent physiological activity.Modern science proves that fucoidin is not a kind of compound of single structure, but has not
With a compounds of group of chemical constituent.Common fucoidin molecular weight is about 20000Da, have it is antiviral, adjust it is immune and
The effects of anticancer.Fucoidin content is about 1~2% in kelp.
In recent years, small molecule fucoidin receives the close attention of people, the especially production of molecular weight 500Da or so
Product, activity are higher than common fucoidin by 40% or more.At present it has been confirmed that small molecule fucoidin in preventive medicine, exempt from
The effect of epidemic disease, tumor suppression and improvement cancer cachexia etc., safety etc..In addition, also having been reported that small molecule fucoidin
Can be used for curing U.S. field, the growth of activated fiber mother cell promotes endogenous collagen synthesis, adjust MMP to skin damage,
High antioxidant capacity, adjust inflammation and allergic reaction, promote wound healing etc..
But it is limited by extractive technique, the fucoidin produced now is mostly the heteroglycan of macromolecular, complicated component, knot
Structure is unclear, limits fucoidin research and market development progress.It is disclosed in patent CN106866834B and a kind of prepares brown alga
The method of gum products, the fucoidin molecular weight prepared by this method is low, but its molecular weight ranges 2000~
10000Da, molecular weight is still higher, and the mobility of product is not good enough, is unfavorable for subsequent applications.
Summary of the invention
Present invention aim to provide the extraction process of small molecule fucoidin and the application in cosmetics, molecule
It measures low, and there is appropriate good mobility.
To achieve the above object, the present invention is achieved by the following scheme:
The extraction process of small molecule fucoidin, the specific steps are as follows:
(1) kelp is cleaned, microwave irradiation drying, crushes, and obtains kelp powder；
(2) kelp powder is added in the distilled water of 10~15 times of weight, stirring is high-pressure homogeneous, obtains kelp broken wall slurry
Liquid；
(3) chitosan-acetic acid solution is added into kelp broken wall slurries, the two volume ratio is 6~8:1, stirs 10~15 points
Clock is added the sodium perborate of 0.08~0.1 times of kelp powder weight, continues stirring 20~30 minutes, is centrifuged, and collects precipitating；
(4) precipitating obtained by step (3) is added in the distilled water of 6~8 times of weight while stirring, supersonic oscillations 30 are divided
Clock obtains dispersion liquid；
(5) to the lysozyme that 0.02~0.03 times of kelp powder weight is added in step (4) in gained dispersion liquid, 35~45
DEG C hydrolysis 3~5 hours, it is subsequently added into the trifluoroacetic acid of 0.01~0.02 times of kelp powder weight, is warming up to 90~100 DEG C, it is close
20~30 minutes and enzyme deactivation are hydrolyzed under the conditions of closing, vacuum recycles trifluoroacetic acid, obtains extracting solution；
(6) extracting solution obtained by step (5) is freezed in -40~-50 DEG C, after freezing completely, under the conditions of room temperature (25 DEG C) slowly
To melt, 0~4 DEG C of centrifugation takes supernatant, repeated freezing, thawing and centrifugal process, until until without precipitating generation, gained supernatant
Liquid freeze-drying, obtains small molecule fucoidin.
Preferably, in step (1), the condition of microwave irradiation drying are as follows: the processing of 300~450W microwave intermitant irradiation, every time
The processing time is 5~10s, is continued after being spaced 30s, and cumulative processing time is 3~4 minutes.
Preferably, in step (2), the process conditions of stirring are: small with 500~800 revs/min of revolving speed stirring 3~4
When.
Preferably, in step (2), high-pressure homogeneous using high pressure Microfluidizer, process conditions are: pressure 100~
120MPa, 45~48 DEG C of temperature, the time 5~8 minutes.
Preferably, in step (3), the chitosan-acetic acid solution the preparation method is as follows: by molecular weight 30~400,000, take off
1~2g Chitosan powder of acetyl degree >=90% is added in the aqueous acetic acid of mass concentration 1%, is heated to 40 DEG C, stirring
To being completely dissolved to obtain the final product.
Preferably, in step (3), the process conditions of centrifugation are as follows: 5000~7000 revs/min of revolving speed, centrifugation time 5~7
Minute.
Preferably, in step (4), the process conditions of centrifugation are as follows: 8000~10000 revs/min of revolving speed, centrifugation time 5~7
Minute.
Preferably, in step (5), the recovery method of trifluoroacetic acid is that vacuum recycles at 40~60 DEG C.
Preferably, in step (6), the process conditions of centrifugation are as follows: 15000~20000 revs/min of revolving speed, centrifugation time 2~
3 minutes.
Preferably, in step (6), the process conditions of freeze-drying are as follows: -10~-15 DEG C of precoolings are solid-state, are then shifted
Into vacuum freeze drier, temperature is down to -40~-50 DEG C, is evacuated to 15~20Pa, and then temperature rises to 20~30 DEG C,
Continuation is handled 10~15 hours under current vacuum degree condition.
The small molecule fucoidin obtained using said extracted technique, molecular weight are 200~300Da.Infrared spectroscopy point
Analysis, in 3420cm-1There is strong absorption at place, indicates the stretching vibration of-OH and O-H key；In 3000-2800cm-1Between weaker peak
For the C-H absorption peak of-CH2 or-CH3,1200-1100cm-1Between less sharp peak be C-H angle vibration, this two groups of peaks are sugar
The characteristic absorption peak of class；1200-1100cm-1Between bigger absorption peak be as caused by two kinds of C-O stretching vibrations, it is one of
Belong to C-O-H, another kind is the stretching vibration of the C-O-C of saccharide ring；1610-1620cm-1The absorption at place is the spy of uronic acid
Levy absorption peak；1250cm-1、850cm-1、820cm-1The peak of left and right is the characteristic absorption peak of sulfate.Product has fucoidin
Each characteristic peak, it may be determined that be fucoidin.
Application of the above-mentioned small molecule fucoidin in cosmetics, preferably moisturizing or antioxidation cosmetic product.Of the invention
Beneficial effect is:
1, after kelp broken wall slurries are made in kelp by the present invention, first with chitosan-acetic acid solution and to be aided with sodium perborate heavy
Polysaccharide is dropped, after distillation water dispersion, lysozyme combination trifluoroacetic acid is recycled to degrade, is handled finally by multigelation real
The separation of existing small molecule fucoidin, products obtained therefrom molecular weight is low (200~300Da), purity is high, and has appropriate good
Mobility can be used for preparing cosmetics, and imparting cosmetics have effects that following: the growth of activated fiber mother cell promotes interior raw
Property collagen synthesis, adjust MMP (matrix metalloproteinase) to skin damage, high antioxidant capacity, adjusts inflammation and allergy is anti-
Answer, promote wound healing etc..
2, kelp broken wall slurries are made in kelp and are conducive to the release of kelp internal component, so that being easier to sink in subsequent step
Polysaccharide component out drops.
3, compared with common ethanol water sedimentation, the present invention is aided with sodium perborate using chitosan-acetic acid solution and settles
Polysaccharide (while a small amount of degradation has occurred), ensure that the abundant sedimentation of polysaccharide component.It does not need to add other calcium chloride i.e. yet
It can inhibit sodium alginate water-swellable, this is because a small amount of sodium perborate has degradation, in conjunction with the winding of chitosan molecule
Effect, just inhibits expansion.
4, compared with common ethanol water extracts, the present invention utilizes lysozyme combination trifluoroacetic acid degradation of polysaccharide, and one
Aspect promotes polysaccharide to be degraded to lower molecular weight, on the other hand effectively prevents polysaccharide and is degraded to monosaccharide, leads to low yield.
5, the present invention handles the separation for realizing small molecule fucoidin finally by multigelation, ensure that the high-purity of product
Degree, meanwhile, so that product has appropriate good mobility.
Specific embodiment
The technical scheme in the embodiments of the invention will be clearly and completely described below, it is clear that described implementation
Example is only a part of the embodiment of the present invention, instead of all the embodiments.Based on the embodiments of the present invention, this field is common
Technical staff's every other embodiment obtained without making creative work belongs to the model that the present invention protects
It encloses.
Since the fucoidin content that different zones salvage kelp is not quite similar, kelp of the present invention is derived from same area
The domain same time salvages, in order to extract the comparison of effect.Since the fucoidin content of kelp different parts is different, it is
Facilitate and compare, the present invention uses Fresh Laminaria Japonica complete stool.
Embodiment 1
The extraction process of small molecule fucoidin, the specific steps are as follows:
(1) kelp is cleaned, microwave irradiation drying, crushes, and obtains kelp powder；
(2) kelp powder is added in the distilled water of 10 times of weight, stirring is high-pressure homogeneous, obtains kelp broken wall slurries；
(3) chitosan-acetic acid solution is added into kelp broken wall slurries, the two volume ratio is 6:1, stirs 10 minutes, is added
The sodium perborate of 0.08 times of kelp powder weight, continues stirring 20 minutes, and precipitating is collected in centrifugation；
(4) precipitating obtained by step (3) is added in the distilled water of 6 times of weight while stirring, supersonic oscillations 30 minutes, is obtained
Dispersion liquid；
(5) lysozyme of 0.02 times of kelp powder weight is added in gained dispersion liquid into step (4), 35 DEG C of hydrolysis 3 are small
When, it is subsequently added into the trifluoroacetic acid of 0.01 times of kelp powder weight, is warming up to 90 DEG C, hydrolyzes 20 minutes and goes out under confined conditions
Enzyme, vacuum recycle trifluoroacetic acid, obtain extracting solution；
(6) extracting solution obtained by step (5) is freezed in -40 DEG C, after freezing completely, is slowly melted under the conditions of room temperature (25 DEG C), 0
DEG C centrifugation, takes supernatant, repeated freezing, thawing and centrifugal process, until the freezing of gained supernatant is done until generating without precipitating
It is dry, obtain small molecule fucoidin.
Wherein, in step (1), the condition of microwave irradiation drying are as follows: the processing of 300W microwave intermitant irradiation, time per treatment
For 5s, continue after being spaced 30s, cumulative processing time is 3 minutes.
In step (2), the process conditions of stirring are: being stirred 3 hours with 500 revs/min of revolving speed.
High-pressure homogeneous using high pressure Microfluidizer in step (2), process conditions are: pressure 100MPa, temperature 45
DEG C, the time 5 minutes.
In step (3), the chitosan-acetic acid solution the preparation method is as follows: by molecular weight 300,000, deacetylation >=
90% 1g Chitosan powder is added in the aqueous acetic acid of mass concentration 1%, is heated to 40 DEG C, and stirring is to being completely dissolved i.e.
?.
In step (3), the process conditions of centrifugation are as follows: 5000 revs/min of revolving speed, centrifugation time 5 minutes.
In step (4), the process conditions of centrifugation are as follows: 8000 revs/min of revolving speed, centrifugation time 5 minutes.
In step (5), the recovery method of trifluoroacetic acid is that vacuum recycles at 40 DEG C.
In step (6), the process conditions of centrifugation are as follows: 15000 revs/min of revolving speed, centrifugation time 2 minutes.
In step (6), the process conditions of freeze-drying are as follows: -10 DEG C of precoolings are solid-state, and it is dry to be then transferred to vacuum refrigeration
In dry machine, temperature is down to -40 DEG C, is evacuated to 15Pa, and then temperature rises to 20 DEG C, continues under current vacuum degree condition
Reason 10 hours.
The small molecule fucoidin obtained using said extracted technique, average molecular weight 270Da.
Embodiment 2
The extraction process of small molecule fucoidin, the specific steps are as follows:
(1) kelp is cleaned, microwave irradiation drying, crushes, and obtains kelp powder；
(2) kelp powder is added in the distilled water of 15 times of weight, stirring is high-pressure homogeneous, obtains kelp broken wall slurries；
(3) chitosan-acetic acid solution is added into kelp broken wall slurries, the two volume ratio is 8:1, stirs 15 minutes, is added
The sodium perborate of 0.1 times of kelp powder weight, continues stirring 30 minutes, and precipitating is collected in centrifugation；
(4) precipitating obtained by step (3) is added in the distilled water of 8 times of weight while stirring, supersonic oscillations 30 minutes, is obtained
Dispersion liquid；
(5) lysozyme of 0.03 times of kelp powder weight is added in gained dispersion liquid into step (4), 45 DEG C of hydrolysis 5 are small
When, it is subsequently added into the trifluoroacetic acid of 0.02 times of kelp powder weight, is warming up to 100 DEG C, hydrolyzes 30 minutes and goes out under confined conditions
Enzyme, vacuum recycle trifluoroacetic acid, obtain extracting solution；
(6) extracting solution obtained by step (5) is freezed in -50 DEG C, after freezing completely, is slowly melted under the conditions of room temperature (25 DEG C), 4
DEG C centrifugation, takes supernatant, repeated freezing, thawing and centrifugal process, until the freezing of gained supernatant is done until generating without precipitating
It is dry, obtain small molecule fucoidin.
Wherein, in step (1), the condition of microwave irradiation drying are as follows: the processing of 450W microwave intermitant irradiation, time per treatment
For 10s, continue after being spaced 30s, cumulative processing time is 4 minutes.
In step (2), the process conditions of stirring are: being stirred 4 hours with 800 revs/min of revolving speed.
High-pressure homogeneous using high pressure Microfluidizer in step (2), process conditions are: pressure 120MPa, temperature 48
DEG C, the time 8 minutes.
In step (3), the chitosan-acetic acid solution the preparation method is as follows: by molecular weight 400,000, deacetylation >=
90% 2g Chitosan powder is added in the aqueous acetic acid of mass concentration 1%, is heated to 40 DEG C, and stirring is to being completely dissolved i.e.
?.
In step (3), the process conditions of centrifugation are as follows: 7000 revs/min of revolving speed, centrifugation time 7 minutes.
In step (4), the process conditions of centrifugation are as follows: 10000 revs/min of revolving speed, centrifugation time 7 minutes.
In step (5), the recovery method of trifluoroacetic acid is that vacuum recycles at 60 DEG C.
In step (6), the process conditions of centrifugation are as follows: 20000 revs/min of revolving speed, centrifugation time 3 minutes.
In step (6), the process conditions of freeze-drying are as follows: -15 DEG C of precoolings are solid-state, and it is dry to be then transferred to vacuum refrigeration
In dry machine, temperature is down to -50 DEG C, is evacuated to 20Pa, and then temperature rises to 30 DEG C, continues under current vacuum degree condition
Reason 15 hours.
The small molecule fucoidin obtained using said extracted technique, average molecular weight 280Da.
Embodiment 3
The extraction process of small molecule fucoidin, the specific steps are as follows:
(1) kelp is cleaned, microwave irradiation drying, crushes, and obtains kelp powder；
(2) kelp powder is added in the distilled water of 10 times of weight, stirring is high-pressure homogeneous, obtains kelp broken wall slurries；
(3) chitosan-acetic acid solution is added into kelp broken wall slurries, the two volume ratio is 8:1, stirs 10 minutes, is added
The sodium perborate of 0.1 times of kelp powder weight, continues stirring 20 minutes, and precipitating is collected in centrifugation；
(4) precipitating obtained by step (3) is added in the distilled water of 8 times of weight while stirring, supersonic oscillations 30 minutes, is obtained
Dispersion liquid；
(5) lysozyme of 0.02 times of kelp powder weight is added in gained dispersion liquid into step (4), 45 DEG C of hydrolysis 3 are small
When, it is subsequently added into the trifluoroacetic acid of 0.02 times of kelp powder weight, is warming up to 90 DEG C, hydrolyzes 30 minutes and goes out under confined conditions
Enzyme, vacuum recycle trifluoroacetic acid, obtain extracting solution；
(6) extracting solution obtained by step (5) is freezed in -40 DEG C, after freezing completely, is slowly melted under the conditions of room temperature (25 DEG C), 4
DEG C centrifugation, takes supernatant, repeated freezing, thawing and centrifugal process, until the freezing of gained supernatant is done until generating without precipitating
It is dry, obtain small molecule fucoidin.
Wherein, in step (1), the condition of microwave irradiation drying are as follows: the processing of 300W microwave intermitant irradiation, time per treatment
For 10s, continue after being spaced 30s, cumulative processing time is 3 minutes.
In step (2), the process conditions of stirring are: being stirred 3 hours with 800 revs/min of revolving speed.
High-pressure homogeneous using high pressure Microfluidizer in step (2), process conditions are: pressure 120MPa, temperature 45
DEG C, the time 8 minutes.
In step (3), the chitosan-acetic acid solution the preparation method is as follows: by molecular weight 300,000, deacetylation >=
90% 2g Chitosan powder is added in the aqueous acetic acid of mass concentration 1%, is heated to 40 DEG C, and stirring is to being completely dissolved i.e.
?.
In step (3), the process conditions of centrifugation are as follows: 5000 revs/min of revolving speed, centrifugation time 7 minutes.
In step (4), the process conditions of centrifugation are as follows: 8000 revs/min of revolving speed, centrifugation time 7 minutes.
In step (5), the recovery method of trifluoroacetic acid is that vacuum recycles at 40 DEG C.
In step (6), the process conditions of centrifugation are as follows: 20000 revs/min of revolving speed, centrifugation time 2 minutes.
In step (6), the process conditions of freeze-drying are as follows: -15 DEG C of precoolings are solid-state, and it is dry to be then transferred to vacuum refrigeration
In dry machine, temperature is down to -40 DEG C, is evacuated to 20Pa, and then temperature rises to 20 DEG C, continues under current vacuum degree condition
Reason 15 hours.
The small molecule fucoidin obtained using said extracted technique, average molecular weight 250Da.
Embodiment 4
The extraction process of small molecule fucoidin, the specific steps are as follows:
(1) kelp is cleaned, microwave irradiation drying, crushes, and obtains kelp powder；
(2) kelp powder is added in the distilled water of 15 times of weight, stirring is high-pressure homogeneous, obtains kelp broken wall slurries；
(3) chitosan-acetic acid solution is added into kelp broken wall slurries, the two volume ratio is 6:1, stirs 15 minutes, is added
The sodium perborate of 0.08 times of kelp powder weight, continues stirring 30 minutes, and precipitating is collected in centrifugation；
(4) precipitating obtained by step (3) is added in the distilled water of 6 times of weight while stirring, supersonic oscillations 30 minutes, is obtained
Dispersion liquid；
(5) lysozyme of 0.03 times of kelp powder weight is added in gained dispersion liquid into step (4), 35 DEG C of hydrolysis 5 are small
When, it is subsequently added into the trifluoroacetic acid of 0.01 times of kelp powder weight, is warming up to 100 DEG C, hydrolyzes 20 minutes and goes out under confined conditions
Enzyme, vacuum recycle trifluoroacetic acid, obtain extracting solution；
(6) extracting solution obtained by step (5) is freezed in -50 DEG C, after freezing completely, is slowly melted under the conditions of room temperature (25 DEG C), 0
DEG C centrifugation, takes supernatant, repeated freezing, thawing and centrifugal process, until the freezing of gained supernatant is done until generating without precipitating
It is dry, obtain small molecule fucoidin.
Wherein, in step (1), the condition of microwave irradiation drying are as follows: the processing of 450W microwave intermitant irradiation, time per treatment
For 5s, continue after being spaced 30s, cumulative processing time is 4 minutes.
In step (2), the process conditions of stirring are: being stirred 4 hours with 500 revs/min of revolving speed.
High-pressure homogeneous using high pressure Microfluidizer in step (2), process conditions are: pressure 100MPa, temperature 48
DEG C, the time 5 minutes.
In step (3), the chitosan-acetic acid solution the preparation method is as follows: by molecular weight 400,000, deacetylation >=
90% 1g Chitosan powder is added in the aqueous acetic acid of mass concentration 1%, is heated to 40 DEG C, and stirring is to being completely dissolved i.e.
?.
In step (3), the process conditions of centrifugation are as follows: 7000 revs/min of revolving speed, centrifugation time 5 minutes.
In step (4), the process conditions of centrifugation are as follows: 10000 revs/min of revolving speed, centrifugation time 5 minutes.
In step (5), the recovery method of trifluoroacetic acid is that vacuum recycles at 60 DEG C.
In step (6), the process conditions of centrifugation are as follows: 15000 revs/min of revolving speed, centrifugation time 3 minutes.
In step (6), the process conditions of freeze-drying are as follows: -10 DEG C of precoolings are solid-state, and it is dry to be then transferred to vacuum refrigeration
In dry machine, temperature is down to -50 DEG C, is evacuated to 15Pa, and then temperature rises to 30 DEG C, continues under current vacuum degree condition
Reason 10 hours.
The small molecule fucoidin obtained using said extracted technique, average molecular weight 250Da.
Embodiment 5
The extraction process of small molecule fucoidin, the specific steps are as follows:
(1) kelp is cleaned, microwave irradiation drying, crushes, and obtains kelp powder；
(2) kelp powder is added in the distilled water of 12 times of weight, stirring is high-pressure homogeneous, obtains kelp broken wall slurries；
(3) chitosan-acetic acid solution is added into kelp broken wall slurries, the two volume ratio is 7:1, stirs 12 minutes, is added
The sodium perborate of 0.09 times of kelp powder weight, continues stirring 25 minutes, and precipitating is collected in centrifugation；
(4) precipitating obtained by step (3) is added in the distilled water of 7 times of weight while stirring, supersonic oscillations 30 minutes, is obtained
Dispersion liquid；
(5) lysozyme of 0.025 times of kelp powder weight is added in gained dispersion liquid into step (4), 40 DEG C of hydrolysis 4 are small
When, it is subsequently added into the trifluoroacetic acid of 0.015 times of kelp powder weight, is warming up to 95 DEG C, hydrolyzes 25 minutes and goes out under confined conditions
Enzyme, vacuum recycle trifluoroacetic acid, obtain extracting solution；
(6) extracting solution obtained by step (5) is freezed in -45 DEG C, after freezing completely, is slowly melted under the conditions of room temperature (25 DEG C), 2
DEG C centrifugation, takes supernatant, repeated freezing, thawing and centrifugal process, until the freezing of gained supernatant is done until generating without precipitating
It is dry, obtain small molecule fucoidin.
Wherein, in step (1), the condition of microwave irradiation drying are as follows: the processing of 300W microwave intermitant irradiation, time per treatment
For 8s, continue after being spaced 30s, cumulative processing time is 4 minutes.
In step (2), the process conditions of stirring are: being stirred 3 hours with 600 revs/min of revolving speed.
High-pressure homogeneous using high pressure Microfluidizer in step (2), process conditions are: pressure 110MPa, temperature 46
DEG C, the time 7 minutes.
In step (3), the chitosan-acetic acid solution the preparation method is as follows: by molecular weight 350,000, deacetylation >=
90% 1.5g Chitosan powder is added in the aqueous acetic acid of mass concentration 1%, is heated to 40 DEG C, and stirring is to being completely dissolved
To obtain the final product.
In step (3), the process conditions of centrifugation are as follows: 6000 revs/min of revolving speed, centrifugation time 6 minutes.
In step (4), the process conditions of centrifugation are as follows: 9000 revs/min of revolving speed, centrifugation time 6 minutes.
In step (5), the recovery method of trifluoroacetic acid is that vacuum recycles at 50 DEG C.
In step (6), the process conditions of centrifugation are as follows: 18000 revs/min of revolving speed, centrifugation time 3 minutes.
In step (6), the process conditions of freeze-drying are as follows: -12 DEG C of precoolings are solid-state, and it is dry to be then transferred to vacuum refrigeration
In dry machine, temperature is down to -45 DEG C, is evacuated to 18Pa, and then temperature rises to 25 DEG C, continues under current vacuum degree condition
Reason 12 hours.
The small molecule fucoidin obtained using said extracted technique, average molecular weight 220Da.
Comparative example 1
A kind of extraction process of fucoidin, the specific steps are as follows:
(1) kelp is cleaned, microwave irradiation drying, crushes, and obtains kelp powder；
(2) kelp powder is added in the distilled water of 12 times of weight, stirring is high-pressure homogeneous, obtains kelp broken wall slurries；
(3) chitosan-acetic acid solution is added into kelp broken wall slurries, the two volume ratio is 7:1, it stirs 37 minutes, from
The heart collects precipitating；
(4) precipitating obtained by step (3) is added in the distilled water of 7 times of weight while stirring, supersonic oscillations 30 minutes, is obtained
Dispersion liquid；
(5) lysozyme of 0.025 times of kelp powder weight is added in gained dispersion liquid into step (4), 40 DEG C of hydrolysis 4 are small
When, it is subsequently added into the trifluoroacetic acid of 0.015 times of kelp powder weight, is warming up to 95 DEG C, hydrolyzes 25 minutes and goes out under confined conditions
Enzyme, vacuum recycle trifluoroacetic acid, obtain extracting solution；
(6) extracting solution obtained by step (5) is freezed in -45 DEG C, after freezing completely, is slowly melted under the conditions of room temperature (25 DEG C), 2
DEG C centrifugation, takes supernatant, repeated freezing, thawing and centrifugal process, until the freezing of gained supernatant is done until generating without precipitating
It is dry, obtain small molecule fucoidin.
Wherein, in step (1), the condition of microwave irradiation drying are as follows: the processing of 300W microwave intermitant irradiation, time per treatment
For 8s, continue after being spaced 30s, cumulative processing time is 4 minutes.
In step (2), the process conditions of stirring are: being stirred 3 hours with 600 revs/min of revolving speed.
High-pressure homogeneous using high pressure Microfluidizer in step (2), process conditions are: pressure 110MPa, temperature 46
DEG C, the time 7 minutes.
In step (3), the chitosan-acetic acid solution the preparation method is as follows: by molecular weight 350,000, deacetylation >=
90% 1.5g Chitosan powder is added in the aqueous acetic acid of mass concentration 1%, is heated to 40 DEG C, and stirring is to being completely dissolved
To obtain the final product.
In step (3), the process conditions of centrifugation are as follows: 6000 revs/min of revolving speed, centrifugation time 6 minutes.
In step (4), the process conditions of centrifugation are as follows: 9000 revs/min of revolving speed, centrifugation time 6 minutes.
In step (5), the recovery method of trifluoroacetic acid is that vacuum recycles at 50 DEG C.
In step (6), the process conditions of centrifugation are as follows: 18000 revs/min of revolving speed, centrifugation time 3 minutes.
In step (6), the process conditions of freeze-drying are as follows: -12 DEG C of precoolings are solid-state, and it is dry to be then transferred to vacuum refrigeration
In dry machine, temperature is down to -45 DEG C, is evacuated to 18Pa, and then temperature rises to 25 DEG C, continues under current vacuum degree condition
Reason 12 hours.
A kind of fucoidin obtained using said extracted technique, average molecular weight 2100Da.
Comparative example 2
A kind of extraction process of fucoidin, the specific steps are as follows:
(1) kelp is cleaned, microwave irradiation drying, crushes, and obtains kelp powder；
(2) kelp powder is added in the distilled water of 12 times of weight, stirring is high-pressure homogeneous, obtains kelp broken wall slurries；
(3) chitosan-acetic acid solution is added into kelp broken wall slurries, the two volume ratio is 7:1, stirs 12 minutes, is added
The sodium perborate of 0.09 times of kelp powder weight, continues stirring 25 minutes, and precipitating is collected in centrifugation；
(4) precipitating obtained by step (3) is added in the distilled water of 7 times of weight while stirring, supersonic oscillations 30 minutes, is obtained
Dispersion liquid；
(5) lysozyme of 0.025 times of kelp powder weight is added in gained dispersion liquid into step (4), 40 DEG C of hydrolysis 4 are small
When, 95 DEG C are warming up to, enzyme deactivation obtains extracting solution；
(6) extracting solution obtained by step (5) is freezed in -45 DEG C, after freezing completely, is slowly melted under the conditions of room temperature (25 DEG C), 2
DEG C centrifugation, takes supernatant, repeated freezing, thawing and centrifugal process, until the freezing of gained supernatant is done until generating without precipitating
It is dry, obtain small molecule fucoidin.
Wherein, in step (1), the condition of microwave irradiation drying are as follows: the processing of 300W microwave intermitant irradiation, time per treatment
For 8s, continue after being spaced 30s, cumulative processing time is 4 minutes.
In step (2), the process conditions of stirring are: being stirred 3 hours with 600 revs/min of revolving speed.
High-pressure homogeneous using high pressure Microfluidizer in step (2), process conditions are: pressure 110MPa, temperature 46
DEG C, the time 7 minutes.
In step (3), the chitosan-acetic acid solution the preparation method is as follows: by molecular weight 350,000, deacetylation >=
90% 1.5g Chitosan powder is added in the aqueous acetic acid of mass concentration 1%, is heated to 40 DEG C, and stirring is to being completely dissolved
To obtain the final product.
In step (3), the process conditions of centrifugation are as follows: 6000 revs/min of revolving speed, centrifugation time 6 minutes.
In step (4), the process conditions of centrifugation are as follows: 9000 revs/min of revolving speed, centrifugation time 6 minutes.
In step (6), the process conditions of centrifugation are as follows: 18000 revs/min of revolving speed, centrifugation time 3 minutes.
In step (6), the process conditions of freeze-drying are as follows: -12 DEG C of precoolings are solid-state, and it is dry to be then transferred to vacuum refrigeration
In dry machine, temperature is down to -45 DEG C, is evacuated to 18Pa, and then temperature rises to 25 DEG C, continues under current vacuum degree condition
Reason 12 hours.
A kind of fucoidin obtained using said extracted technique, average molecular weight 3200Da.
Comparative example 3
A kind of extraction process of fucoidin, the specific steps are as follows:
(1) kelp is cleaned, microwave irradiation drying, crushes, and obtains kelp powder；
(2) kelp powder is added in the distilled water of 12 times of weight, stirring is high-pressure homogeneous, obtains kelp broken wall slurries；
(3) chitosan-acetic acid solution is added into kelp broken wall slurries, the two volume ratio is 7:1, stirs 12 minutes, is added
The sodium perborate of 0.09 times of kelp powder weight, continues stirring 25 minutes, and precipitating is collected in centrifugation；
(4) precipitating obtained by step (3) is added in the distilled water of 7 times of weight while stirring, supersonic oscillations 30 minutes, is obtained
Dispersion liquid；
(5) lysozyme of 0.025 times of kelp powder weight is added in gained dispersion liquid into step (4), 40 DEG C of hydrolysis 4 are small
When, it is subsequently added into the trifluoroacetic acid of 0.015 times of kelp powder weight, is warming up to 95 DEG C, hydrolyzes 25 minutes and goes out under confined conditions
Enzyme, vacuum recycle trifluoroacetic acid, obtain extracting solution；
(6) it is freeze-dried, obtains small molecule fucoidin.
Wherein, in step (1), the condition of microwave irradiation drying are as follows: the processing of 300W microwave intermitant irradiation, time per treatment
For 8s, continue after being spaced 30s, cumulative processing time is 4 minutes.
In step (2), the process conditions of stirring are: being stirred 3 hours with 600 revs/min of revolving speed.
High-pressure homogeneous using high pressure Microfluidizer in step (2), process conditions are: pressure 110MPa, temperature 46
DEG C, the time 7 minutes.
In step (3), the chitosan-acetic acid solution the preparation method is as follows: by molecular weight 350,000, deacetylation >=
90% 1.5g Chitosan powder is added in the aqueous acetic acid of mass concentration 1%, is heated to 40 DEG C, and stirring is to being completely dissolved
To obtain the final product.
In step (3), the process conditions of centrifugation are as follows: 6000 revs/min of revolving speed, centrifugation time 6 minutes.
In step (4), the process conditions of centrifugation are as follows: 9000 revs/min of revolving speed, centrifugation time 6 minutes.
In step (5), the recovery method of trifluoroacetic acid is that vacuum recycles at 50 DEG C.
In step (6), the process conditions of freeze-drying are as follows: -12 DEG C of precoolings are solid-state, and it is dry to be then transferred to vacuum refrigeration
In dry machine, temperature is down to -45 DEG C, is evacuated to 18Pa, and then temperature rises to 25 DEG C, continues under current vacuum degree condition
Reason 12 hours.
A kind of fucoidin obtained using said extracted technique, average molecular weight 220Da.
Test example
The product yield (being calculated with the weight of raw material kelp) of Statistics Implementation example 1~5 and comparative example 1~3, purity and stop
Angle the results are shown in Table 1.
1. extraction effect of table compares
| Yield (%) | Purity (%) | Angle of repose (°) | |
| Embodiment 1 | 1.82 | 99.92 | 27 |
| Embodiment 2 | 1.81 | 99.93 | 27 |
| Embodiment 3 | 1.85 | 99.95 | 27 |
| Embodiment 4 | 1.85 | 99.95 | 27 |
| Embodiment 5 | 1.90 | 99.98 | 27 |
| Comparative example 1 | 1.11 | 99.81 | 29 |
| Comparative example 2 | 1.45 | 99.82 | 29 |
| Comparative example 3 | 1.82 | 90.12 | 37 |
As it can be seen from table 1 the extraction process high income of Examples 1 to 5, purity is high, angle of repose is small, has good stream
Dynamic property.Comparative example 1 omits sodium perborate, and comparative example 2 omits trifluoroacetic acid, and yield is substantially reduced, and the molecule of product
It measures also larger；Last multigelation step is replaced with freeze-drying by comparative example 3, purity is substantially reduced, and product stops
Angle is big, and mobility is not good enough.
Examples 1 to 5 and 1~3 gained fucoidin of comparative example respectively take 0.1g, are added into 100mL distilled water, ultrasound
Wave vibrates 30 minutes, and as a control group with distilled water, (skin of face was contained using 24 hours moisture for progress inoxidizability and moisture retention
Amount detection) detection, it the results are shown in Table 2.
2. inoxidizability of table and moisture retention testing result
As known from Table 2, Examples 1 to 5 and 1~3 gained fucoidin of comparative example have good inoxidizability and moisturizing
Property, there is anti-oxidant or moisturizing cosmetic application prospect, far superior to comparative example 1~3.
It is obvious to a person skilled in the art that invention is not limited to the details of the above exemplary embodiments, Er Qie
In the case where without departing substantially from spirit or essential attributes of the invention, the present invention can be realized in other specific forms.Therefore, no matter
From the point of view of which point, the present embodiments are to be considered as illustrative and not restrictive, and the scope of the present invention is by appended power
Benefit requires rather than above description limits, it is intended that all by what is fallen within the meaning and scope of the equivalent elements of the claims
Variation is included within the present invention.
In addition, it should be understood that although this specification is described in terms of embodiments, but not each embodiment is only wrapped
Containing an independent technical solution, this description of the specification is merely for the sake of clarity, and those skilled in the art should
It considers the specification as a whole, the technical solutions in the various embodiments may also be suitably combined, forms those skilled in the art
The other embodiments being understood that.
Claims (10)
1. the extraction process of small molecule fucoidin, which is characterized in that specific step is as follows:
(1) kelp is cleaned, microwave irradiation drying, crushes, and obtains kelp powder；
(2) kelp powder is added in the distilled water of 10~15 times of weight, stirring is high-pressure homogeneous, obtains kelp broken wall slurries；
(3) chitosan-acetic acid solution is added into kelp broken wall slurries, the two volume ratio is 6~8:1, it stirs 10~15 minutes,
The sodium perborate of 0.08~0.1 times of kelp powder weight is added, continues stirring 20~30 minutes, is centrifuged, collects precipitating；
(4) precipitating obtained by step (3) is added in the distilled water of 6~8 times of weight while stirring, supersonic oscillations 30 minutes, is obtained
Dispersion liquid；
(5) lysozyme of 0.02~0.03 times of kelp powder weight, 35~45 DEG C of water are added in gained dispersion liquid into step (4)
Solution 3~5 hours, is subsequently added into the trifluoroacetic acid of 0.01~0.02 times of kelp powder weight, is warming up to 90~100 DEG C, closed item
20~30 minutes and enzyme deactivation are hydrolyzed under part, vacuum recycles trifluoroacetic acid, obtains extracting solution；
(6) extracting solution obtained by step (5) is freezed in -40~-50 DEG C, after freezing completely, is slowly melted under room temperature, 0~4 DEG C
Centrifugation takes supernatant, and repeated freezing, thawing and centrifugal process, until generating without precipitating, gained supernatant is freeze-dried,
Obtain small molecule fucoidin.
2. extraction process according to claim 1, which is characterized in that in step (1), the condition of microwave irradiation drying are as follows:
The processing of 300~450W microwave intermitant irradiation, time per treatment are 5~10s, are continued after being spaced 30s, cumulative processing time 3
~4 minutes.
3. extraction process according to claim 1, which is characterized in that high using high pressure Microfluidizer in step (2)
Homogeneous is pressed, process conditions are: 100~120MPa of pressure, 45~48 DEG C of temperature, the time 5~8 minutes.
4. extraction process according to claim 1, which is characterized in that in step (3), the system of the chitosan-acetic acid solution
Preparation Method is as follows: 1~2g Chitosan powder of molecular weight 30~400,000, deacetylation >=90% is added to mass concentration 1%
Aqueous acetic acid in, be heated to 40 DEG C, stirring is to being completely dissolved to obtain the final product.
5. extraction process according to claim 1, which is characterized in that in step (3), the process conditions of centrifugation are as follows: revolving speed
5000~7000 revs/min, centrifugation time 5~7 minutes.
6. extraction process according to claim 1, which is characterized in that in step (5), the recovery method of trifluoroacetic acid is 40
Vacuum recycles at~60 DEG C.
7. extraction process according to claim 1, which is characterized in that in step (6), the process conditions of centrifugation are as follows: revolving speed
15000~20000 revs/min, centrifugation time 2~3 minutes.
8. extraction process according to claim 1, which is characterized in that in step (6), the process conditions of freeze-drying are as follows:-
10~-15 DEG C of precoolings are solid-state, are then transferred in vacuum freeze drier, temperature is down to -40~-50 DEG C, is evacuated to
15~20Pa, then temperature rises to 20~30 DEG C, and continuation is handled 10~15 hours under current vacuum degree condition.
9. the small molecule fucoidin obtained using extraction process described in any one of claim 1~8, which is characterized in that its
Molecular weight is about 200~300Da.
10. application of the small molecule fucoidin in cosmetics described in claim 9.
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