CN103087214B - The preparation method of low-molecular-weightsea sea cucumber fucosan sulfate - Google Patents
The preparation method of low-molecular-weightsea sea cucumber fucosan sulfate Download PDFInfo
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Abstract
The invention discloses a kind of preparation method of low-molecular-weightsea sea cucumber fucosan sulfate, using the sea cucumber polysaccharide that will enzymolysis process utilized to obtain after sea cucumber pulverizing as raw material, comprise the following steps: 1), by sea cucumber polysaccharide be dissolved in sodium chloride solution, then ethanol is added, centrifugal removing precipitation, obtains supernatant liquor; In supernatant liquor, add ethanol, centrifugal, the precipitation of gained, after dialysis and lyophilize, obtains sea cucumber fucoidan; 2), by sea cucumber fucoidan be dissolved in solvent and carry out supersound process at the temperature of 0 ~ 45 DEG C, the treatment time is 10 ~ 180min, and ultrasonic sound field intensity is 60 ~ 800W/cm
2; 3), by step 2) solution dialysis after the process of gained, after vacuum lyophilization, obtain low-molecular-weightsea sea cucumber fucosan sulfate.The present invention utilizes sonication techniques, can degraded macromolecular amount sea cucumber fucoidan rapidly, and reaction conditions is gentle, environmental protection.<!--1-->
Description
Technical field
The present invention relates to the preparation method of low-molecular-weightsea sea cucumber fucosan sulfate, specifically, relate to a kind of method utilizing ultrasonic degradation to prepare low-molecular-weight sea cucumber fucoidan.
Background technology
Sea cucumber (seacucumber, holothurians) Echinodermata (Echinodermata) Holothuroidea (Holothuroidea) Parapet hand order (Aspidocita) animal is belonged to, mainly be distributed in the torrid zone and ocean, temperate zone, the sea cucumber of China's record has 101 kinds, and wherein more than 20 kinds can be edible.Sea cucumber is exactly the excellent tonic product of famous and precious medicine-food two-purpose since ancient times, and rank first of " sea ", Traditional Chinese Medicine is thought, sea cucumber taste is salty, warm in nature, enters the heart, kidney channel, has tonifying kidney and benefiting sperm, enriching blood and moistening for dryness, hemostasis and anti-inflammation, and stomach such as to quench the thirst at the effect.Research in recent years thinks that sea cucumber is a kind of high protein, lower fat, low sugar, not containing the heath food of cholesterol, and be rich in the compositions such as the various sea cucumber polysaccharide useful to human body, amino acid, indispensable fatty acid, VITAMIN, mineral substance, colloid, selenka, make sea cucumber have antitumor, anticoagulation, raising immunizing power, reducing blood-fat, the effect such as control rheumatoid arthritis and osteoarthritis.
Sea cucumber polysaccharide is mainly present in wall of sea cucumber Stichopus japonicus and discarded internal organ thereof, accounts for the 7-10% of wall of sea cucumber Stichopus japonicus, is mainly divided into two classes: sea cucumber fucoidan and fucosylation chondroitin sulfate.Sea cucumber fucoidan is the straight-chain polysaccharide be made up of L-fucose, early-stage Study shows, be separated from a kind of U.S. meat ginseng and obtain fucoidan, it is the repeating unit formation of [→ 3Fuc (2S, 4S) α 1 → 3Fuc (2S) α 1 → 3Fuc (2S) α 1 → 3Fuc α 1 →] n through resolving its structure.Sea cucumber fucoidan has multiple biological activity, comprises anti-oxidant, anticoagulation, Tumor suppression, reducing blood-fat, raising immunizing power, anti-inflammatory, stomach protection, antivirus action etc.
Sea cucumber fucoidan has many activity, but due to its molecular weight comparatively large, be not easily absorbed by the body utilizations, thus studying and be restricted in utilizing.Low-molecular-weightsea sea cucumber fucosan sulfate, owing to having the advantages such as low, the easy absorption of viscosity, bioavailability be good, shows biological activity more better than macromolecular polysaccharide.The exploitation in fields such as healthcare products, medicine, foodstuff additive of the degraded of polysaccharide and low molecular weight product thereof and application are very extensive, and degradation method conventional at present mainly contains chemical degradation method, biological degradation method and physical degradation methods.The method of the degraded fucoidan of current bibliographical information concentrates on chemical degradation method mostly, although chemical method is swift in response, reaction is violent, destroy comparatively large, and product molecular weight distribution is wide to polysaccharide structures.Although biological degradation method reaction temperature and, specificity is high, and can be applicable to polysaccharide, the especially single-minded enzyme for fucoidan degraded is very rare, and reaction conditions is comparatively harsh, and cost is high, and industrialized production has certain difficulty.Physical degradation methods is due to little to its lytic activity structure deteriorate, molecular weight is easy to control, reaction efficiency is high and become a kind of desirable degradation method, polysaccharose substance commonly uses physical degradation methods ultrasonic degradation, irradiation-induced degradation, microwave degradation etc., wherein irradiation-induced degradation is most effective, the research of konjak glucomannan is shown, after 5.0kGy and 100.0kGy irradiation, molecular weight of product is down to 370kDa and 39.8kDa respectively by 481kDa, but because radiation treatment is destroyed very violent to molecular structure, and security is still disputable, so its application is restricted.
Wu Xiaolin is studied the characteristic of digesting and assimilating of different molecular weight sea cucumber fucoidan to the comparison of Rats adiposis hepatica improvement result and different molecular weight sea cucumber fucoidan in " molecular weight active to sea cucumber fucoidan and the influence research digested and assimilated " and " preparation of different molecular weight sea cucumber fucoidan and digest and assimilate the preliminary study of characteristic " (Chinese Sea medicine the 30 volume 3 phase in 2011,20-24).
Summary of the invention
The technical problem to be solved in the present invention is to provide a kind of preparation method of low-molecular-weightsea sea cucumber fucosan sulfate, the present invention utilizes sonication techniques, can degraded macromolecular amount sea cucumber fucoidan rapidly, and reaction conditions is gentle, simple to operate, environmental protection.
In order to solve the problems of the technologies described above, the invention provides a kind of preparation method of low-molecular-weightsea sea cucumber fucosan sulfate, using the sea cucumber polysaccharide that will enzymolysis process utilized to obtain after sea cucumber pulverizing as raw material, comprise the following steps:
1), by sea cucumber polysaccharide be dissolved in 2.8 ~ 3.2mol/L(and be preferably 3mol/L) sodium chloride solution (for strong brine) in, obtain sea cucumber polysaccharide solution, in sea cucumber polysaccharide solution, the mass concentration of sea cucumber polysaccharide is that 0.8 ~ 1.2%(is preferably 1%);
In sea cucumber polysaccharide solution, add ethanol, until the volume final concentration of ethanol is 34 ~ 40%, centrifugal removing precipitation, obtains supernatant liquor;
In supernatant liquor, add ethanol, until the volume final concentration of ethanol is 60 ~ 90%, centrifugal, the precipitation of gained, after dialysis and lyophilize, obtains sea cucumber fucoidan;
2), by sea cucumber fucoidan be dissolved in solvent and carry out supersound process at the temperature of 0 ~ 45 DEG C (being preferably 5 ~ 35 DEG C), the treatment time is that 10 ~ 180min(is preferably 10 ~ 80min), ultrasonic sound field intensity is 60 ~ 800W/cm
2(be preferably 180 ~ 550W/cm
2); Ultrasonic frequency is 21 ~ 25kHz;
3), by step 2) solution dialysis after the process of gained, after vacuum lyophilization, obtain low-molecular-weightsea sea cucumber fucosan sulfate.
Improvement as the preparation method of low-molecular-weightsea sea cucumber fucosan sulfate of the present invention: the molecular weight cut-off of the dialysis tubing that the dialysis in step 1) is used is 8000kDa.
Further improvement as the preparation method of low-molecular-weightsea sea cucumber fucosan sulfate of the present invention: step 2) solvent is deionized water, mass concentration is 0.5 ~ 5% acetum, mass concentration be 0.5 ~ 5% hydrochloric acid soln or mass concentration be the sulphuric acid soln of 0.5 ~ 5%.
Further improvement as the preparation method of low-molecular-weightsea sea cucumber fucosan sulfate of the present invention: step 2) in the solid-liquid ratio of sea cucumber fucoidan and solvent be 1g/20-200ml.I.e. 1g sea cucumber fucoidan adapted 20-200ml solution.Preferably solid-liquid ratio is 1g/50-70ml.
Further improvement as the preparation method of low-molecular-weightsea sea cucumber fucosan sulfate of the present invention: the molecular weight cut-off of the dialysis tubing that the dialysis in step 3) is used is 1000kDa.
Further improvement as the preparation method of low-molecular-weightsea sea cucumber fucosan sulfate of the present invention:
Step 2) be: be dissolved in 30ml deionized water by 0.5g sea cucumber fucoidan and carry out supersound process at the temperature of 13.0713 DEG C, the treatment time is 80min, and ultrasonic sound field intensity is 508.28W/cm
2.
Further improvement as the preparation method of low-molecular-weightsea sea cucumber fucosan sulfate of the present invention:
Step 2) be: mass concentration 0.5g sea cucumber fucoidan being dissolved in 30ml is the acetic acid of 2%, the mass concentration of 30ml be the hydrochloric acid of 2% or the mass concentration of 30ml is at the temperature of 13.0713 DEG C, carry out supersound process in the sulfuric acid of 2%, treatment time is 40min, and ultrasonic sound field intensity is 508.28W/cm
2.
The technical solution adopted in the present invention is that (this is for known technology with the sea cucumber polysaccharide that will enzymolysis process utilized to obtain after sea cucumber pulverizing, such as can be prepared according in containing the enzymolysis process delivered in gentle and quiet Master's thesis " different sea cucumber polysaccharide extraction and isolation and chemical composition analysis compare ") obtain sea cucumber fucoidan as raw material, break glycosidic link by supersound process and obtain low-molecular-weightsea sea cucumber fucosan sulfate.
Compared with prior art, outstanding advantages of the present invention is:
1, use physical method to break glycosidic link, degraded sea cucumber fucoidan, destroys little to product structure, without side reaction, and noresidue material, molecular weight is easy to control;
2, preparation process is simple, and with low cost, reaction efficiency is high, pollution-free;
3, the destruction in the past made to product structure in chemically degradation of polysaccharide process is overcome;
4, conventionally known: gained low-molecular-weightsea sea cucumber fucosan sulfate of the present invention has good Scavenging ability and anticoagulant active, and be easily absorbed by the body, can be applicable to the preparation of antithrombotic, anticoagulation, Tumor suppression, the healthcare products of strengthening immunity, functional food or medicine, there is high society, economic implications and application potential.
Embodiment
The preparation method of embodiment 1-1, a kind of low-molecular-weightsea sea cucumber fucosan sulfate, using the sea cucumber polysaccharide that will enzymolysis process utilized to obtain after sea cucumber pulverizing as raw material, carries out following steps successively:
1), by sea cucumber polysaccharide be dissolved in the sodium chloride solution (strong brine) of 3mol/L, obtain sea cucumber polysaccharide solution, in sea cucumber polysaccharide solution, the mass concentration of sea cucumber polysaccharide is 1%;
In sea cucumber polysaccharide solution, add ethanol, until the volume final concentration of ethanol is 37%, centrifugal (6000r/min, 15min) removes precipitation, obtains supernatant liquor;
Ethanol is added in described supernatant liquor, until the volume final concentration of ethanol is 75%, centrifugal (6000r/min, 15min), the precipitation of gained through dialysis (crossing molecular weight cut-off is the dialysis tubing of 8000kDa) and lyophilize (in-45 DEG C, the dry 40h of vacuum tightness 0.1Mpa) after, obtain sea cucumber fucoidan.
2), by 0.5g sea cucumber fucoidan be dissolved in 30ml deionized water and carry out supersound process at the temperature of 5 DEG C, the treatment time is 10min, and ultrasonic sound field intensity is 181.53W/cm
2; Now ultrasonic frequency is more for 21-25kHz(remarks illustrate: instrument regulates voluntarily within the scope of this).
3), by step 2) solution dialysis (crossing interception is the dialysis tubing of 1000kDa) after the process of gained, after vacuum lyophilization (vacuum tightness ,-45 DEG C of dry 40h in 0.1Mpa), obtain the low-molecular-weightsea sea cucumber fucosan sulfate of about 0.38g.
Adopt gel permeation chromatography to detect molecular weight of product the low-molecular-weightsea sea cucumber fucosan sulfate of above-described embodiment 1-1 gained, its molecular weight (kDa) is 217.48.
Embodiment 1-2 ~ 1-5,
In order to verification step 2) in different ultrasonic sound field intensity carry out the impact of the low-molecular-weightsea sea cucumber fucosan sulfate that ultrasonic degradation obtains preparation, change step 2) in ultrasonic sound field intensity, all the other are with embodiment 1-1, thus obtain embodiment 1-2 ~ 1-5; The low-molecular-weightsea sea cucumber fucosan sulfate molecular weight of concrete ultrasonic sound field intensity and corresponding gained is as shown in table 1.
Table 1, different ultrasonic sound field intensity gained low-molecular-weightsea sea cucumber fucosan sulfate molecular weight
Can be found by table 1, ultrasonic sound field intensity is 181.53-544.59W/cm
2time, after effect 10min, along with the increase of ultrasonic sound field intensity, sea cucumber fucoidan molecular weight constantly reduces, and concentrates on 138.47-217.48kDa.When ultrasonic sound field intensity is 544.59, sea cucumber fucoidan molecular weight is down to 138.47kDa by 547.11kDa.Within the scope of this sound field, ultrasonic degradation sea cucumber fucoidan Be very effective.
Embodiment 2-1 ~ 2-3,
In order to verification step 2) in carry out the impact of the low-molecular-weightsea sea cucumber fucosan sulfate that ultrasonic degradation obtains preparation at differential responses temperature, change embodiment 1-4 step 2) in temperature of reaction, all the other are with embodiment 1-4, thus obtain embodiment 2-1 ~ 2-3(namely, treatment time is 10min, and ultrasonic sound field intensity is 423.57W/cm
2); The low-molecular-weightsea sea cucumber fucosan sulfate molecular weight of concrete temperature of reaction and corresponding gained is as shown in table 2.
Table 2, differential responses temperature gained low-molecular-weightsea sea cucumber fucosan sulfate molecular weight
| Embodiment 1-4 | Embodiment 2-1 | Embodiment 2-2 | Embodiment 2-3 | |
| Temperature of reaction (DEG C) | 5 | 15 | 25 | 35 |
| Molecular weight (kDa) | 141.72 | 139.06 | 152.36 | 173.43 |
Can be found by table 2, when temperature of reaction is 5-45 DEG C, after ultrasonication 10min, on the whole, degraded trend strengthens along with the reduction of temperature, and sea cucumber fucoidan molecular weight concentrates on 139.06-212.97kDa, when temperature is 15 DEG C, molecular weight is minimum, is 139.06kDa.In this temperature range, ultrasonic degradation sea cucumber fucoidan Be very effective.
Embodiment 3, on the basis of above-described embodiment, response surface design analysis optimization low-molecular-weightsea sea cucumber fucosan sulfate ultrasonic degradation condition, carry out following steps:
Get 0.5g sea cucumber fucoidan and be dissolved in 30ml deionized water, the ultrasonication time is 10min, select have 2 factors of remarkably influenced (temperature of reaction, ultrasonic sound field intensity) to carry out response surface experiments on peak molecular weight, thus optimize the condition of ultrasonic degradation fucoidan.
Minitab data system analysis software is adopted to carry out regression analysis to testing data.Use response surface central composite design method (centralcompositedesign, CCD) to design 2 factor 3 hydraulic tests, the basic, normal, high test level of each independent variable(s) is encoded with-1,0,1 respectively, experimental design and the results are shown in Table 3.Utilize Minitab his-and-hers watches 3 testing data to carry out multiple regression matching, obtain fucoidan retention time (Y) to temperature of reaction (X
1), ultrasonic power (X
2) secondary multiple regression simulation equation be:
Y=59.4595+8.24809X
1+5.48866X
2+0.139715X
1 2-0.0775104X
2 2-0.283303X
1X
2
Table 3, CCD response surface design test design and result
Carry out variance analysis to response surface design result, find model P<0.01, model returns highly significant; Lose and intend item P=0.285>0.05, not significantly, illustrate that in selected two factor range of test, gained model can reflect true relation between parameter; R
2=0.9328 show this model and actual tests matching better.From regression model coefficient test of significance result, the once item X of model
1(temperature of reaction), X
2(ultrasonic sound field intensity) is all remarkable to the degraded of sea cucumber fucoidan, wherein X
1(P<0.001), very significantly; Quadratic term is all not remarkable, and mutual item X
1x
2(P<0.05) remarkable to Degradation.To model by analysis, obtain optimum parameter: temperature: 13.1398 DEG C, sound field intensity: 508.28W.cm
2.
Embodiment 4,
In order to verification step 2) in carry out the impact of the low-molecular-weightsea sea cucumber fucosan sulfate that ultrasonic degradation obtains preparation, the step 2 by embodiment 1-1 under the different ultrasonication time) make following content into, all the other are with embodiment 1-1:
2), by 0.5g sea cucumber fucoidan be dissolved in 30ml deionized water and carry out supersound process at the temperature of 13.0713 DEG C, the treatment time is 2.5min(or is 0min, 5min, 20min, 40min, 80min), ultrasonic sound field intensity is 508.28W/cm
2.
The low-molecular-weightsea sea cucumber fucosan sulfate molecular weight of gained is as shown in table 4.
Table 4, different gained low-molecular-weightsea sea cucumber fucosan sulfate molecular weight action time (embodiment 4)
Can be found by table 4, before supersound process in 20min, molecular weight and molecular weight is very rapid, 5min especially, and molecular weight drops to 140.97kDa from 547.11kDa, and degradation efficiency is very high; After 20min, along with the prolongation of ultrasonic time, molecular weight and molecular weight tends towards stability.After process 80min, molecular weight is down to 78.92kDa.
Embodiment 5,
In order to verification step 2) in carry out the impact of the low-molecular-weightsea sea cucumber fucosan sulfate that ultrasonic degradation obtains preparation, the step 2 by embodiment 1-1 under different solvents) make following content into, all the other are with embodiment 1-1:
2) mass concentration, 0.5g sea cucumber fucoidan being dissolved in 30ml is at the temperature of 13.0713 DEG C, carry out supersound process in the acetic acid of 2% (or hydrochloric acid, mass concentration are the sulfuric acid of 2% for mass concentration is 2%), treatment time is 40min(, the ultrasonication time is 40min), ultrasonic sound field intensity is 508.28W/cm
2.
The low-molecular-weightsea sea cucumber fucosan sulfate molecular weight of gained is as shown in table 5.
Table 5, differential responses solvent gained low-molecular-weightsea sea cucumber fucosan sulfate molecular weight
| Solvent | Acetic acid (2%) | Hydrochloric acid (2%) | Sulfuric acid (2%) |
| Molecular weight (kDa) | 79.68 | 77.53 | 81.60 |
Can be found by table 5, deionized water is replaced with the acetic acid of lower concentration, hydrochloric acid and sulfuric acid, aggravation effect is had to ultrasonic degradation, after ultrasonication 40min, molecular weight is down near 80kDa, uses different types of acid to carry out ultrasonic degradation sea cucumber fucoidan Be very effective as reaction solvent.
Finally, it is also to be noted that what enumerate above is only several specific embodiments of the present invention.Obviously, the invention is not restricted to above embodiment, many distortion can also be had.All distortion that those of ordinary skill in the art can directly derive from content disclosed by the invention or associate, all should think protection scope of the present invention.
Claims (6)
1. the preparation method of low-molecular-weightsea sea cucumber fucosan sulfate, using the sea cucumber polysaccharide that will enzymolysis process utilized to obtain after sea cucumber pulverizing as raw material, is characterized in that comprising the following steps:
1), by sea cucumber polysaccharide be dissolved in the sodium chloride solution of 2.8 ~ 3.2mol/L, obtain sea cucumber polysaccharide solution, described sea cucumber is many
In sugar soln, the mass concentration of sea cucumber polysaccharide is 0.8 ~ 1.2%; In described sea cucumber polysaccharide solution, add ethanol, until the volume final concentration of ethanol is 34 ~ 40%, centrifugal removing precipitation, obtains supernatant liquor; In described supernatant liquor, add ethanol, until the volume final concentration of ethanol is 60 ~ 90%, centrifugal, the precipitation of gained, after dialysis and lyophilize, obtains sea cucumber fucoidan;
2), by sea cucumber fucoidan be dissolved in solvent and carry out supersound process at the temperature of 0 ~ 45 DEG C, the treatment time is 10 ~ 180min, and ultrasonic sound field intensity is 60 ~ 800W/cm
2; Described solvent to be mass concentration be 0.5 ~ 5% acetum, mass concentration be 0.5 ~ 5% hydrochloric acid soln or mass concentration be the sulphuric acid soln of 0.5 ~ 5%;
3), by step 2) solution dialysis after the process of gained, after vacuum lyophilization, obtain low-molecular-weightsea sea cucumber fucosan sulfate.
2. the preparation method of low-molecular-weightsea sea cucumber fucosan sulfate according to claim 1, is characterized in that: the molecular weight cut-off of the dialysis tubing that the dialysis in described step 1) is used is 8000kDa.
3. the preparation method of low-molecular-weightsea sea cucumber fucosan sulfate according to claim 2, is characterized in that: described step 2) in the solid-liquid ratio of sea cucumber fucoidan and solvent be 1g/20 ~ 200ml.
4. the preparation method of low-molecular-weightsea sea cucumber fucosan sulfate according to claim 3, is characterized in that: the molecular weight cut-off of the dialysis tubing that the dialysis in described step 3) is used is 1000kDa.
5. the preparation method of low-molecular-weightsea sea cucumber fucosan sulfate according to claim 4, is characterized in that: described step 2) in: temperature is 5 ~ 35 DEG C, and the treatment time is 10 ~ 80min, and ultrasonic sound field intensity is 180 ~ 550W/cm
2.
6. the preparation method of low-molecular-weightsea sea cucumber fucosan sulfate according to claim 5, it is characterized in that: step 2) be: mass concentration 0.5g sea cucumber fucoidan being dissolved in 30ml is the acetic acid of 2%, the mass concentration of 30ml be the hydrochloric acid of 2% or the mass concentration of 30ml is at the temperature of 13.0713 DEG C, carry out supersound process in the sulfuric acid of 2%, treatment time is 40min, and ultrasonic sound field intensity is 508.28W/cm
2.
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| CN106866834B (en) * | 2017-03-16 | 2018-01-30 | 山东省食品发酵工业研究设计院 | A kind of method and its application for preparing the efficiently fucoidin of customization molecular weight |
| CN109705232A (en) * | 2018-12-28 | 2019-05-03 | 金耀仑 | A kind of extraction process of low-molecular-weight algal carbohydrate gum and its application in cosmetics |
| CN111548427A (en) * | 2019-02-12 | 2020-08-18 | 湖北洪森生物科技有限公司 | Preparation method of high-activity rice polysaccharide powder |
| CN110357983B (en) * | 2019-08-14 | 2021-08-31 | 中国海洋大学 | Preparation method of sea cucumber fucosan sulfate and chondroitin sulfate oligosaccharide |
| CN111713654A (en) * | 2020-07-27 | 2020-09-29 | 大连工业大学 | Leisure food containing polysaccharide and fishbone calcium and preparation method thereof |
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