CN105001348A - Extraction method for fucoidin with high yield and high ratio of fucose - Google Patents
Extraction method for fucoidin with high yield and high ratio of fucose Download PDFInfo
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Abstract
The present invention discloses an extraction method for fucoidin with a high yield and a high ratio of fucose, belongs to the technology field of algae processing and utilization. According to the present invention, nemacystis is used as a raw material, a high-pressure homogenisation method is used to extract the fucoidin, and therefore extraction time is short, extraction efficiency is high, and retention of natural components content in the fucoidin is maximized. By combining a chemical degradation method and a enzymatic degradation method to degrade the fucoidin, and by ultrafiltration technology, finally low-molecular-weight fucoidin with outstanding anti-tumor activity is obtained. According to the present invention, the yield of the obtained fucoidin is 21%-34%, the purity is 85%-90%, the fucose content reaches 70%-75%, and the organic sulfate ion content reaches 20%-30%.
Description
Technical field
The present invention relates to a kind of extracting method of fucoidin of high yield pulp1 height Fucose ratio, belong to marine alga process and utilization technology field.
Background technology
China's algae resource enriches, and especially giving prominence to brown alga resource, is marine alga production and consumption big country.Containing abundant polysaccharide component in brown alga, as algin, laminarin, carrageenin etc., have all become important food or medical material and have been widely used.At China, Okinawa Japan, coastally all there is distribution.
Fucoidin (Fucoidan), is iuntercellular polysaccharide intrinsic in brown alga, is present in cell wall matrix.Recent studies shows, fucoidin have anticoagulation, antitumor, antiviral, anti-oxidant, promote the physiological function more than 20 such as skin regeneration, skin moisture-keeping, low weight molecular fucoidan is especially outstanding with antitumor efficacy.Compared with ganoderan, curcumine etc., fucoidin can not only strengthen body immunity, and has the special efficacy of inducing apoptosis of tumour cell, has very large development potentiality at functional foodstuff and medicine trade.
Fucoidin main component is L-fucose, and linear backbone is generally that Fucose is formed by connecting simultaneously with semi-lactosi with α-1,3 or α-Isosorbide-5-Nitrae glycosidic link, seminose, glucose, the sulfation mixed polysaccharide of the monosaccharide components such as wood sugar.The medium research of Wu Xing finds that hepatoma cell migration can play restraining effect in fucosylation, ectogenic when adding Fucose sugar chain, cancer cell migration suppresses in concentration dependent, it can thus be appreciated that the content of Fucose and the biologic activity of fucoidin have correlative connection in this polysaccharide.Be generally that the fucose content of the fucoidin in source is at 10-50% with brown alga.In sulfated polysaccharide, the content of sulfate radical is the very important index determining its active size, is also the importance of research structure activity relationship simultaneously.To the puritan filler polysaccharide fucoidin deriving from brown alga, the existence of sulfate radical has material impact equally to its physicochemical property and biological activity, and therefore the content of sulfate radical is qualification fucoidin sample and the important parameter analyzing structure activity relationship.In the traditional extraction process of fucoidin with water extraction and sour formulation the most common, but it is many to take traditional extraction to send out the fucoidin impurity of method gained, and yield is low, and quality is low.The fucoidin quality that current market is sold is not high, and the marine alga processing technology that containing part algin composition, mass deficiency for health products trade, therefore is sought " high added value, high-level efficiency, low cost " has become new developing direction.Because protolith polysaccharides molecular mass is large, poorly soluble, be not easy to be absorbed, seriously limit its application, but through the low molecule quality fucoidin of Partial digestion, biological activity is more remarkable, and research shows, the fucoidin anti-tumor activity of molecular weight 8000-30000 is higher.
Summary of the invention
In order to overcome the problems referred to above, the invention provides a kind of extracting method of fucoidin of high yield pulp1 height Fucose ratio, be from Hai Yunzhong prepare antitumor fucoidin method, the method efficiently can prepare environmental protection, pollution-free, highly purified fucoidin product.
The object of this invention is to provide a kind of extracting method of fucoidin of high yield pulp1 height Fucose ratio, described method comprises: accumulate for raw material with sea, through high-pressure homogeneous, the heavy sugar of centrifuging and taking supernatant, ethanol, filtration, drying, obtains fucoidin.
Described high-pressure homogeneous, in one embodiment of the invention, be under 10-20Mpa, by material-water ratio 1:30-1:160, homogeneous 1-3 time.
Described material-water ratio, refers to the mass ratio of the dry weight that sea accumulates and water in the present invention.
Described sea accumulates, and in one embodiment of the invention, is that dry sea accumulates (humidity can be 8-10%) or fresh sea accumulates.When using dry sea to accumulate as raw material, first adding bubble one bubble, carrying out high-pressure homogeneous after water absorption and swelling again.
The heavy sugar of described ethanol in one embodiment of the invention, is use the ethanol that volume fraction is 70-80%.
The heavy sugar of described ethanol, in one embodiment of the invention, be get centrifugal after supernatant liquor, i.e. extracting solution, adds the dehydrated alcohol of 3-4 times of volume, mixing, leaves standstill, and obtains throw out.
Described centrifugal, in one embodiment of the invention, be centrifugal 10-15min under 4000-5000rpm.
Described filtration In one embodiment of the present invention, is adopt centrifugal or Plate Filtration.
Described drying In one embodiment of the present invention, is vacuum-drying.
Described method, in one embodiment of the invention, centrifuging and taking supernatant and ethanol sink between sugared step, also comprise chemical degradation or enzymic degradation, ultrafiltration, to obtain the fucoidin of corresponding molecular weight ranges.
Described method, in one embodiment of the invention, comprising: (1) is high-pressure homogeneous: get sea and accumulate, and adds water by material-water ratio 1:30-1:160, then homogeneous 1-3 time under 10-20Mpa; (2) centrifuging and taking supernatant: the material after high-pressure homogeneous is carried out centrifugal, gets supernatant liquor, be extracting solution; (3) chemical degradation: concentrated extracting solution, to the 1/4-1/2 of original volume, then adds the H of 30%
2o
2, 50-60 DEG C of water-bath, degraded 1-4h, regulates degradation solution pH to neutral, then adds catalase reaction 10-20min; (4) ultrafiltration: degradation solution flows through the ultrafiltration post of PSPP, obtains trapped fluid; (5) the heavy sugar of ethanol: the dehydrated alcohol adding 3-4 times of volume in trapped fluid obtained in the previous step, mixing, leaves standstill; (6) throw out after filtration, dry, obtain fucoidin.
Described step (3), in one embodiment of the invention, 30%H
2o
2addition be add 5-8mL in every L.
Described step (3), in one embodiment of the invention, the work of catalase enzyme is 10,000-5 ten thousand U/mL, addition 0.1-0.4mL/L.
Described step (3), in one embodiment of the invention, is adopt molecular weight cut-off to be the ultrafiltration post of 30KD, 10KD, thus obtains the fucoidin of molecular weight between 10000-30000.
Described step (3), in one embodiment of the invention, be adopt concentrating under reduced pressure, condition is temperature 70-75 DEG C, and vacuum tightness is-0.1MPa.
Described method, in one embodiment of the invention, comprising: (1) is high-pressure homogeneous: get sea and accumulate, and adds water by material-water ratio 1:30-1:160, then homogeneous 1-3 time under 10-20Mpa; (2) centrifuging and taking supernatant: the material after high-pressure homogeneous is carried out centrifugal, gets supernatant liquor, be extracting solution; (3) enzymic degradation: concentrated extracting solution, pH to 5-6 adjusted by adding citric acid-Trisodium Citrate, adds the fucoidanase of 50-55U according to every L concentrated extracting solution, degraded 2-5h, and degradation solution crosses ultrafiltration post, obtains trapped fluid; (5) the heavy sugar of ethanol: the dehydrated alcohol adding 3-4 times of volume in trapped fluid obtained in the previous step, mixing, leaves standstill; (6) throw out after filtration, dry, obtain fucoidin.
Described fucoidanase, in one embodiment of the invention, extracts from aspergillus niger.
Described fucoidanase, in one embodiment of the invention, obtain in the following manner: aspergillus niger is carried out liquid fermentation and culture, the supernatant liquor after fermented liquid is centrifugal is crude enzyme liquid 1, and the supernatant of thalline after redissolution, ultrasonication, frozen centrifugation is crude enzyme liquid 2, get crude enzyme liquid, 4 DEG C add ammonium sulfate solids to saturation ratio is 80%, stir 2h, hold over night, 10000rpm/min, frozen centrifugation 20min, collecting precipitation, T-buffer dissolves, 10000rpm/min, supernatant is got after frozen centrifugation 20min, and the 24h that dialyses at 4 DEG C, concentrated, cross DEAE-sepharose FF anion-exchange column, T-buffer balances, with 0-2M NaCl gradient elution, collect elution peak, merge component, concentrated, dialyse 24h at 4 DEG C, concentrated, cross gel chromatographic columns sphacryl S-300, phosphoric acid buffer balances, wash-out collects active ingredient, concentrated, freeze-drying obtains pure enzyme.
Described fucoidanase, in one embodiment of the invention, its enzyme activity determination method is: enzyme liquid to be measured is added to 0.5% fucoidin solution (with buffer), at 50 DEG C, reaction 1h.Boiling water bath goes out enzyme 15min, and measure reducing sugar content by DNS method, blank is by enzyme liquid to be measured after boiling water bath 15min, then carries out aforesaid operations; The enzyme amount that enzyme lives that unit definition is 50 DEG C, release per hour 1 μm of ol Fucose needs under pH value 6.0 condition.
Described method, in one embodiment of the invention, immediately aqueous extraction step after high-pressure homogenization step; Described water extraction is carried out under ultrasonic or agitation condition.
Described water extraction in one embodiment of the invention, is by the material after high-pressure homogeneous, extracts 1-6h, and then carry out next step centrifugally operated under ultrasonic or agitation condition.
Described water extraction in one embodiment of the invention, is carry out under 50-60 DEG C of water-bath.
Described water extraction in one embodiment of the invention, is repeatedly extract 2-3 time, and extracting solution merges.
The advantage that the present invention has:
1. the present invention adopts sea to accumulate for starting material, and adopt the method for high-pressure homogenization to extract fucoidin, extraction time is short, and extraction efficiency is high, and farthest remains the content of fucoidin natural constituents.
2. the present invention utilizes high-pressure homogenization, and combine with chemical degradation method or enzymic degradation method degraded fucoidin, by ultra-filtration technique, and low-molecular-weight fucoidin during final acquisition anti-tumor activity is obvious.
3. extraction conditions of the present invention is gentle, without acid-base class, ensure that the biological activity of fucoidin to greatest extent, and without the discharge of acid-base class waste liquid in leaching process, is conducive to environmental protection.
4. the yield of fucoidin that obtains of the present invention is at 21%-35%, and purity 85%-90%, wherein fucose content reaches 70%-75%, and organic sulfuric acid reaches 20%-23%, molecular weight 10000-30000 with content, reaches market one-level and top grade product standard.
Embodiment
Fucoidin measures: adopt Phenol sulfuric acid procedure to survey polysaccharide, do typical curve with Fucose standard; With Fucose concentration for X-coordinate, light absorption value is ordinate zou drawing standard curve, typical curve equation y=0.0135x+0.0066, R
2=0.9991.
The monosaccharide component analysis of fucoidin: chromatography of ions-pulsed amperometric method (IC-PAD), chromatographic column is CarboPac PA20; Moving phase: H
2o, 250mmol/LNaOH, 1mol/L NaAc; Flow velocity: 0.5mL/min; Detector: pulsed amperometry.
Sample pre-treatments: take polysaccharide sample 1mg and be dissolved in 1.0mL 4mol/L trifluoroacetic acid, inflated with nitrogen tube sealing, 90 DEG C of hydrolysis 1h, dry up with nitrogen after cooling, the 10mL that adds water dissolves, and supplies sample introduction with after millipore filtration membrane filtration.
Sulfate radical content measures and uses gelatin-barium chloride method, turbidimetry for Determination.
Embodiment 1: high pressure homogenization method extracting directly fucoidin
Take dry sea and accumulate 50g, pulverizer is pulverized, and 100 orders sieve, and accumulate according to dry sea: the mass ratio of water=1:30 adds water, and soaks for some time, stirs evenly, under 15Mpa, and homogeneous 1 time.
After high-pressure homogeneous, centrifugal 15min under 4000rpm, get centrifugal after supernatant liquor (i.e. extracting solution), extracting solution concentrates, and adds the dehydrated alcohol of 3 volumes by every L extracting solution, mixing, leave standstill, obtain throw out.Throw out after filtration, vacuum-drying, obtain refining fucoidin.
Obtain fucoidin 15.48g, yield is 30.96% (product/raw material dry weight × 100), and purity is 88.22%, organic sulfate radical content 22.03%.Wherein in fucoidin, the molar content of Fucose is 73.84%.
Embodiment 2: high pressure homogenization method extracts fucoidin (1) in conjunction with chemical degradation method
Take fresh sea and accumulate 0.5kg (dry weight is about 20-25g), clean up, add 1.5kg water (i.e. material-water ratio 1:60-1:75), under 20Mpa, homogeneous 1 time.Then centrifugal 10min under 5000rpm, get centrifugal after supernatant liquor (i.e. extracting solution).Extracting solution is concentrated into 1/2 of original volume, then according to the H adding 30% of 8mL in every L liquid
2o
2, 60 DEG C of water-baths, degraded 1h, 1M NaOH solution regulates degradation solution pH to neutral, then adds catalase reaction 20min.Degradation solution flows through the ultrafiltration post that molecular weight cut-off is 30KD, 10KD, and the concentrated solution collecting 10-30KD obtains trapped fluid, adds the dehydrated alcohol of 4 times of volumes in trapped fluid, and mixing leaves standstill 24h, collects solid sediment; Throw out, through Plate Filtration, 50 DEG C of drying under reduced pressure, obtains fucoidin.
Obtain fucoidin 6.84g, yield is 27.4-34%, and purity is 84.26%, molecular weight 10000-30000, organic sulfate radical content 20.35%.Wherein in fucoidin, the molar content of Fucose is 75.19%.
Embodiment 3: high pressure homogenization method extracts fucoidin (2) in conjunction with chemical degradation method
Take fresh sea and accumulate 0.5kg (dry weight is about 20-25g), clean up, the 4kg that adds water (i.e. material-water ratio 1:160-1:200), under 10Mpa, homogeneous 3 times.Centrifugal, get supernatant liquor (i.e. extracting solution).Extracting solution is concentrated into 1/4 of original volume, then according to the H adding 30% of 5mL in every L liquid
2o
2, 50 DEG C of water-baths, degraded 4h, 1M NaOH solution regulates degradation solution pH to neutral, then adds catalase reaction 10min.Degradation solution flows through the ultrafiltration post that molecular weight cut-off is 30KD, 10KD, and the concentrated solution collecting 10-30KD obtains trapped fluid, adds the dehydrated alcohol of 4 times of volumes in trapped fluid, and mixing leaves standstill, and collects solid sediment; Throw out, through Plate Filtration, 50 DEG C of drying under reduced pressure, obtains fucoidin.
Obtain fucoidin 5.18g, yield is 20.72-25.9%, and purity is 82.97%, molecular weight 10000-30000, organic sulfate radical content 20.18%.Wherein in fucoidin, the molar content of Fucose is 72.36%.
Embodiment 4: " high pressure homogenization method+water extraction+enzymic degradation " extracts fucoidin
Take fresh sea and accumulate 0 (dry weight is 20-25g), clean up, add the water (i.e. material-water ratio 1:100) of 2.5kg, under 10Mpa, homogeneous 2 times.Centrifugal, get supernatant liquor (i.e. extracting solution).Material after high-pressure homogeneous is placed in 50-60 DEG C of water-bath, under ultrasonic or agitation condition, extracts 1-6h, centrifugally obtain extracting solution.
Enzymic degradation: concentrated extracting solution, pH to 5-6 adjusted by adding citric acid-Trisodium Citrate, the fucoidanase of 50-55U is added according to every L concentrated extracting solution, degraded 2-5h, degradation solution flows through the ultrafiltration post that molecular weight cut-off is 30KD, 10KD, and the concentrated solution collecting 10-30KD obtains trapped fluid, the dehydrated alcohol of 3 times of volumes is added in trapped fluid, mixing, leaves standstill, and collects solid sediment; Throw out, through Plate Filtration, 50 DEG C of drying under reduced pressure, obtains fucoidin.
Obtain fucoidin 6.9g, yield is 27.6%-34.5%, and purity is 89.46%, organic sulfate radical content 22.09%.Wherein in fucoidin, the molar content of Fucose is 73.18%.
Embodiment 5: the high-pressure homogeneous impact on fucoidin quality
Take fresh sea to accumulate, clean up, add water 4kg, under 5-25Mpa, and homogeneous 1 time.Centrifugal, get supernatant liquor, concentrated, then add the H of 30%
2o
2, 50 DEG C of water-bath degradeds, regulate degradation solution pH to neutral, then add catalase reaction 10min.Degradation solution flows through the ultrafiltration post of PSPP, obtains trapped fluid, adds the dehydrated alcohol of 4 times of volumes in trapped fluid, mixing, leaves standstill, and collects solid sediment; Throw out after filtration, drying under reduced pressure, obtain fucoidin.
Change high-pressure homogeneous pressure conditions, other steps are consistent, compare different pressure to the impact of extracting the fucoidin obtained.Result is as shown in table 1.Result shows, pressure too low (below 5MPa) can affect the yield of fucoidin, and pressure too high (25MPa) can affect SO
4 2-content, thus the biological activity affecting fucoidin.
The high-pressure homogeneous pressure of table 1 is on the impact of product
Embodiment 6: raw material type is on the impact of fucoidin quality
Feed change kind, adopt 10Mpa, other steps are consistent with embodiment 5, compare and adopt same procedure to extract the result of the fucoidin obtained from different material, as shown in table 2.Result shows, by the inventive method, accumulate for raw material extracts fucoidin with sea, yield can reach 21.36%, far away higher than sea-tangle and wakame.Polysaccharide content and SO simultaneously
4 2-content also significantly improves, and fucose content can reach 74%.
Table 2 raw material is on the impact of product
Although the present invention with preferred embodiment openly as above; but it is also not used to limit the present invention, any person skilled in the art, without departing from the spirit and scope of the present invention; all can do various changes and modification, what therefore protection scope of the present invention should define with claims is as the criterion.
Claims (10)
1. an extracting method for the fucoidin of high yield pulp1 height Fucose ratio, to be characterised in that, described method comprises: accumulate for raw material with sea, through high-pressure homogeneous, the heavy sugar of centrifuging and taking supernatant, ethanol, filtration, drying, obtains fucoidin.
2. the method according to claim, is characterized in that, described high-pressure homogeneous, is under 10-20Mpa, by material-water ratio 1:30-1:160, and homogeneous 1-3 time.
3. method according to claim 1, is characterized in that, described ethanol sink sugar be get centrifugal after supernatant liquor, i.e. extracting solution, adds the ethanol that volume fraction is 70-80%, mixing, leave standstill, obtain throw out.
4. method according to claim 1, is characterized in that, described centrifuging and taking supernatant and ethanol sink between sugared step, also comprises chemical degradation or enzymic degradation, ultrafiltration, to obtain the fucoidin of corresponding molecular weight ranges.
5. method according to claim 4, is characterized in that, described method comprises: (1) is high-pressure homogeneous: get sea and accumulate, and adds water by material-water ratio 1:30-1:160, then homogeneous 1-3 time under 10-20Mpa; (2) centrifuging and taking supernatant: the material after high-pressure homogeneous is carried out centrifugal, gets supernatant liquor, be extracting solution; (3) chemical degradation: concentrated extracting solution, adds the H of 30% at extracting solution
2o
2, 50-60 DEG C of water-bath, degraded 1-4h, regulates degradation solution pH to neutral, then adds catalase reaction 10-20min; (4) ultrafiltration: degradation solution flows through the ultrafiltration post of PSPP, obtains trapped fluid; (5) the heavy sugar of ethanol: the dehydrated alcohol adding 3-4 times of volume in trapped fluid obtained in the previous step, mixing, leaves standstill; (6) throw out after filtration, dry, obtain fucoidin.
6. method according to claim 4, is characterized in that, described method comprises: (1) is high-pressure homogeneous: get sea and accumulate, and adds water by material-water ratio 1:30-1:160, then homogeneous 1-3 time under 10-20Mpa; (2) centrifuging and taking supernatant: the material after high-pressure homogeneous is carried out centrifugal, gets supernatant liquor, be extracting solution; (3) enzymic degradation: concentrated extracting solution, pH to 5-6 adjusted by adding citric acid-Trisodium Citrate, adds the fucoidanase of 50-55U according to every L concentrated extracting solution, degraded 2-5h, and degradation solution crosses ultrafiltration post, obtains trapped fluid; (5) the heavy sugar of ethanol: the dehydrated alcohol adding 3-4 times of volume in trapped fluid obtained in the previous step, mixing, leaves standstill; (6) throw out after filtration, dry, obtain fucoidin.
7. according to the arbitrary described method of claim 1-6, it is characterized in that, immediately aqueous extraction step after described high-pressure homogenization step; Described water extraction is carried out under ultrasonic or agitation condition.
8. method according to claim 7, is characterized in that, described water extraction is by the material after high-pressure homogeneous, extracts 1-6h, and then carry out next step centrifugally operated under ultrasonic or agitation condition.
9. according to the fucoidin that the arbitrary described method of claim 1-8 obtains.
10. the application of fucoidin described in claim 9.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN106749439A (en) * | 2016-12-30 | 2017-05-31 | 山东省科学院生物研究所 | New fucoidan oligosaccharide and preparation method and application |
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| CN107903330A (en) * | 2017-11-22 | 2018-04-13 | 广东丸美生物技术股份有限公司 | Degradation of Polysaccharides and low-molecular-weight polysaccharide |
| CN109430412A (en) * | 2018-12-25 | 2019-03-08 | 光明乳业股份有限公司 | A kind of functional form liquid milk and preparation method thereof containing fucoidin |
| CN109430399A (en) * | 2018-12-25 | 2019-03-08 | 光明乳业股份有限公司 | A kind of functional form acidified milk and preparation method thereof |
| CN109699745A (en) * | 2019-02-28 | 2019-05-03 | 福建农林大学 | A kind of fucosylated oligosaccharide baby formula milk powder and preparation method thereof |
| CN115417934A (en) * | 2022-09-02 | 2022-12-02 | 威海迪普森生物科技有限公司 | Preparation method of kelp extract with high fucooligosaccharide content |
| CN115417934B (en) * | 2022-09-02 | 2023-09-26 | 威海迪普森生物科技有限公司 | Preparation method of high-content fucoidin kelp extract |
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