CN106883309A - The multiplex-enzyme extraction method of laminarin and the laminarin for obtaining - Google Patents
The multiplex-enzyme extraction method of laminarin and the laminarin for obtaining Download PDFInfo
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- CN106883309A CN106883309A CN201710240346.2A CN201710240346A CN106883309A CN 106883309 A CN106883309 A CN 106883309A CN 201710240346 A CN201710240346 A CN 201710240346A CN 106883309 A CN106883309 A CN 106883309A
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- Prior art keywords
- laminarin
- enzyme
- ethanol
- kelp
- cellulase
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- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
- C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
- C08B37/0003—General processes for their isolation or fractionation, e.g. purification or extraction from biomass
Abstract
The invention discloses the multiplex-enzyme extraction method and the laminarin that obtains of a Polysaccharides From Laminaria Japonica, with kelp processing leftover bits and pieces as raw material, first dries pulverizing takes appropriate Kelp Powder, full-automatic hydrolysis reactor is added, by solid-liquid ratio 1：35 45ml add pH6.0 buffer solutions, add complex enzyme after stirring evenly, by Kelp Powder quality for 1g based on, the addition of cellulase, hemicellulase, pectase and protease is respectively 0.004 0.006g, 0.006 0.008g, 0.004 0.006g, 0.01 0.014g；Regulation pH value is 5.5 6.5,45 55 DEG C, digests 3 4h, and intensification is gone out enzyme, takes out hydrolyzate, and suction filtration removes precipitation and insoluble matter, and it is 65 75% to add ethanol to system volume fraction of ethanol to supernatant, in 4 DEG C of refrigerator cold-storages, overnight.3000g 4000g/min are centrifuged 20min, and sediment is freeze-dried, obtain final product kelp raw polysaccharide.The present invention can in a mild condition be effectively improved polysaccharide yield, purity, complete to retain polysaccharide structures and bioactivity, during will not produce noxious material and pollution is produced to environment.
Description
Technical field
It is the multiplex-enzyme extraction method of a Polysaccharides From Laminaria Japonica specifically the present invention relates to laminarin and its extracting method
And the laminarin for obtaining.
Background technology
The sea-tangle yield of China occupies first place in the world.At present, kelp processing industry is based on roughing；Brown alga industry is producing
Based on sodium alginate, mannitol and iodine.A large amount of residue waste liquids and leftover bits and pieces etc., brown alga are produced when brown alga industry and sea-tangle roughing
Carbohydrate gum and Sargassum protein and dietary fiber etc. are contained therein, and not only pollute environment waste of resource but also be also a kind of huge
Waste.Numerous studies find that there are laminarin various physiology such as anti-oxidant, antitumor, antiviral, anticoagulation, hypotensive to live
Property, improve the immunity of body.Find out a kind of laminarin in high efficiency extraction sea-tangle that can be used in industrialized production
Method, is a kind of the efficient using new approach is opened up of brown alga resource, is that the high-valued exploitation of brown alga industry set up one
Perfect technology platform, preferably develops marine resources, further gos deep into Development of Marine economy.
The method for extracting laminarin at present is a lot, wherein mainly including Hot water extraction, acid extraction method, alkaline extraction, super
Sound wave extraction method, microwave loss mechanisms and enzyme extraction method etc..But the laminarin that these methods are extracted is general due to degradation problem yield
All over relatively low.
Hot water extraction is to extract the more traditional and important method of laminarin.Specific way is first clean sea-tangle,
Then it is put into baking oven and is baked to dry, then grind into powder, finally pour into hot water Kelp Powder, is extracted.Extracting temperature
70-90 DEG C, extraction time 6-8h, solid-liquid ratio be 1：40-60.The method is with low cost, simple to operate, but the recovery rate of polysaccharide compared with
It is low, if while temperature is too high, it will the activity of destruction polysaccharide, consume energy higher.
Sour formulation is higher than the polysaccharide yield of water extraction, but can cause the degraded of polysaccharide molecule, therefore as water extraction, all
There is the deficiency that recovery rate is low, purity is not high.
Alkaline extraction is similar with sour formulation, and in the basic conditions, the presence of laminarin is its esters state, therefore it dissolves
Degree becomes big, so laminarin can be extracted using alkaline extraction.Equally, alkali extraction method not high, the alkali environment that equally exists recovery rate
The degradation problem of polysaccharide molecule may be caused.
Ultrasonic extraction can significantly increase the content of laminarin than sour formulation, can with the time-consuming and energy, but
Thick many candies yield amplification is smaller, and higher to extraction equipment requirement, is not suitable for producing in enormous quantities.Zhou Junming etc. is carried to supercritical ultrasonics technology
The technique for taking fucoidin is optimized, and optimal extraction process is determined：Sonication times 60min, ultrasonic power
800W.Gained laminarin yield is 8.64%.
Zhang Haiyan, Cui Haiping etc. use multistage microwave amplifier laminarin, and orthogonal examination is set up with time, power, solid-liquid ratio etc.
Test, determine that optimum process condition is：Time is 3min, and microwave power is 480W, and solid-liquid ratio is l:20, recovery rate with this understanding
It is 17.5%.
At present, laminarin method major part is extracted in world wide and belongs to pure chemistry method, this method extracts laminarin
Due to degradation problem, not only yield is universal relatively low (typically 20% or so), and the substantial amounts of chemical drugs using acid or alkalescence
Pollution of the product to environment is than larger.Utilization of the enzyme in the industries such as fruit syrup, wine, weaving, feed, leather, production than
It is relatively wide, and with its it is efficient, nontoxic, selectivity the features such as and enjoy favor.Current zymohydrolysis extracting method extracts laminarin also not
Enough maturations, the method that only a small amount of document report is hydrolyzed using single enzyme.The a variety of compositions of sea-tangle cell membrane are constituted,
Connection method between them is varied, is difficult to decompose cell membrane with single kind of enzyme, it is impossible to make intracellular material all release
Release.Also have researcher to be extracted using combined-enzyme method, but it is combined using 2 kinds or 3 kinds of enzymes, does not have removing protein
Or removing protein is not thorough, the purity of polysaccharide of extraction is not high enough.
The content of the invention
The technical problems to be solved by the invention are, for the existing extraction process recovery rate of current laminarin, purity, work
Property low, or pollution environment, the shortcomings of time-consuming, there is provided the multiplex-enzyme extraction method of a Polysaccharides From Laminaria Japonica, using combined-enzyme method
Extract the polysaccharide in sea-tangle, it is intended to polysaccharide yield and purity are improved under temperate condition, it is complete to retain polysaccharide structures and bioactivity.
In order to solve the above-mentioned technical problem, the technical solution adopted by the present invention is：The multiplex-enzyme extraction of one Polysaccharides From Laminaria Japonica
Method, comprises the following steps：
(1) with kelp processing leftover bits and pieces as raw material, first dry, crush, take Kelp Powder, add full-automatic hydrolysis reactor,
By solid-liquid ratio 1g：35-45ml adds 0.1mol/L, and the citric acid-sodium citrate buffer solution of pH6.0 adds complex enzyme after stirring evenly,
Complex enzyme is made up of cellulase, hemicellulase, pectase and protease, by Kelp Powder quality for 1g based on, cellulase
Addition is 0.004-0.006g, hemicellulase 0.006-0.008g, pectase 0.004-0.006g and proteinase-10 .01-
0.014g；
(2) regulation pH value is 5.5-6.5,45-55 DEG C, digests 3-4h, and heat up the enzyme that goes out, and takes out hydrolyzate, and suction filtration removes heavy
Form sediment and insoluble matter；
(3) it is 65%-75% to add ethanol to system volume fraction of ethanol to supernatant, in 4 DEG C of refrigerator cold-storages, overnight,
3000g-4000g/min is centrifuged 20min, and sediment is freeze-dried, obtains final product kelp raw polysaccharide；
(4) Thick many candies are made into the aqueous solution that mass concentration ratio is 25%-35%, add ethanol to system ethanol volume integral
Number is 60%-80%, and repeating precipitation must refine laminarin three times.
Specifically, comprise the following steps：
(1) with kelp processing leftover bits and pieces as raw material, first dry, crush, take Kelp Powder, add full-automatic hydrolysis reactor,
By solid-liquid ratio 1g：40ml adds 0.1mol/L, and the citric acid-sodium citrate buffer solution of pH6.0 adds complex enzyme after stirring evenly, multiple
Synthase is made up of cellulase, hemicellulase, pectase and protease, by Kelp Powder quality for 1g based on, cellulase plus
Enter amount for 0.005g, hemicellulase 0.007g, pectase 0.005g and proteinase-10 .012g；
(2) regulation pH value is 6.0,50 DEG C, digests 3.5h, and heat up the enzyme that goes out, and takes out hydrolyzate, and suction filtration is removed and precipitated and insoluble
Thing；
(3) it is 65% to add ethanol to system volume fraction of ethanol to supernatant, in 4 DEG C of refrigerator cold-storages, overnight, 3500g/
Min is centrifuged 25min, and sediment is freeze-dried, obtains final product kelp raw polysaccharide；
(4) Thick many candies are made into the aqueous solution that mass concentration ratio is 30%, addition ethanol to system volume fraction of ethanol is
70%, repeating precipitation must refine laminarin three times.
The cellulase, hemicellulase, pectase and protease are the cellulase of DSM company production
Validase TRL, hemicellulase Bakezyme HSP6000BG, pectase Klzrzyme150, protease ValidaseFP
Concentrate。
Laminarin obtained in the multiplex-enzyme extraction method of above-mentioned laminarin.
The beneficial effects of the invention are as follows：The present invention extracts the polysaccharide in sea-tangle using combined-enzyme method can be had in a mild condition
Effect ground improves polysaccharide yield, purity, complete reservation polysaccharide structures and bioactivity, during will not produce noxious material and right
Environment produces pollution.Thick many candies content is 45.0%, and purity is 43.7%.
Specific embodiment
The present invention is described in further detail with reference to specific embodiment：
The multiplex-enzyme extraction method of laminarin of the invention, comprises the following steps：
(1) with kelp processing leftover bits and pieces as raw material, first dry, crush, take Kelp Powder, add full-automatic hydrolysis reactor,
By solid-liquid ratio 1g：35-45ml adds 0.1mol/L, and the citric acid-sodium citrate buffer solution of pH6.0 adds complex enzyme after stirring evenly,
Complex enzyme is made up of cellulase, hemicellulase, pectase and protease, by Kelp Powder quality for 1g based on, cellulase
Addition is 0.004-0.006g, hemicellulase 0.006-0.008g, pectase 0.004-0.006g and proteinase-10 .01-
0.014g；
(2) regulation pH value is 5.5-6.5,45-55 DEG C, digests 3-4h, and heat up the enzyme that goes out, and takes out hydrolyzate, and suction filtration removes heavy
Form sediment and insoluble matter；
(3) it is 65%-75% to add ethanol to system volume fraction of ethanol to supernatant, in 4 DEG C of refrigerator cold-storages, overnight,
3000g-4000g/min is centrifuged 20min, and sediment is freeze-dried, obtains final product kelp raw polysaccharide；
(4) Thick many candies are made into the aqueous solution that mass concentration ratio is 25%-35%, add ethanol to system ethanol volume integral
Number is 60%-80%, and repeating precipitation must refine laminarin three times.
Specifically, comprise the following steps：
(1) with kelp processing leftover bits and pieces as raw material, first dry, crush, take Kelp Powder, add full-automatic hydrolysis reactor,
By solid-liquid ratio 1g：40ml adds 0.1mol/L, and the citric acid-sodium citrate buffer solution of pH6.0 adds complex enzyme after stirring evenly, multiple
Synthase is made up of cellulase, hemicellulase, pectase and protease, by Kelp Powder quality for 1g based on, cellulase plus
Enter amount for 0.005g, hemicellulase 0.007g, pectase 0.005g and proteinase-10 .012g；
(2) regulation pH value is 6.0,50 DEG C, digests 3.5h, and heat up the enzyme that goes out, and takes out hydrolyzate, and suction filtration is removed and precipitated and insoluble
Thing；
(3) it is 65% to add ethanol to system volume fraction of ethanol to supernatant, in 4 DEG C of refrigerator cold-storages, overnight, 3500g/
Min is centrifuged 25min, and sediment is freeze-dried, obtains final product kelp raw polysaccharide；
(4) Thick many candies are made into the aqueous solution that mass concentration ratio is 30%, addition ethanol to system volume fraction of ethanol is
70%, repeating precipitation must refine laminarin three times.
The cellulase, hemicellulase, pectase and protease are the cellulase of DSM company production
Validase TRL, hemicellulase Bakezyme HSP6000BG, pectase Klzrzyme150, protease ValidaseFP
Concentrate。
Laminarin obtained in the multiplex-enzyme extraction method of above-mentioned laminarin.
The function of cellulase and hemicellulase is to decompose cell membrane, makes cell wall rupture；Pectase, can decompose fruit
Colloid, the ICC content of sea-tangle is more, and polysaccharide can be discharged；And compound protease can make cell membrane and thin
Intracellular protein is degraded into the oligopeptide fragments or amino acid of small molecule and removes, and can improve purity of polysaccharide.The most frequently used removing protein
Method is trifluorotrichloroethane method, trichloroacetic acid method etc..In addition, protease method with its it is efficient, safe the characteristics of, now
As a kind of new paragon for removing isolating protein.
In following embodiments, complex enzyme is selected
The measure of Thick many candies content:The quality of kelp raw polysaccharide is calculated using dry mass method, the content of polysaccharide is with phenol-sulphur
Acid system detection, determines purity of polysaccharide.
Embodiment 1
Kelp processing leftover bits and pieces is raw material, and first dries pulverizing takes appropriate Kelp Powder, adds full-automatic hydrolysis reactor, plus
Enter certain volume (solid-liquid ratio 1g：0.1mol/L 35ml), pH6.0 citric acid-sodium citrate buffer solution, add compound after stirring evenly
Enzyme by Kelp Powder quality for 1g based on, the addition of cellulase is 0.004g, hemicellulase 0.006g, pectase 0.004g and
Proteinase-10 .01g；
Regulation pH value is 5.5,45 DEG C, digests 3h, and heat up the enzyme that goes out, and takes out hydrolyzate, and suction filtration removes precipitation and insoluble matter, to
It is 60% that supernatant adds ethanol to system volume fraction of ethanol, in 4 DEG C of refrigerator cold-storages, overnight.3000g/min is centrifuged 20min,
Sediment is freeze-dried, obtain final product kelp raw polysaccharide.Thick many candies are made into 25% aqueous solution, ethanol to system ethanol is added
Volume fraction is 60%, and repeating precipitation must refine laminarin three times.
Embodiment 2
Kelp processing leftover bits and pieces is raw material, and first dries pulverizing takes appropriate Kelp Powder, adds full-automatic hydrolysis reactor, plus
Enter certain volume (solid-liquid ratio 1g：0.1mol/L 40ml), pH6.0 citric acid-sodium citrate buffer solution, add compound after stirring evenly
Enzyme by Kelp Powder quality for 1g based on, the addition of cellulase is 0.005g, hemicellulase 0.007g, pectase 0.005g and
Proteinase-10 .012g；
Regulation pH value is 6.0,50 DEG C, digests 3.5h, and heat up the enzyme that goes out, and takes out hydrolyzate, and suction filtration removes precipitation and insoluble matter,
It is 65% to add ethanol to system volume fraction of ethanol to supernatant, in 4 DEG C of refrigerator cold-storages, overnight.3500g/min is centrifuged
20min, sediment is freeze-dried, obtain final product kelp raw polysaccharide.Thick many candies are made into 30% aqueous solution, ethanol to body is added
It is that volume fraction of ethanol is 70%, repeating precipitation must refine laminarin three times.
Embodiment 3
Kelp processing leftover bits and pieces is raw material, and first dries pulverizing takes appropriate Kelp Powder, adds full-automatic hydrolysis reactor, plus
Enter certain volume (solid-liquid ratio 1g：0.1mol/L 45ml), pH6.0 citric acid-sodium citrate buffer solution, add compound after stirring evenly
Enzyme by Kelp Powder quality for 1g based on, the addition of cellulase is 0.006g, hemicellulase 0.008g, pectase 0.006g and
Proteinase-10 .014g；
Regulation pH value is 6.5,55 DEG C, digests 4h, and heat up the enzyme that goes out, and takes out hydrolyzate, and suction filtration removes precipitation and insoluble matter, to
It is 70% that supernatant adds ethanol to system volume fraction of ethanol, in 4 DEG C of refrigerator cold-storages, overnight.4000g/min is centrifuged 20min,
Sediment is freeze-dried, obtain final product kelp raw polysaccharide.Thick many candies are made into 35% aqueous solution, ethanol to system ethanol is added
Volume fraction is 80%, and repeating precipitation must refine laminarin three times.
The data test of embodiment 4 compares
Using laminarin obtained in the method for the present invention, with the extraction method laminarin such as Hot water extraction and ultrasonic wave
Yield, is compared as follows table：
Comparing of several extracting methods to laminarin yield
Embodiment described above is merely to illustrate technological thought of the invention and feature, in the art its object is to make
Technical staff it will be appreciated that present disclosure and implementing according to this, it is impossible to patent model of the invention is only limited with the present embodiment
Enclose, i.e., equal change or modification that all disclosed spirit is made still fall in the scope of the claims of the invention.
Claims (4)
1. the multiplex-enzyme extraction method of a Polysaccharides From Laminaria Japonica, it is characterised in that comprise the following steps：
(1) with kelp processing leftover bits and pieces as raw material, first dry, crush, take Kelp Powder, full-automatic hydrolysis reactor is added, by material
Liquor ratio 1g：35-45ml adds 0.1mol/L, and the citric acid-sodium citrate buffer solution of pH6.0 adds complex enzyme after stirring evenly, and is combined
Enzyme is made up of cellulase, hemicellulase, pectase and protease, by Kelp Powder quality for 1g based on, the addition of cellulase
Measure is 0.004-0.006g, hemicellulase 0.006-0.008g, pectase 0.004-0.006g and proteinase-10 .01-
0.014g；
(2) regulation pH value is 5.5-6.5,45-55 DEG C, digests 3-4h, and intensification is gone out enzyme, takes out hydrolyzate, suction filtration remove precipitation and
Insoluble matter；
(3) it is 65%-75% to add ethanol to system volume fraction of ethanol to supernatant, in 4 DEG C of refrigerator cold-storages, overnight,
3000g-4000g/min is centrifuged 20min, and sediment is freeze-dried, obtains final product kelp raw polysaccharide；
(4) Thick many candies are made into the aqueous solution that mass concentration ratio is 25%-35%, addition ethanol to system volume fraction of ethanol is
60%-80%, repeating precipitation must refine laminarin three times.
2. the multiplex-enzyme extraction method of laminarin according to claim 1, it is characterised in that comprise the following steps：
(1) with kelp processing leftover bits and pieces as raw material, first dry, crush, take Kelp Powder, full-automatic hydrolysis reactor is added, by material
Liquor ratio 1g：40ml adds 0.1mol/L, and the citric acid-sodium citrate buffer solution of pH6.0 adds complex enzyme, complex enzyme after stirring evenly
Be made up of cellulase, hemicellulase, pectase and protease, by Kelp Powder quality for 1g based on, the addition of cellulase
It is 0.005g, hemicellulase 0.007g, pectase 0.005g and proteinase-10 .012g；
(2) regulation pH value is 6.0,50 DEG C, digests 3.5h, and heat up the enzyme that goes out, and takes out hydrolyzate, and suction filtration removes precipitation and insoluble matter；
(3) it is 65% to add ethanol to system volume fraction of ethanol to supernatant, in 4 DEG C of refrigerator cold-storages, overnight, 3500g/min
25min is centrifuged, sediment is freeze-dried, obtain final product kelp raw polysaccharide；
(4) Thick many candies are made into the aqueous solution that mass concentration ratio is 30%, it is 70% to add ethanol to system volume fraction of ethanol,
Repeating precipitation must refine laminarin three times.
3. the multiplex-enzyme extraction method of laminarin according to claim 1, it is characterised in that the cellulase, half
Cellulase, pectase and protease are the cellulase Validase TRL of DSM company production, hemicellulase
Bakezyme HSP6000BG, pectase Klzrzyme150, protease ValidaseFP Concentrate.
4. laminarin obtained in the multiplex-enzyme extraction method of the laminarin as described in claim any one of 1-3.
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Cited By (5)
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| CN107722129A (en) * | 2017-09-18 | 2018-02-23 | 赵方铭 | A kind of method for extracting fucoidin from sea-tangle using ultrasonic wave |
| CN108997511A (en) * | 2018-09-07 | 2018-12-14 | 南阳理工学院 | One main laminaria active polysaccharide and preparation method thereof |
| CN109123493A (en) * | 2018-07-18 | 2019-01-04 | 广州市凯虹香精香料有限公司 | The preparation process of one main laminaria enzymolysis liquid |
| CN111419743A (en) * | 2020-03-03 | 2020-07-17 | 齐鲁工业大学 | Preparation method and application of vitis amurensis extract |
| CN112521430A (en) * | 2020-11-23 | 2021-03-19 | 自然资源部第三海洋研究所 | Large-scale preparation method of sulfated kelp oligosaccharide suitable for cosmetics |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
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| CN108997511A (en) * | 2018-09-07 | 2018-12-14 | 南阳理工学院 | One main laminaria active polysaccharide and preparation method thereof |
| CN111419743A (en) * | 2020-03-03 | 2020-07-17 | 齐鲁工业大学 | Preparation method and application of vitis amurensis extract |
| CN112521430A (en) * | 2020-11-23 | 2021-03-19 | 自然资源部第三海洋研究所 | Large-scale preparation method of sulfated kelp oligosaccharide suitable for cosmetics |
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Application publication date: 20170623 |