CN1229393C - Process for preparing low molecular weight fucidan by using microbial enzyme method - Google Patents
Process for preparing low molecular weight fucidan by using microbial enzyme method Download PDFInfo
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- CN1229393C CN1229393C CN 03112609 CN03112609A CN1229393C CN 1229393 C CN1229393 C CN 1229393C CN 03112609 CN03112609 CN 03112609 CN 03112609 A CN03112609 A CN 03112609A CN 1229393 C CN1229393 C CN 1229393C
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Abstract
The present invention utilizes microorganisms to prepare fucoidin enzyme, and the enzyme is utilized for further hydrolyzation to prepare fucoidin which has the low molecular weight. The technology comprises the following steps: step a, brown algae is used as raw material, the raw material is pulverized and sieved, and the raw material is lixiviated with water; when the temperature is from 40 to 100 DEG C, the raw material is stirred and lixiviated for 6 to 10 hours, and the solid content is removed in a centrifugation mode; filter liquid is collected and concentrated, starch is removed, precipitation is carried out with ethanol in a grading mode, precipitate is frozen and dried, and fucoidin powder is obtained; step b, enzymolysis reaction is carried out on the fucoidin; step c, after the reaction is finished, the temperature is raised, which leads the enzyme activity to be lost; step d, through separation of a gel chromatography molecular sieve and freeze drying operation, fucoidin which has the low molecular weight is obtained. The present invention leads the preparation of fucoidin enzyme to be easy and overcomes the problem that when a chemical method is utilized, harsh preparation conditions are required, for example, high temperature and high pressure are required, and energy is consumed. Simultaneously, the defects that the polymerization degrees of the fucoidin are inconsistent, the purification difficulty is high, and the yield is low existing in the chemical method are avoided. The enzymic degradation is also good for protecting side chain redicals which contain sulfur and other branched chains.
Description
Technical field
The present invention relates to the fucoidin in the brown alga is raw material, utilizes microorganism fucoidin enzyme enzyme process to prepare low weight molecular fucoidan technology.
Background technology
Fucoidin belongs to different poly carbohydrate, and its main component is the L-Fucose, and its linear backbone is that rock algae pyranoid ring is with α-1, the 3-glycosidic link is formed by connecting, wherein also be mingled with other carbohydrates, as galacturonic acid or glucuronic acid etc., because of material source different.Sulfation takes place at the 4-O base in this sugar usually, also can be on other site, and therefore, this polysaccharide belongs to the sulfur-bearing polysaccharide.Find in the animal experiment in early days that fucoidin is easy to generate antigenicity and obvious toxic and side effects, if through partial hydrolysis, the viscosity of solution can reduce, and can reduce this sugar issuable antigenicity and toxic side effect in application.The low weight molecular fucoidan of molecular weight size appropriateness can keep the physiologically active of former polysaccharide, and some also has specific physiologically active.Outside the Pass the biologic activity of low weight molecular fucoidan is removed and to be had with the molecular weight size, also relevant with sulphur content and sulfur-containing group site and branched structure.
The acquisition of low weight molecular fucoidan is many based on chemical degradation method (acid hydrolyzation) at present, adopt chemical process to carry out partial hydrolysis, working condition requires harsh (high temperature, high pressure, power consumption), and the sacchariferous polymerization degree is not of uniform size, the purifying difficulty is big, and yield is low.Adopt enzymic degradation, then can overcome these shortcomings, enzymic degradation also helps some sulfur-containing side chain groups of protection and other side chain simultaneously.Fucoidin can produce the fucoidin of lower molecular weight by the fucoidin enzymic hydrolysis that marine microorganism and shellfish produce, and further passes through the hydrolysis of fucosidase, produces Fucose.Japan extracts the fucoidin enzyme and prepares and have the physiologically active low weight molecular fucoidan and succeed from the shellfish hepatopancreas, adopt the fucoidin enzyme that extracts purifying in the scallop hepatopancreas as Japanese scholar, successfully from being 480000 fucoidin, molecular weight produces the active fucoidin of molecular weight about 50000, but, because hepatopancreas collection and storage are comparatively difficult in the shellfish course of processing, thereby cause the preparation difficulty of this kind enzyme big, limited its application in suitability for industrialized production.
The research work of China's fucoidin is less, only has several pieces of articles to relate to its extraction and sepn process at present.At present, also do not retrieve the report for preparing low weight molecular fucoidan with microorganism fucoidin enzyme enzyme process both at home and abroad.
Technology contents
Technical task to be solved by this invention is to utilize microorganisms producing fucoidin enzyme, utilizes the further hydrolytic action of this enzyme to produce low weight molecular fucoidan.
For solving above-mentioned technical task, the present invention has adopted following technical matters:
A, be raw material with the brown alga, with raw material pulverizing, the back flooding of sieving, temperature in the time of 40～100 ℃, stirring and leaching 6～10 hours, centrifugal removal solid substance is collected filtrate, is concentrated, and adopts α-Dian Fenmei to remove starch, ethanol precipitation, the throw out lyophilize obtains the fucoidin powder;
B, above-mentioned fucoidin is implemented enzyme digestion reaction, temperature is in the time of 30～50 ℃, and the reaction times was controlled in 2 hours, and the pH value is controlled at pH6～pH8;
Enzyme in c, the enzyme digestion reaction is respectively by the enzyme based on the fucoidin enzyme solid-state or that the liquid state fermentation method makes by microorganism; The microbiological culture media raw material that is used for producing the fucoidin enzyme comprises one or more of brown alga and extract and agriculture organic waste materials;
Heat after d, enzyme digestion reaction finish and make enzyme forfeiture alive;
E, enzymolysis solution obtain low weight molecular fucoidan through gel chromatography molecular sieving, lyophilize.
The raw material of above-mentioned preparation low weight molecular fucoidan during with flooding the lixiviate ratio of raw material and water be 1: 7～15, lixiviate, filtration, add α-Dian Fenmei after concentrating, temperature of reaction was 1～2 hour 40～60 ℃ of reaction times, and the α-Dian Fenmei amount is controlled at the 40Iu/ml concentrated solution.
The microbial fermentation temperature of preparation fucoidin enzyme is controlled between 25～32 ℃, and the time was controlled at 12～48 hours, obtains crude enzyme liquid through separating again.
The brown alga that is used to produce fucoidin comprises many capsules black wrack, sea-tangle, Sargassum fusiforme etc.
The temperature that is used to produce fucoidin is controlled at 70～90 ℃.
The fucoidin concentration of enzyme digestion reaction is controlled at≤and 10%.
Described enzyme digestion reaction temperature is at 35～45 ℃, and the reaction back temperature that finishes is heated to 100 ℃ and made the enzyme forfeiture of living.Fucoidin enzyme amount is controlled at≤the 40Iu/g substrate in the described enzyme digestion reaction.
Can prepare any one section low weight molecular fucoidan of fucoidin molecular weight ranges in 900～50000 dalton by technique scheme, the physiologically active that can keep former polysaccharide, also has specific physiologically active simultaneously, what the present invention adopted is to prepare fucoidin with microorganisms producing fucoidin enzyme, i.e. the fucoidin enzymic hydrolysis that produces by marine microorganism produces the fucoidin of lower molecular weight.Compared with prior art, solved on the one hand and adopted scallop to prepare that hepatopancreas in the fucoidin enzyme process is difficult to gather and difficulty that storage, transport point run into, made the preparation of fucoidin enzyme be more prone to; On the other hand, overcome the harsh preparation condition that utilizes chemical method to need high temperature, high pressure, power consumption, avoided simultaneously that the sacchariferous polymerization degree of chemical method is not of uniform size, the purifying difficulty reaches the low defective of yield greatly; Enzymic degradation also helps some sulfur-containing side chain groups of protection and other side chain.
The production of enzyme is easy to control among the preparation technology of low weight molecular fucoidan disclosed by the invention, and turnout is big.
Summary of drawings
Fig. 1 is preparation technology's schema of fucoidin enzyme;
Fig. 2 is preparation technology's schema of low weight molecular fucoidan.
Embodiment
1, the preparation of fucoidin enzyme
The preparation of fucoidin enzyme is referring to Fig. 1, and specific embodiment is as follows:
1. the raw materials for production of fucoidin enzyme
The microorganism culturing raw material that is used to produce the fucoidin enzyme mainly utilizes brown alga (many capsules black wrack, sea-tangle, Sargassum fusiforme etc.) and extract and agriculture organic waste materials (wheat bran).Substratum all can with single or composite parts.
2. the preparation of fucoidin enzyme
The zymin that is used to produce low weight molecular fucoidan mainly is the fucoidin enzyme, and it can be produced by solid-state or liquid state fermentation method respectively by microorganism.Temperature is controlled between 25～32 ℃, and the time was controlled at 12～48 hours, obtains crude enzyme liquid through separating again.
With the thalassiomycetes is example, comparatively it is desirable to temperature and is controlled under 28 ℃ of conditions and cultivated 24 hours, through separating, obtains crude enzyme liquid again.
Data that record under all temps and the time conditions such as following table one, two:
Table one
| Temperature | Time (hour) | Enzyme activity (the dried substratum of unit: Iu/g) |
| 25℃ | 24 | 24.5 |
| 28℃ | 24 | 32.5 |
| 30℃ | 24 | 27 |
| 32℃ | 24 | 17.5 |
Table two
| Temperature | Time (hour) | Enzyme activity (the dried substratum of unit: Iu/g) |
| 28℃ | 12 | 17.5 |
| 28℃ | 24 | 33 |
| 28℃ | 36 | 24.3 |
| 28℃ | 48 | 18 |
2, the preparation of fucoidin
The preparation of fucoidin is referring to Fig. 2, and specific embodiment is as follows:
1. the processing of raw material
Above-mentioned raw materials for production at first should screen and remove the composition that goes mouldy, and kept dry, and after the classified again pulverizing, it is standby to cross 360 mesh sieves.
2. fucoidin preparation
With the raw material of pulverizing and sieving, use flooding, during 70～90 ℃ of temperature, stirring and leaching 6～10 hours.Centrifugal removal solid substance is collected filtrate, concentrates, and adds α-Dian Fenmei, and temperature of reaction was 1～2 hour 40～60 ℃ of reaction times, and the α-Dian Fenmei amount is controlled at the 40Iu/ml concentrated solution, and amylase is removed starch, ethanol precipitation.The throw out lyophilize obtains the fucoidin powder.
When the raw material flooding of above-mentioned preparation fucoidin, 70～90 ℃ of temperature, stirring and leaching 6～10 hours specific embodiment such as table three:
Table three
| Temperature | Extraction time (hour) | The yield of sugar |
| 40℃ | 10 | 0.5698g/20g sea-tangle ≈ 2.8% |
| 60℃ | 8 | 0.5892g/20g sea-tangle ≈ 2.9% |
| 80℃ | 10 | 0.6823g/20g sea-tangle ≈ 3.4% |
| 100℃ | 10 | 0.6845g/20g sea-tangle ≈ 3.4% |
3, enzyme process prepares low weight molecular fucoidan, referring to Fig. 2.
1. enzyme digestion reaction
The enzyme digestion reaction condition is: 40 ℃ of temperature, and pH6.5, the reaction times was controlled in 2 hours, was foundation with the product polymerization degree.Heat to 100 ℃ after reaction finishes, enzyme is lived lose.
2. the preparation of low weight molecular fucoidan
Through the gel chromatography molecular sieving, obtain the low weight molecular fucoidan of different stage, lyophilize obtains powder.
Claims (8)
1, a kind of enzyme process prepares low weight molecular fucoidan technology, and this technology may further comprise the steps:
A, be raw material with the brown alga, with raw material pulverizing, the back flooding of sieving, temperature in the time of 40～100 ℃, stirring and leaching 6～10 hours, centrifugal removal solid substance is collected filtrate, concentrated, ethanol precipitation, the throw out lyophilize obtains the fucoidin powder;
B, above-mentioned fucoidin is implemented enzyme digestion reaction, temperature is in the time of 30～50 ℃, and the reaction times was controlled in 2 hours, and the pH value is controlled at pH6～pH8;
Enzyme in c, the enzyme digestion reaction is respectively by the enzyme based on the fucoidin enzyme solid-state or that the liquid state fermentation method makes by microorganism; The microbiological culture media raw material that is used for producing the fucoidin enzyme comprises one or more of brown alga and extract and agriculture organic waste materials;
Heat after d, enzyme digestion reaction finish and make enzyme forfeiture alive;
E, enzymolysis solution obtain low weight molecular fucoidan through gel chromatography molecular sieving, lyophilize.
2, enzyme process according to claim 1 prepares low weight molecular fucoidan technology, it is characterized in that: the raw material of described preparation low weight molecular fucoidan during with flooding the lixiviate ratio of raw material and water be 1: 7～15, lixiviate, filtration, add α-Dian Fenmei after concentrating, temperature of reaction was 1～2 hour 40～60 ℃ of reaction times, and the α-Dian Fenmei amount is controlled at the 40Iu/ml concentrated solution.
3, enzyme process according to claim 1 prepares low weight molecular fucoidan technology, it is characterized in that: the microbial fermentation temperature of preparation fucoidin enzyme is controlled between 25～32 ℃, and the time was controlled at 12～48 hours, obtains crude enzyme liquid through separating again.
4, enzyme process according to claim 1 prepares low weight molecular fucoidan technology, it is characterized in that: the brown alga that is used to produce fucoidin comprises many capsules black wrack, sea-tangle, Sargassum fusiforme.
5, enzyme process according to claim 1 prepares low weight molecular fucoidan technology, it is characterized in that: the temperature in producing the fucoidin process during with the flooding brown alga is controlled at 70～90 ℃.
6, enzyme process according to claim 1 prepares low weight molecular fucoidan technology, it is characterized in that: the fucoidin concentration of enzyme digestion reaction is controlled at≤and 10%.
7, enzyme process according to claim 1 prepares low weight molecular fucoidan technology, it is characterized in that: described enzyme digestion reaction temperature is at 35～45 ℃, and the reaction back temperature that finishes is heated to 100 ℃ and made the enzyme forfeiture of living.
8, prepare low weight molecular fucoidan technology according to claim 1 or 3 described enzyme process, it is characterized in that: fucoidin enzyme amount is controlled at≤the 40Iu/g substrate in the described enzyme digestion reaction.
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Families Citing this family (11)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN100457783C (en) * | 2005-08-25 | 2009-02-04 | 北京市绿泉科技发展有限公司 | Seaweed product for agriculture and preparation method of fucoidin |
| CN100429318C (en) * | 2005-10-28 | 2008-10-29 | 大连水产学院 | Process for preparing fucoidan by enzymatic hydrolysis of brown algae |
| CN101012286B (en) * | 2006-11-23 | 2010-05-19 | 郑婵颖 | Process for producing water soluble seaweed powder |
| US8748595B2 (en) * | 2007-02-23 | 2014-06-10 | Baxter International Inc. | Methods for fucoidan purification from sea weed extracts |
| CN101999517B (en) * | 2010-10-28 | 2013-11-27 | 北京资源亚太饲料科技有限公司 | Preparation method and application of low-molecular weight seaweed polysaccharide trace element chelate |
| CN102030838B (en) * | 2010-10-30 | 2012-08-01 | 中国海洋大学 | High-pressure water vapor degradation method for marine sulfated polysaccharides |
| CN106832041A (en) * | 2017-04-07 | 2017-06-13 | 安徽天安生物科技股份有限公司 | A kind of method that biologic enzymolysis method extracts fucoidin |
| CN107014657A (en) * | 2017-05-24 | 2017-08-04 | 合肥学院 | A kind of quick, easily fine and close der Pilz flaking method |
| CN109055460B (en) * | 2018-09-13 | 2021-06-29 | 青岛创通生物科技有限公司 | Low molecular weight fucoidin and application thereof in preparing cosmetics |
| CN113599302B (en) * | 2021-08-23 | 2024-04-16 | 无限极（中国）有限公司 | Application of rhodophyta polysaccharide as ceramide expression promoter and preparation method thereof |
| CN114532500A (en) * | 2022-02-18 | 2022-05-27 | 福建农林大学 | Preparation method of active kelp matter |
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