JPS6157520A - Solution having high fucoidan purity, or preparation of fucoidan - Google Patents

Abstract

PURPOSE:To obtain the titled solution by filtering and removing impurities from a fucoidan-containing solution or low-viscosity fucoidan-containing solution using an ultrafiltration membrane. CONSTITUTION:A fucoidan-containing solution obtained from algae containing fucoidan (e.g. brown algae such as tangle, wakame seaweed, gulfweed, etc.), or a fucoidan-containing solution having a viscosity decreased by beta-1,3-glucanase and/or protease, is treated with an ultrafiltration membrane (having a fractionation molecular weight of 5,000-60,000, preferably 10,000-15,000 and/or 60,000- 100,000). The impurities are filtered and removed by this process to obtain a solution having high fucoidan purity. If necessary, the solution is dried as it is, or fucoidan is coagulated and precipitated by adding a polar solvent to a concentration of 40-55(v/v)%, and the precipitated fucoidan is dried to obtain high- purity dried fucoidan.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention involves separating impurities from a solution containing 7-coidan obtained from seaweed containing fucoidan using an ultraf filter membrane,
A method for obtaining a solution (hereinafter abbreviated as a high-purity fucoidan solution) in which the fucoidan content (substantially determined by the fufus content, which is the main constituent sugar of fucoidan) is increased relative to the soluble i solid content in a fucoidan-containing solution. provide,
Furthermore, by creating a solution with a high fucoidan concentration,
The present invention relates to a highly pure solution of fucoidan or a method for producing fucoidan, which provides advantages such as savings in coagulation and precipitant and direct drying of the solution, such as savings in drying energy.

Conventionally, 7 coidan has been recognized to have medicinal effects such as antilipidemic activity and antilipidemic activity, and various production and purification methods have been proposed. For the purpose of obtaining a concentrated fucoidan solution, there are examples in which a vacuum concentration method has been used, but as seen in the comparative example of the present application, the purity of the isolated fucoidan decreases. Other methods include precipitation separation of trivalent metal ions, etc., redissolution to liberate and remove trivalent metal ions, etc., and re-precipitation with a polar solvent such as methanol for concentration, purification, and isolation. However, the solution containing fucoidan 7 has a high viscosity similar to fresh egg white, making it difficult to work with. It has been difficult to completely prevent contamination by metal salts.

As a result of various studies on methods to solve the above-mentioned drawbacks, the present inventor has discovered that fucoidan can be obtained by filtering and removing impurities from a fucoidan-containing solution obtained from fucoidan-containing seaweed using an ultrafiltration membrane. A highly pure solution can be obtained, and if necessary, for example, if the fucoidan-containing solution has high viscosity and poor workability, β-1,3 glucanase and/or protease can be dissolved in the fucoidan-containing solution. By temporarily removing impurities from a fucoidan-containing solution whose viscosity has been lowered by using an ultra-f filtration membrane, a solution with high purity of 7-coidan can be easily obtained. By adding or adding water to a fucoidan-containing solution and temporarily removing impurities using an ultrafiltration membrane, a solution with even higher purity of fucoidan can be obtained. As the outer membrane, fractionated molecules ff1s, oo.

to 10,000 to 15,000, preferably 10,000 to 15,000
0 and/or 60,000 to 100,000 to remove impurities by passing through and obtain a solution with high fucoidan purity. impurities are removed by filtration, and the fucoidan is dried using a conventional method from the resulting highly pure fucoidan solution.
When collecting the material, either dry it as it is, or use a polar solvent concentration of 40 to 55% (v/v') to coagulate and precipitate the fucoidan, dry the precipitated fucoidan, and collect highly pure dried fucoidan. They discovered that it is possible and completed the present invention.

Next, embodiments of the present invention will be explained.

The seaweed used as a raw material is not particularly limited as long as it contains fucoidan, but examples of seaweed containing a large amount of fucoidan include kelp, wakame, solderwara, hibamata,
Brown algae such as Arame, Kajime, and Letsonia may be used. As the extraction solvent, water, alkaline solution, acid solution or +
2. There are solutions containing acetone, methanol, ethanol, etc. at a low concentration of 40% (v/v) or less in these solvents, and these solvents are added at least 4 times the dry weight of the seaweed.
Normally, extract using 5 to 30 times the amount, or
A dried product of 7-coidan of purity may be dissolved in an extraction solvent and used as a fucoidan-containing solution. Note that it is desirable to use the fucoidan-containing solution as a clarified solution by passing through, centrifuging, or the like. In addition, it is desirable to remove contaminants such as protein alginic acid from the resulting fucoidan-containing solution from the viewpoint of membrane lifespan, deterioration of fucoidan purity, etc. As a removal method, fractionated molecular nails = lF0.000-100,000
It can be removed by using an ultrafiltration membrane, or by using a known precipitant such as trichloroacetic acid, isoelectric point, iontophoresis, etc.

Ultrafon membranes include polyacrylonitrile, polyamide, polysulfone, and ceramic membranes, and modulus membranes include flat membranes, periosteum, spiral windings, etc., which can be selected as appropriate. This is preferable from the viewpoints of humidity and humidity, and can be carried out satisfactorily by using a spiral module (ULVAC). Filtration pressure is 0.5 k! 7/ctA or more usually 1-7 kg/
cnl is sufficient. The humidity is preferably 70° C. or lower from the viewpoint of fucoidan depolymerization, but it is not necessary to limit the humidity to 70° C. or lower since the transient rate increases if the temperature is high. Next, some fucoidan-containing solutions have low fucoidan purity and concentration. For example, the content and viscosity of washing wastewater such as letunia in the production of alginic acid is very low, so the molecular weight cut-off is 60,000 to 100,000.
After fucoidan is recovered into liquid F using a 0.0 ultrafiltration membrane, a solution with high fucoidan purity can be produced by performing ultrafiltration with a molecular weight cutoff of 5.00 o=o, OOO. Usually, for konbu, hibamata, wakame, kajime, arame, etc., which have a high 7-koidan content, extraction with high viscosity is performed by -times of extraction, several times of repeated extraction (see Example 6), or countercurrent extraction. A liquid is obtained. In the case of the latter high viscosity liquid, the molecular weight cut-off is 60,000 to 100,
000 and s, ooo~.

By performing filtration twice, io and ooo, the fucoidan purity can be further increased. The viscous fucoidan-containing solution has viscoelasticity similar to fresh egg white, and when the viscosity is measured with a B-type viscometer, due to the Weisenberg effect,
It rolls up along the axis of rotation and cannot be measured. The concentration is usually 0.02% or more as fucose. Such a highly viscous 7-koidan-containing solution is difficult to concentrate by ultrafiltration and has poor workability;
Alternatively, when protease is dissolved, the viscosity decreases rapidly. Enzyme preparations containing this enzyme as the main component include β-1
- Since chitalase is easily available as the 3-glucanase and protease "Amano" as the protease, it can be easily carried out by obtaining these enzymes. The practice of the present invention is not limited to the enzyme preparations described above, and the purpose can be achieved even by using other enzyme preparations or other enzyme agents containing the enzymes described above as impurities, and the purpose of the present invention is not limited to the above-mentioned enzyme preparations. Organisms, ground products, powders, or enzyme extracts can also be used. It is effective to use the enzyme in an amount of 0.001% by weight or more (hereinafter simply referred to as %) based on the dry weight of raw seaweed, and usually 0.01 to 1% is sufficient. Of course, even if it is used in an amount of 1% or more, no inconvenience will occur.

The enzyme may be added in such a way that the added enzyme is dissolved in a solution containing 7-coidan extracted from fucoidan-containing seaweed. For better understanding, to explain further, when extracting fucoidan, when adding and dissolving the enzyme, there are two methods: adding and mixing it to the algae and/or its pieces and/or its powder, and adding and dissolving it in the extraction solvent. The method of adding and dissolving during extraction, the method of adding and dissolving after extraction, the method of separating fucoidan-extracted seaweed residue or concentrating this extract in vacuum, separating the fucoidan fraction by fucoidan precipitation method or adsorption method. There is a method of dissolving the fucoidan by adding it to the fucoidan solution obtained by drying it and then redissolving it, or by drying it once and then redissolving it. In short, if the added β-1.3 glucanase and/or protease are dissolved in the fucoidan solution, the purpose of reducing viscosity can be achieved. This method is effective as the fucoidan precipitation/adsorption method described above.

Enzyme reaction conditions are not particularly limited, but the concentration is 0 to 70°C.
1. Preferably, the reaction time at 30 to 70° C. is 5 minutes or more, preferably 10 minutes to 2 hours.

The decrease in viscosity is more pronounced in higher viscosity liquids. The degree of viscosity reduction could not be measured by normal methods due to the Weisenberg effect, so we measured it using a large hole ($ 1.5 mm).
When comparing viscosity using 100C and measuring the time for 10cc to be sucked in and 5cc to flow out (hereinafter, the viscosity measured by this method is referred to as comparative viscosity), the viscosity may normally reach π or less of the original viscosity. be.

A decrease in the viscosity of a fucoidan solution also occurs depending on temperature and/or pH. For example, it is possible to reduce the viscosity of a highly viscous liquid by heating it at 100°C for 30 minutes or by leaving it at a pH of 2 or less, so even without using the enzyme, the viscosity can be reduced by such physical conditions. A solution with high fucoidan purity can also be produced from the reduced solution. However, in the case of a fucoidan-containing solution whose viscosity has been lowered due to physical and chemical conditions, even if it is flocculated at an ethanol concentration of 60% (v/'v), it will be in the form of small fluffy lumps or light precipitates rather than flocculation. If such a small amount of precipitate is produced, it becomes even more difficult to precipitate. However, the fucoidan-containing solution whose viscosity was lowered using the enzyme had an ethanol concentration of 45% (v/
v) is a state of tangled filaments or heavy agglomerated precipitates in the form of lumps, which is similar to the state of precipitation from a highly viscous fucoidan-containing solution to which no enzyme is added. Furthermore, the separated and collected fucoidan had the following content in terms of L-7 course content:
L-7 with high purity and recovered as fucoidan
Since the course yield increases, it is presumed that the viscosity decrease is not due to the depolymerization of 7-fidan but due to the decomposition of the high viscosity imparting substance contained as an impurity.

When a treated or untreated solution containing 7-coidan is ultrafiltered as described above, impurities such as minerals, amino acids, sugars, etc. contained in the solution are removed together with the f solution, and 7-coidan in the solution is It remains in the containing solution and gradually becomes a highly concentrated solution, increasing the fucoidan content relative to the total soluble solids. That is, it is considered that the purity of 7-koidan in the solution increases. Furthermore, fucoidan-containing solutions, especially ultra-d
Water is added to the highly purified fucoidan solution concentrated by filtration to reduce the concentration of impurities, and then ultrafiltration is performed again, or water is added to sequentially dilute the impurities while concentrating by extraocular filtration. By carrying out this process, it is possible to obtain a solution with an even higher purity of fucoidan, and by freeze-drying, vacuum concentration drying, foam drying, or spray drying such a high-purity solution, a high-purity 7-coidan dried product can be obtained. can be obtained.

Furthermore, when collecting fucoidan from a solution with increased fucoidan purity by the coagulation precipitation method, a polar solvent such as methanol or ethanol that can be easily removed by drying or the like may be added. The concentration of the polar solvent is preferably 40 to 55% (v/v) from the viewpoint of economy and fucoidan purity.

Next, examples will be shown to facilitate understanding of the present invention.

In addition, the L-7 course measurement in the examples was performed by the cysteine sulfuric acid method or gas chromatography.

Example 1 500 # of finely chopped Rishiri konbu (moisture 13%) and 10 parts of water
1 was added, fucoidan was extracted at 40°C for 5 hours, the extract was separated, water of 51 was further added to the remaining seaweed residue, fucoidan was extracted at 40°C for 14 hours, and the seaweed extraction residue was separated in the same manner. Further, 51 water was added and fucoidan was extracted at 40°C for 5 hours, and the three fucoidan extracts were combined to yield 18.311 (soluble solids content 1.44%, 7 courses 0.
014%, the purity of L-7 (hereinafter referred to as L-) based on the soluble solid content.
Make a clear F solution, add 1.8 to 9 (konbu dried matter 42.7
91) was fractionated, and the fractionated molecular weight was 15,00.
It was concentrated through an ultrafiltration machine equipped with a polysulfone membrane of 480# (soluble solids content 1.67%,
．． A concentrated solution with a purity of 2.40% (L-7 course purity 2.40%) was obtained. (At this point, the viscosity of the concentrate increased and pump circulation of the concentrate became impossible.) This concentrate 1. :99.5%
Add 3861 nl of (v/v) ethanol, stir and mix for 3 hours to separate 7 coidan aggregates, add 15 mA of 99.5% (v/v) ethanol to the separated fucoidan aggregates, and dehydrate. After that, collect 7 fidans and get 60
℃, dried for 16 hours, fucoidan 0.54F (L-7
The course yielded 3.4.68%). The dry matter yield for kelp is 1
．． The yield was 26% (L-7 course yield 0.44%).

Comparative Example 1 1.8 kg of the extract obtained in Example 1 was concentrated in vacuum to give 463 g (soluble solids 5.60%, L-7 course 0).
．． 054%, L-7 course purity 0.96%) was obtained. To this concentrated solution, 358 mJ of 99.5% (V/V) ethanol was added and stirred for 3 hours to separate the fucoidan aggregates.
% (v//) of ethanol after processing and dehydration at 60℃
, dried for 16 hours, 0.81 g (L-7 course 20.
66%). The yield per dried kelp is 1 fucoidan.
．． The yield was 89% (L-7 course yield 0.39%).

Comparison Example 2 99.5 to 1.8 ky of the extract obtained in Example 1
% (v/v) x Tanur L451 mli processing, 3
Stir for hours to aggregate fucoidan, separate the aggregates,
Thereafter, in the same manner as in Example 1, dried fucoidan o, 53f
1 (7 courses 34.75%) was obtained. The yield per dry matter of kelp is 7 coidan 1.24% (L-fucose yield 0.
43%).

The above experiment shows that when the extract obtained by removing 7-coidan from the extract is used as a seasoning, it is most preferable to use ethanol to separate fucoidan, Example 1 and 7-coidan purity (L-7 course content) The L-7 course yield is approximately the same, but approximately 4 times as much ethanol is required. or,
In Comparative Example 1, the amount of ethanol used was almost the same as in Example 1, but the L-fucose content was lower;
Moreover, the L-fucose yield is decreasing. Also, Example 1
The L-fucose purity relative to the soluble solid content in the L-fucose concentrate obtained in Comparative Example 1 was 2.4 in Example 1.
%, whereas in Comparative Example 1 it was 0.96%,
Purity is increased by 2.5 times by ultrafiltration.

Example 2 Add 0.1 N hydrochloric acid 21 containing 2 g of pepsin to shredded Aran 300I, extract at 40'C for 16 hours, neutralize with caustic soda, and extract 1.4 kg of neutralized liquid (L-7 course o
, o o s%). This was subjected to ultrafiltration in the same manner as in Example 1 to obtain 150! i (L-7 course 0
．． A concentrated solution of 0.044%) was obtained. Eta for this / /'
Add 115 at to make ethanol concentration 45% (V/V)
Example 1
Fucoidan 0.28.9 (L-7 course 2)
3.57%).

Comparative Example 3 An extract neutralized solution of 1.38 kj7 was obtained in the same manner as in Example 2. This was concentrated in vacuo to obtain 170 g of concentrated liquid. To this, 12 o 1 nl of ethanol was added to make a fucoidan aggregate, and the following procedure was carried out in the same manner as in Example 2.
．． It was 9.5' (7 feet 1.81%).

Although the alane used in the above experiment had a low fucoidan content, it showed the same tendency as that obtained in Example 1 and Comparative Example 1, and in terms of saving flocculant, ultrafiltration concentration was more effective than simple concentration. The results showed that the purity of fucoidan and the yield of L-fucose were improved.

Example 3 Cut 2kg of i11 grade Atsuba 1st grade konbu into thin strips and add 50
Add 301 °C of water and extract for 3 hours with occasional stirring.
Separate from the residue and centrifuge to obtain a clear extract 21.
4ki9 (soluble solids 3.54%, fucose 0.08
%, comparative viscosity 140 sec/5 cc) was obtained. In addition to the viscosity of fresh egg white, the above extract has rubber-like viscoelasticity, and even if it is passed through an ultrafilter, the liquid cannot be circulated through a pump.
It was determined that θ reduction using an actual machine was also impossible. Furthermore, when the viscosity was measured using a B-type viscometer, the so-called Weisenberg effect, in which the material wrapped around the rotor and rolled up along the rotor shaft, was noticeable, and the viscosity could not be measured. There, a large hole (961.5mm)
Aspirate l Q cc using a 10 cc pipette and
The time for cc outflow was measured and expressed as comparative viscosity. Further, L-fucose was measured by the cysteine sulfate method. Enzyme chitalase (β) is added to the above extract 4,000.9
Negative - 1.3 Glue force nase main body) 0.3g (relative to liquid 0.
015%) was added and allowed to react at 45°C for 190 minutes while stirring uniformly, and the viscosity decreased to 9.8 seconds/5 cc.

This low viscosity extract 2,000F has a molecular weight cutoff of 15,000
Concentrate using an ultrafilter equipped with a polysulfone membrane of
Add the low viscosity extract from the evening and continue filtration. When the total amount of P liquid from the extract 4,000.9 reaches 3,870.9, stop the filtration, take out the concentrate, and concentrate. Combined with the cleaning liquid, the total amount is 196. ! i+ (Soluble solids content 7.26%
, L-7 course 1.31%, L-7 course purity 18.0
A concentrated liquid with a comparative viscosity of 45 sec/5 cc) was obtained. 149 ml of 99.5% (V/V) ethanol was added to this 'eJ condensate, stirred and mixed for 3 hours, and the aggregated 7-coidan was separated.
) After adding 30ml of ethanol and dehydrating, heat at 60°C1.
Dry for 16 hours, 6.2F (L-fucose 39.56%
) dried fucoidan was obtained. The recovery rate of L-fucose in extract liquid 4.000.9 is 76.65%.

Example 4 Protease “Amano A” was used instead of chitalase in Example 3.
J4N was added and ultrafiltration was carried out in exactly the same manner as in Example 3, and the concentrate and the concentrate washing liquid were combined to a total of 220! A concentrated liquid of 15 cc (soluble solids content: 7.02%, L-7 course: 1.17%, L-7 course: 16.67%, comparative viscosity: 62 seconds, 15 cc) was obtained. This concentrate contains 99.5% (v/v)
The mixture was stirred for 3 hours, the aggregated fucoidan was separated, dehydrated with ethanol, and dried at 60°C for 116 hours.

The dry fucoidan obtained was 6.3.9 (L-7 course 38.63%). L- in extract 4,000#
The 7-course recovery rate is 76.05%.

Comparative Example 4 Chitalase (
After adding 0.3.9 (main component of β-1.3 glue) and stirring for 190 minutes at 45°C, the viscosity was 9.8.
sec/5 cc. To this, 99.5% (v/v
) 1,580cc of ethanol was added, and the ethanol concentration was 45% (V/V) (Examples 2 and 3 also had an ethanol concentration of 4.
5% (V/V)), the aggregated fucoidan was separated, and then dehydrated and dried in the same manner as in Example 3.
46 F7 coidan (L-fuco-7, (35,10%
) was obtained. The recovery rate of L-fucose in 2.0 OoI of the extract is 75.90%.

Comparative studies of Examples 3 and 4 and Comparative Example 4 show that in order to obtain an ethanol concentration of 45% (V'V) for fucoidan flocculation, Examples 3 and 4 required one of the extracts and the fucose content of the recovered fucoidan. It was found that the purity was high, and that ultrafiltration had a significant effect.

Example 5 Extract liquid of Example 3 3. Chitalase 0.5 to OOOg
1, and after mixing, add protease [Amano AJ3
17 was added and reacted at 45°C for 2 hours, resulting in a comparative viscosity of 5.
A low viscosity extract of 8 seconds/5 cc was prepared, and using the same ultrafilter as in Example 3, the low viscosity extract 2. ooooo,
P was concentrated, and 1,000 J of the low viscosity extract was further concentrated to obtain 2,900 J of the f solution. ! i', add 30.0.9 I of water to the low viscosity liquid charge tank, continue filtration, wash the concentrated liquid until the total amount of lachrymal fluid reaches 3.200 fI, and then add 300 I of water to the charge tank. Filter the total amount of P liquid 3.
It was washed until it reached 460.9. In the same way, using 300 g of water, the water was washed 5 times in total, the washed concentrate was taken out, and the concentrated liquid adhering to the container was further washed out with water. , L-7 course purity 45.02%) was obtained. The obtained water-washed concentrate was dried using a freeze-dryer @4.2.9 (L)
Fucoidan containing -Fucose 45, 31%) was obtained. The recovery rate of L-fucose in the extract was 79.3%. This experiment shows that fucoidan of high purity can be obtained in good yield by concentrating the extract by ultrafiltration and washing the concentrated liquid with water.

Example 6 Leaves of Letsonia nigretuscens (hereinafter abbreviated as leaves)
500g, 0. Add 8 kg of IN hydrochloric acid solution,
Extract with stirring at 0°C for 3 hours, add new leaves to the first extract separated from the algae, extract at 40°C for 3 hours, add new leaves to the resulting second extract, and heat at 40°C. 16
After time extraction, add thallus 5001 to the obtained 3rd extraction solution, extract at 40°C for 3 hours, and add 500 g of thallus and pepsin 3 to the obtained 4th extraction solution! 7 and extracted at 40°C for 3 hours, repeating 5 times using a total of 2.5 kg of leaflets.
2.4 kg of extract was obtained.

The clarified extract obtained by centrifuging this extract was concentrated by ultrafiltration in the same manner as in Example 1, and further washed with water 5 times in the same manner as in Example 5, and the obtained concentrate was freeze-dried. Fucoidan 5.6F (L-fucose 34.62%) was obtained.

Claims (1)

[Claims] 1. From a fucoidan-containing solution obtained from fucoidan-containing seaweed, or from a fucoidan-containing solution whose viscosity has been lowered by dissolving β-1,3 glucanase and/or protease, if necessary. , is characterized in that a solution with high purity of fucoidan is collected by filtering and removing impurities using an ultrafiltration membrane, or a dried product of fucoidan is collected by a conventional method from a solution with high purity of fucoidan. Fucoidan-containing solution or fucoidan production method 2, the production method according to claim 1, in which impurities are filtered out using an ultrafiltration membrane while adding or adding water to the fucoidan-containing solution. 3. Molecular weight cutoff 60,000-100 as an ultrafiltration membrane
,000 to remove the portions that cannot pass through the filtration membrane. Molecular weight cutoff 5,000-60,0
00 is used to filter impurities and remove the filtrate.Production method 5 according to claims 1 and 2, when dry fucoidan is collected by a conventional method from a solution with high purity of fucoidan, The manufacturing method according to claims 1 to 4, which comprises drying the solution as it is, or coagulating and precipitating the fucoidan at a polar solvent concentration of 40 to 55% (v/v), and drying the precipitated fucoidan.