KR100500700B1 - Preparation Method of Purified Laminaran from Brown Algae - Google Patents

Abstract

The present invention comprises the steps of extracting the dry powder of laminaran-containing brown algae with an acidic or neutral solution; Adding lower alcohol to the brown algae extract to precipitate crude lamina; Adding a quaternary ammonium salt solution to the aqueous solution of the crude lamina sediment and separating it into an insoluble acidic polysaccharide precipitate and a soluble neutral polysaccharide supernatant; Adding a lower alcohol to the neutral polysaccharide solution to precipitate laminaran; And it provides a method for producing laminaran comprising the step of removing the remaining salts and low molecular impurities and drying.

Description

Preparation Method of High Purity Laminaran from Brown Algae {Preparation Method of Purified Laminaran from Brown Algae}

The present invention relates to a method for producing high purity laminaran from brown algae extract, more specifically, the step of extracting a dry powder of laminaran-containing brown algae raw material of laminaran-containing brown algae with an acidic or neutral solution; Adding lower alcohol to the brown algae extract to precipitate crude lamina; Adding a quaternary ammonium salt solution to the aqueous solution of the crude lamina sediment and separating it into an insoluble acidic polysaccharide precipitate and a soluble neutral polysaccharide supernatant; Adding a lower alcohol to the neutral polysaccharide solution to precipitate laminaran; And it relates to a method for producing laminaran comprising the step of removing the residual salts and low molecular impurities and drying.

Laminaran has a basic structure of β-1,3-glucan, a storage polysaccharide contained in a large amount of brown algae as shown in the following chemical structural formula, and is a linear neutral polysaccharide containing 1,6 chain bonds.

Such laminaran is currently actively studied as an immune activator of fish, but almost no research as a food. Polysaccharides similar to laminaran include schizophyllan, which is currently used as an adjuvant for anticancer drugs in Japan, and these have been reported to have strong immune enhancing effects. In addition, strong immune activity, also known in yeast and mushrooms, is known to be due to β-glucan. To date, it has been reported in relation to β-1,3-glucan (containing 1,6 chains) that the polyhelix chain has a triple helix structure, the position of substitution of the 1,6 bond chain, and the size of the molecular weight. Therefore, the difference in activity is known to be very large, and the polysaccharide's immune activity is considered to be the main factor is the activity of macrophage (macrophage), a non-specific immune cell. Laminaran, β-glucan having such bioactive properties, is known to be contained in a large amount of various brown algae, such as kelp, seaweed, E. coli and rhubarb, which are mass-produced on the coast of Korea or mass-produced by aquaculture. However, only the basic research of the content analysis level has yet to be developed an effective separation and purification method that can be provided as an anti-cancer material as a physiologically active material.

Conventional studies related to the separation and purification of laminarans include the results of previous studies (Table 2, Black, 1965), which analyzed the contents of crude laminaran of major brown algae (Table 2, Black, 1965). Since it contains a large amount of impurities such as acidic sugars such as uronic acid and sulfuric acid and minerals and proteins, it is difficult to expect the expression of laminaran.

Table 1 Yield and general composition of Crude laminaran (%)

Seaweed Kelp Gompi rhubarb 톳 yield 2.6 11.3 11.0 19.6 9.4 Ash 42.4 28.8 31.0 7.5 39.3 protein 1.7 2.5 17.2 14.0 12.6 The shrine 51.2 66.2 52.2 82.3 43.0 Uronic acid 17.2 21.3 11.9 8.9 9.9 Sulfate 9.1 9.7 17.5 3.5 11.4 Per composition Fuc 25.0 22.1 28.7 3.1 27.3 Rib 0.0 0.0 0.0 0.0 0.0 Xyl 0.0 3.2 3.7 0.0 0.0 Man 58.8 59.7 16.5 4.9 7.4 Gal 16.2 9.1 8.8 8.5 49.6 Glc 0.0 5.8 42.4 83.5 15.7

The most commonly known method of extracting and purifying crude laminaran is the most commonly known method. The dried powder of brown algae containing laminaran is extracted by stirring for 2 hours at 20 ° C. with 0.09N HCl solution and centrifugation (8,000g). The residue was extracted again with 0.05N HCl solution, and the supernatant obtained by centrifugation was combined with the mother liquor and ethanol was added thereto to precipitate the crude laminaran to obtain an aqueous solution of the crude laminaran. The aqueous solution of Zoramiran was treated with Zeo-Karb 225 (H ⁺ ) column, a strong acid cation exchange resin, and ethanol was added to the separated eluted solution to precipitate purified Laminaran, washed with ethanol and ether and dried under reduced pressure. How to Obtain Laminaran [WAP Black, Methods carbohyd. Chem., 5, 159, (1965)], but it is known that the quality problems such as the cumbersome treatment process, high treatment cost, low purity of the purified purified laminaran.

In addition, the dried powder of brown algae is immersed in 1 L of 0.4% hydrochloric acid containing formaldehyde (1 ml / L), stored at room temperature for 4 hours, squeezed algae in 0.5 L water and heated at 50 to 60 ° C. for 3 hours. A process patent for separating and purifying only lamanran with a special ion exchange resin called polychromium-1 after obtaining an extract of zona ramiran (TN Zvyagintseva, EV Sundukova, LA Elyakova, Russian Federation Patent no. 1642725. Chem. Abstr ., 126 ( 1997) (590. patents), but there are significant problems in terms of treatment process cost, safety, and quality of laminaran, resulting in low industrial applicability.

In addition, after extracting brown algae algae with a phosphate solution, alkali carbonate or caustic alkali is added to the alkali metal salt of phosphoric acid to change the pH value of the solution. Prior art relating to a process for separating fucoidan and laminaran and finally adding dilute sodium carbonate solution to extract alginic acid (isolation of fucoidan and laminaran and alginic acid and purification of crude alginic acid, Korean Patent 1981-0000029, 1981 2 2) has been reported, but according to this technique, the purity of the separated polysaccharides is too low to expect polysaccharide characteristics.

The present invention has been made to solve the problems of the prior art as described above, the object of the present invention is to provide a method for effectively producing a high purity laminaran with a low content of impurities such as acidic polysaccharides from laminaran containing seaweed.

In order to achieve the above object, the present invention comprises the steps of (1) extracting the dry powder of laminaran-containing brown algae with an acidic or neutral solution; (2) adding low alcohol to the brown algae extract to precipitate crude laminaran; (3) adding a quaternary ammonium salt solution to the aqueous solution of the crude lamina sediment and separating the mixture into an insoluble acidic polysaccharide precipitate and a soluble neutral polysaccharide supernatant; (4) precipitating laminaran by adding lower alcohol to the neutral polysaccharide solution; And (5) provides a method for producing laminaran comprising the step of removing the remaining salts and low molecular impurities and drying.

The step (1) is preferably a step of extracting the brown algae in the step of extracting the brown algae raw material with fresh water, and then crushed to a particle size of 20 ~ 80 mesh size and then heat treatment to maintain 60 ~ 150 ℃.

The step (1) preferably comprises the step of extracting the brown algae dry powder at a solution of pH 2.0 to 8.0 at 20 ~ 60 ℃.

In the step (2), the lower alcohol is preferably ethanol or alcohol containing 95% or more of ethanol.

The quaternary ammonium salt of step (3) does not require special limitation, but is preferably selected from cetylpyridinium chloride, cetylpyridinium bromide, cetyltrimethylammonium bromide.

The step (3) preferably comprises adding a 0.2 to 10% quaternary ammonium solution to the aqueous solution of Zoramiran and stirring at 10 to 50 ℃.

The lower alcohol of step (4) is preferably ethanol or alcohol containing 95% or more of ethanol

It is preferable that the low molecular weight impurity of said step (4) is 5000 Daltons or less.

Hereinafter, the content of the present invention will be described in more detail with reference to preferred purification examples.

The extraction step of the step (1) is preferably a first heat treatment step of washing the laminaran-containing brown algae raw material with fresh water, dried to grind to a particle size of 20 to 80 mesh size and then maintained at 60 to 150 ℃ for 2 to 30 minutes After the first heat treatment, the brown algae powder was extracted with 20 to 60 times the solution of pH 2.0 to 8.0 at 20 to 60 ° C. for 1 to 6 hours, and then centrifuged or filtered to separate the extract and the residue. It can be achieved by the process of extracting the solution once or twice and then centrifuging or filtering to obtain the extract.

Precipitating step of the crude laminaran of the step (2), preferably, 1 to 5 times lower alcohol, preferably ethanol is added to the extract of the step (1) to precipitate the crude laminaran to obtain the crude laminaran Can be achieved by the process.

Obtaining the soluble neutral polysaccharide solution of step (3) is preferably, by adding a quaternary ammonium solution, such as 0.2 to 10% CPC (cetypyridinium chloride) to 0.1-10% aqueous solution of Zoramiran Stirring at -50 ° C for 30 minutes to 6 hours to separate the insoluble salt precipitate of acidic polysaccharide combined with quaternary ammonium salt such as CPC and supernatant of neutral polysaccharide, and then separating it into neutral and acidic polysaccharide by filtration or centrifugation. It can be achieved through.

In the precipitation step of laminaran of step (4) and the drying step of step (5), 1 to 5 times lower alcohol, preferably ethanol, is added to the neutral polysaccharide solution obtained in step (3) to precipitate laminaran. The laminaran obtained by filtration can be achieved by dissolving in distilled water and then dialysis (MWCO 5,000) or ultrafiltration to remove residual salts and low molecular impurities, followed by drying. (See Figure 1)

The separation and purification method of high purity laminaran from the brown algae of the present invention mentioned above will be described in more detail according to the process.

1. Selection of raw algae

As raw material seaweed, brown algae with high laminaran content are used. Such brown algae include kelp, seaweed, gompi, Ecklonia cava, rhubarb, sesame seeds, and soybean paste. Preferably, the algae are harvested at a high polysaccharide content. Then, the side of the fire, the immature crude, etc. are selectively removed, washed with fresh water, and dried and stored sufficiently to have a water content of 15% or less as a raw material. Depending on the type of raw materials, Ecklonia cava and rhubarb do not differ in the timing of harvesting and the contents of the crude ingredients, but other brown algae, such as seaweed and kelp, have a large difference in the polysaccharide content according to maturity. It is recommended to use the raw material collected.

2. Grinding and heat treatment of raw algae

The dried seaweed is coarsely pulverized to a particle size of 20 to 80 mesh and then heat treated at 60 to 150 ° C. for 2 to 30 minutes to coagulate chlorophyll and protein components, thereby facilitating extraction of the water-soluble sugar component in the subsequent extraction process.

3. First Extraction

20 to 60 times the amount of acidic or neutral solution (a dilute solution of pH 2.0-8.0 natural water or hydrochloric acid or organic acid for food industry) is added to the raw seaweed powder, and then the mixture is stirred for 1 to 6 hours at 20 to 60 ° C. After extraction, the mixture was centrifuged (3,000 g × 10 minutes) or filtered to obtain a primary extract and a residue.

4. Secondary Extraction

The residue obtained from the primary extraction is used as a raw material, and extracted under the same conditions as the primary extraction, followed by centrifugation or filtration to obtain a secondary extract and a residue.

5. Alcohol Precipitation

After combining the primary extract and the secondary extract, 1 to 5 times the amount of ethanol (or alcohol concentration of 95% or more alcohol) is added and allowed to stand to precipitate the polymer fraction consisting of acidic polysaccharide and neutral polysaccharide in the extract. At this time, the fat-soluble pigment and various low molecular weight substances are present in the dissolved state in the supernatant without being precipitated.

6. Recovery of Zoramanaran

The polymer fraction obtained by alcohol precipitation is centrifuged (3,000 g × 10 minutes) or filtered to recover crude laminaran consisting mostly of acidic and neutral polysaccharides. The recovered laminaran is dried (hot air drying at 60 ° C. or lower, vacuum drying or vacuum freeze drying) to volatilize the remaining ethanol component.

7. Solubilization of eggs

Water is added to prepare an aqueous solution of Zoramiran with a concentration of 1 to 10% solids.

8. Separation and Purification of Laminaran

A quaternary ammonium salt solution (CPC: cetylpyridinium chloride, C ₂₁ H ₃₈ ClN.H ₂₀ , FW 358.0) was added to an aqueous solution of Zoraminaran, followed by 30 to 5 hours at 20 to 40 ° C. After stirring, the insoluble precipitate formed by combining with the coexisting acidic polysaccharide component in the aqueous solution and the neutral polysaccharide component as a supernatant are separated and filtered, and 1-5 times of ethanol is added to the filtrate to separate and precipitate the neutral polysaccharide laminaran. In this case, quaternary ammonium salts used as a promoter for binding reactions with acidic polysaccharides may include CTAB (cetyltrimethylammonium bromide: CH ₃ (CH ₂ ) 15 N (Br) (CH 3) _3, FW364.5) or CPB (in addition to CPC). Cetylpyridinium bromide: C ₂₁ H ₃₈ NBr, FW 384.4) may also be used.

9. Removal of trace impurities from purified laminaran

After dissolving laminaran separated from acidic polysaccharide in distilled water, dialysis membrane (MWCO 5,000) is used to remove various low molecular impurities such as small amount of salts remaining after dialysis or ultrafiltration using dialysis membrane (MWCO 5,000) for 5 to 24 hours, followed by drying or lyophilization. Pure Laminaran is obtained.

Hereinafter, the content of the present invention will be described in more detail with reference to Examples. However, these examples are only presented to understand the content of the present invention, and the scope of the present invention should not be construed as being limited to these embodiments.

Example 1 Extraction of Zoramiran Egg from Kelp

This embodiment relates to a process for separating laminaran using kelp as a raw material for separating and purifying high purity laminaran from brown algae. Using fresh cultured shimama (Wando, July 25, width 1.8m, thickness 3mm or so) as a raw material, 5kg was immersed in 75L of clean fresh water and desalted for 3 hours with occasional stirring. Thereafter, the resultant was dried for 4 hours using a hot air dryer at 60 ° C., and coarsely pulverized to obtain 550 g of dried Kashima powder having a water content of 12% and a particle size of 30 mesh. 15 L of hydrochloric acid solution of pH 3.0 was added to 500 g of dried shimama powder, followed by stirring and leaching at 20 ° C. for 4 hours, centrifugation, and the residue was extracted secondarily under the same conditions. About 22 L of the acidic extract obtained by the secondary extraction was added 60 L of alcohol to precipitate crude lamina. The precipitate was centrifuged at 3,000 g for 10 minutes and lyophilized to obtain 51.5 g of crude laminaran with 2.5% water content.

Example 2 Purification of Laminaran

This example discloses a process of separating and purifying pure laminaran from crude laminaran obtained from Ecklonia cava as a raw material. To 500 g of desalted dried finely prepared Ecklonia cava powder, 18 L of hydrochloric acid solution for the food industry of pH 3.0 was added, followed by stirring and leaching at 25 ° C. for 3 hours, followed by centrifugation. 80 L of 95% ethanol (alcohol) was added to about 30 L of the primary and secondary acidic seaweed extracts obtained by centrifugation and stirred at room temperature to precipitate crude lamina. The precipitate was filtered through a filter cloth and lyophilized to give 57 g of crude lamina. Then, after dissolving Zoramiran in 1.5 L of water, a 5% solution of CPC, a kind of tetravalent ammonium salt, was added until no precipitate was formed, followed by stirring at 35 ° C. for 3 hours to separate the precipitate into acidic polysaccharide and the supernatant neutral polysaccharide. It was. Thereafter, the filtrate was filtered and 3 times the amount of ethanol was added to the filtrate to precipitate laminaran. The laminaran was dissolved in a small amount of distilled water, dialyzed for 12 hours (MWCO 5,000), and lyophilized to obtain 23.5 g of purified laminaran.

Experimental Example 1 Extraction Yield by Extraction Solvent Ratio

In order to determine the proper extraction solvent ratio and the number of extraction in extracting laminaran from the seaweed containing laminaran, the extraction test was carried out as follows. That is, a diluted hydrochloric acid (pH 2) solution was added to the ground algae (Tashima) sample so that the ratio of the sample and the solvent was 1:20 to 1:50 (w / v), followed by stirring extraction at 20 ° C for 3 hours. . The extract was filtered under reduced pressure using a primary filter cloth and a secondary filter pad to obtain a primary extract. The filtered residue was subjected to secondary and tertiary extraction under the same conditions as the primary extraction. Three times of ethanol was added to each extract, and the mixture was left to stand overnight, followed by centrifugation (3,000 g x 15 minutes). Precipitated crude bran was dissolved in a small amount of distilled water, lyophilized to prepare crude bran, and then the yields of the extractive conditions were examined. When the solvent ratio was 1:20, 1:30, 1:40 and 1:50, The yields of the total crude laminaran combined with the primary, secondary and tertiary extracts were 15.7%, 23.9%, 23.8%, and 24.9%, respectively. Above 1:30, the increase in extraction yield was minimal.

The yield of crude lamina according to the extraction frequency is 56.7%, 48.5%, 52.5% of the total yield when the extraction ratio is 1:20, 1:30, 1:40, 1:50 , 52.2% were extracted, and 30.6%, 38.5%, 35.3%, 39.4% at the time of the second extraction, and 8.4-13.0% at the third extraction.

In view of the above results, in consideration of extraction yield and extraction time, it was considered appropriate to extract twice at the solvent ratio of 1:30 to 1:40. (See Figure 2, Table 2)

<Table 2> Extraction yield (%) of coarse eggs by extract solvent ratio and number of extracts of dried kelp

Extraction yield Kelp: Extraction solvent ratio (W / V) 1: 20 1: 30 1: 40 1: 50 1st extraction yield 8.9 11.6 12.5 13.0 2nd extraction yield 4.8 9.2 8.4 9.8 3rd extraction yield 2.0 3.1 2.9 2.1   Total yield 15.7 23.9 23.8 24.9

Experimental Example 2 Examination of Extraction Time

In order to set the optimum extraction time from brown algae, the crude lamina was prepared by varying the extraction time from 2 to 5 hours and the yield was examined. That is, the extraction solvent ratio was 1:40, the extraction temperature was 30 ℃, and the extraction pH was adjusted to pH 2, and the extraction time was prepared by varying the extraction time to 2 to 5 hours to examine the yield. The yield was 7.7% at 2 hours of extraction, and the yields at 3, 4, and 5 hours of extraction were 12.4%, 12.7%, and 12.9%, respectively. There was no increase. Therefore, 3 hours extraction seems to be effective in the shortest time. (See Fig. 3)

Experimental Example 3 Effect of pH on the Yield of Zoramiran Egg Extract

The extraction yield and chemical composition of crude lamina were investigated when extracted from brown algae at different pHs. That is, when the extraction solvent ratio is 1:40 and the extraction time is adjusted for 3 hours at 30 ° C. and then the extraction test is performed while changing the pH, when the pH is extracted differently from 1 to 7, as shown in FIG. 4. The extraction yield of the crude lamina was 10.3%, 14.5%, 15.8%, 11.0%, 8.5%, 7.8% and 7.2%, respectively, and the extraction yield was the highest when extracted at pH 3. From the neutral pH range of pH 7.0 to the acidic region, the extraction yield increased due to hydrolysis. From below pH 3, the hydrolysis proceeded excessively and the extraction yield tended to decrease. As a result, the extraction yield of zona vera in the acidic region is higher than that in the neutral region, but the extraction yield decreases again in the strongly acidic region, which is related to the fact that it contains a large amount of acid polysaccharide (Fucose-containing polysaccharide). It is also thought to be closely related to the composition of raw seaweeds.

Experimental Example 4 Influence of Extraction Temperature on the Yield of Zoramiran Extract

As a raw material of rhubarb belonging to the brown algae, the yield and chemical composition of the chopped lamina were examined when the extraction temperature was changed at different extraction temperatures. That is, the extraction solvent ratio is 1:40 and the extraction time is adjusted to 3 hours at pH 3 and then extracted while varying the pH, as shown in Table 4 below when the extraction yield of the chopped lamina is extracted at 50 ℃ It was the best at 14.3% and the yield increased and decreased with little difference in the yield up to 30 ~ 70 ℃. The yield decreased as the temperature increased from 80 ° C. or higher to over temperature.

In addition, as the extraction temperature increases, the composition of the extract component generally decreases the protein content, increases the concentration of acidic polysaccharides such as uronic acid and sulfuric acid group, and decreases the content of glucose, which is a constituent sugar of laminaran, in the total sugar. Seemed. In view of these characteristics, it is considered that the optimum temperature range for extracting Joraminaran is preferably 50 ° C. or lower in consideration of the composition of the extract and the extraction yield.

<Table 3> Yield and Composition of Crude Laminar Extracts of Rhubarb Extract by Extraction Temperature

  division Extraction temperature (℃) 30 50 70 90 Zoraminaran yield (%) 13.2 14.3 12.9 11.5 protein(%) 8.3 8.1 7.9 7.5 Total sugar (%) 70.8 73.4 71.4 70.6 Uronic acid (%) 10.3 12.3 13.1 14.2 Sulfuric acid group (%) 7.7 8.0 8.2 8.3 Composition per composition (%) Fucose 7.0 7.5 8.1 8.7 Ribose 1.0 1.2 1.2 1.2 Xylose 1.1 1.2 1.2 1.4 Mannose 1.9 2.2 2.1 2.3 Galactose 0.0 0.0 0.0 0.0 Glucose 89.0 86.2 85.7 85.3 Rhamnose 0.0 0.0 0.0 0.0 Arabinose 0.0 0.0 0.0 0.0

Experimental Example 5 Purification Condition

To the dry powder of rhubarb in brown algae, add 18 L of hydrochloric acid solution for the food industry at pH 3.0, stir and leach at 25 ° C for 3 hours, and add 75 L of alcohol to the primary and secondary extracts obtained by repeating the centrifugation twice. It left to stand for a minute and precipitated crude laminaran. Thereafter, the precipitate obtained by centrifugation was lyophilized to obtain 89 g of crude lamina. Subsequently, after dissolving Zoramirane in 2 L of water, 5% aqueous solution of 3 kinds of quaternary ammonium salt solutions, such as CPC, was added slowly, and it stirred for 3 hours at 35 degreeC, and the coexisting acidic polysaccharide was precipitated. About 5 liters of alcohol was added to the filtrate of the neutral polysaccharide obtained by filtration, and the precipitated separated laminaran was collected by filtration, dissolved in 200 ml of distilled water, and dialyzed in a dialysis membrane (MWCO 5,000) for 12 hours. After removing impurities such as salts and low molecular weight substances to be lyophilized to obtain purified laminaran. At this time, quaternary ammonium salts used as a co-precipitation accelerator for acidic polysaccharides were CPC, CPB and CTAB, and the yield of purified laminaran according to the salt type was 33.2% (CPC), 34.5% (CPB) and 35.4% ( CTAB) showed higher values in order of CTAB> CPB> CPC, and the yield of laminaran in the supernatant was slightly different from CPC (44.6%), CPB (44.5%) and CTAB (43.7%), respectively. There was. In addition, the purification purity of laminaran obtained by purification by alcohol precipitation and dialysis from the neutral polysaccharide obtained by quaternary ammonium salt precipitation (supernatant not precipitated after quaternary ammonium treatment) was 92.7% (based on total sugar content). ), 92.3% in CPB treatment and 93.4% in CTAB treatment. At this time, organic components other than laminaran were trace proteins, acidic polysaccharides such as uronic acid and sulfuric acid. As a result of these experiments, there was no significant difference in the removal of acid polysaccharides and separation recovery of laminaran, a neutral polysaccharide, according to the kinds of ammonium salts. It is considered that suitable salts can be used in consideration of this (Table 4).

Table 4 Preparation yield and chemical composition of laminaran of rhubarb extract treated with quaternary ammonium salt (unit: dry base,%)

ingredient Precipitate after ammonium salt treatment Supernatant after Ammonium Salt Treatment CPC treatment CPB treatment CTAB processing CPC treatment CPB treatment CTAB processing  Yield 33.2 34.5 35.4 44.6 44.5 43.7 Total sugar 33.4 33.8 34.6 92.7 92.3 93.4 Protein 10.8 11.7 12.2 1.8 1.6 1.8 Uronic acid 27.6 28.0 29.6 1.5 1.4 1.5 Sulphate 10.5 11.2 12.4 1.2 1.1 1.2 Rhamnose 1.3 1.4 1.4 - - - Fucos 49.1 50.0 50.7 - - -   Ribose - - - 3.5 3.3 3.2 Arabinose - - - 1.0 1.0 0.9 Xylose 5.0 5.2 5.3 - - - Mannose 13.4 14.3 13.9 2.5 2.6 2.5 Galactose 8.1 7.8 8.5 - - - Glucose 20.5 21.0 19.4 93.3 94.7 93.8

Note: CPC: C ₂₁ H ₃₈ NCl, FW: 340.0 _, CPB: C ₂₁ H ₃₈ NBr, FW 384.4, CTAB: CH ₃ (CH ₂ ) 15N (Br) (CH3) _3, FW364.5

Molecular weight of laminran derived from CPC purified brown algae was determined by HPLC equipped with RI detector using dehydroxel as a standard for molecular weight measurement using Ultrahydrogel 1000 column. It was composed of glucan (molecular weight 26,000 Daltons) and laminaran (molecular weight 12,900 Daltons) consisting of glucose and protein. Chromatograms of CPC purified brown alga laminaran identified by gas chromatography are shown in FIG. 6, and ion chromatograms showing molecular binding positions of CPC purified laminaran are shown in FIG. 7.

According to the present invention, high purity laminaran with low content of impurities such as acid polysaccharide can be effectively separated and purified from laminaran containing seaweed. Therefore, laminaran, which has a strong immune activity and cytotoxic activity against cancer cell lines, can be used as a raw material of an anticancer supplement or a functional food material having a similar physiological function, from brown algae resources that are widely absent in Korea or cultured in large quantities. Separation with high purity is expected to contribute greatly to the creation of high value-added by the effective utilization of resources and to the construction of a technology base for the effective utilization of natural seaweeds as alternative medicine materials.

1 is a process chart of producing high purity Laminaran from brown algae as a preferred embodiment of the present invention.

Figure 2 is a graph showing the extraction yield of the coarse bran contained in dried shimama according to the number of extraction and the ratio of the extraction solvent as a preferred embodiment according to the present invention

Figure 3 is a graph showing the extraction yield of the chopped lamina contained in dried shimama according to the extraction time as a preferred embodiment according to the present invention

Figure 4 is a graph showing the effect of the pH on the extraction yield when extracting the choramiran from kelp as a preferred embodiment according to the present invention

5 is a graph showing the effect of the extraction temperature on the extraction yield when extracting the coarse lanthanum contained in rhubarb as a preferred embodiment according to the present invention

Figure 6 is a gas chromatogram of brown alga laminaran purified by CPC as a preferred embodiment according to the present invention [a: glucose, b: myo-inositol (internal standard)]

Figure 7 shows a glucose bound to the carbon atom 1 position in the total ion chromatogram of purified laminaran [A: 2,3,4,6-Me ₄ -Glc (laminaran molecule) as a preferred embodiment according to the present invention ), B: 2,4,6-Me ₃ -Glc (which represents the unit constituent of laminaran as β-1,3 conjugate of glucose), C: 2,3,4-Me ₃ -Glc (of glucose unit sugar of laminaran as β-1,6 conjugate), D: 2,4-Me ₂ -Glc (the laminaran constituent sugar as β-1,3 and β-1,6 conjugates of glucose) ), E: Myo-inositol- (OAC) _6: Internal Standards]

Claims (8)

(1) extracting a dry powder of laminaran-containing brown algae with an acidic or neutral solution; (2) adding low alcohol to the brown algae extract to precipitate crude laminaran; (3) adding a quaternary ammonium salt solution to the aqueous solution of the crude lamina sediment and separating the mixture into an insoluble acidic polysaccharide precipitate and a soluble neutral polysaccharide supernatant; (4) precipitating laminaran by adding lower alcohol to the neutral polysaccharide solution; And (5) removing the remaining salts and low molecular impurities and drying the laminaran. The method of claim 1, wherein the step of extracting the brown alga in step (1) comprises the step of washing and drying the brown algae raw material with fresh water, pulverizing to a particle size of 20 ~ 80 mesh size and then maintaining the heat treatment at 60 ~ 150 ℃ Laminaran production method characterized by The method of claim 1, wherein the step (1) comprises the step of extracting the brown algae dry powder with a solution of pH 2.0 to 8.0 at 20 ~ 60 ℃ The method of claim 1, wherein the step (2) is a lower alcohol is ethanol or a process containing a ethanol containing 95% or more ethanol The method of claim 1, wherein the quaternary ammonium salt of step (3) is selected from cetylpyridinium chloride, cetylpyridinium bromide and cetyltrimethylammonium bromide. [6] The laminaran according to claim 1 or 5, wherein step (3) comprises adding 0.2 to 10% of a quaternary ammonium solution to an aqueous solution of crude laminaran and stirring the solution at 10 to 50 ° C. Manufacturing method  The method for producing laminaran according to claim 1, wherein the lower alcohol of step (4) is made of ethanol or alcohol containing 95% or more of ethanol.  The method for producing laminaran according to claim 1, wherein the low molecular weight impurity of step (4) is 5000 Daltons or less.