KR101004835B1 - Manufacturing Method of Fucoidan by Enzyme Treatment at Ultra High Pressure - Google Patents

Abstract

The present invention relates to a method for producing fucoidan using ultra-high pressure enzyme treatment, and to a method for producing fucoidan that can obtain fucoidan with excellent anticoagulant activity in high yield by performing ultra-high pressure enzyme treatment when extracting fucoidan from brown algae.

In the present invention, fucoidan can be obtained by lowering the molecular weight and further excellent in antithrombotic activity and antioxidant activity by ultra-high-pressure enzyme treatment at the time of low molecular weight of fucoidan.

Fucoidan, Ultra High Pressure Enzyme Treatment, Antithrombosis, Antioxidant

Description

Manufacturing Method of Fucoidan by Enzyme Treatment at Ultra High Pressure}

The present invention relates to a method for producing fucoidan using ultra-high pressure enzyme treatment, characterized in that the ultra-high pressure enzyme treatment in the process of extracting or low molecular weight fucoidan contained in brown seaweeds (褐藻 類, brown seaweeds).

One of the major causes of death in modern society is the lesion of the heart and blood vessels caused by thrombosis.

Heparin, a form of antioxidant polysaccharide, has been used for the treatment of thrombosis for almost 50 years. In addition, heparin is used as an external circulation solvent in hemodialysis and vascular surgery. Heparin is the second most popular natural therapeutic admixture after insulin.

Heparin, on the other hand, has side effects such as thrombocytopenia development, bleeding effect, ineffectiveness against congenital or acquired antithrombin deficiency, and inability to bind thrombin and fibrin. Moreover, heparin is mainly extracted from pig intestines or bovine lungs, which have significantly lower yields. Accordingly, there is a need for research on heparin substitutes having an anticoagulant effect.

Recently, fucoidan, which has emerged as a substitute for heparin, is a viscous polymer having a weight average molecular weight of 40,000 to 2,000,000 daltons, and is mainly used for mucus of mollusks such as abalone and squid or brown algae such as seaweed or kelp. Included. In particular, about 20% of fucoidan is present in the sporophyll of brown seaweed (Undaria pinnatifida).

In addition, fucoidan has various physiologically active functions such as anticoagulant effect, anticancer action, arteriosclerosis, thrombosis prevention, and antioxidant action, and thus can be applied to various food fields and pharmaceutical fields.

Therefore, there is a need for a method for producing fucoidan using ultrahigh pressure enzyme treatment with higher yield.

In addition, fucoidan is composed of a hardly degradable polysaccharide of a polymer that is impossible for humans to digest, and when used without lowering molecular weight, there is a problem that it is almost impossible for the beneficial bioactive component to be utilized in the human body. Accordingly, there is a need for a method for producing low molecular weight fucoidan with excellent yield and physiological activity.

An object of the present invention is to provide a method for producing fucoidan using ultra-high pressure enzyme treatment that can be obtained with high yield of fucoidan having excellent anticoagulant activity by extracting fucoidan from brown algae.

It is another object of the present invention to provide a method for producing fucoidan using an ultrahigh pressure enzyme treatment, which is capable of obtaining a low molecular fucoidan which has more excellent anticoagulant activity and can be used in the body by performing ultrahigh pressure enzyme treatment when low molecular weight of fucoidan.

The present invention for solving the above object is preparing a brown algae; Adding a polysaccharide degrading enzyme to the prepared brown algae and subjecting it to an ultra-high pressure enzyme treatment; Filtering the ultrahigh-pressure enzymatically digested solution; Adding salt to the filtrate to remove alginic acid in the form of insoluble salts; Precipitating crude fucoidan by adding alcohol to the filtrate from which the alginic acid has been removed; And desalination and lyophilization of the crude fucoidan to provide fucoidan using the ultra-high pressure enzyme treatment.

The present invention also comprises the steps of preparing brown algae; Hot water extraction of the prepared brown algae; Filtering the hot water extract; Adding salts to the filtrate to remove coexisting alginic acid in the form of insoluble salts; Precipitating crude fucoidan by adding alcohol to the filtrate from which the alginic acid has been removed; Desalting the crude fucoidan; And it provides a method for producing fucoidan using ultra-high pressure enzyme treatment comprising the step of adding a polysaccharide degrading enzyme to the crude fucoidan and ultra-high pressure enzyme treatment.

In addition, the brown algae provides a fucoidan production method using ultra-high pressure enzyme treatment, characterized in that the seaweed spores.

In addition, the polyglycolytic enzyme provides a method for producing fucoidan using ultra-high pressure enzyme treatment, characterized in that beta-glucanase (β-glucanase).

In addition, the salt provides a method for producing fucoidan using ultra-high pressure enzyme treatment, characterized in that the calcium chloride.

In addition, the ultra-high pressure enzyme treatment provides a fucoidan production method using an ultra-high pressure enzyme treatment, characterized in that carried out at a pressure of 80MPa ~ 500MPa.

Hereinafter, the present invention will be described in more detail.

The method for producing fucoidan using the ultrahigh pressure enzyme treatment according to the present invention has the effect of obtaining a high yield of fucoidan with useful activities such as preventing blood coagulation and antioxidant activity by extracting the fucoidan from brown algae. have.

In addition, the present invention can be obtained not only low molecular weight fucoidan without structural modification, such as there is no difference in sulfuric acid group content that plays an important role in physiological activity by performing ultra-high pressure enzyme treatment when low molecular weight of fucoidan, anti-thrombosis There is an effect to obtain more fucoidan with excellent activity and antioxidant activity.

Hereinafter, preferred embodiments of the present invention will be described with reference to the accompanying drawings.

First, a preferred embodiment of the present invention comprises the steps of preparing brown algae; Adding a polysaccharide degrading enzyme to the prepared brown algae and subjecting it to an ultra-high pressure enzyme treatment; Removing the alginic acid in the form of an insoluble salt by adding a salt to the digested solution subjected to the ultrahigh pressure; Precipitating crude fucoidan by adding alcohol to the brown algae extract from which the alginic acid has been removed; And desalting and lyophilizing the crude fucoidan. Figure 1a is a flow diagram showing a method for producing fucoidan using ultra-high pressure enzyme treatment according to an embodiment of the present invention.

1. Preparing the brown algae

Brown algae are first washed with fresh water to remove salt and debris. Although it does not specifically limit as said brown algae, For example, seaweed, seaweed, seaweed, kelp, mozukuku, big horse thread, etc. are preferable. In particular, about 20% of fucoidan is contained in the spores of wakame seaweed, which is more preferable as a raw material of the present invention.

After removing foreign substances and salts, the lower the moisture content of the fucoidan, the lower the molecular weight is better because of the physical properties of the fucoidan, so it is dried to 3 to 7% moisture content.

The dried brown algae is coarsely ground to an appropriate size using a grinder. Preferably pulverized to 80 ~ 120 mesh (mesh). When the size of the crushed brown algae is larger than 80 mesh, the volume of the enzyme can be reduced, so the efficiency of the enzyme is lowered, and if it is smaller than 120 mesh, the cost is not economical.

2. Ultra High Pressure Enzyme Treatment Step

Add distilled water corresponding to 30 to 40 times the volume of the prepared brown algae, and adjust the pH to 5 to 8 using 0.1N NaOH, and then add polysaccharide enzyme. The polysaccharide is not particularly limited, but beta-glucanase (β-glucnase), cellulase (celulase), hemicellulase (protein), proteinase, alpha-amylase (α-amylase) , Pentosanase, xylanase, arabanase, arabanase, pectin transeliminase, polygalacturonase, pectinesterase It is preferable to mix and use the above. Among the polysaccharides, beta-glucanase is more preferable because the yield of fucoidan is the highest.

At this time, it is preferable to use the polysaccharide enzyme in 0.2 ~ 1.0% (w / v) because the yield and physiological activity of the fucoidan is good.

Then, it is placed in an ultrahigh pressure device, and subjected to an ultrahigh pressure enzyme treatment for 20 to 30 hours at 30 to 50 ° C and 800 atm (80 MPa) to 5,000 atm (500 MPa). When the pressure range of the ultra-high pressure enzyme treatment is less than 800 atm (80 MPa), the extraction efficiency of the fucoidan can be lowered, and when the pressure exceeds 5,000 atm (500 MPa), there is a risk of changing the structure of the extracted fucoidan, the range is desirable.

The ultra-high pressure technology is such a non-thermal treatment as the consumer's interest in foods that maintain their natural taste and aroma through minimal processing and researches to minimize heat treatment for nutrient preservation of food are widely conducted. It is a new technology that is attracting attention among construction methods.

The ultra-high pressure enzyme treatment according to the present invention has a high extraction rate of fucoidan, and has a low heat cost because it works at a relatively low temperature of 30 ~ 50 ℃.

3. Filtration step

The insoluble residue in the ultra-high pressure enzyme-treated extract was effectively filtered using a dense filter cloth or filter cloth of 100 mesh or more, centrifuged for 20-30 minutes with a centrifugal force of 2,000 × G or more, or by using a continuous centrifuge for solid-liquid separation. Can be separated.

4. Alginate Removal Step

The extract separated through the filtration step contains a large amount of alginic acid in addition to fucoidan, and 1-5% (w / v) of food additive-grade calcium chloride (CaCl ₂ ) is added to effectively remove the alginic acid. At this time, the water-soluble alginic acid in the extract is converted to insoluble calcium alginate, and all precipitated after about 1 hour, the precipitated calcium alginate is separated and removed by conventional filtration or centrifugation. Preferably, the insoluble calcium alginate precipitate in the extract formed by the calcium chloride treatment is filtered by a filter cloth, a membrane, or other conventional filtration method, or centrifuged for 20 to 30 minutes with a centrifugal force of 2,000 × G or more.

5. Alcohol precipitation  step

The alginic acid is extracted is concentrated using a rotary evaporator (Rotary evaporator) at 70 ~ 80 ℃ until the content of solids is 20% to 30%.

Then, three times (v / v) of the alcohol to the concentrate is added and left for 8 to 12 hours at room temperature, centrifuged for 20 to 30 minutes with a centrifugal force of 2,000 × G or more to precipitate the fucoidan.

6. Desalination  And lyophilization step

After dissolving by adding a small amount of water to the fucoidan obtained by alcohol precipitation, it is dialyzed for 8 to 12 hours using a dialysis membrane.

The dialysis solution is then lyophilized to obtain purified fucoidan.

According to the method for producing fucoidan using the ultrahigh pressure enzyme treatment according to the present invention, it is possible to obtain fucoidan from brown alga with a high yield.

And another preferred embodiment of the present invention comprises the steps of drying the brown algae; Grinding the dried brown algae; Hydrothermal extraction of the ground brown algae; Adding salts to the brown algae extract to remove coexisting alginic acid in insoluble salt form; Precipitating crude fucoidan by adding alcohol to the brown algae extract from which the alginic acid has been removed; Desalting the crude fucoidan; And adding a polysaccharide degrading enzyme to the crude fucoidan and treating the ultra-high pressure enzyme. Figure 1b is a flow chart showing a method for producing fucoidan using ultra-high pressure enzyme treatment according to an embodiment of the present invention.

1. Preparing the brown algae

As described above.

2. Hydrothermal extraction  step

To the prepared brown algae, distilled water corresponding to 30 to 40 times the volume of brown algae was added, and the pH was adjusted to 5 to 8 using 0.1 N NaOH, followed by 3 to 5 at 80 to 90 ° C. and 120 to 150 rpm in a shaker. Extract hot water for hours.

3. Filtration step

As described above.

4. Alginate Removal Step

As described above.

5. Alcohol precipitation  step

As described above.

6. Desalination  step

Treat as described above and lyophilize to obtain crude fucoidan.

7. Ultra High Pressure Enzyme Treatment Step

Distilled water is added to the obtained crude fucoidan to be 2-10% (w / v). After adjusting the pH to 5 ~ 8 using 0.1N NaOH, add 0.1 ~ 0.3% (w / v) of polysaccharide enzyme, and put it in ultra high pressure device at 30 ~ 50 ℃, 800atm (80MPa) ~ 5,000 At high pressure (500MPa) it is subjected to ultra-high pressure enzyme treatment for 20-30 hours. When the pressure range of the ultra-high pressure enzyme treatment is less than 800 atm (80 MPa), the effect of low molecular weight of fucoidan is insignificant, and when the pressure exceeds 5,000 atm (500 MPa), there is a possibility that the structure of the fucoidan is changed. Do. Then, it is lyophilized to obtain low molecular fucoidan. At this time, the polyglycolytic enzyme is as described above.

According to the fucoidan production method using the ultra-high pressure enzyme treatment according to the present invention, it is possible to obtain a low molecular fucoidan excellent in anti-blood coagulation activity and available in the body.

Fucoidan prepared according to the method of the present invention is anti-blood by antithrombotic activity (TT), activated partial thromboplastin time (APTT) anticoagulant activity, and collagen-induced platelet aggregation test by collagen. Coagulation activity was measured and found to have excellent antithrombotic effect.

The invention can be better understood by the following examples, which are only illustrative of the invention and are not intended to limit the scope of protection defined by the appended claims.

[Example 1: Fucoidan Production Method Using High Yield Ultra High Pressure Enzyme Treatment]

Example  1-1: Fucoidan  Screening of Enzymes for Extraction

Seaweed spores were prepared, dried, and ground to a size of 80 to 120 mesh. Then, after adding distilled water corresponding to 40 times the volume of wakaepocots, the pH was adjusted to 8 using 0.1N NaOH. This exemplary polysaccharides of pen tryptophan ^TM lyase in (PENTOPAN), serenity mix ^TM (CEREMIX), Hatori atom ^TM (VISCOZYME), Ultra Flow ^TM (ULTRAFLO), pekti Annex ^TM (PECTINEX), cellulose Cloud host ^TM (CELLUCLAST), a Carr Tunisia claim ^TM (TUNICASE) enzyme of seven was respectively added at 1% (w / v). Each sample was placed in an ultrahigh pressure device (DSF-10L, Japan) and subjected to ultrahigh-pressure enzyme treatment for 24 hours at 40 ° C and 5000 atmospheres (500 MPa).

After the ultra-high pressure enzyme treatment, each sample was centrifuged (2,230 × G) to remove the residue, and 2% (w / v) of calcium chloride (for food additives) was added to the separated supernatant, and left for 1 hour, followed by precipitation. Alginic acid was removed by centrifugation (2,230 x G). The remaining supernatant was concentrated to 20-30% solids content. Three times (v / v) alcohol was added to the concentrate, and left for 12 hours. At this time, the precipitated fucoidan was separated by centrifugation, and then dissolved by adding a small amount of water, followed by dialysis (MW 3500) for 12 hours, and freeze-dried to obtain a purified fucoidan.

At this time, the extraction yield of fucoidan according to the type of polysaccharide degrading is shown in Table 1 below.

Table 1: Extraction yield of fucoidan according to polysaccharide enzyme type

Enzyme Treatment Condition Raw material Fucoidan production amount (g) % Recovery Enzyme-free treatment 7.5g 1.29 8.62 Pentophan 500 BG 7.5g 1.46 9.74 Ceremix 6X MG 7.5g 1.61 10.79 Bizcozyme 7.5g 1.74 11.56 Ultraflow L 7.5g 1.68 11.20 Pectinex 100 L 7.5g 1.52 10.13 Cellulast 7.5g 1.24 8.29 Tunicase FN 7.5g 1.92 12.83

* Pentopan 500 BG ^TM (Pentopan 500 BG) is a single enzyme of xylanase.

* Celebrity mix 6X MG ^TM (Ceremix 6X MG) is a beta-cellulase (celulase), protein xylanase (proteinase), alpha glucanase claim (β-glucanase) - amylase (alpha-amylase), pentosan xylanase (pentosanase) It is an enzyme which mixed four types of enzymes, such as each by the same weight ratio.

* Visco atom ^TM (Viscozyme L) is a beta-4 species such as cellulase (celulase), Hemicellulase (hemicellulase), xylene Rana claim (xylanase), Ara bar or the (arabanase) the glucanase the (β-glucanase) Are enzymes mixed in the same weight ratio.

4, such as cellulase (celulase), xylene Rana claim (xylanase), pentosan xylanase (pentosanase), Ara bar or the (arabanase) the glucanase the (β-glucanase) - * ultra flow L ^TM (Ultraflo L) is a beta It is an enzyme which mixed the enzyme of a species in the same weight ratio, respectively.

* Pectinex 100L ^TM (pectinex 100L) is a polygalacturonase, pectinesterase, cellulase, hemicellulase, etc. in pectintranseliminase. It is an enzyme which mixed four types of enzyme in the same weight ratio, respectively.

Cellullast ^TM (celluclast) is a cellulase homoenzyme.

Tunicase FN ^™ is a beta-glucanase homoenzyme.

As shown in Table 1, the yield was the highest as 12.83% when the tunicaase FN ^TM (beta-glucanase) was added and ultra-high pressure enzyme treatment was used.

Example  1-2: pH According to the change of Fucoidan  Extraction yield

40 times distilled water was added to the seaweed spores pulverized with 80 to 120 mesh, and the pH was adjusted to 4, 5, 6, 7, 8 with 0.1N NaOH and 0.1N HCl, and then in Example 1-1. 1% (w / v) was added to the polysaccharide enzyme, tunicaase FN ^TM (β-glucanase), which had the highest yield of fucoidan extraction. Each sample was placed in an ultrahigh pressure device (DSF-10L, Japan) and subjected to ultrahigh-pressure enzyme treatment for 24 hours at 40 ° C and 5000 atmospheres (500 MPa). After the ultrahigh pressure enzyme treatment, purified fucoidan was obtained in the same manner as in Example 1-1.

At this time, the yield of fucoidan according to each pH is calculated and shown in Table 2 below.

Table 2: Extraction yield of fucoidan with pH change

pH 4 5 6 7 8 yield(%) 10.38 9.08 9.77 14.07 16.01

As shown in Table 2, when pH was 8, the yield of fucoidan was the highest at 16.01.

Example  1-3: Depending on enzyme concentration and grinding size Fucoidan  Extraction yield

40 times distilled water was added to the seaweed spores cut into 2cm × 3cm size and the seaweed spores ground into 120 mesh, and the pH was adjusted to 8. Tunicase FN (β-glucanase) was added to 0, 0.2, 0.3, 0.6 and 1% (w / v), respectively, and placed in an ultrahigh pressure device (DSF-10L, Japan), and then 40 ° C and 5000 atm ( Ultrahigh pressure enzyme treatment for 24 hours at 500MPa) conditions. After the ultrahigh pressure enzyme treatment, purified fucoidan was obtained in the same manner as in Example 1-1.

At this time, the extraction yield of the fucoidan according to the concentration of polysaccharide degrading enzyme and the grinding size of the raw materials are shown in Table 3 and FIG.

Table 3: Extraction yield of fucoidan according to the concentration of polysaccharide degrading enzyme and grinding size of raw materials

Tunicase FN (β-glucanase)
(%) Fucoidan Extraction Yield (%) Cutting (2cm × 3cm) 120 mesh Enzyme-free treatment 2.59 6.31 0.2 4.31 9.69 0.3 6.25 11.00 0.6 11.45 12.90 One 15.59 19.78

As shown in Table 3 and Figure 2, the yield of fucoidan showed a tendency to increase in a concentration-dependent manner for the polysaccharide degrading enzyme. In addition, when the concentration of tunicaase FN (β-glucanase) was 1%, the yield of fucoidan in seaweed spores pulverized with 120 mesh was 20%, and the yield of fucoidan in wakame spores cut into 2 cm x 3 cm sizes. 5% higher results. It is analyzed that the efficiency is increased when the size of the pulverized raw material is smaller because the volume to which the polysaccharide enzyme can react increases.

[Example 2: Low molecular weight fucoidan production method using ultra-high pressure enzyme treatment]

Seaweed spores were prepared, dried, and ground to a size of 80 to 120 mesh. Then, after adding distilled water corresponding to 40 times the volume of wakaepocots, the pH was adjusted to 8 using 0.1N NaOH. Then, hot water was extracted for 4 hours at 85 ° C. and 140 rpm in a shaker (Woori Science, Korea).

The hot water extract was centrifuged (2,230 × G) to remove the residue, 2% (w / v) of calcium chloride (for food additives) was added to the separated supernatant, and left for 1 hour, followed by centrifugation of the precipitated alginic acid. (2,230 x G). The remaining supernatant was concentrated using a distillation concentrator (EYELA, Japan) at 80 ° C. until the solid content was 20-30%. Three times (v / v) alcohol was added to the concentrate, and left for 12 hours. At this time, the precipitated fucoidan was separated by centrifugation, dissolved by adding a small amount of water, and then dialyzed with a dialysis membrane (MW 3500, Biodesign Dialysis, USA) for 12 hours and freeze-dried to obtain crude fucoidan.

The crude fucoidan obtained above was dissolved in distilled water to a solution having a concentration of 5% (w / v), and the pH was adjusted to 8 using 0.1N NaOH. Tunicaase FN (β-glucanase) was added to 0.0%, 0.3%, 0.6%, 1.0%, 2.0% (w / v), respectively, and placed in an ultrahigh pressure device (DSF-10L, Japan) at 40 ° C and 5000 atmospheres. Low molecular weight fucoidan was obtained by performing ultrahigh pressure enzyme treatment for 24 hours at (500 MPa) and then freeze-drying.

Example  2-1: Conditions for Ultra High Pressure Enzyme Treatment Fucoidan  Molecular Weight

The molecular weight of the obtained fucoidan was analyzed using a HPLC (Summit HPLC system, Dionex, USA) method using a High-Performance Size Exclusion Chromatography (HPSEC) instrument. The obtained fucoidan was hydrolyzed with trifluoroacetic acid (TFA), and then analyzed by Shodex OHpac SB-804 HQ, Shodex OHpac SB-802.5 HQ column, and analyzed using an RI detector. At this time, the flow rate was 1.0 mL / min, and 100 μl of the sample was injected. The molecular weight distribution of fucoidan according to the conditions of ultrahigh pressure enzyme treatment is shown in Table 4 below.

Table 4: Molecular weight distribution of fucoidan according to conditions of ultra high pressure enzyme treatment

Molecular weight (Da) ratio (%) 300-3,000 20,000- 100,000 100,000-200,000 500,000- 600,000 800,000- 1,000,000 1,000,000- 1,200,000 Fucoidan Sigma ^TM - 10.1 - 89.9 - - Joe Fucoidan (C.F) 7.6 - 9.5 - - 82.9 C.F + Tunicase (1.0%) 17.3 13.4 - - - 69.3 C.F + Tunicase (0.0%) + Ultra High Pressure 13.0 - 10.1 - - 77.2 C.F + Tunicase (0.3%) + Ultra High Pressure 20.7 - 10.2 - 69.2 - C.F + Tunicase (0.6%) + Ultra High Pressure 16.2 - 9.0 - 74.8 - C.F + Tunicase (1.0%) + Ultra High Pressure 20.7 - 7.8 - - 63.2 C.F + Tunicase (2.0%) + Ultra High Pressure 30.2 - 8.7 - - 53.5 C.F + acid treatment 66.6 33.4 - - - -

As shown in Table 4, the crude fucoidan without ultra-high pressure enzyme treatment is a mixed molecular weight in a ratio of 300-3,000 Da: 100,000-200,000 Da: 1,000,000-1,200,000 Da = 7.6: 9.5: 83, with an average molecular weight of about 1,000,000 Da.

In addition, fucoidan obtained by adding 0.3% of tunicaase FN (β-glucanase) and ultra-high-pressure enzyme treatment was mixed in the ratio of 300-3,000 Da: 100,000-200,000 Da: 800,000-1,000,000 Da = 20.7: 10.2: 69.2 As molecular weight form, the average molecular weight was 900,000 Da. Compared with the crude fucoidan without the ultra-high pressure enzyme treatment, it can be seen that the ratio of the polymer is reduced, and the ratio of the low molecules of 300-3,000 Da and 100,000-200,000 Da is increased.

On the other hand, the acid-treated fucoidan was found to increase the ratio of low molecular weight of less than 3000 Da at the ratio of 300-3,000 Da: 20,000-100,000 = 66.6: 33.4. I could confirm it.

Example  2-2: According to the conditions of ultrahigh pressure enzyme treatment Fucoidan Sulfate  content

The fucoidan obtained in Example 2 was hydrolyzed at 110 ° C. for 4 hours with 0.5-2N hydrochloric acid solution, and then 0.2 ml of the hydrolyzate was taken. To this, 3.8 ml of 4% TCA solution and 1 ml of BaCl ₂ -gelatin reagent (Deajung, South Korea) were added, and the absorbance was measured at 360 nm after being left at room temperature for 20 minutes for analysis. In this case, K ₂ SO ₄ was used as a standard sample. The sulfuric acid group content of fucoidan according to the conditions of the ultrahigh pressure enzyme treatment is shown in Table 5 below.

Table 5: Sulfuric acid content of fucoidan according to the conditions of ultra-high pressure enzyme treatment]

% Per total Sulfuric acid content (%) Sigma ^TM Fucoidan (Pucoidan Standard) 60 6.26 ± 0.009 Joe Fucoidan (C.F) 52 7.17 ± 0.011 C.F + Tunicase (1.0%) 37 4.83 ± 0.021 C.F + Tunicase (0.0%) + Ultra High Pressure 33 6.41 ± 0.014 C.F + Tunicase (0.3%) + Ultra High Pressure 52 5.66 ± 0.015 C.F + Tunicase (0.6%) + Ultra High Pressure 52 5.40 ± 0.014 C.F + Tunicase (1.0%) + Ultra High Pressure 73 5.20 ± 0.015 C.F + Tunicase (2.0%) + Ultra High Pressure 79 1.09 ± 0.004 C.F + acid treatment 28 2.78 ± 0.004

As shown in Table 5, the fucoidan according to the present invention showed a result of lowering the sulfuric acid group content which plays an important role in physiological activity compared to the polymer fucoidan before the ultra-high pressure enzyme treatment, but the degree of decrease is about 1 to 2% It was found that the change was not large.

Example  2-3: Ultra High Pressure Enzyme Treatment Fucoidan  Elemental analysis

The major elements of fucoidan, carbon, hydrogen, oxygen, nitrogen and sulfur, were analyzed using an elemental analyzer (Elemental Analyzer, EA1108, Fisons instrument, Italy). The content analysis results are shown in Table 6 below.

Table 6: Elemental Analysis of Fucoidan Treated with Ultra High Pressure

% Of element Carbon (C) Hydrogen (H) Oxygen (O) Nitrogen (N) Sulfur (S) Ash system Sigma ^TM Fucoidan (Pucoidan Standard) 23.82 3.98 45.01 0.09 6.33 20.77 100 Joe Fucoidan (C.F) 23.97 4.33 48.66 0.79 9.07 13.18 100 C.F + Tunicase (1.0%) 25.30 4.57 45.89 0.74 8.05 15.45 100 C.F + Tunicase (0.0%) + Ultra High Pressure 23.56 4.21 42.48 0.83 8.49 20.43 100 C.F + Tunicase (0.3%) + Ultra High Pressure 24.04 4.44 45.96 0.83 7.53 17.2 100 C.F + Tunicase (0.6%) + Ultra High Pressure 24.97 4.72 47.06 0.75 6.91 15.59 100 C.F + Tunicase (1.0%) + Ultra High Pressure 25.99 4.57 47.64 0.80 7.40 13.6 100 C.F + Tunicase (2.0%) + Ultra High Pressure 28.05 4.88 47.13 0.66 5.97 13.31 100 C.F + acid treatment 12.06 2.17 22.53 0.42 4.57 58.25 100

As shown in Table 6, the sulfuric acid group content by the ultra-high pressure enzyme treatment showed a result of lowering by 1 to 2% similar to the result of Example 2-2, but the content of sulfur element that plays an important role in physiological activity It can be seen that the change is not large.

Example  2-4: ultra high pressure enzyme treatment Fucoidan FT - IR  Spectral results

Fucoidan (FIG. 3a) before the ultra-high pressure enzyme treatment, Fucoidan (FIG. 3b), ultra-high-pressure enzyme treatment (FIG. 3C), and Fucoidan standard (FIG. 3D) prepared by the general enzyme extraction method (FIG. -IR, Spectrum GX, Perkin Elmer, USA) absorption spectrum results are shown in Figures 3a to 3d, respectively, and are shown in Figure 3e to summarize the results.

As shown in FIG. 3a and FIG. 3b, the FT-IR spectra were similar before and after the ultrahigh pressure enzyme treatment, and the structure of the standard fucoidan also showed similar FT-IR spectra.

In particular, the strong absorption bands around 1220 and 830 cm ⁻¹ exhibit S = O and COS bonds, respectively. This indicates that the COS is bound to the axial position, and it can be seen that most of the ultra-high pressure-treated fucoidans have a sulfate group bonded to the C-4 position (Anno et al, 1966; Nishino, 1989). Thus, it can be seen that the fucoidan according to the method of the present invention does not cause structural deformation.

However, fucoidan acid-treated with 1N HCl for 24 hours showed a structural change, resulting in an absorption spectrum in which the structure of the binding site of S═O and C—O—S was modified.

Example  2-5: ultra high pressure enzyme treatment Fucoidan Antithrombotic activity  analysis

Test Example 1 Measurement of Antithrombotic Activity Using TT (thrombin Time)

Blood clotting time was measured by measuring the blood clotting time using a coagulometer (Coatron M1, TECO, Germany), a blood coagulation analyzer. As a positive control, heparin (Sparma, Sigma, USA), a natural blood coagulation inhibitor, and the fucoidan (fucoidan, Sigma, USA) were used as standard fucoidans. All the analyzes were expressed as mean ± standard deviation (mean ± SD) of the results of three replicates.

First, in order to measure the antithrombotic activity of the ultrahigh-pressure enzyme-treated fucoidan, the effect of prolonging blood coagulation time was measured by using a TT (thrombin time) analysis. TT is a common method of testing common blood coagulation pathways and is appropriate for comparison of blood coagulation by different mechanisms.

If the clotting time shows a large value, it means that the time for fibrinogen to be converted to fibrin by thrombin is delayed. That is, in the present invention, such anticoagulant activity was analyzed as anticoagulant activity.

After dissolving 2 ml of 10 mM Tris-buffer (pH 7.5) in a TT (Thrombin time) reagent ampoule, the solution was further diluted with 6 ml of the buffer to prepare a thrombin solution. The sample obtained in Example was dissolved in 10 mM Tris-buffer (pH 7.5) at a concentration of 25, 50 and 100 µg / ml to prepare and use a measurement sample. In addition, fibrinogen was dissolved in 0.125% (w / v) in 10 mM Tris-buffer (pH 7.5) to prepare and use a fibrinogen solution. 12 μl of the sample solution and 228 μl of the fibrinogen solution (0.125%, w / v) were mixed and stirred. 25 μl of the mixed solution and the thrombin solution were put into cuvettes, respectively, and preheated at 37 ° C. for 3 minutes. Then, 50 μl of the thrombin solution was added to 25 μl of the mixed solution, mixed, and the blood coagulation reaction was measured. A blood coagulation analyzer was performed.

For comparison, heparin (heparin, Sigma, USA) and the special reagent fucoidan (fucoidan, Sigma, USA) for the comparison through the same process and the results of the TT was shown in Table 7 and FIG.

Table 7: Antithrombotic Activity (TT) of Heparin, Standard Fucoidan and Ultra High Pressure Enzyme Treated Fucoidan

density
(Μg / ml) Antithrombotic activity TT (seconds) Heparin 0.45 262 ± 3.8 Sigma ^TM Fucoidan (Pucoidan Standard) 12.5 42 ± 0.9 Joe Fucoidan (C.F) 12.5 158 ± 13.3 C.F + Tunicase (1.0%) 12.5 124 ± 2.3 C.F + Tunicase (0.0%) + Ultra High Pressure 12.5 77 ± 8.9 C.F + Tunicase (0.3%) + Ultra High Pressure 12.5 253 ± 10.3 C.F + Tunicase (0.6%) + Ultra High Pressure 12.5 167 ± 7.4 C.F + Tunicase (1.0%) + Ultra High Pressure 12.5 193 ± 28.8 C.F + Tunicase (2.0%) + Ultra High Pressure 12.5 96 ± 12.8 C.F + acid treatment 12.5 13 ± 0.3

As shown in Table 7 and FIG. 4, the crude fucoidan extracted normally showed anticoagulant activity of 158 seconds, while the ultrahigh pressure enzyme-treated fucoidan was added with 0.3% of tunicaase FN (β-glucanase) as 253 seconds. The anticoagulant activity was similar to that of the positive control heparin. Accordingly, it was found that by treating ultra-high pressure enzymes with fucoidan, the effect of inhibiting the activity of thrombin, a factor involved in the common pathway of antithrombotic activity, was increased.

Test Example 2: Determination of antithrombotic activity using activated partial thromboplastin time (aPTT)

In order to measure the antithrombotic activity of the ultra-high pressure enzyme-treated fucoidan, the effect of prolonging the coagulation time was measured by using an activated partial thromboplastin time (aPTT) assay. The aPTT is a blood coagulation test method derived from the endogenous blood coagulation pathway and is suitable for comparison between samples having different mechanisms in blood coagulation action.

First, as a positive control for comparison with the ultrahigh-pressure enzyme-treated fucoidan prepared in Example, heparin (heparin, Sigma, USA) was used to mix the plasma (plasma) in a 1: 9 ratio, the blank test sample was used as a sample Unmixed plasma was used. In addition, the special reagent fucoidan (fucoidan, Sigma, USA) was used as the standard fucoidan. The aPTT (TECO, Germany) reagent and 25 mM calcium chloride (CaCl2) reagent are preheated at 37 ° C for at least 5 minutes, and then 25 µl of plasma is dispensed into the cuvette, 25 µl of the pre-heated aPTT reagent is added, and then at 37 ° C. The reaction was carried out for exactly 5 minutes, and 25 μl of 25 mM calcium chloride was added thereto, and the time until the mixture was coagulated was measured and shown in Table 8 and FIG. 5. The unit of measurement is expressed in seconds.

Table 8: Antithrombotic Activity (aPTT) of Heparin, Standard Fucoidan and Ultra High Pressure Enzyme Treated Fucoidan

density
(Μg / ml) Antithrombotic activity aPTT (seconds) Heparin 4 229 ± 10.7 Sigma ^TM Fucoidan (Pucoidan Standard) 25 161 ± 1.7 Joe Fucoidan (C.F) 25 106 ± 5.1 C.F + Tunicase (1.0%) 25 124 ± 2.3 C.F + Tunicase (0.0%) + Ultra High Pressure 25 133 ± 8.1 C.F + Tunicase (0.3%) + Ultra High Pressure 25 156 ± 3.1 C.F + Tunicase (0.6%) + Ultra High Pressure 25 148 ± 4.7 C.F + Tunicase (1.0%) + Ultra High Pressure 25 135 ± 2.1 C.F + Tunicase (2.0%) + Ultra High Pressure 25 94 ± 5.4 C.F + acid treatment 25 40 ± 1.0

As shown in Table 8 and FIG. 5, Fucoidan treated with ultra high pressure by adding 0.3% of tunicaase FN (β-glucanase) had anticoagulant activity of 156 seconds, 106 of crude fucoidan before ultrahigh enzyme treatment. The results were 50 seconds higher than the second, and showed anticoagulant activity similar to that of standard fucoidan. Accordingly, it was found that the ultrahigh pressure enzyme treatment inactivates factors involved in the endogenous pathway, thereby inhibiting coagulation, thereby inhibiting coagulation.

Example  2-6: ultra high pressure enzyme treatment Fucoidan  Antioxidant activity

The crude fucoidan before the ultrahigh pressure enzyme treatment and the fucoidan treated with the ultrahigh pressure enzyme were prepared at a concentration of 2,000 µg / ml, respectively, and DPPH radical scavenging ability (%) experiment was performed. 160 μl of dissolved DPPH solution and 40 μl of sample dissolved in methanol at 0.4mM were added and allowed to stand in the dark for 30 minutes, and then absorbance was measured at 515 nm using a microplate reader (VERSAmax, Molecular Device, CA, USA). It was. Ascorbic acid and alpha tocopherol were used as reference materials. The experimental results are shown in Table 9, Figure 6a and Figure 6b.

Table 9: Antioxidant Activity of Ultrahigh Pressure Enzyme-treated Fucoidan (DPPH Radical Scavenging Activity)

DPPT radical scavenging activity (%) Joe Fucoidan (C.F) 16.31 ± 2.33 C.F + Tunicase (1.0%) 19.55 ± 1.25 C.F + Tunicase (0.0%) + Ultra High Pressure 18.46 ± 3.14 C.F + Tunicase (0.3%) + Ultra High Pressure 24.75 ± 3.88 C.F + Tunicase (0.6%) + Ultra High Pressure 16.95 ± 1.77 C.F + Tunicase (1.0%) + Ultra High Pressure 15.49 ± 2.14 C.F + Tunicase (2.0%) + Ultra High Pressure 7.20 ± 4.54 C.F + acid treatment not detected

As shown in Table 9, Figure 6a and Figure 6b, the antioxidant activity of the ultra-high-pressure enzyme-treated fucoidan by adding 0.3% of the tunicaase FN (β-glucanase) was 24.75%, 16.31 of the crude fucoidan before the ultra-high pressure enzyme treatment It showed higher antioxidant activity than%.

According to the present invention, by the ultra-high pressure enzyme treatment during the low molecular weight of the fucoidan, there is no difference in sulfuric acid group content that plays an important role in physiological activity, as well as low molecular weight fucoidan can be obtained without structural modification, There is an effect that can be obtained fucoidan more excellent antithrombotic activity and antioxidant activity.

Figure 1a is a flow chart showing a method for producing fucoidan using the ultrahigh pressure enzyme treatment according to a preferred embodiment of the present invention, Figure 1b is a flow chart showing a method for producing fucoidan using an ultrahigh pressure enzyme treatment according to another preferred embodiment of the present invention. ,

2 is a graph showing the extraction yield of fucoidan according to the concentration of polysaccharide degrading enzyme and the grinding size of the raw material,

Figure 3a is a fucoidan before the ultra-high pressure enzyme treatment, Figure 3b is a fucoidan treated with ultra-high pressure enzyme in accordance with a preferred embodiment of the present invention, Figure 3c is an acid-treated fucoidan, Figure 3d is a standard fucoidan absorption absorption analyzed by an infrared spectrophotometer 3E is a graph showing the spectral results, and FIG. 3E is a graph showing the contents of FIGS. 3A to 3D in a comprehensive view.

Figure 4 is a graph showing the antithrombotic activity (TT) of heparin, crude fucoidan, standard fucoidan and ultrahigh pressure enzyme-treated fucoidan,

5 is a graph showing the antithrombotic activity (aPTT) of heparin, crude fucoidan, standard fucoidan and ultrahigh pressure enzyme treated fucoidan,

Figure 6a is a graph showing the antioxidant activity of the reference material, Figure 6b is a graph showing the antioxidant activity of crude fucoidan, ultra-high pressure enzyme-treated fucoidan, ultra-high pressure enzyme-treated fucoidan, acid-treated fucoidan.

Claims (6)

Preparing brown algae; Adding a polysaccharide degrading enzyme to the prepared brown algae and subjecting it to an ultra-high pressure enzyme treatment; Filtering the ultrahigh-pressure enzymatically digested solution; Adding salt to the filtrate to remove alginic acid in the form of insoluble salts; Precipitating crude fucoidan by adding alcohol to the filtrate from which the alginic acid has been removed; And Desalting and lyophilizing the crude fucoidan to obtain fucoidan, The ultra-high pressure enzyme treatment is a fucoidan production method using an ultra-high pressure enzyme treatment, characterized in that carried out at a pressure of 80MPa ~ 500MPa. Preparing brown algae; Hydrothermal extraction of the prepared brown algae; Filtering the hot water extract; Adding salts to the filtrate to remove coexisting alginic acid in the form of insoluble salts; Precipitating crude fucoidan by adding alcohol to the filtrate from which the alginic acid has been removed; Desalting the crude fucoidan; And Adding a polysaccharide degrading enzyme to the crude fucoidan and treating the ultra-high pressure enzyme, The ultra-high pressure enzyme treatment is a fucoidan production method using an ultra-high pressure enzyme treatment, characterized in that carried out at a pressure of 80MPa ~ 500MPa. The method according to claim 1 or 2, wherein the brown alga is a method for producing fucoidan using ultrahigh pressure enzyme treatment, characterized in that seaweed spores. The method of claim 1 or 2, wherein the polysaccharide is beta-glucanase (β-glucanase), characterized in that the fucoidan production method using ultra-high pressure enzyme treatment. The method according to claim 1 or 2, wherein the salt is calcium chloride. delete

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