KR102247715B1 - Manufacturing method of fucoidan having high purity and low molecular - Google Patents

Abstract

The present invention is a washing step of washing the spore leaves of Jeju-sandol seaweed, which are brown algae, to remove salt and foreign substances, and to dry them to contain a predetermined moisture; Extrusion and coarse pulverization step of coarse pulverization after extruding the washed spore leaves into an extruder; An extraction step of extracting with an extractor by adding purified water 10 to 15 times by weight to the coarsely pulverized product to obtain an extract containing fucoidan; A first fermentation step of fermenting for 12 to 36 hours by adding a culture medium containing Bacillus methylotropicus to the extract to decompose sugar to make a small molecule; A filtration step of filtering the primary fermentation broth with a filter to obtain and store a filtrate obtained by separating the cells; A purification step of precipitation and removal of alginic acid from the filtrate and desalting; A concentration step of concentrating the purified liquid with a concentrator; A second fermentation step of mixing the cell culture broth containing Bacillus methylotropicus with the fermentation object obtained in the previous step and fermenting for 12 to 36 hours to decompose sugar to make ultra-low molecular weight; Freeze-drying step of sterilizing the secondary fermentation broth and then freeze-drying it; An ultra-fine pulverization step of pulverizing the dried product into ultra-fine powder of 2000 mesh or more with a pulverizer; And it provides a method for producing a high-purity low molecular weight fucoidan comprising a quality inspection and packaging step of packaging after the quality inspection of the fucoidan powder.

Description

Manufacturing method of high purity low molecular weight fucoidan {MANUFACTURING METHOD OF FUCOIDAN HAVING HIGH PURITY AND LOW MOLECULAR}

The present invention relates to a method for producing fucoidan, and more particularly, a method for producing a high-purity low-molecular fucoidan capable of extracting an extract containing fucoidan from spore leaves of a brown algae, Jeju-sandol seaweed, and obtaining a low-molecularized fucoidan from the extract. It is about.

Fucoidan, derived from brown algae, is a slippery component contained in Tot, seaweed, kelp, Ecklonia cava, mozuku, and large yam, that is, a viscous polysaccharide and is composed of fibrous polysaccharides of 2 to 4% of the amount of dry liquor.

These fucoidans contain a lot of nutrients such as essential minerals and vitamins that are beneficial to the human body, such as calcium and jade, although protein and fat are weak, and alginic acid, which acts to inhibit the absorption of cholesterol in the slippery component, lowers blood pressure. It is known to contain laminin, which is attracting attention.

Fucoidan contains a lot of lactic acidified fugos, fugos, galactose, etc. In 1995, after the Japanese Cancer Society reported that lactic acidized fugos and fugos were effective against cancer, brown algae-derived fucoidan began to attract attention. . However, since fucoidan is composed of non-degradable polysaccharides with a molecular weight of about 3 million to 5 million that cannot be digested by humans, it is almost impossible to utilize organic components in the human body when used without making it low molecular weight.

As a related art, Patent Document 1 describes a method of isolating and purifying fucoidan, laminarin, and alginic acid from brown algae using phosphate.

However, the above-described prior art is only a degree to use as a product by extracting high molecular weight fucoidan as it is in its natural state, or simply improving the palatability or flavor of the extracted product, and it is difficult to extract fucoidan with high purity. There is a problem that it is difficult to be easily absorbed into the interior and it is difficult to expect utility.

In addition, until recently, many research institutes at home and abroad are actively conducting research on the manufacturing method of fucoidan, but fucoidan is extracted and recovered without loss or deterioration of the active ingredient containing fucoidan, can be well absorbed into the human body, and mass production is possible. It is a situation that does not suggest an efficient manufacturing method.

Patent Notice No. 1981-0000029 (published on February 2, 1981)

The present invention was conceived to solve all the above-described problems, and more than 2,000 mesh of brown algae, in particular, for easy absorption into the human body through primary and secondary fermentation and purification using microorganisms for the extract extracted from the spore leaves of Jeju-sandol seaweed. An object thereof is to provide a method for producing a high-purity low-molecular fucoidan capable of obtaining a low molecular weight fucoidan in an ultrafine powder state with high purity and capable of mass-producing fucoidan without loss or deterioration of active ingredients.

In order to solve the above problems, the present invention is a washing step of removing salt and foreign substances by washing the spore leaves of the brown algae Jeju-sandol seaweed with water; Extrusion and coarse pulverization step of coarse pulverization after extruding the washed spore leaves into an extruder; An extraction step of extracting with an extractor by adding purified water of 10 to 15 times by weight to the coarsely pulverized product to obtain an extract containing fucoidan; A first fermentation step in which a cell culture solution containing Bacillus methylotropicus is added to the extract and fermented for 12 to 36 hours to decompose sugar to reduce molecular weight; A filtration step of filtering the primary fermentation broth with a filter to obtain and store a filtrate obtained by separating the cells; A purification step of sedimenting and desalting alginic acid from the filtrate; A concentration step of concentrating the purified liquid with a concentrator; A second fermentation step of mixing the cell culture broth containing Bacillus methylotropicus with the fermentation object obtained in the previous step and fermenting it for 12 to 36 hours to decompose sugar to make ultra-low molecular weight; Freeze-drying step of sterilizing the secondary fermentation broth and then freeze-drying it; An ultra-fine pulverization step of pulverizing the dried product into ultra-fine powder of 2000 mesh or more with a pulverizer to pulverize it; And it provides a method for producing a high-purity low molecular weight fucoidan comprising a quality inspection and packaging step of packaging after the quality inspection of the fucoidan powder.

According to the present invention, a low-molecular weight fucoidan of 2,000 mesh or more is easily absorbed into the human body through primary and secondary fermentation and purification of brown algae, especially from spore leaves of Jeju-sandol seaweed. It can be obtained with high purity, and there is an effect of mass-producing fucoidan without loss or degradation of active ingredients.

1 is a flowchart of a method of manufacturing a high-purity low-molecular fucoidan according to an embodiment of the present invention.
2 is a graph showing the relationship between molecular weight and elution time based on a chromatogram of a molecular weight standard product.

Hereinafter, with reference to the drawings, specific details for carrying out the method for manufacturing a high-purity low-molecular fucoidan according to the present invention will be described in detail with reference to embodiments.

The method for producing high-purity low-molecular fucoidan according to the present invention extracts an extract containing fucoidan from the spore leaves of the brown algae, Jeju-san-dol seaweed, and obtains low-molecularized fucoidan from the extract with high purity.Referring to FIG. 1, the washing step ( S10), extrusion and coarse pulverization step (S20), extraction step (S30), first fermentation step (S40), filtration step (S50), purification step (S60), concentration step (S70), secondary fermentation step (S80) ), freeze drying step (S90), ultra-fine pulverization step (S100), quality inspection and packaging step (S110).

The washing step (S10) is a process of removing salt and foreign substances by washing the spore leaves of Jeju-sandol seaweed, which is a brown algae.

Jeju-san-dol seaweed grows naturally in rocks along the coast of Jeju Island, and contains more various nutrients such as protein, vitamins, minerals, calcium, and iodine compared to farmed seaweed. The spore leaves of Jeju-san-dol seaweed make spores of seaweed and erupt. It corresponds to the reproductive organs that spread seaweed as a result, and for this reason, active ingredients for self-protection and vigorous activity are condensed in the spore lobe, and physiologically active substances such as fucoidan are also contained.

First, by immersing the spore leaves of Jeju-sandolmidae in fresh water or washing with fresh water to remove salt and foreign substances, and to elute salt, the washing process can be repeated several times.After removing foreign substances and salt, the washed spore leaves are removed. After natural drying using solar heat and sea breeze, it can be artificially dried using a dryer until the desired moisture content is reached. At this time, it is preferable to dry so that the moisture content of the spore leaves is 10 to 15%. If the moisture content is less than 10%, it may be deteriorated due to excessive drying or difficult to extrude, and if it exceeds 15%, it may not be suitable for coarse pulverization or low molecular weight of fucoidan may be difficult.

After performing the washing step (S10), the washed spore leaves are put into an extruder, extruded, and coarsely pulverized, followed by extrusion and coarse pulverization (S20).

In the extrusion and coarse pulverization step (S20), isothiocyanate is added and pressure is applied in an extruder to extrude the spore leaves at a temperature of 60 to 90°C, and if it is less than 60°C, molding may be difficult, and it exceeds 90°C. Doing so can destroy active ingredients.

In addition, the extruded product is coarsely pulverized into 100 to 300 mesh with a grinder. At this time, if the coarse pulverized product is less than 100 mesh, the particle size is large, so the extraction yield may decrease, and if the coarse pulverized product exceeds 300 mesh, the surface area increases. There is a problem in that excessive purified water must be added in the extraction step.

Isothiocyanates are irritating substances extracted from cruciferous plants, are natural volatile antibacterial substances, are known as sour ingredients of mustard seeds and horseradish, and are added during the extrusion process to inhibit the growth of bacteria, etc., and spore leaves. It stimulates and opens the pores of the cell wall, increases the solubility of the cell wall, and increases the gap so that soluble components can be easily extracted, and a large number of polysaccharides can be extracted.

After the extrusion and coarse pulverization step (S20) is performed, an extraction step (S30) of obtaining an extract containing fucoidan is performed by adding purified water 10 to 15 times by weight to the coarse pulverized product and extracting it with an extractor.

The extraction step (S30) extracts the extract containing fucoidan while stirring the coarse pulverized product at a temperature of 50 to 70°C for 2 to 5 hours using a rotary stirrer. At this time, it is preferable to increase the extraction efficiency by hitting the coarse pulverized product of the spore leaves of Jeju-san-dol seaweed by generating a vortex in the purified water using a blower.

After the extraction step (S30) is performed, a first fermentation step (S40) is performed in which a cell culture solution containing Bacillus methylotropicus is added to the extract and fermented for 12 to 36 hours to decompose sugar and make it low molecular weight. .

Bacillus methylotrophicus (Bacillus methylotrophicus) is a fermentation strain that grows most vigorously in a temperature range of 35 to 39°C. It has excellent high heat resistance, excellent viability in strong acids, and has an effect of inhibiting the growth of pathogens.

In order to cultivate Bacillus methylotropicus, the culture medium is made by mixing brown rice vinegar with water and a nutrient supply source to create a condition of pH 3.0 to 4.5, and brown rice vinegar is an organic acid produced during fermentation as well as various nutrients contained in brown rice. , Amino acids, etc. are abundantly contained, so it functions as a nutrient supply source.In particular, it inhibits the proliferation of bacteria, softens the cell wall of the spore lobe so that soluble components can be easily extracted so that a large amount of polysaccharides can be extracted from within the human body. It can be easily absorbed and dissolved to increase digestion and absorption rate. At this time, the brown rice vinegar is preferably mixed with 1 to 5 parts by weight per 100 parts by weight of water, and if it exceeds 5 parts by weight, the activity of the cells may decrease.

In the first fermentation step (S40), 0.3 to 0.5 g of the cell culture broth obtained by culturing Bacillus methylotropicus with respect to 1 L of the extract was added to a temperature of 35 to 39°C and a pH of 3.0 to 4.5. The sugar can be decomposed by fermentation for 12 to 36 hours. At this time, microbial decomposition is performed by an acid-resistant strain under the acidic condition of pH, the low molecular weight efficiency of fucoidan is increased, and contaminants such as bacteria can be removed. However, in a strong acid with a pH of less than 3.0, safety to the human body may be deteriorated, so it is preferable to proceed under a condition of pH 3.0 or higher.

Such microbial fermentation reduces the molecular weight of the fucoidan indicator material with a molecular number of 200,000 to 1,000,000 Da to less than 20,000 Da, removes proteins, fats, ash, etc. to relatively increase the yield of the fucoidan indicator material, which is easy to separate and purify. It improves the quality by purifying the product.

After performing the first fermentation step (S40), a filtration step (S50) in which the first fermentation broth is filtered through a filter to obtain and store the filtrate obtained by separating the cells may be performed.

After performing the filtration step (S50), a purification step (S60) of sedimenting and removing alginic acid from the filtrate and desalting is performed. In the purification step (S60), 5 to 10 parts by weight of an aqueous calcium chloride solution is added to 100 parts by weight of the filtrate and stirred to precipitate and remove alginic acid to recover the filtrate. Then, the filtrate is pressurized to permeate the semipermeable membrane to demineralize.

After performing the purification step (S60), a concentration step (S70) of concentrating the purified liquid with a concentrator is performed.

After performing the concentration step (S70), a second fermentation step of mixing the cell culture broth containing Bacillus methylotropicus with the fermentation object obtained in the previous step and fermenting it for 12 to 36 hours to decompose sugar to make ultra-low molecular weight ( S80) is performed.

The second fermentation step (S80) is fermented for 12 to 36 hours at a temperature of 35 to 39°C by adding 0.5 to 0.7 g of the cell culture broth obtained by culturing Bacillus methylotropicus to 1 L of a fermentation object based on the amount of dry matter. By decomposing sugar, fucoidan is further reduced in molecular weight, so that it becomes ultra-low molecular weight with a molecular weight of 1,000 Da or less, which is a state that can be well absorbed by the human body.

After the secondary fermentation step (S80) is performed, a freeze-drying step (S90) of sterilizing the secondary fermentation broth and then freeze-drying is performed.

After the freeze-drying step (S90) is performed, an ultra-fine pulverization step (S100) of obtaining a fucoidan powder is performed by pulverizing the dried product into ultrafine powders of 2000 mesh or more with a grinder.

After the ultra-fine pulverization step (S100) is performed, a quality inspection and packaging step (S110) of packaging after quality inspection of the fucoidan powder are performed.

<Example>

10kg of spore leaves of Jeju Sandolmiyeop, which grows wild in the rocks along the coast of Jeju Island, were immersed in fresh water for 1 hour to remove foreign substances, eluted salt, and then immersed again for 2 hours to elute salt. Then, after natural drying using solar heat and sea breeze, it was dried again in a dryer and slowly dried until the moisture content of the spore leaves reached about 12%. Then, a small amount of isothiocyanate was added to the dried spore leaves, and pressure was applied in an extruder, extruded at a temperature of 70° C., and coarsely pulverized into 100 to 300 mesh with a grinder.

Then, the coarse pulverized product was put in an extract bag, 12 times of purified water was added at a weight ratio to the coarse pulverized product, and then stirred in an extractor at a temperature of 60° C. for 5 hours to obtain an extract containing fucoidan. At this time, by creating a vortex in the purified water using a blower, the coarse pulverized product of the spore leaves of Jeju-san-dol seaweed was hit.

After the extraction is complete, prepare a culture solution of Bacillus methylotropicus cultured in a mixture of water, a nutrient source, and brown rice vinegar, and 20g of a culture solution containing Bacillus methylotropicus in 50ℓ of the extract is used as a dry matter. Was added and stirred for 24 hours at a temperature of 37° C. and a pH of 3.5 for the first microbial fermentation.

Next, the first fermentation broth was filtered with a filter to obtain a filtrate obtained by separating the cells, and 6 parts by weight of an aqueous calcium chloride solution was added to 100 parts by weight of the filtrate, followed by stirring to precipitate and remove alginic acid to recover the filtrate, and then pressurize the filtrate. After passing through a semipermeable membrane, desalting was purified, and then concentrated with a concentrator.

In addition, 6 g of the culture medium containing Bacillus methylotropicus was added to 10 L of the concentrated fermented product based on the amount of dry matter, and the microbial fermentation was performed for 24 hours at a temperature of 37°C.

Next, the secondary fermentation broth was sterilized, freeze-dried, and pulverized into ultra-fine powders of 2000 mesh or more with a grinder to obtain fucoidan powder, followed by quality inspection and packaging.

The thus obtained fucoidan was measured for molecular weight distribution using size exclusion chromatography using a TSKgel GMPWXL column.

That is, molecular weight standards (such as fluran) as shown in Table 1 were dissolved in 0.1 mol/L sodium acetate solution to obtain a 0.05 W/V% solution.

<Table 1>

0.5 g of fucoidan prepared in the above example was collected, and 10 ml of a 0.1 mol/L sodium acetate solution was added. The solution was allowed to stand at room temperature overnight, heated in boiling water for 10 minutes, cooled, and filtered through a membrane filter having a pore diameter of 0.45 μm, and the obtained solution was used as a test solution.

Standard solution and test solution were injected into a high-speed liquid chromatography apparatus using a size exclusion column, and the obtained results were analyzed using the 480II data station GPC program [SYSTEM INSTRUMENTS, Inc.]. On the other hand, the molecular weight estimation of each peak was carried out using the calibration curve of Fig. 3 prepared based on the elution time and molecular weight of the molecular weight standard product.

The results of measuring the molecular weight distribution are shown in Table 2 below.

<Table 2>

In the end, the method for producing high-purity low-molecular fucoidan according to the present invention is an ultra-fine particle of 2,000 mesh or more to facilitate absorption into the human body through primary and secondary fermentation and purification using cells for the extract extracted from spore leaves of brown algae, especially Jeju-san-dol seaweed. It is possible to obtain a low molecular weight fucoidan in a powder state with high purity, and to mass-produce fucoidan without loss or deterioration of active ingredients.

In the present invention, the above embodiments are only examples, and the present invention is not limited thereto. Anything that has substantially the same configuration as the technical idea described in the claims of the present invention and achieves the same operation and effects is included in the technical scope of the present invention.

Claims (6)

Washing step of washing the spore leaves of the brown algae, Jeju-san-dol seaweed, to remove salt and foreign matter; Extrusion and coarse pulverization step of coarse pulverization after extruding the washed spore leaves into an extruder; An extraction step of extracting with an extractor by adding purified water 10 to 15 times by weight to the coarsely pulverized product to obtain an extract containing fucoidan; A first fermentation step of fermenting for 12 to 36 hours by adding a culture medium containing Bacillus methylotropicus to the extract to decompose sugar to make a small molecule; A filtration step of filtering the primary fermentation broth with a filter to obtain and store a filtrate obtained by separating the cells; A purification step of sedimenting and desalting alginic acid from the filtrate; A concentration step of concentrating the purified liquid with a concentrator; A second fermentation step of mixing the cell culture broth containing Bacillus methylotropicus with the fermentation object obtained in the previous step and fermenting for 12 to 36 hours to decompose sugar to make ultra-low molecular weight; Freeze-drying step of sterilizing the secondary fermentation broth and then freeze-drying it; An ultra-fine pulverization step of pulverizing the dried product into ultra-fine powder of 2000 mesh or more with a pulverizer to obtain fucoidan powder; And a quality inspection and packaging step of packaging after quality inspection for the fucoidan powder,
In the washing step, the washed spore leaves are dried to a moisture content of 10 to 15%,
The extrusion and coarse pulverization step is a method for producing a high-purity low-molecular fucoidan, characterized in that the spore leaf is extruded at a temperature of 60 to 90°C by adding isothiocyanate, and then coarsely pulverized into 100 to 300 mesh.
Washing step of washing the spore leaves of the brown algae, Jeju-san-dol seaweed, to remove salt and foreign matter; Extrusion and coarse pulverization step of coarse pulverization after extruding the washed spore leaves into an extruder; An extraction step of extracting with an extractor by adding purified water 10 to 15 times by weight to the coarsely pulverized product to obtain an extract containing fucoidan; A first fermentation step of fermenting for 12 to 36 hours by adding a culture medium containing Bacillus methylotropicus to the extract to decompose sugar to make a small molecule; A filtration step of filtering the primary fermentation broth with a filter to obtain and store a filtrate obtained by separating the cells; A purification step of sedimenting and desalting alginic acid from the filtrate; A concentration step of concentrating the purified liquid with a concentrator; A second fermentation step of mixing the cell culture broth containing Bacillus methylotropicus with the fermentation object obtained in the previous step and fermenting for 12 to 36 hours to decompose sugar to make ultra-low molecular weight; Freeze-drying step of sterilizing the secondary fermentation broth and then freeze-drying it; An ultra-fine pulverization step of pulverizing the dried product into ultra-fine powder of 2000 mesh or more with a pulverizer to obtain fucoidan powder; And a quality inspection and packaging step of packaging after the quality inspection of the fucoidan powder,
In the first fermentation step, 0.3 to 0.5 g of a cell culture solution containing Bacillus methylotropicus and brown rice vinegar was added to 1 L of the extract, based on the amount of dry matter, at a temperature of 35 to 39°C and a pH of 3.0 to 4.5. A method for producing high-purity low-molecular fucoidan, characterized in that the sugar is decomposed by fermentation for 12 to 36 hours.
The method according to claim 1 or 2,
The extraction step is characterized in that the extraction is performed while stirring for 2 to 5 hours at a temperature of 50 to 70° C. with respect to the coarse pulverized product using a rotary stirrer, and the purified water is flowed using a blower to enable smooth extraction. Method for producing high-purity low-molecular fucoidan.
The method according to claim 1 or 2,
In the purification step, 5 to 10 parts by weight of an aqueous calcium chloride solution is added to 100 parts by weight of the filtrate and stirred to precipitate and remove alginic acid to recover the filtrate, and then the filtrate is pressurized to permeate the semipermeable membrane to demineralize. Manufacturing method.
The method according to claim 1 or 2,
In the second fermentation step, 0.5 to 0.7 g of the cell culture broth containing Bacillus methylotropicus is added to 1 L of the fermentation target and fermented at a temperature of 35 to 39° C. for 12 to 36 hours to obtain sugar. A method for producing high-purity low-molecular fucoidan, characterized in that decomposition.
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