JP2007290971A - Method for extracting glycerol galactoside - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for safely and efficiently extracting glycerol galactoside from a marine alga (including laver) belonging to red algae without using an organic solvent such as ethanol, an ether, etc., and a method for safely and efficiently extracting glycerol galactoside from a low-quality laver which has an especially low protein content, is hardly used and called "color faded" or "color faded laver". <P>SOLUTION: The method for selectively extracting glycerol galactoside from a marine alga belonging to red algae, especially laver as a raw material by using only water at 0-30°C as a solvent. A laver having ≤30 wt.% protein content is preferably used as the raw material. The extracted glycerol galactoside is easily purified by ultrafiltration followed by ion exchange resin treatment or electrodialysis. <P>COPYRIGHT: (C)2008,JPO&INPIT

Description

  The present invention relates to a novel method for extracting glycerol galactoside. Specifically, the present invention relates to a method for extracting glycerol galactoside contained in red algae such as laver more safely and efficiently than before.

Japanese Patent Laid-Open No. 5-139988 Seiseisha Koseikaku, 391-392 pages of the revised 4th edition “Suisan Kagaku” issued on October 5, 1973

  Glycerol galactoside (hereinafter sometimes referred to as “GG”) in which galactose is α-bonded to glycerol is also referred to as fluroidoside, and is widely distributed in seaweeds of red algae such as Nori, Tsunomata, and Dulse (Non-patent Document 1). ). The inventor of the present invention is particularly referred to as “color loss” or “color loss nori” among the nodules in which the glycerol galactoside belongs to the red alga Amori genus, and the protein content is 30% by weight or less and is almost used. It is contained in a large amount of low-quality paste, and glycerol galactoside has strong bifidobacterial growth promoting ability, is not digested by digestive enzymes, and is not absorbed from the intestinal tract. Elucidated in collaboration with other researchers.

  In order to use glycerol galactoside for foods and pharmaceuticals, it is necessary to extract and purify glycerol galactoside from seaweeds belonging to red algae. The conventional extraction method of glycerol galactoside is a method using an organic solvent such as ethanol or ether. For example, after extraction by heating with 75% ethanol aqueous solution from the above-mentioned “color loss paste” etc., degrease with diethyl ether, A process of removing impurities through an ion exchange resin is employed.

  Thus, in the conventional extraction method of glycerol galactoside, an organic solvent such as ethanol or ether is used, which is expensive and requires special equipment and equipment. In addition, solvents such as diethyl ether cannot be used in foods, and special considerations are necessary to ensure the safety of extraction work from the viewpoint of flammability, explosiveness, and toxicity. Furthermore, the ion exchange resin treatment is a general means used for the purification of sugar solution, etc. However, if the ion exchange resin is used to remove impurities of the extracted glycerol galactoside, a large amount of resin is regenerated and washed. Since an acid or alkali solution is required, there is a concern in terms of environmental load. Under these circumstances, it is necessary to develop a method for extracting glycerol galactoside from seaweeds belonging to red algae, without using organic solvents such as ethanol and ether, in a safe and efficient manner and with as little pollution as possible. It has been.

  In Example 2 of Patent Document 1, distilled water was added to the dried powder of ogonori and heated and extracted repeatedly in a boiling water bath. The extract was collected, dialyzed against a cellophane membrane, and then freeze-dried. A method for obtaining a dried product is disclosed. However, since this extraction method is performed at a high temperature, it is considered that a large amount of a high molecular polysaccharide component (for example, porphyran, which is a kind of galactan) is extracted together with glycerol galactoside. Since the polymer component is a major obstacle in the purification process after extraction, it is difficult to extract and purify a single glycerol galactoside by this method.

  In view of the above situation, the present invention provides a method for safely and efficiently extracting glycerol galacside from seaweeds (including seaweeds) belonging to red algae without using an organic solvent such as ethanol or ether. Is the first problem. The present invention also provides a method for safely and efficiently extracting glycerol galacside from low-quality paste called “color loss” or “color loss paste” that has low protein content and is rarely used. Providing is a second problem.

  The present inventor aims to solve the above-mentioned problems, paying attention to a method using only cheap and safe water as an extraction solvent, and as a result of intensive research, as a result of using low-temperature water, glycerol galactosid is efficiently used. The present invention has been completed by finding that it can be extracted automatically and the subsequent purification is easy.

  That is, among the present invention for solving the above problems, the invention described in claims 1 and 2 is directed to selectively using glycerol galactoside from seaweed belonging to red algae using only water at 0 to 30 ° C. as a solvent. This is the extraction method.

  The invention described in claim 2 is the method for extracting glycerol galactoside according to claim 1, wherein a seaweed belonging to the red alga is used as a raw material.

  Furthermore, the invention described in claim 3 is the method for extracting glycerol galactoside according to claim 2, wherein a paste having a protein content of 30% by weight or less is used as a raw material of the genus Amanori.

  Furthermore, the invention described in claim 4 is purified by subjecting the glycerol galactoside extracted by the method described in any one of claims 1 to 3 to an ultrafiltration treatment followed by an ion exchange resin treatment or an electrodialysis treatment. It is a method of obtaining the obtained glycerol galactoside.

  According to the present invention, glycerol galactoside having the activity of promoting the growth of bifidobacteria from seaweeds belonging to red algae can be obtained using only inexpensive and safe water without using expensive and dangerous organic solvents such as ethanol and ether. And can be extracted. Moreover, according to the present invention, glycerol galactoside after extraction can be easily purified. That is, in the glycerol galactoside extracted according to the present invention, the content of a high molecular weight component such as porphyran that hinders subsequent filtration and purification is suppressed to such an extent that it does not hinder the purification of glycerol galactoside alone. Therefore, since the extract of glycerol galactoside obtained by the present invention has low viscosity, high molecular weight components mixed in a small amount can be easily removed by ultrafiltration treatment, and low molecular weight ionic contaminants can be treated by ion exchange resin treatment. Of course, it can be removed easily by electrodialysis. The electrodialysis treatment is economical because there is no need to use an acid agent or an alkali agent for washing and regeneration of the resin as in the case of an ion exchange resin treatment, and there is no risk of burdening the environment. It is.

  The present invention can be applied to a method for extracting glycerol galactoside from a paste belonging to the genus Red seaweed Amanori, and among the pastes, in particular, glycerol from “color-loss paste” having a protein content of 30% by weight or less and low quality. It can be applied to a method for extracting galactoside. Since the “color loss paste” has a high content of glycerol galactoside, the present invention has hardly been used in the past, and the “color loss paste”, which is also referred to as industrial waste, can be used as a useful substance without placing a burden on the environment. Can be converted into raw materials. In other words, the present invention is a fundamental technology that is indispensable for effectively utilizing low-quality “color loss”.

  As described above, according to the present invention, glycerol galactoside, which is a bifidobacteria growth promoting component, can be extracted inexpensively, safely, and without burdening the environment from paste, in particular, “color faded paste”. Galactoside can be supplied as a useful additive to various foods and beverages as a bifidobacteria growth promoter. Further, the glycerol galactoside extracted by the present invention can be safely used as a bifidobacteria growth promoter for supplements, nutritional supplements, and health foods. Furthermore, it can be used with peace of mind for fish and livestock feed and feed.

  In the present invention, glycerol galactoside is formed by α-bonding the hydroxyl group at the 1-position of galactose to the hydroxyl group of glycerol. Glycerol galactoside is contained in red seaweeds of seaweed, but it has been found that it is contained in a lot of seaweed having a low protein content, which is called “color fading seaweed”. Therefore, as a raw material for glycerol galactoside, it is preferable to use a discolored paste having a protein content of 30% by weight or less, preferably 25% by weight or less.

Hereinafter, preferred examples of the method for extracting glycerol galactoside according to the present invention will be described.
In the present invention, 0 to 30 ° C. water, preferably 0 to 10 ° C. water is added to a dried product of red algae Amano seaweed containing laver (nori) and extracted without heating. The extraction time is preferably about 10 to 16 hours. This extract can remove high molecular components (proteins, polysaccharides, etc.) by ultrafiltration with a molecular weight cut-off of about 5000. The filtrate obtained by ultrafiltration contains low-molecular ionic impurities such as amino acids and organic acids, which can be removed by treatment with an ion exchange resin. Moreover, it can remove easily also by performing an electrodialysis process instead of an ion exchange resin process. The electrodialysis treatment is more economical and less burdensome on the environment because there is no need to use a large amount of acid / alkaline solution for resin regeneration or washing unlike ion exchange resin treatment. The solution obtained by the ion exchange resin treatment or the electrodialysis treatment is treated with a reverse osmosis membrane to remove water and concentrate glycerol galactoside. Furthermore, glycerol galactoside crystals can be obtained if moisture is completely removed by freeze-drying or the like. In the case of “color loss”, the same method as described above may be used.

That is, the present invention is characterized in that glycerol galactoside is selectively extracted from seaweeds belonging to red algae using only water at 0 to 30 ° C. as a solvent. In general, when water is extracted from seaweeds, a heat extraction method is employed to improve the extraction efficiency. When this method is applied to a paste, a high-molecular substance such as a polysaccharide porphyran is combined with glycerol galactoside. A large amount is extracted together and the viscosity of the extract is remarkably increased, which becomes an obstacle to the filtration / purification operation after the extraction operation. In the present invention, since the water extraction of glycerol galactoside is performed at a low temperature (0 to 30 ° C.) below room temperature, the amount of polysaccharide porphyran extracted can be suppressed.
Hereinafter, the present invention will be described in more detail with reference to examples and test examples.

<Example of a method for extracting GG from discolored paste>
(1) Cold Water Extraction 200 mL of water cooled to 10 ° C. was added to 10 g of a dry powder of discolored paste having a protein content of 20%, and extracted by shaking overnight while maintaining the temperature at 10 ° C. The obtained extract was No. Suction filtered through one filter paper. To this residue, 10 ° C. water was added, stirred for 1 hour, extracted again, and combined with the previous extract. When glycerol galactoside contained in the extract was subjected to HPLC analysis by the method shown in (4), the recovery rate was 94.9%.
(2) Ultrafiltration In order to remove 50 mL from the obtained extract and remove the polymer component, ultrafiltration was performed using a Millipore Pericon XL ultrafiltration module (Biomax-5, molecular weight cut-off 5000). Filter and collect the filtrate. When glycerol galactoside in the filtrate was subjected to HPLC analysis by the method shown in (4), the recovery rate was 88.0%.
(3) Electrodialysis In order to remove ionic substances from the obtained filtrate, electrodialysis was performed. For electrodialysis, an electrodialyzer microacylator S-1 (manufactured by Asahi Kasei) was used. As the dialysis membrane cartridge, Neoceptor AC-220-10 (fractionated molecular weight 300: manufactured by Astom Co., Ltd.) was used.
The filtrate was made up to 50 mL with distilled water, and electrodialyzed with V-PATTERN set to P1 and an end conductivity of 100 μS / cm. After completion of dialysis, the dialysate was recovered and the dialysate was lyophilized to recover glycerol galactoside. The recovered glycerol galactoside was subjected to HPLC analysis by the method shown in (4), and the purity and recovery rate were measured. As a result, the purity was 96.4% and the recovery rate was 85.0%. The HPLC chromatogram of glycerol galactoside is shown in FIG.
(4) HPLC analysis of GG The extract of (1) was filtered through an ultrafree MC unit (fractionated molecular weight 5000) to remove the polymer component, and then the sample was analyzed. The filtrate of (2) was analyzed as it was. The glycerol galactoside of (3) was analyzed after dissolving in water to a constant volume.
In the HPLC analysis, Coregel 87MM manufactured by TRANSGENOMICS was used as the column, the column temperature was 80 ° C., distilled water was used as the mobile phase, the flow rate was 0.6 mL / min, and the injection amount was 20 μL. The elution of glycerol galactoside was detected with a differential refractometer (manufactured by Shimadzu Corporation).

<< Test Example 1 >>
<Extraction test of GG with varying extraction water temperature>
A test was conducted to confirm how the extraction amount of porphyran and glycerol galactoside, which hinders purification of glycerol galactoside, changes when the extraction water temperature is changed.
(1) Test method 10 mL of water was added to 0.25 g of a color-falling dry powder having a protein content of 20% by weight, heated to 5 ° C, 10 ° C, 25 ° C, 37 ° C, and 95 ° C, then cooled and shaken overnight. And extracted. Centrifugation was performed at 3500 rpm for 15 minutes, and the supernatant was collected. 5 mL of water was added to each precipitate and further extracted by shaking at the same temperature for 1 hour. The extract was centrifuged in the same manner, and the supernatant was combined with the previous supernatant. The precipitate was similarly shaken and extracted, and the supernatants were combined (extracted 3 times in total). The obtained supernatant was filled up to 25 mL, and a part thereof was taken and ultrafiltered with an ultra-free MC unit (molecular weight cut off 5000). The sugar content of the extract supernatant and the ultrafiltrate was quantified by the phenol-sulfuric acid method, and the amount of porphyran extract and the amount of glycerol galactoside extracted per gram of dry powder was calculated according to the following formula.
Porphyran extract amount (mg / g paste)
= (Sugar content of the extraction supernatant-sugar content of the ultrafiltrate) /0.25 g
GG extract amount (mg / g paste) = sugar content of ultrafiltrate / 0.25g

(2) Test results The test results are shown in FIG. From FIG. 2, it was revealed that porphyran was hardly extracted under the extraction temperature of 30 ° C. or less, while glycerol galactoside was extracted regardless of the extraction temperature.
(3) Findings From the test results, it became clear that by suppressing the extraction water temperature to 30 ° C. or less, it is possible to increase the viscosity of the extract and suppress the extraction of porphyran, which is an impediment to the purification operation. In actual extraction, it is considered preferable to perform extraction at a low water temperature of 10 ° C. or lower in order to suppress the propagation of various bacteria during the extraction.

<< Test Example 2 >>
<Extraction test of GG with hot and cold water>
The extraction efficiency of glycerol galactoside was compared between hot water extraction usually used for extraction of seaweed components and cold water extraction extracted with water at 10 ° C.
(1) Test method 10 g of dry-colored paste with a protein content of 20% by weight (GG content of 11.2%) and 200 mL of water were added to two heat-resistant screw-mouth bottles, respectively, and one of them was shaken overnight at 10 ° C. The other was heated at 95 ° C. for 1 hour. Thereafter, each extract was designated as No.1. Suction filtered through one filter paper. Water was similarly added to the residue, stirred for 1 hour and extracted again, and the extract was combined with the previous extract. Each obtained extract was filled up to 500 mL with water, and glycerol galactoside in the extract was quantified by the HPLC method used in Example 1 to measure the extraction efficiency of glycerol galactoside. The extraction efficiency of glycerol galactoside was calculated by the following equation.
Extraction efficiency (%) of GG = extracted GG amount / GG amount contained in paste × 100

(2) Test results Table 1 shows the test results. In the extraction of glycerol galactoside using hot water, the extraction efficiency (recovery rate of GG in laver) is 88.8%, whereas in the extraction of glycerol galactoside using cold water at 10 ° C., 94.9. % Extraction efficiency was obtained.
(3) Findings From the test results, it was found that when glycerol galactoside is extracted from paste, higher extraction efficiency can be obtained by extraction with low-temperature water rather than extraction with hot water. It was confirmed that at least the extraction efficiency with low-temperature water was practically sufficient.

<< Test Example 3 >>
<Ultrafiltration test of hot water and cold water extract>
A test was conducted to purify glycerol galactoside by ultrafiltration of the hot water extract and the cold water extract of Nori.
(1) Test method 50 mL of each of the hot water extract and the cold water extract obtained in Test Example 2 were weighed and used with Millipore Pericon XL ultrafiltration module (Biomax-5, molecular weight cut-off 5000), respectively. Ultrafiltration was performed. The ultrafiltration operation was performed until filtration could not be performed due to a pressure loss due to an increase in the viscosity of the filtrate residue, or until the filtrate residue reached the dead volume of the apparatus and filtration could not be performed. Since the molecular weight of glycerol galactoside is 254.23, glycerol galactoside is recovered in the filtrate, so the glycerol galactoside in the filtrate and the filtrate residue was quantified by the HPLC method used in Example 1. Further, the recovery rate of glycerol galactoside was calculated by the following formula.
GG recovery rate (%)
= GG content in recovered filtrate / GG content of extract before filtration × 100

(2) Test results Table 2 shows the test results. As shown in Table 2, 45 mL of 50 mL of the cold water extract was recovered by ultrafiltration. Filtration does not stop due to pressure loss, and the filtrate residue is probably due to the dead volume of the device. Glycerol galactoside was recovered 88.0% in the filtrate. On the other hand, only 35 mL of 50 mL of hot water extract was recovered by ultrafiltration. During the ultrafiltration operation, the ultrafiltration operation became impossible due to an increase in viscosity that was thought to be due to porphyran in the filtrate. For this reason, the recovery rate of glycerol galactoside was decreased and remained at 67.0%.
(3) Findings From the test results, in the hot water extract of Nori, the efficiency of ultrafiltration is reduced due to the increase in viscosity, which seems to be due to porphyran in the extract, and the recovery rate of glycerol galactoside is reduced. It was revealed that the purification efficiency was greatly impaired. On the other hand, it was clarified that the cold water extract did not increase in viscosity and a high recovery rate of glycerol galactoside was obtained. Therefore, when extracting glycerol galactoside from paste, it has become clear that a method of extracting with low-temperature water is preferable in order to perform the ultrafiltration operation in the subsequent step with high efficiency.

<< Test Example 4 >>
<Ionic substance removal test of ultrafiltration filtrate of cold water extract>
As a purification method for removing ionic substances, which are impurities other than glycerol galactoside, from the ultrafiltration filtrate of the cold water extract, ion exchange resin treatment and electrodialysis treatment were performed, and the results were compared.
(1) Ion-exchange resin treatment 20 mL of the ultrafiltration filtrate of the paste cold water extract obtained in Test Example 3 was weighed and filled with amphoteric ion-exchange resin MB-2 (manufactured by Organo) swollen with water. The column was loaded on a column (2.5 × 25 cm), developed with distilled water at a flow rate of 3 mL / min, and the ionic substance was removed by fractionating the eluate. The eluates (0-400 mL) were combined and lyophilized to give a solid. The solid was weighed and dissolved in 25 mL of distilled water, and glycerol galactoside was quantified by the HPLC method used in Example 1 to calculate the content and purity of glycerol galactoside in the solid.
(2) Electrodialysis treatment 20 mL of the ultrafiltration filtrate of the paste cold water extract obtained in Test Example 3 was weighed and electrodialyzed. As the electrodialyzer, a microacylator S-1 (manufactured by Asahi Kasei) was used. As the dialysis membrane cartridge, Neoceptor AC-220-10 (fractionated molecular weight 300: manufactured by Astom Co., Ltd.) was used. Electrodialysis was performed with V-PATTERN of P1 and an end conductivity of 100 μS / cm. After completion of dialysis, the dialysate was collected. The dialysate was lyophilized to obtain a solid. The solid was weighed and dissolved in 25 mL of distilled water, and glycerol galactoside was quantified by the HPLC method used in Example 1 to calculate the content and purity of glycerol galactoside in the solid.

(3) Test results Table 3 shows the test results. As shown in Table 3, 80.9% of glycerol galactoside was recovered by the ion exchange resin treatment. The purity of the collected glycerol galactoside was 89.0%. On the other hand, the recovery rate of glycerol galactoside by electrodialysis was 94.0%, and the purity of the recovered glycerol galactoside was 85.0%.
(4) Observations Glycerol galactoside was purified to a high purity of 89.0% from the ultrafiltrated filtrate of Nori's cold water extract by ion exchange resin treatment. It was clarified that it can be purified to a sufficient purity for application. In addition, the electrodialysis treatment showed a high recovery rate of glycerol galactoside of 94.0% compared with the ion exchange resin treatment, and it was revealed that the electrodialysis treatment was excellent as a method for removing ionic substances. In conclusion, it was confirmed that both the ion exchange resin treatment and the electrodialysis treatment can be used for purification of glycerol galactoside extracted by the present invention.

  As is apparent from the examples and test examples described above, according to the present invention, glycerol galactoside, which is contained in a lot of paste, particularly low-quality “color faded paste” and has a bifidobacteria growth promoting activity, such as ethanol and diethyl ether, is included. It is possible to extract only the cheap and safe water without using expensive and dangerous organic solvents, and to suppress the elution of polysaccharide porphyran, which hinders filtration and purification operations in the subsequent process, to a level that does not hinder it. it can. Therefore, since the obtained extract has a small amount of porphyran, the viscosity is low, and high molecular weight compounds such as proteins and porphyran contained in the extract can be easily removed by subsequent ultrafiltration. Furthermore, the filtrate obtained by ultrafiltration contains low-molecular ionic contaminants such as amino acids and salts, which can be removed by treatment with an ion exchange resin. The same effect can be obtained by performing electrodialysis instead of ion exchange resin treatment, and electrodialysis treatment uses a large amount of acid / alkaline necessary for resin regeneration and washing as in ion exchange resin treatment. Since it is not necessary to use a solution, it is more economical and can reduce the burden on the environment.

  As described above in detail, according to the present invention, glycerol galactoside, which is a bifidobacteria growth promoting component, can be extracted inexpensively and safely from paste, particularly low-quality discolored paste. Moreover, the glycerol galactoside extracted by the present invention is easy to purify, and there is no risk of contaminating the environment during the purification. Therefore, the glycerol galactoside extracted according to the present invention can be purified and supplied to various foods and beverages as a safe additive having a bifidobacteria growth promoting action. They can also be used as supplements, health foods, and dietary supplements, and can also be applied to pharmaceuticals, cosmetics, feeds, and the like.

HPLC chromatogram of glycerol galactoside extracted and purified from paste by the method of the present invention (corresponding to Example 1) Extraction rate of glycerol galactoside and porphyran when extracted from paste with water at various temperatures (corresponding to Test Example 1)

Claims (4)

  A method for selectively extracting glycerol galactoside from seaweed belonging to red algae using only water at 0 to 30 ° C. as a solvent.   The method for extracting glycerol galactoside according to claim 1, wherein a seaweed belonging to the red algae is used as a raw material.   The method for extracting glycerol galactoside according to claim 2, wherein a paste having a protein content of 30% by weight or less is used as a raw material from a genus Amanori. A method for obtaining a purified glycerol galactoside by subjecting the glycerol galactoside extracted by the method according to any one of claims 1 to 3 to an ultrafiltration treatment followed by an ion exchange resin treatment or an electrodialysis treatment.