JPH03141288A - Fractionation of phospholipid component in lecitin and collection of oligosaccharide - Google Patents

Abstract

PURPOSE:To improve workability of fractionation of the subject component by bringing a plant-derived lecitin into contact with an aqueous alcohol solution, subsequently preparing an organic solution of the above mentioned lecitin and treating the resultant solution using the chromatography method. CONSTITUTION:For example, a plant-derived lecitin is initially dissolved in a nonpolar solvent such as n-hexane or chloroform, subsequently brought into contact with an aqueous alcohol solution (e.g. ethanol) of 30-70(vol/vol)% and subjected to filtration, etc., for removal of aqueous alcohol soluble components in the above-mentioned lecitin. The obtained lecitin is then dissolved in an organic solvent (e.g. hexane/2-propanol/water solution) in >=1(wt/vol)% concentration and the resultant solution is subjected io the high-performance liquid chromatography, thus carrying out fractionation of the objective phospholipid component. In addition, oligosaccharides are obtained by purifying the removed aqueous alcohol solution soluble components using an active carbon, etc.

Description

Detailed Description of the Invention (Field of Industrial Application) The present invention provides a method for efficiently fractionating phospholipid components in plant-derived lecithin, and a method for collecting oligosaccharides from by-products produced in the above fractionation process. Regarding.

(Prior art and problems to be solved by the present invention) Lecithin is full of surfactant and anti-aggressive effects,
In addition to being widely used as an emulsifier in the food industry, it also has a wide range of industrial uses, such as as an additive to lubricating oils and fuel oils, and as a surface treatment agent for magnetic recording media. It also has physiological activity that lowers blood cholesterol, and is attracting attention for its use in the health food and pharmaceutical fields.

Recently, attention has been focused on the characteristics of phospholipid components in lecithin, and attempts have been made to separate and fractionate phospholipids into individual components in order to utilize them to a higher degree.

The fi fat '31 in lecithin is phosphatidyl.

Choline, phosphatidyl ethanolamine, phosphatidyl serine, phosphatidyl inositol, phosphatidic acid (rPcJ, "PE", r
PsJ, "PI", "PA") and other glycerophospholipids are contained in many dishes.

To separate and fractionate these components in lecithin, column chromatography using silica gel, alumina, or an ion exchanger as an adsorbent is generally used. In this case, lecithin is usually diluted in advance with a large amount of organic solvent and loaded onto a column, and each component is eluted from the column while sequentially changing the composition of the solvent.

In high-performance liquid chromatography using silica gel as an adsorbent, a mixed solvent such as hexane/2-propanol/water is used as a solvent system that allows good separation of each component (Paul Van der Meeren: 5th Lecithin Colloquium. April 10-12, 1989, Cannes, France).

Lecithin becomes stable micelles in chloroform, hexane, etc., forming a homogeneous system even at high concentrations, but its solubility in other solvents is extremely low (particularly in plant-based lecithin, it is generally 0. .1 (wt/vo
l) It is about % or less.

Therefore, in order to fractionate lecithin into each phospholipid component using the above-mentioned mixed solvent system, it is essential to use a large excess of solvent in order to completely dissolve and disperse lecithin. This requires large-scale facilities and equipment. In addition, a series of steps including recovery of the solvent requires a great deal of effort, time, and cost, posing a major economic problem.

Therefore, if it were possible to increase the solubility of cytin in each solvent, the workability would be significantly improved and the economic burden would be greatly reduced, which would have extremely great advantages.

(Means for solving the problem) The present inventors have improved the solubility of lecithin in a solvent,
As a result, we conducted intensive research into methods for efficiently separating and fractionating phospholipids into their respective components.

As a result, we obtained the totally unexpected finding that by bringing lecithin into contact with an aqueous alcoholic solution to remove the aqueous alcoholic solution-soluble components in lecithin, the solubility of lecithin in solvents can be dramatically improved. The inventors have discovered that by using lecithin whose solubility has been improved through such treatment, it is possible to efficiently fractionate phospholipids by chromatography, and have completed the present invention. Furthermore, the present inventors found that the components dissolved and extracted in the hydroalcoholic solution were mostly raffinose,
They discovered that it is an oligosaccharide such as stachyose, and completed the present invention.

Thus, the present invention provides that lecithin of plant origin is contacted with a hydroalcoholic solution in lecithin to remove the hydroalcoholic solution soluble components in lecithin, and then the lecithin is reduced to 1 (wt/vol). )% or more as an organic solvent solution, which is subjected to column chromatography, and is an invention of a method for removing phospholipid components in lecithin, which is characterized by subjecting the phospholipid components in lecithin to column chromatography as an organic solvent solution, and purifying a hydroalcoholic solution that is removed by the above-mentioned method. This is an invention of a method for collecting oligosaccharides characterized by the following.

Oligosaccharides have attracted attention because they have physiological activities such as bifidobacteria growth activity.

The present invention will be explained in more detail below.

The lecithin of plant origin used in the present invention may be commercially available lecithin in powder or paste form made from soybean, corn, rapeseed, or other oil seeds, or the process of refining oils and fats from the oil seeds. It is also possible to use, in particular, a degummed soapstock obtained in the degumming step.

Therefore, "lecithin" in the present invention also includes the above-mentioned degummed soapstock.

In the present invention, lecithin of plant origin can be used as it is, or with aliphatic hydrocarbons such as pentane, hexane, heptane, isooctane, and cyclohexane, aromatic hydrocarbons such as benzene and toluene, tetraroform, tetracarbon, dichloromethane, A substance selected from the group of halogenated hydrocarbons such as methylene chloride is dissolved in a nonpolar solvent and brought into sufficient contact with the solvent by stirring or the like. When used for foods, it is preferable to use n-hexane for food additives.

Alcohols include aliphatic alcohols having 1 to 4 carbon atoms, such as methanol, ethanol, 1-propanol, 2-
Either propa, nol, 1,2 or 3-butanol, or mixtures thereof can be used, although ethanol is preferred for food grade use.

The alcohol concentration in aqueous alcohol 'f8) &
When the raw material lecithin is dissolved or dispersed in a non-polar solvent, it is 30-70 (vol/vol)%, and when it is not dissolved or dispersed in these in advance (when it is brought into direct contact, it is 20-85 (vol/vol)%). If the concentration exceeds these ranges, the sugar removal efficiency may decrease or the lecithin's aqueous alcohol 'fs
Solubilization into the liquid phase occurs, neither of which is practical.

After sufficient contact with the aqueous alcoholic solution, the lecithin or the nonpolar solvent phase containing lecithin may be separated from the aqueous alcoholic solution by centrifugation, filtration, static separation, countercurrent distribution, or other suitable means.

The lecithin obtained by such treatment has significantly increased solubility not only in the mixed solvent system used for column chromatographic separation of phospholipids but also in solvents in general. For example, the solubility in hexane/2-propertool/water (58:39:31) was 0.0% before treatment.
1 (wt/vol) or less, whereas
After treatment, it becomes 50 (wt/vol)% or more, and the solubility increases dramatically.As a result, what was previously only used for analysis and experiments can now be used in the universal method of the present invention. This makes it possible to use it in industry.

In the present invention, the lecithin subjected to the above treatment is dissolved in an appropriate universal solvent to a concentration of 1 (wt/vol)% or more,
Subject to chromatographic fractionation. In this case, it is efficient to use high performance liquid chromatography. 1(
The reason why the concentration was set at more than 1% wt/vol. is that if the concentration was lower than this, the advantage of the present invention that the solubility of lecithin would dramatically increase would not be fully utilized, and a large amount of solvent would be used. This is because the amount becomes 1% to 20% (wt/yol).

In the method of the present invention, the solubility of lecithin in a solvent is dramatically increased as a result of saccharides being dissolved and separated in the hydroalcoholic solution in contact with lecithin.

In view of this point, another object of the present invention is to collect and recover oligosaccharides from a hydroalcoholic solution treated with lecithin.

In the process of the present invention, oligosaccharides (sucrose, raffinose, stachyose, etc.) corresponding to about 50-85% of the total solids are obtained in the hydroalcoholic solution obtained by treating soybean lecithin.

Since oligosaccharides accounted for only about 5 to 8% in raw lecithin, it was found that oligosaccharides were selectively transferred into the hydroalcoholic solution.

In the present invention, the aqueous alcohol solution is purified using activated carbon, ion exchange resin, etc. as necessary,
The purified oligosaccharide can be collected by drying as appropriate.

(Example) Example 1 10 g of powdered soybean lecithin was dissolved in 50 ml of chloroform, 100 mj of 50 (wt/vo 1)% ethanol was added thereto, and after shaking for 5 minutes in a separatory funnel, the mixture was divided into two layers. It was left standing until it separated. The lower chlorotetraform layer was separated, and the solvent was distilled off to obtain 8.2 g of paste lecithin. 60 ice crystals were carefully mixed with en-hexane/2 propatool/water (58:39:3) 300 μI. and subjected to 4-speed chromatography under the following conditions.

Column Nijiri gel column 4.7mwaφ, 30c
m eluent: Same column as the dissolving solution 28°C: 25°C Detection 1): UV detector (205 m layer) PESPI, PC, and Risopc were eluted in this order, and the separation was very good.

On the other hand, activated carbon corresponding to 0.5% of the liquid volume was added to the ethanol layer Cζ, heated to 60°C, stirred for 10 minutes, and dried to form a slightly colored powdery substance 1. 2g was obtained.

What is the composition of the substance? Sucrose 48.2%, raffinose 6.0%, stachyose 26.6%, other 19.2%
Met.

Example 2 10 g of pasty soybean lecithin of ffT was dissolved in 50 mZ of n-hexane, and 30% of 2propanol 25
-l was added and treated in the same manner as in Example 1 to obtain 8.9 g of lecithin from the upper n-hexane layer. The lecithin 1
．． 0g was dissolved in 10ml of the same solution as in Example 1,
It was subjected to high performance liquid chromatography under the following conditions.

Column: Semi-preparative silica gel column 30mφ, 30
cm The solution a solution, column temperature, and detector were all the same as in Example 1.

The chromatogram of this example is shown in FIG. 1, and the analysis results of each portion by thin layer chromatography are shown in FIG. 2.

(Example 3) Add water equivalent to 1% oil to soybean grain crude oil obtained by hexane extraction, stir at 70°C for 10 minutes, and then centrifuge to obtain soybean extract soapstock (32% water, insoluble in acetone). 54%
) Add 200 mJ of 70% ethanol aqueous solution to 100 g, mix at 60°C for 20 minutes, and centrifuge 5Ii (3,0
00 rpm for 120 minutes), the lower layer was separated and concentrated under reduced pressure to obtain 62 g of a paste-like substance. The substance was dissolved in a mixed solvent of n-hexane/2-propanol/water (58:39:3) to a concentration of 100 g/wal, and 25 μl of the solution was subjected to high performance liquid chromatography under the following conditions. did.

Column: Analytical silica gel column 4.7mmφ, 0
cm Elution night: n-hexane/2-propertool/water (58
: 39: 3 → 58: 3 7: Gradient elution column temperature of 5): f! Chamber detection M: UV detection '54 (205 layer m) Each phospholipid component was well separated.

In addition, activated carbon was added to the upper ethanol layer, heated,
The oligosaccharide was purified by stirring and drying.

(Effects of the Invention) Lecithin treated by the method of the present invention has significantly increased solubility in organic solvents. Therefore, when separating phospholipids into each component by chromatography, it is possible to load the column with a high concentration, and the amount of dissolved JII solution can be reduced compared to the conventional method. This not only saves labor and time, but also makes it possible to significantly reduce equipment investment costs and running costs such as solvents.

In addition, since the aqueous alcoholic solution brought into contact with lecithin in the present invention has a high concentration of oligosaccharides, physiologically active oligosaccharides can be easily collected from it, and its economic value is Extremely large.

show. The numbers on the horizontal axis correspond to the peak numbers in FIG. 1, and the numbers on the vertical axis correspond to PA and PE.

PC, PI, LysoPC is a phospholipid component,
Phosphatidic acid, phosphatidyl ethanolamine, choline, and inositol lysophosphatidylcholine are shown respectively.

FIG. 3 is a chromatogram obtained by high performance liquid chromatography in Example 3. The horizontal and vertical axes are the same as in FIG. 1, and the abbreviations are the same as in FIG. 2, except that LysoPE is lysophosphatidyl ethanolamine.

[Brief explanation of the drawing]

FIG. 1 is a chromatogram obtained by high performance liquid chromatography in Example 2. The horizontal axis shows the elapsed time (minutes), and the vertical axis shows the relative sensitivity of the LJV detection type.

Claims (1)

[Scope of Claims] (1) After contacting plant-derived lecithin with an aqueous alcohol solution to remove the aqueous alcohol solution-soluble components in the lecithin, the lecithin is chromatographed as a 1 (wt/vol)% or more organic solvent solution. 1. A method for fractionating phospholipid components in lecithin, the method comprising subjecting the phospholipid components to a graph. (2) The fractionation method according to claim (1), wherein the chromatography is high performance liquid chromatography (3) Claim (1) or (2), wherein the lecithin is dissolved or dispersed in a nonpolar solvent and brought into contact with an aqueous alcohol solution. fractionation method. (4) The fractionation method according to claim (3), wherein the nonpolar solvent is one or a mixture of two or more selected from n-hexane, chloroform, carbon tetrachloride, isooctane, and heptane. (5) The fractionation method according to claim (3) or (4), wherein the nonpolar solvent is n-hexane for food additives. (6) The fractionation method according to any one of claims (1) to (3), wherein the alcohol in the aqueous alcoholic solution is one or a mixture of two or more selected from aliphatic alcohols having 1 to 4 carbon atoms. (7) The alcohol concentration in the aqueous alcoholic solution is 30~
The fractionation method according to claims (1) to (3) or (6), wherein the fractionation amount is 70 (vol/vol)%. (8) Claim (1) in which the lecithin is brought into contact with the hydroalcoholic solution without being dissolved or dispersed in a non-polar solvent.
Or (2) fractionation method. (9) The alcohol concentration in the aqueous alcohol solution is 20~
The fractionation method according to claim 8, wherein the fractionation amount is 85 (vol/vol)%. (10) A method for collecting oligosaccharides, which comprises purifying a component soluble in an aqueous alcohol solution obtained by the fractionation method according to claim (1).