JP3240249B2 - Phospholipid fractionation method - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a method for fractionating ampholytic phospholipids from a mixture of acidic and amphiphilic phospholipids, which is commercially available, for example, as crude lecithin. And a method for fractionating lecithin into a soluble fraction and an insoluble fraction.

[0002]

2. Description of the Related Art Phospholipids obtained from natural products such as soybean and egg yolk are commercially available as "lecithin (crude lecithin)",
Mainly phosphatidylcholine (hereinafter referred to as "PC"),
It is a mixture of phosphatidylethanolamine (hereinafter referred to as “PE”), phosphatidylinositol (hereinafter referred to as “PI”), and phosphatidic acid (hereinafter referred to as “PA”). Each of the phospholipids has the following chemical structural formula. In addition, "lecithin" may be another name of phosphatidylcholine (PC),
The “lecithin (crude lecithin)” referred to here is mainly a mixture of phospholipids such as PC, PE, PI, and PA.

[0003]

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[0004] Lecithin, lecithin fractions and derivatives thereof are widely used as foods, cosmetics, pharmaceuticals, and the like as natural products having excellent emulsifying properties, physiological functions, surfactant activities, antibacterial activities and the like.
Used for industrial products. Fractionation of lecithin into its constituent phospholipids can be said to be an indispensable technique to further enhance these functions and utilize them more highly. Therefore, techniques for fractionating lecithin into its components have been developed.

[0005] A number of fractionation methods have been attempted to enrich lecithin into its constituent phospholipids, especially PC, which has the highest industrial value. Ethanol extraction of lipids was a classic method, and when chloroform was not yet synthesized and ethers were rare, ethanol was used to extract brain lipids (LNVanquelin: 1811).
However, at that time, phospholipids in lipids could not be identified, and ethanol was not used for the purpose of fractionating phospholipids in extracted lipids. Gobley called phospholipids soluble in warm ethanol as "lecithin" (1846-47), and called Thudicum (Thudicum).
chum) (1828-1901) referred to a phospholipid insoluble in warm ethanol as kephalin.

[0006] These fractions were later found to be PC-rich and PE- and PI-rich fractions. This classical ethanol fractionation is used without exception for the purpose of roughly classifying phosphatidylcholine (PC) from other phospholipids, and it has been common sense to carry out the ethanol fractionation at room temperature.

On the other hand, as an example of performing ethanol fractionation at a high temperature, Japanese Patent Application Laid-Open No. 01-289455 has been proposed. However, in this case, too, the purpose is to obtain a high PC content fraction, and the lecithin (in this case, crude lecithin) is stirred and extracted in ethanol at 60 to 70 ° C. for 30 minutes, and further left at the same temperature for 30 minutes. The resulting insoluble fraction is obtained. In this case, the insoluble fraction is used to prepare a powdering agent for mixing with the PC fraction to form a powder. This insoluble fraction contains almost no PC, and PE, P
It consists of phospholipids such as I and PA.

In a conventional ethanol fractionation method performed at normal temperature,
PC is mainly extracted and considerable PE remains in the insoluble fraction, so that the purity of PC in the soluble fraction increases, but the ratio of acidic phospholipid in the insoluble fraction is relatively low. Was.

In the above-mentioned patent, although ethanol is fractionated by heating, it is intended to produce a powdering aid of the PC concentrated fraction, so that it is stirred at 65 ° C. for 30 minutes to collect only a high-temperature precipitate. However, it takes time, the yield of acidic phospholipids is halved, the enrichment is not sufficient, and it is not economical.

[0010]

Further, for example, Japanese Patent Publication No.
As described in JP-A-38594, not only PC but also PE as a substrate for causing a phosphatidyl group transfer reaction is required, so that an economical method of concentrating and separating PE has been desired.

[0011] The present invention is a very simple operation, PC and PE
A bipolar phospholipids and then fractionated into a soluble fraction, PA and PI
Of phospholipids that can be recovered in the insoluble fraction
The purpose is to obtain a fractionation method . In addition, this PC and PE
Can be used as a substrate for causing a phosphatidyl group transfer reaction, and the insoluble fraction is also P
Starting materials for producing I, leather tanning agents, or sugar
Ru can be used for cooking oil, etc. with the exception of the lipid component.

[0012]

According to the method for fractionating phospholipids according to the first aspect of the present invention, acidic and ambipolar phospholipids are used.
From a mixture of phospholipids, ambipolar and acidic phospholipids
In the fractionation method, the mixture of the acidic and bipolar phospholipids is suspended in an 85% or more aqueous ethanol solution heated to 50 ° C. or more, and then the suspension is added. Is cooled to a predetermined temperature to fractionate into a soluble fraction composed of bipolar phospholipids and an insoluble fraction containing most of acidic phospholipids.

In the method for fractionating phospholipid according to the second aspect of the present invention, phosphatidylcholine (P
C) A method for fractionating an amphoteric phospholipid mixture of phosphatidylcholine and phosphatidylethanolamine from a phospholipid mixture consisting of phosphatidylethanolamine (PE), phosphatidylinositol, and phosphatidic acid. The suspension is suspended in an 85% or more aqueous ethanol solution heated as described above, and then the suspension is cooled to 50 ° C. or lower, and the soluble fraction comprising the phosphatidylcholine and phosphatidylethanolamine and the phosphatidylinositol,
It is fractionated into an insoluble fraction containing most of phosphatidic acid.

[0014]

In the present invention, a mixture of acidic and ambipolar phospholipids is suspended in an aqueous ethanol solution heated to 50 ° C. or higher, and then the suspension is cooled to a predetermined temperature to dissolve the soluble fraction. And the insoluble fraction, it is possible to very easily fractionate amphoteric phospholipids of PC and PE from lecithin, for example.

The point that the present invention is crucially different from the above-mentioned technology is that the present invention intends to simultaneously separate not only PC but also another base-containing phospholipid, PE, from other phospholipids. Therefore, instead of dissolving and extracting lecithin in ethanol at a high temperature, the lecithin is cooled to 50 ° C. or lower to remove the insoluble fraction generated.

[0016] By this cooling operation, most of the PA and PI that were insufficiently separated by the high-temperature fractionation can be recovered as an insoluble fraction. Thus, the soluble fractions PC and PE obtained
Of the soluble fraction in this case, the majority of PC (> 75%) and approximately half of PE (4%).
(5% or more), and PA and PI are hardly detected. On the other hand, the corresponding insoluble fraction contains about half of PE and P
Almost all of A and PI are recovered.

Specifically, first, the extraction temperature of lecithin with aqueous ethanol is set at 50 ° C. or higher. Although extraction can be performed at 50 ° C. or less, the yield and separation are insufficient. Higher temperatures are advantageous in terms of solubilization and yield of PE. Temperatures higher than the boiling point may be used as long as the device is devised so that ethanol does not evaporate, but it is economical and prevents oxidation of phospholipids. For example, 60 to 70 ° C. is preferable.

The concentration of hydrous ethanol is shown in the table below.
As shown in FIG. 2, if it is 75% or more, perform at the conventional room temperature
Removes PI that could not be sufficiently removed by ethanol extraction
And more preferably at least 85%. If it is less than 85%, the recovery is low, and the PA is not removed.

Furthermore, the concentration of lecithin has a great influence on the resolution. That is, the lower the lecithin concentration, the higher the recovery rate, and conversely, the higher the lecithin concentration, the lower the recovery rate. The lecithin concentration is preferably selected in the range of 5 to 40%, but is preferably in the range of 10 to satisfy the resolution, recovery and economy.
30% is more preferred.

No special stirrer / emulsifier is required for extraction.
An ordinary stirrer may be used as long as it can simply disperse and dissolve lecithin. The temperature at which lecithin and ethanol are mixed is not particularly limited, and addition at room temperature or at a high temperature does not affect the results. Further, it is not necessary to strictly manage the stirring extraction holding time at a high temperature, and several minutes after the temperature reaches a predetermined temperature is sufficient. It may be as long as 10 minutes, and even if it is kept unnecessarily long, not only the separation ability and the yield are not improved, but also the oxidation of the lipid proceeds uneconomically.

Next, the mixture is cooled to collect the insoluble fraction.
Although sufficient separation and recovery can be achieved up to an ambient temperature of 0 ° C. or less and room temperature, from the viewpoint of recovery, 10
~ 50 ° C is desirable. When the temperature is cooled to 10 ° C. or less, the yield of the insoluble fraction increases, but the separation gradually becomes insufficient.

[0022]

【Example】

Embodiment 1 FIG. Influence of Temperature FIG. 1 is an explanatory diagram showing the operation steps for examining the influence of temperature on the method for fractionating an amphoteric phospholipid of the present invention. As shown in FIG. 1, crude soybean lecithin (PC: PE: PI: PA = 3
0: 30: 20: 12) 10 g are added to 40 g of ethanol (99.5%) at a predetermined temperature of 25 to 70 ° C.,
This was stirred with a magnetic stirrer for 5 minutes, and then left at a predetermined temperature for 30 minutes to form a supernatant (S1) and a precipitate (P1).
1) and decantation or centrifugation (1,500
(Rotation, 15 minutes). Phospholipids were quantitatively analyzed by developing a sample by thin-layer chromatography and spots colored with a detection reagent for phosphorus (Dittmer-Lester reagent) using an image analysis processor (trade name: Dr. Gel; Mitani). . The results are shown in Table 1 below.

[0023]

[Table 1]

As can be seen from Table 1, the dissolution temperature was set at 50
The temperature of (PE) in the supernatant (S1) cannot be increased unless the temperature is raised to at least ° C. In addition, the lower the fractionation temperature, the higher the concentration of (PC + PE) in the supernatant (S1). If the precipitate is separated at a high temperature without cooling, the supernatant (S1)
The mixing of acidic phospholipids (PI and PA) therein increases and the yield is poor. Therefore, the temperature is raised to 50 ° C. or more,
It was confirmed that it is desirable to separate the mixture by cooling the mixture to a temperature lower than 0 ° C.

Embodiment 2 FIG. Influence of Water Content of Ethanol FIG. 2 is an explanatory diagram showing an operation process in which the influence of the water content of ethanol was examined. As shown in the figure, 99.5, 95, 9
Six types of aqueous ethanol solutions of 0, 85, 80, and 75% were prepared, and crude lecithin was subjected to warm ethanol fractionation at 70 ° C., and the phospholipid composition of the S1, S2, P1, and P2 fractions was examined. The results for the S2 fraction are shown in Table 2 below.

[0026]

[Table 2]

As shown in Table 2, most of the S2 fraction is enriched with more than 70% of the neutral lipid (NL) and PC and about half of the PE, and the conventional ethanol extraction performed at room temperature. (PI), which could not be sufficiently removed by the method, was hardly detected, and most of (PA) was also removed, especially when the water content was 5% or less. On the other hand, in the P1 and P2 fractions, the remaining half of PE and most of PI and PA were separated and concentrated. Although the concentration effect of (PC + PE) on the S2 fraction was sufficiently recognized even in the case of 85% ethanol, it was shown that the higher the water content, the smaller the amount of the recovered phospholipid in the S2 fraction. The recovery rate was defined as the weight% based on the crude lecithin (measured with phospholipid-saturated acetone).

Embodiment 3 FIG. Effect of phospholipid concentration Heated ethanol fractionation was carried out in the same manner as in Example 2 by changing the concentration of crude lecithin, and lecithin on the recovery rate of S2 fraction and the phospholipid composition (%) by the heated ethanol fractionation method The effect of concentration was verified. The results are shown in Table 3 below. The recovery rate shown in Table 3 is the percentage of the solid content recovered in the S2 fraction relative to the crude lecithin, and PC + PE is the percentage of (PC + PE) in the phospholipid recovered in the S2 fraction.

[0029]

[Table 3]

As shown in Table 3, although the concentration of (PC + PE) did not greatly differ between 10% and 50% crude lecithin, the recovery rate was found to be significantly lower as the concentration of crude lecithin was increased. If it is less than 5%, the recovery rate is high, but efficiency is poor from the viewpoint of industrial productivity. Since the recovery rate greatly affects the cost, a concentration of 10 to 30% (W / W) is considered to be preferably suitable.

Embodiment 4 FIG. Effect of pH Add 1N hydrochloric acid or caustic soda to crude lecithin
H was adjusted and an experiment was performed (pH 4, 5.5, 7, 8:
Those without pH adjustment have a pH of 5.5). As a result, there is no difference in the target (PC + PE) concentration difference even when the pH is changed.
The sample with no pH adjustment was the best in recovery.

Embodiment 5 FIG. Influence of stirring The ethanol extraction at 70 ° C. was compared with that obtained by a homomixer capable of strong stirring and that obtained by only weakly stirring with a magnet rotor. There was almost no difference between the recovery rate and the composition of (PC + PE).

Embodiment 6 FIG. FIG. 3 is an explanatory view showing the operation steps of the scale-up of the heated ethanol method shown in FIG. 2. As shown in the figure, 4 kg (about 5 liters) of ethanol was heated to about 72 ° C. in a warm bath, and 1 kg of crude lecithin was gradually added and dissolved while stirring (60 ° C. after completion of the addition). The mixture was allowed to stand for 10 minutes, and the supernatant was separated by decantation into a 5-liter beaker. The supernatant was allowed to cool at room temperature for 1 hour and fractionated again by decantation. As a result, 588 g of the S2 fraction was obtained. The same test was repeated three more times. Table 4 shows the test results of four times in total.

[0034]

[Table 4]

As shown in Table 4, the recovery rate is stable.
It was confirmed that the decantation was easy, and that the composition of phospholipids and other components by thin-layer chromatography was stable. As described above, it was confirmed that the soluble fraction enriched in amphoteric phospholipid (PC + PE) and the insoluble fraction enriched in acidic phospholipid (PI + PA) could be fractionated by a simple operation. The obtained soluble fraction is used, for example, as a substrate for phosphatidyl group transfer reaction or cooking oil, and the insoluble fraction is used as a starting material for PI production, a leather tanning agent, or a cooking oil except glycolipid components. Available.

Embodiment 7 FIG. The (PC + PE) -rich S1 fraction (PC (50
%), PE (38%), PI (1%), PA (2%),
A phosphatidyl group transfer reaction was performed with phosphatidyl group donor) and glycerol (phosphatidyl group acceptor).
That is, 10 parts by weight of the substrate (S1 fraction)
0 parts by weight of ethyl acetate and 40 parts by weight of glycerol, 40 parts by weight of an aqueous phospholipase D enzyme solution (40 units / g)
(Substrate) was added and the mixture was kept at 45 ° C. for 20 hours to perform a phosphatidylglycerol (PG) generation reaction. The reaction product was developed by thin-layer chromatography and spots colored with a phosphorus detection reagent (Dittmer-Lester reagent) were quantified with an image analysis processor (Dr. Gel; Mitani Corporation). As a result, a phospholipid composition containing 78% of PG in the phospholipid was obtained (transfer reaction rate: 81%). The recovery rate of PG to PC + PE in the crude lecithin starting material was 4%.
It was 8.9%.

On the other hand, the PC65 obtained by the conventional method of separating and dissolving crude lecithin in ethanol at an environmental temperature of 25 ° C.
%, And a substrate containing 15% PE, the reaction product phosphatidylglycerol was added to the phospholipid in 70%.
Was obtained (transfer reaction rate: 81%), and the production and recovery rate of PG with respect to (PC + PE) in crude lecithin as a starting material was 32.1%.

[0038]

As described above, according to the present invention, a mixture of acidic and bipolar phospholipids is suspended in a heated aqueous ethanol solution, and then the suspension is cooled to a predetermined temperature to dissolve the soluble fraction. And the insoluble fraction.
Was fractionated bipolar phospholipids PC and PE from very simply as lecithin, turn the majority of PA and PI in the insoluble fraction
There is an effect that can be collected .

[Brief description of the drawings]

BRIEF DESCRIPTION OF DRAWINGS FIG. 1 is an explanatory diagram showing operation steps for examining the influence of temperature on the method for fractionating an ambipolar phospholipid of the present invention.

FIG. 2 is an explanatory diagram showing operation steps in which the influence of the water content of ethanol was examined.

FIG. 3 is an explanatory view showing an operation step of scale-up of the heated ethanol method shown in FIG. 2;

──────────────────────────────────────────────────続 き Continued on the front page (58) Field surveyed (Int. Cl. ⁷ , DB name) A23J 7/00 C07F 9/10

Claims (2)

(57) [Claims] 1. A fractionation method for separating an ambipolar phospholipid and an acidic phospholipid from a mixture of acidic and ambipolar phospholipids, wherein the mixture of acidic and ambipolar phospholipids is heated to 50 ° C. or more. Suspended in a warm 85% or more aqueous ethanol solution,
Then, the suspension is cooled to a predetermined temperature and fractionated into a soluble fraction comprising an ambipolar phospholipid and an insoluble fraction containing most of the acidic phospholipids. . 2. Mainly phosphatidylcholine, phosphatidylethanolamine, phosphatidylinositol,
A fractionation method for fractionating an amphoteric phospholipid mixture of phosphatidylcholine and phosphatidylethanolamine from a phosphatidic acid-containing phospholipid mixture, comprising: converting the phospholipid mixture to an 85% or more aqueous ethanol solution heated to 60 to 70 ° C. The suspension is then cooled to 50 ° C. or lower, and fractionated into a soluble fraction comprising the phosphatidylcholine and phosphatidylethanolamine and an insoluble fraction containing most of the phosphatidylinositol and phosphatidic acid. A method for fractionating a phospholipid characterized by the following.