JPS6130555B2 - - Google Patents

Description

Detailed Description of the Invention The present invention provides natural lysolecithin (lysolecithin), which is characterized by reacting bovine brain, pig brain, chicken egg yolk, or soybean homogenate with pancreatin, an enzyme extracted from pig pancreas, and heat-treating it after the reaction. phosphatidylcholine).

It has been known that lysolecithins have useful properties such as immunostimulatory effects and inhibitory effects on smooth muscle contraction, and that these compounds have α-type and β-type structures. The following method is known as a method for producing natural lysolecithin.

(i) A method for obtaining lysolecithin by using natural lecithin as a raw material and treating it with datoxin phospholipase. [Ansell, GB &
Howthome, JN), 1964 “Phospholipids”, Amsterdam, 95: Elserier] (ii) A method for obtaining lysolecithin by treating natural lecithin (reagent lecithin) with pancreatin. . DJ Hanahan, Journal of Biological Chemistry (J.Biol.Chem.) No. 195
Vol., p. 199 (1952)] However, in the above method (i), the datoxin phospholipase A used is very expensive and erosive to the skin, making it difficult to handle, and post-reaction operations are also complicated. (ii)
The commercially available lecithin used is extracted and separated from soybeans or eggs, but its composition is extremely poor.
Lecithin, which is a raw material for the present invention, has a low content and is very expensive if high purity lecithin is to be obtained. Furthermore, even if highly purified lecithin is used, a considerable amount of unsaturated fat residues in lecithin itself are oxidized, which significantly reduces the activity of pancreatin and significantly slows down the reaction rate of pancreatin-induced deacylation. It has disadvantages such as being slow and requiring a long time to complete the reaction. Furthermore, when commercially available lecithin is used as a raw material in the operation after the completion of the reaction, the reaction solution becomes an emulsified state, and subsequent isolation and purification must be performed using methods such as Folch partitioning and column chromatography. However, it has drawbacks such as being extremely complicated.

Therefore, from the above, both methods (i) and (ii) are unsuitable as industrial production methods.

As a result of intensive research on a method for industrially obtaining lysolecithin from lecithin by applying pancreatin, the present inventors found that it is possible to obtain lysolecithin from fresh lecithin-containing tissues of animals and plants, namely pig brain, cow brain, soybean, and especially chicken egg yolk. When used as such, in the enzymatic decomposition reaction of pancreatin, as a result of the synergistic effect of the coenzyme contained in the tissue, the deacylation reaction proceeds very smoothly, and the desired lysolecithin is produced in a high yield. was also found to be generated. In addition, since the reaction solution contains proteins, oligopeptides, etc., it is difficult to isolate lysolecithin using operations such as Folch partitioning and column chromatography, but post-reaction heat treatment ,
In particular, by introducing a thermal denaturation process for proteins heated to 50 to 120°C, surprisingly, the target lysolecithin is adsorbed quantitatively into the denatured coagulated proteins, and then through operations such as solvent extraction, It has the advantage of being easily separated.

The method of the present invention has been completed based on the above findings, and is an extremely excellent method for industrially producing natural lysolecithin.

The present invention will be further explained in detail below.

In carrying out the present invention, in addition to the phospholipase A ₂ necessary for the reaction, it is thought that a small amount of phospholipase A ₁ is also present in commercially available pancreatin, but in general, this consideration has little effect, so phospholipase There is no need to perform a heat treatment in advance to deactivate the activity of _A1 . In the method of the present invention, pancreatin is first added to cow brain, pig brain, chicken egg yolk, or soybean homogenate and reacted in water. Enzymes other than those mentioned above, such as ribonuclease, amylase, trypsin, chymotrypsin, lipase, and carboxypeptidase A
In order to suppress the activities of B and B, it is preferable to add calcium chloride and deoxycholic acid to the reaction system. And the above reaction is generally 5 to 50
The reaction is carried out at a temperature of 35°C, preferably 35°C, and a pH of 7.0 to 8.0, and the reaction is generally completed in 30 minutes to 12 hours. For example, when chicken egg yolk is used as a raw material and 0.1 to 2.0 g of pancreatin is used per chicken egg yolk (18 to 20 g), the reaction is completed in about 3 hours. After the reaction, the reaction solution is in a milky state, mostly consisting of proteins and fatty acids. By heating this reaction solution at 100 to 120°C, the protein is coagulated and the target substance is adsorbed therein. The target product can be isolated and collected by taking this coagulated protein and using conventional methods. For example, the coagulation protein obtained above can be mixed with methanol, ethanol,
Suspend in alcohol such as isopropanol to remove insoluble proteins, remove the solvent from the resulting extraction solution under reduced pressure, add a mixed solvent of halogenated hydrocarbons, alcohol, and water to the residue, and add organic Separate the layers. Then, the solvent was distilled off under reduced pressure.
Furthermore, in order to remove fatty acids, neutral fat sphingomyelin and oligopeptides, methanol,
Highly purified lysolecithin can be easily obtained by performing an extraction process with an alcohol such as ethanol and a hydrocarbon solvent such as n-hexane or cyclohexane, followed by solvent separation with an alcohol such as propanol and ethanol. .

Next, the present invention will be explained with reference to Examples.

Example 1 Only the egg yolk is separated from 100 commercially available chicken eggs and dispersed in 2.5 liters of distilled water. Dissolve 4.4 g of calcium chloride dihydrate in 200 ml of distilled water and 5.0 g of sodium deoxycholate in distilled water.
200 ml of pancreatin (Wako Pure Chemical Industries, Ltd.) and 100 g of pancreatin (manufactured by Wako Pure Chemical Industries, Ltd.) suspended in 200 ml of distilled water were added to the above dispersion and heated to 30-35°C.
Stir for 3 hours. Next, the reaction solution is heated at 105 to 110°C for 3 hours on a water bath in which calcium chloride is dissolved. After cooling, centrifugal dehydration is performed to obtain 1.1 kg of a yellow-brown substance. Add 1.2 methanol to this,
After stirring at room temperature for 30 minutes, filter out insoluble materials. Furthermore, methanol 1.6, 1.6, 1.0 is added to this, the extraction is repeated, and the extracts are collected and concentrated under normal pressure to a volume of 2. Next, 2 parts of chloroform and 1 part of water are added, and the chloroform layer is separated. Furthermore, 400 ml of chloroform is added to the aqueous layer, the chloroform layer is separated, and the obtained chloroform solution is combined and the solvent is distilled off under reduced pressure to obtain 480 g of a brown substance. (This brown substance is referred to as crude product 1.) Next, add 1 part of 80% methanol aqueous solution to this crude product 1.
Add 400ml of n-hexane, separate the lower layer, and add 500ml of 80% methanol aqueous solution to the upper layer solution.
Add and separate the lower layer. The resulting methanol aqueous solution was combined and the solvent was distilled off under reduced pressure. Add 200 ml of ethanol to the residue, filter out the insoluble materials, and distill off the solvent from the filtrate under reduced pressure. Next, add 200 ml of isopropanol and filter out the insoluble materials. If the solvent is distilled off from the filtrate under reduced pressure and then dehydrated by azeotroping with toluene, 133g of pale yellow substance is obtained.
get. This is recrystallized once each from pyridine and ethanol to obtain 29 g of white amorphous crystalline lysolecithin. (In addition, in one chicken egg yolk,
It is said that 500 to 700 mg of lecithin is contained, so if one chicken egg yolk contains 600 ml of lecithin, the yield in this example is 71.4%. ) IR (KBr) cm ^-1 1720, 1460, 1235, 1080, 1050, 960 NMR (CDC1 ₈ ) τ value 6.10 (9H) 6.80 (9H) 7.65 (2H) 8.70 (27H) 9.10 (3H) Optical rotation [α ] ^20/D = -2.75 (C = 11.5, CHC1 ₃ :
CH ₃ OH = 4:1) [α] ^20/D = -0.66 (C = 10.6, CHC1 ₃ :
CH ₃ OH = 1:1) Example 2 To 1480 g of the crude product obtained in Example 1, 800 ml of pyridine was added and dissolved by heating. After standing overnight, filter out the precipitated crystals and add 200 ml of ethanol to it.
ml, filter out the insoluble matter and add ether to the filtrate.
When 500ml is quickly added, 37g of white crystals precipitate out. This is collected and recrystallized once each from pyridine and ethanol to obtain 2 g of lysolecithin in the form of white amorphous crystals. (Example 1
Considering the same way, the yield is 78.8%. )

Claims (1)

[Scope of Claims] 1. A natural lysolecithin (lysophosphatidylcholine) characterized in that cow brain, pig brain, chicken egg yolk or soybean homogenate is reacted with pancreatin, an enzyme extracted from pig pancreas, and heat-treated after the reaction. manufacturing method. 2. The method for producing natural lysolecithin according to claim 1, using chicken egg yolk homogenate. 3. The method for producing natural lysolecithin according to claim 1, wherein the temperature at which the porcine pancreas extract enzyme pancreatin is applied is 15 to 50°C. 4. The method for producing natural lysolecithin according to claim 1, wherein the heat treatment is performed at a temperature of 50 to 120°C.