JPS6336792A - Production of phospholipid by enzyme - Google Patents

Abstract

PURPOSE:To reduce water content in a reaction system and to suppress formation of by-products, by adding a water phase containing a receptor and phospholipase D in a state wherein the water phase is sealed in a reverse micelle to an organic solvent having dissolved a raw material phospholipid and carrying out reaction. CONSTITUTION:A water phase containing a hydroxyl group-containing receptor such as serine, ethanolamine, trehalose, etc., and phospholipase D in a state wherein the water phase is sealed in reverse micelle is added to an organic solvent having dissolved a raw material phospholipid such as phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, etc., and reaction is carried out. The reverse micelle is formed by a surface active agent and the raw material phospholipid itself is used as the surface active agent. In order to suppress formation of by-products, the water content in the reaction system is about 6-15mol based on 1mol raw material phospholipid or surface active agent. The formed aimed phospholipid is purified by chromatography, etc.

Description

DETAILED DESCRIPTION OF THE INVENTION (Industrial Application Field) The present invention relates to a method for producing phospholipids using an enzyme, and particularly to a method for producing phospholipids whose base structure has been converted.

(Prior Art) In recent years, phospholipids have attracted attention not only for their use as emulsifiers but also for their use as base materials for liposomes, drug carriers, artificial blood, artificial cells, etc.
As substances with pharmacological effects, various uses in the medical, pharmaceutical, and engineering fields are being considered. In order to meet such diverse demands, it is of great industrial significance to develop a method for efficiently producing phospholipids having structures suitable for each use.

As a method for producing phospholipids using enzymes, a technique is known in which phospholipase D is applied to phospholipids in the presence of an arbitrary receptor, and a phospholipid having a target base is produced using a phosphatidyl group transfer reaction. (S, F, Yan
g, et al., J. Biol. Chem.

242, (3) 477-484 (1967))
: (R, M, C, Dawson.

Biochem, J., 102.205-210 (
1967) ).

When attempting to exchange the base moiety of a phospholipid using a phosphatidyl group transfer reaction by phospholipase D, a two-phase system of an aqueous phase and an organic solvent phase is generally used. namely, enzymes, receptors, pH buffers, which are primarily water soluble;
This is a reaction system in which an aqueous solution containing inorganic salts and the like and an organic solvent phase containing mainly lipophilic raw material phospholipids are stirred and mixed. Including the aforementioned technology, subsequent research (K, Bruz
ik andM, Tsai, Biochemist
y23. (8) 1656-1661 (1984
) etc.] is also widely used.

(Problems to be Solved by the Invention) However, in such a conventionally used reaction system, the presence of a large amount of water as a solvent for water-soluble components essentially causes phospholipase D to have no hydrolytic activity. Because of this, hydrolysis occurs as a side reaction, resulting in the production of phosphatidic acid (hereinafter abbreviated as PA).

The generation of PA through hydrolysis not only makes it difficult to separate and purify the target phospholipid after the reaction, but also because the raw material phospholipid is consumed by the hydrolysis reaction, it is difficult to separate and purify the target phospholipid after the reaction. When attempting to transfer a phosphatidyl group to a receptor, it has been virtually impossible to obtain the desired product because the reaction rate is extremely low compared to the hydrolysis reaction rate.

Such problems are unavoidable as long as water is present, since phospholipase D itself is originally a hydrolytic enzyme.

In order to solve this problem by reducing the water content in the reaction system to the limit without deactivating the enzyme, the present inventors have conducted various studies and found that the reaction system The present invention was achieved by devising a new reaction system in which the components previously added to the microemulsion are added by encapsulating them in reverse micelles (Wh-type microemulsion).

As used herein, the term "reverse micelle" refers to a state in which amphipathic molecules are associated in a low polar solvent with lipophilic groups facing outward and hydrophilic groups facing inward, centering around a very small aqueous phase.

(Means for Solving the Problems) The present invention involves adding an aqueous phase containing a hydroxyl group-containing receptor and phospholipase D to an organic solvent in which a raw material phospholipid is dissolved, in the form of a reverse micelle encapsulated in the reaction. It is characterized by doing.

The raw material phospholipid used in the present invention may be any phospholipid extracted from nature, purified after extraction, or synthesized, as long as it can serve as a substrate for phospholipase D. Moreover, commercially available products or products prepared by known methods may be used.

Examples include defatted soybean lecithin, egg yolk lecithin, phosphatidylcholine (hereinafter abbreviated as PC), phosphatidylethanolamine (hereinafter abbreviated as PE), phosphatidylserine (hereinafter abbreviated as PS), phosphatidylglycerol (hereinafter abbreviated as PC), etc., or mixtures thereof. can be given. In order to maximize the effects of the present invention, it is more convenient to use purified phospholipids or those with a simple composition as raw material phospholipids in terms of purification of the reaction product. In addition, from the viewpoint of raw material cost, easy availability, and reactivity with enzymes, PC, PE, or PS are particularly preferred because they are industrially effective.

Preferably, the reverse micelles are formed using a surfactant.

When most of the raw material phospholipids are PC, PE, PC or PS or a mixture thereof having two acyl groups with a carbon chain length of 12 or more, a surfactant for the raw material phospholipids themselves to form reverse micelles. Acts as a. but,
PC, PE5PS, PC or lyso-PC, lyso-PE in which the raw material phospholipid has two acyl groups with a carbon chain length of 10 or less,
When it is difficult to form reverse micelles with the raw material phospholipid itself, as in the case of Lyso PS and Lyso PG, it is preferable to separately add a surfactant that satisfies the following conditions.

(a) It can be dissolved in the reaction solvent at a concentration above the critical micelle concentration.

(b) Reverse micelles can be formed.

(c) There is no significant loss in enzyme activity.

(d) It is desirable not to have alcohol residues.

Examples include sodium lauryl sulfate, sodium di(2-ethylhexyl)sulfosuccinate, sodium di-n-octylsulfosuccinate, polyoxyethylene nonylphenyl ether, and the like. The amount of these surfactants added is 0.5 to 5 mol, preferably 1 to 3 mol, per 1 mol of raw material phospholipid.

As the reaction solvent, any solvent that satisfies the following conditions can be used.

(a) The raw material phospholipid can be dissolved at a concentration higher than the critical micelle concentration.

(b) hardly miscible or soluble in water;

(c) There is no significant loss in enzyme activity.

In other words, it does not matter if it has a double bond or an ether bond in the molecule, it does not matter if it has a functional group such as an alkyl group, and it is linear, branched, or cyclic and has 5 to 10 carbon atoms. A hydrocarbon compound, a halogenated hydrocarbon compound having 1 to 2 carbon atoms, or a mixture thereof,
Examples include n-hexane, isooctane, diethyl ether, diisopropyl ether, cyclohexane, benzene, xylene, chloroform, carbon tetrachloride, dichloroethane, and mixtures of two or more of these. Since a compound having an alcohol structure becomes a receptor for a phosphatidyl group transfer reaction, it is not very preferable to add it for purposes other than use as a substrate.

As phospholipase D, any phospholipase D can be used, as long as it has phosphatidyl group transfer activity, regardless of whether it is commercially available or prepared by a known method. For example, Boehringer Manheim
cabbage-derived phospholipase D (PLDP) manufactured by Toyo Jozo Co., Ltd. (Nheim Gmbt+); microbial-derived phospholipase D (PLDP) manufactured by Toyo Jozo Co., Ltd. (Ichiku);

``Methods in E
zymology” (J, M.

Lowenstein, ed.), vol. 1
4+ pp197-203゜8chademic P
For example, enzyme preparations extracted and purified or partially purified by the method of A. ress, New York (1969)), or extracted crude enzymes can be mentioned.

Receptors include not only compounds conventionally known as receptors for phosphatidyl group transfer reactions such as choline, methanol, ethanol, ethanolamine, serine, glycerol, and glucose, but also 1-amino-2-propanol, 1-ortho Compounds having a primary or secondary alcohol structure containing sugars, such as methyl glucoside and trehalose, which were conventionally considered not to be receptors for phosphatidyl group transfer reactions, can also be used.

In order to encapsulate enzymes, receptors, etc. in reverse micelles, a solution of enzymes, receptors, etc. is added dropwise into a solvent containing raw material phospholipid dissolved above the critical micelle concentration, and immediately shaken, stirred, or ultrasonicated. A known method such as can be used.

In order to form reverse micelles and at the same time suppress the by-product of PA during the phosphatidyl group transfer reaction, the water content in the reaction system should be adjusted to 6% per mole of raw material phospholipid or surfactant.
~15 moles, preferably 8 to 12 moles gives best results.

The reaction temperature may be any optimum temperature for the enzyme used, and is usually 3.
It is in the range of 0 to 40°C. Preferably, the stability of the reverse micelles is improved by keeping the temperature constant from the time the reverse micelles are formed until the end of the enzyme reaction.

However, this does not apply when the solvent used has a low boiling point.

The reaction time is 0.5 to 36 hours, preferably 4 to 2.1 hours.
It's time.

The target phospholipid having any base produced in this manner can be easily purified by appropriately using known means such as solvent fractionation, silicic acid or silica gel chromatography, alumina chromatography, DIEAE-cellulose chromatography, etc. be able to.

Furthermore, since the production of pA is suppressed, unreacted substrates can be easily recovered.

(Effects of the invention) By using the reaction system of the present invention, the generation of PA that was observed in conventional reaction systems was suppressed, and the production of the target phospholipid after the reaction was facilitated. .

Furthermore, it has become possible to produce target phospholipids that could hardly or never be obtained using conventional reaction systems.

(Example) Hereinafter, the present invention will be specifically described based on Reference Examples, Examples, and Comparative Examples.

In addition, the compositional analysis and purity test of phospholipids were performed by thin layer chromatography (TLC). TLC board (Merck N)
115721) with a diameter of 3 to 100 μg.
Spotted on 5mm, chloroform-methanol-water (
120nia 0:5) or chloroform-acetone-acetic acid-methanol-water (50:20:15:10:5). For detection, Jittmer reagent, 50% sulfuric acid, ninhydrin reagent, or Anthrone reagent was used depending on the purpose. For quantitative measurements, the color developed with Jittmer reagent was analyzed using a high-speed thin layer chromatography scanner (C5-9 manufactured by Shimabara Seisakusho).
20 type).

Reference example 1 Soybean PC and PE were prepared by Paldan (Von H.

Pardun's method (Fette 5eifen A)
nstrichmitte dish, (2) 55-62
(1984)).

Commercially available defatted soybean lecithin powder (PC24%, PE21%,
Phosphatidylinositol 14%, PA 8%) 20g
isopropanol-methanol-water (50:45:5
) Dispersed in 100mj and heated and stirred at 40°C to dissolve. The mixture was cooled to 20°C with stirring and kept at 20°C for 1 hour. The insoluble matter was centrifuged or filtered under reduced pressure using a glass filter while maintaining the temperature at 20°C. The collected supernatants were dried under reduced pressure to form PC and PE concentrates (PC68%, PE1
7% PA, 7% PA, PS not included) 9.7 g was obtained.

Reference Example 2 M, Lees method from cow brain
ds in Enzymology” (S, P, Colo
wick and N, 0. Kaplan ed.).

vol, 3. pp328. eight chademic
Press+ New York (1957)
] and purified by DEAE-cellulose column chromatography.

300g of fresh bovine brain membranes and blood vessels removed from a nearby slaughterhouse for 1.2 Jl! Homogenized and extracted in acetone. Transfer the filtration residue to 1.
Extract with 21 acetone. The filter residue is extracted with 1.21 ethanol. Similarly, the filtration residue is 1.2 j2
The extract was extracted twice with petroleum ether, and the extracts were collected and dried under reduced pressure to obtain 3.9 g of a crude cephalin fraction.

D was dissolved in chloroform and prepared in acetic acid form.
IEAE-cellulose column (Whatman DE32
, diameter 2.5 cm x 20 cm). After washing the column with chloroform-methanol (1:4) 1 1, both fractions eluted with 150 ml of acetic acid were collected.

An equal volume of chloroform was added to the acetic acid elution fraction, and the mixture was washed four times with 2 volumes of water. The chloroform layer was dried under reduced pressure and PS (
PS98%) 0.8g was obtained.

Reference Example 3 Commercially available sodium dioctyl sulfosuccinate was purified.

A 50% methanol solution of sodium dioctyl sulfosuccinate (manufactured by Nippon Oil & Fats Co., Ltd., trade name Rapizol B-90) was left standing at 4°C for 3 hours, and then heated to 2,000°C at 4°C.
The precipitate was removed by centrifugation at Xg for 15 minutes.

5 g of special grade activated carbon (manufactured by Wako Pure Chemical Industries, Ltd.) was added to the supernatant, and the mixture was stirred for 18 hours. Activated carbon was filtered off and methanol was extracted.

The dried product was dried in a vacuum desiccator containing phosphorus pentoxide for at least one day and was used as purified sodium dioctyl sulfosuccinate.

Reference example 4 KaLes and Sastrii mentioned above (M, KaLes and
P.S.

Phospholipase D was partially purified from the cabbage soluble fraction according to the method of 5astry).

The inner leaves of fresh cabbage obtained from a nearby farm were washed with water and shredded, 200 rnl of water was added to 100 g, and the mixture was homogenized for 5 minutes under water cooling. The filtrate was filtered through 5 layers of gauze and centrifuged at 4°C for 30 minutes at 12,0OOXg, and the supernatant was 190mj! I got it. Transfer this supernatant to 55°C15
After heat treatment for a minute, the mixture was immediately cooled on ice. 12,0OOX at 4℃
g, centrifuged for 30 minutes to obtain supernatant 175+++f.

Add 350mf of acetone cooled to -15°C little by little while stirring, leave it to stand for 10 minutes, and then heat to 4°C to give 12,0OOX.
g, the precipitate was collected by centrifugation for 30 minutes.

The precipitate was dissolved in 15 ml of 50 mM acetate buffer pl (5,6) and dialyzed three times against 500 ml of the same buffer at 4°C.

The insoluble material was centrifuged at 4° C. and 10,000×g for 15 minutes, and the resultant product was used as cabbage partially purified phospholipase D.

It was confirmed that this enzyme solution did not contain any detectable amount of phospholipid.

Example I L-serine 1.5M, cabbage phospholipase D (Boehringer Mannheim) 7mg/mtt, calcium chloride trihydrate Ion M, Q, 5ml of 5
It was dissolved in mM acetate buffer pH 5,6 to form an aqueous phase. 1.5 g of the concentrate of soybean PC and PE obtained in Reference Example 1 was dissolved in dry diisopropyl ether 40- to form an organic phase.

The organic phase was maintained at 35°C, and 0.35 In1 of the aqueous phase was added dropwise while being treated with ultrasonic waves using an N5100-20 type ultrasonic cleaner (output 100 W, newly manufactured by Nippon Seiki Co., Ltd.). A transparent reverse micelle solution was obtained.

This product was shaken back and forth at 15 rpm for 12 hours at 35°C to react.

Take a portion of the reaction mixture 1. Direct TLC analysis showed a PC of 48%, a PE of 14%, and a PALL% of 24% of the product, which was identified as PS due to its Rf matching with standard PS and positive response to Zittmer and ninhydrin reagents.

A DEAE-cellulose column (Whatman's JA DE32, diameter 2
．． 5×20 cm).

Column with chloroform-methanol (1:4) 500
ml, and dried under reduced pressure to remove unreacted substrate (PC73%).
, PE27%) were recovered.

The column was further eluted with 400ml of acetic acid. The acetic acid elution fractions were collected, added with an equal volume of chloroform, and washed four times with 2 volumes of water. The chloroform layer was dried under reduced pressure, and PS (PS9
8%) 294 mg were recovered.

Example 2 Using isooctane 1- dissolved in 40 mg of commercially available hydrogenated egg yolk lecithin (PS99%) as the organic phase, 1-
Amino-2-propanol 1.5M, cabbage phospholipase D (manufactured by Boehringer Mannheim > 7■/-
Aqueous phase 11 consisting of 10 mM of calcium monochloride trihydrate
pl was added dropwise to form reverse micelles.

This was shaken back and forth at 15 rpm for 8 hours at 38°C.
Made it react.

When a portion of the reaction mixture was taken and directly analyzed by TLC, it was found that
Product 24 was determined to be the target phospholipid in which a phosphatidyl group was introduced into 1-amino-2-propanol because PC was 62% and PA was 5%, standard PE and Rf were close to each other, and the results were positive for Jittmer's reagent and ninhydrin reagent. It contained %.

Example 3 Using cyclohexane 1- in which 40 mg of the bovine brain PS obtained in Reference Example 2 (PS98%) was dissolved as the organic phase, the same procedure as in Example 1 was carried out at 38°C, and 1.5 M of glycerin and cabbage phospholipase D (Boehringer) were prepared.・Manufactured by Manno λim)
7/-1 9 μl of an aqueous phase consisting of 10 mM calcium chloride trihydrate and 5 mM acetate buffer pH 5.6 was added dropwise to form reverse micelles.

This mixture was shaken reciprocally at 15 rpm for 112 hours at 38°C to react.

When a portion of the reaction mixture was taken and directly analyzed by TLC, it was found that
It was 70% PS and 3% PA, and contained 25% of the product, which was identified as PG because it matched the standard PC (!: Rf) and was positive to the Zittmer reagent.

Example 4 1-Orthomethyl glucoside 1.5M, phospholipase D (manufactured by Toyo Jozo a PLDP) 1■/-15m
20 μl of an aqueous phase consisting of M acetate buffer pH 5,6 was added dropwise to form reverse micelles.

This product was shaken back and forth at 15 rpm for 12 hours at 35°C to react.

When a portion of the reaction mixture was taken and directly analyzed by TLC, it was found that
It was 74% PC, 4% PA, and contained 19% unidentified phospholipids.

The reaction mixture was directly fractionated using a 11m5745 preparative TLC plate (manufactured by Merck & Co., Ltd.) using chloroform-methanol-water (120Nia 0:5) as a developing solvent.
16 mg of unidentified phospholipid was obtained.

When this phospholipid was analyzed using a JMS-Dx303 mass spectrometer (manufactured by JEOL Ltd. (4i) under the following conditions, the parent peak on the cation side was m/e 847, and the parent peak on the anion side was m/e 847. It was m/e 823.

Measurement conditions Ionization method r FAB method Impact gas: xe Partial ion acceleration voltage: 6kV Filament current: 20mA Detector: Conversion dynode extrusion voltage: 15kV Matrix Nitriethanolamine (sodium chloride added for cations) Data processing: JMA- DA5000 These values were consistent with the sodium salt (molecular weight 82/1 + 23) and anion (molecule ff 1824-1) with a molecular weight (824) assuming that a phosphatidyl group was introduced into 1-orthomethyl glucoside. It was identified as the target phospholipid.

Example 5 Using 2.5 mf of commercially available dipalmitoylroform-isooctane (35:65) as the organic phase, 1.5 M trehalose, phospholipase D (Toyo Jozo Co., Ltd.
17 μl of an aqueous phase consisting of a 1 mg/ml, 5 mM acetic acid 11 buffer solution (pH 5.6) was added dropwise to form reverse micelles.

This product was shaken back and forth at 15 rpm for 12 hours at 35°C to react.

When a portion of the reaction mixture was taken and directly analyzed by TLC, it was found that
It contained 17% of phospholipid i, which was determined to be the target phospholipid in which a phosphatidyl group was introduced into trehalose, since it simultaneously showed positive results for Jittmer's reagent and Anthrone's reagent at 76% PC and 5% PA.

16 mg of phospholipids obtained by fractionating the reaction mixture in the same manner as in Example 4 was subjected to mass spectrometry in the same manner as in Example 4. As a result, the cation peak m/e was 995, and the anion peak m/e was 995. The peak m/e was 971, which coincided with the calculated molecular weight (972) of a sodium salt (molecules, 1972+23) and an anion (molecular weight 972-1), so it was identified as the target phospholipid.

Example 6 Using 3 ml of n-hexane in which 100 mg of commercially available dioctyl PC and 90 mg of the purified sodium dioctyl sulfosuccinate obtained in Reference Example 3 were dissolved as an organic phase, the mixture was heated to pH 6.0 with hydrochloric acid in the same manner as in Example 1 at 37°C. 1 adjusted to 0
．． 1 mg/ml phospholipase D (Toyo Jozo Co., Ltd. (barrel P'LDP) dissolved in 5M ethanolamine 36
μl was added dropwise as the aqueous phase to form reverse micelles.

This product was shaken back and forth at 15 rpm for 10 hours at 37°C to react.

When a portion of the reaction mixture was taken and directly analyzed by TLC, it was found that
It contained 22% of the phospholipid, which was determined to be the target phospholipid because it had an Rf value close to standard PE with a PC of 71% and a P of 7%, and was simultaneously positive for the Jittmer reagent and ninhydrin reagent.

Example 7 Commercially available 1-stearoyl lyso PC 200 mg, sodium lauryl sulfate (manufactured by Wako Pure Chemical Tri, for biochemistry use) 10
0mg? dichloromethane-isooctane (3
In the same manner as in Example 1 at 35°C using 5mi as the organic phase, 1.5M glycerin and calcium chloride dihydrate 1.0 were added to the cabbage partially purified enzyme solution obtained in Reference Example 4.
A solution of 65 μE of mM was added dropwise as an aqueous phase to form reverse micelles.

This product was shaken back and forth at 15 rpm for 15 hours at 35° C. to react.

A DEAE-cellulose column (DE52 manufactured by Whatman, diameter 1.8
m). Chloroform-methanol (1
: 4) After washing the column with 100 ndl, 50 m
Chloroform-methanol containing M ammonium acetate (
1:4) Both eluted volumes of 100 ml were collected. An equal volume of chloroform was added to the acetic acid elution fraction, and the mixture was washed four times with 2 volumes of water.

Phospholipids obtained by drying the chloroform layer under reduced pressure 26.
When 7 mg was subjected to mass spectrometry in the same manner as in Example 4, the cation side peak m/e was 534, the anion side peak m/e 511, and the calculated molecular weight (512) of each sodium salt (molecules ffl 512+23 ) and an anion (molecule l 512-1), it was identified as the target 1-stearoyl lyso-PC.

Comparative Example 1 A reaction was carried out using a conventional reaction system under conditions as close as possible to those of Example 1.

L-serine 200mM, calcium chloride 40mM, phospholipase D (Boehringer Mannheim) 2.5
2.5 m of 50 mM acetate buffer at pH 5,6 containing mg
l and soybean pc and pE>5 condensate 40 obtained in Reference Example 1
37 mg of diisopropyl ether 5rn1 dissolved in
The mixture was stirred at 500 rpm.

Five identical samples were reacted in parallel for 1, 2, 4, and 12 hours each. After the reaction, the lipids were extracted three times with 5 rnl of diethyl ether and the phospholipid composition was analyzed.

The results are shown in the table below.

Table 1 Composition (%) Reaction time (h) PCPE PA PSl
1.5 31 42 12 Comparative Example 2 A reaction was carried out using a conventional reaction system under conditions as close as possible to those of Example 4.

■-p! in which 1.5M of orthomethyl glucoside and 20 nitrate of phospholipase D (PLDP manufactured by Toyo Jojo ■) were dissolved! (5
, 2.5 ml of 50mM vinegar M91 buffer solution from No.6 and 100mg of dipalmitoyl PC? Capacity) Benzene 2.5n
dl was stirred at 35° C. and 500 rpm to react.

Analysis was performed over time in the same manner as in Comparative Example 1, but the spot corresponding to the target phospholipid observed in Example 4 was not observed on TLC, and the substrate was completely decomposed after 16 hours of reaction. Analysis was discontinued.

Comparative Example 3 A reaction was carried out using a conventional reaction system under conditions as close as possible to those of Example 5.

2.5ml of 50mM acetate buffer (pH 5,6) in which 1.5M trehalose, 20μg of phospholipase D (ElK Yojojo (Horizona PLDP)) were dissolved, and dipalmitoyl PC.
chloroform-isooctane (100 mg)
35:65) 2.5a+j! were stirred at 35° C. and 500 rpm to react.

Analysis was performed over time in the same manner as in Comparative Example 1, but the spot corresponding to the target phospholipid observed in Example 5 was not observed on TLC, and the substrate was completely decomposed after 16 hours of reaction. Analysis was discontinued/

Claims (3)

[Claims] (1) In producing a phospholipid with a converted base structure, an aqueous phase containing a hydroxyl group-containing receptor and phospholipase D is added in the form of a reverse micelle encapsulated in an organic solvent in which a raw material phospholipid is dissolved. A method for producing phospholipids using an enzyme, characterized by carrying out a reaction. (2) The manufacturing method according to claim 1, wherein reverse micelles are formed using a surfactant, and the raw material phospholipid itself is used as the surfactant. (3) The receptor is serine, ethanolamine, 1-amino-2-propanol, 1-orthomethyl-glucoside,
The manufacturing method according to claim 1, which is either glycerol or trehalose.