JPS6225987A - Production of diglyceride by enzymatic method - Google Patents

Abstract

PURPOSE:To obtain in high yield a diglyceride, by blending a specific fatty acid or an ester of it with a lower alcohol with a specified amount of glycerin, dehydrating the blend and treating the blend with an alkali lipase of microorganism. CONSTITUTION:1mol 4-22C saturated or unsaturated fatty acid or ester of it with 1-3C lower alcohol is blended with 0.5-1.0mol glycerin, dehydrated in the presence or absence of an organic solvent (except primary alcohol solvent) substantially without adding water and treated with an alkali lipase of microorganism (preferably lipase produced by a bacterium belonging to the genus Achromobacter or Alclaligenes). Preferably the blend is dehydrated into <=1 water content.

Description

Detailed Description of the Invention [Field of Industrial Application] The present invention relates to a method for producing diglyceride at a high concentration by an enzymatic method. It has been known since then, and many proposals have been made regarding its use and manufacturing technology.

However, diglyceride does not attract much attention (rather, it is treated as a product that is mixed with monoglyceride or triglyceride or is produced as a by-product as an intermediate, and has little utility value. Therefore, there are no methods for producing diglyceride in high concentration. There are no effective proposals so far.

As for the chemical method of producing diglyceride, there is a method in which soap is applied to dichlorohydrin.

A method in which a fatty acid is reacted with a disulfate ester, a method in which a fatty acid is added to monotrityl glycerin, etc. have been proposed, but these methods require complicated reaction processes and thermal energy, and are not economically viable.

On the other hand, it is known that diglyceride ° is produced as an intermediate together with monoglyceride and triglyceride in the glyceride production reaction product caused by ester base by lipase and glycerorinsing. For example, Yamaita et al.・Bal variaripoliteikano =- (Chromobacterium viscosu
m var paralipolytjcum) glue'
Using a bioreactor system that takes advantage of the properties of polyethylene membranes, the synthesis of glyceride from glycerin and oleic acid was carried out in a system containing 3 to 3% water, and approximately 0% The glyceride composition ratio (c mol%) when % of fatty acids were ester bonded was approximately 36% monoglyceride, 3% diglyceride, 70% triglyceride, and θ% unreacted oleic acid. C JAOC8,,, </rg], reporting
72 Otsu (/9zag)].

Kyuta et al. also used three types of lipases, including the lipase produced by Chromobacterium viscosum, and glycerin with different water content as the substrate! Ester synthesis or alcoholysis was carried out in an emulsion system by adding 1 mole of oleic acid or its lower alcohol ester to ~30 moles, and the synthesis rate and composition of the glyceride produced were investigated. As a result, when Chromobacterium lipase, which had the highest synthesis rate, was used, diglyceride of about 3.5% (Wt) was produced in the ester synthesis reaction, and about 1% (Wt) of diglyceride was produced in the alcoholysis using methyl oleate. reported that 7% (wt) of diglycerides were formed together with monoglycerides, triglycerides, and unreacted fatty acids [Journal of the Chemical Society of Japan, /co, /797
C/9.5>3]].

[Problem that the invention seeks to solve]

As mentioned above, many chemical synthesis methods for diglycerides have been proposed, but none of them are economically practical.

On the other hand, enzyme reactivity using lipase is performed at room temperature.

The reaction can be carried out under mild conditions at normal pressure, and there is no harm to glycerin or its product, glyceride.

It has no effect. However, in the conventional proposals for enzymatic glyceride production methods, attention has been paid to diglyceride, and no technical proposal has been made for producing diglyceride as the main component.

The reason for this is that diglyceride has not been actively developed for use until now, and its usefulness has not been recognized.In fact, diglyceride is seen as a useless substance found in monoglycerides and triglycerides.

This is because there was even a tendency to suppress the amount of production.

Further, in all of the conventional proposals for producing diglyceride by enzymatic methods, water is used in the reaction system. However, lipase is originally known as a typical hydrolytic enzyme along with protease and amylase, and its general function is that in the presence of water, it not only reduces its ability to produce esters. , O may cause re-decomposition of the ester that has been produced over time, and may even change the bonding position of the fatty acid. All of the conventional proposals have problems, such as the need for a strong centrifugal separation process to separate the ester product obtained by the water emulsion reaction from the emulsion after the reaction, as well as the low concentration of diglyceride produced. - -m- Therefore, the present invention solves the problem of providing a method for easily obtaining diglyceride at a high production rate using lipase.

[Means for solving problems]

In view of the above circumstances, the present inventors believe that the action of enzymes is essential to the presence of water, and as long as we consider the reaction in an aqueous system, the glyceride production reaction is inevitably related to the decomposition of eggplant water. Based on the idea that we have no choice but to follow the reaction equilibrium side with the reaction, we cannot avoid low synthesis rate, re-decomposition, and re-synthesis that involves replacement of fatty acids, and we do not follow the conventional concept. We have conducted intensive research on lipases that enable the production of diglycerides in the presence or absence of organic solvents without adding water.

Results 1 For example, Achromobacter (Achromobacter)
lipase produced by microorganisms of the genus A.
Alkaline lipases such as lipases produced by microorganisms of the genus S. lcaligenes (Japanese Patent Publication No. 3-3, No. 2093) can be used to treat the above-mentioned fatty acids or their lower alcohol esters and glycerin. without substantially adding water to the mixture in a specified proportion of (,
We have discovered a surprising phenomenon in that when dehydrated and reacted in the presence or absence of an organic solvent, a wide range of fatty acids and glycerin produce highly concentrated glycerides consisting mainly of diglycerides. The present invention was completed based on this discovery.

That is, 1. In the present invention, substantially all of Add water (
, an enzymatic method for producing diglyceride characterized by dehydration in the presence or absence of an organic solvent (excluding primary alcohol solvents) and the action of microbial alkaline lipase - its purpose Where,
The object of the present invention is to provide a method for easily obtaining glyceride mainly consisting of diglyceride at a high production rate which is impossible with conventional enzymatic methods.

In the present invention, diglyceride includes only diesters produced from glycerin and fatty acids by the action of microbial alkaline lipase, as well as diglycerides produced between lower alcohol esters of fatty acids and glycerin.

The present invention will be explained in detail below.

The glycerin used in the present invention can be either natural glycerin or synthetic glycerin.

Furthermore, the fatty acid used in the method of the present invention refers to a saturated or unsaturated fatty acid having 04 to C2□ carbon atoms.

Examples of C4 to C22 fatty acids include butanoic acid, valeric acid, caproic acid, caprylic acid, capric acid,
Examples include lauric acid, myristic acid, valmitic acid, stearic acid, isostearic acid, /2-hydroxystearic acid, oleic acid-lysylureinic acid, υnolic acid, riluic acid, eicosanoic acid, tocosanoic acid, and arachidonic acid.

Furthermore, the above-mentioned fatty acid may form an ester with a C7-C3 lower alcohol (for example, methanol as the C8-C3 lower alcohol).

x Tan-A/, globanol, glycerin, inpropatol, etc., and specific examples of these fatty acid esters include, for example, methyl laurate.

Methyl myristate, methyl palmitate, methyl stearate, ethyl laurate, ethyl palmitate, ethyl stearate, ethyl oleate.

Isopropyl laurate, Isopropyl myristate,
Examples include inglovir palmitate, and glycerin pills include glycerin monolaurate, glycerin monostearate, glycerin monooleate, and glycerin tristearate.

glycerin triolate, glycerin trilaurate,
Examples include glycerin tripalmitate, coconut oil, soybean oil, cottonseed oil, milk butter-balm oil, beef tallow, lard, olive oil, and hydrogenated natural fats such as these.

Examples include the above fatty acids and their lower alcohol esters; however, in the present invention, there is no restriction in the selection of the fatty acids and their lower alcohol esters.

Next, regarding the microbial alkaline lipase used to carry out the present invention, any microbial alkaline lipase that is effective in carrying out the present invention can be used.
lipase (Special Publication No. 4T9-320 and No. 0) produced by Meito-AL- and No. 6ta (Feikoken Bacterial Co., Ltd. No. 1/3) belonging to the genus Mobacter (Special Publication No. 4T9-320 and No. 0)
), Alcaligenes
) Polysaccharide belonging to the genus PL-2 ≦ Otsu No. 3/?
Lipase produced by No. 7) [Tokuko Shota! -36 Wata No. 3 Publication)
Lipase produced by Meito PL-, <79 (Feikoken Bibori No. 37♂3), which belongs to alkaline earth metals
Publication No. 39093] Lipase-PL, hereinafter referred to as y-li, and the like can be cited as specific examples of effective lipases with particularly excellent solvent resistance.

This point will be explained using an experimental example.

Experimental Example Stability in solvent Lipase-PL Otsu 79 [manufactured by Polysaccharide Sangyo Co., Ltd.], Lipase-P TJ 2 (-Otsu [manufactured by Polysaccharide Sangyo Co., Ltd.], Lipase-A-LC Meito Sangyo c strain) Place 1 m of the powdered powder of the same product in a centrifuge tube with a 7 rat stopper, and add various solvents such as n-hexane, n-pentane, n-hebutane, petroleum ether, cyclohexane, and cyclopentane-tertiary butyl alcohol. , diacetone alcohol.

Acetone, acetonitrile, diisobutyl ketone.

Aml of benzene-carbon tetrachloride and water were added, thoroughly stirred, and shaken at 37° C. for 2 hours, and residual activity was measured by reverse zeka titer measurement.

Lipase measurements were performed using Kunio et al.'s i (Ag
riC.

Biol. Chem, <t, <ct), No. 1/Ta 9, /9F2].

Regarding lipase-AL, Kunio et al. [Oil Chemistry 23 (
[U], page 9, /92]. The result is the first
Shown in the table. In addition, the numbers in Table 1 indicate the residual rate of lipase activity (
%).

Table 1 Lipases other than those exemplified above can be used stably without substantially adding water (in the presence or absence of organic solvents (excluding primary alcohol solvents)). Any microbial alkaline lipase can be used as long as it maintains its activity and has a strong ability to produce diglyceride between glycerin and fatty acid or its lower alcohol ester. There are no restrictions on origin or type.

Furthermore, in the present invention, the microbial alkaline lipase added to the reaction system may be either a purified product or a crude product.

As for its form, powdered or granular enzymes or dried bacterial cells can be used.

Furthermore, an immobilization carrier 1, for example a polypropylene membrane.

Various polymers such as ion exchange resins and Celite.

It is also possible to use dry immobilized enzymes immobilized on carriers such as inorganic materials such as glass peas, zeolite, and bentonite. By immobilizing the enzyme on these carriers, the contact surface between the substrate and the enzyme can be expanded, which is more advantageous in terms of progressing the reaction than using enzyme powder.

In the present invention, the organic solvent used in the reaction in the presence of an organic solvent (excluding primary alcohol solvents) is liquid at one reaction temperature, stably maintains microbial alkaline lipase activity, and is insoluble. Any type of lipase may be used as long as it can generate a diester in a higher concentration than glycerin and fatty acids, but as long as the above conditions are satisfied, organic solvents or such solvents that can dissolve both glycerin and fatty acids at the same time may be used. It is preferable to use it in combination with an organic solvent. Examples of the organic solvent used in the present invention include n-hebutane% n-pentane, n-hexane, petroleum ether,
Aliphatic hydrocarbons such as isootane; alicyclic hydrocarbons such as cyclopentane, cyclohexane, cyclobutane, etc.; benzene.

Toluene, xylene, phenol, aromatic hydrocarbons; ketones such as acetone, methyl isobutyl ketone, etc.; acetonitrile, 2-nitropropane,
Nitrogen-containing solvents such as pyridine, quinoline, dimethylformamide, etc.; ethers such as dimethyl ether-diethyl ether, diisopropyl ether, dioxane, etc.; sulfoxide catalysts such as carbon; co-dimethyl-3-pentanol; 2. Secondary alcohols such as O-dimethyl-gooheptatool; tert-butyl alcohol, m3R amyl alcohol, diacetone alcohol, 3-methyl-3-pentanol, 3-ethyl-3
-Like pentanol, eumethyl-cohexanol? Examples include 43B alcohols.

Particularly preferred among these are diimbutyl ketone,
Solvents such as n-hexane, tertiary butanol, diacetone alcohol, and tertiary amyl alcohol are preferred; among them, tertiary alcohols can be used to dissolve glycerin and fatty acids or their lower alcohol esters (because they can be dissolved in one reaction). It is preferable to use soil in which the reaction is carried out - it becomes an extremely effective reaction solvent, especially when the reaction solution is continuously poured into an enzyme-filled tank.

Further, although the reaction solvent can be used alone, two or more kinds of solvents can also be used in combination.

The embodiment of the suspension in which a fatty acid or its lower alcohol ester and glycerin are brought into contact with microbial alkaline lipase in the presence of an organic solvent to generate diglyceride ° is as follows:
The reaction can be carried out under conditions of stirring or shaking, which can be selected as appropriate. Under reaction conditions in which both substrates can be dissolved in an organic solvent, the reaction can be carried out by filling a reaction tower with solid lipase powder or lipase supported and immobilized on bentonite and dehydrated, and circulating the reaction solution through the reaction tower. 0 Next, when an organic solvent is not used in the present invention,
If necessary, the fatty acid may be liquefied by heating, and a microbial alkaline lipase may be applied to the mixture of glycerin and the fatty acid or its lower alcohol ester as a liquid mixture.

-15 In the present invention, the molar ratio of glycerin to 1 mole of fatty acid or its lower alcohol ester is 0.5
~1 mol, preferably θ, go, z, etc. can be selected as appropriate, and conditions that best promote the reaction, obtain a high yield, and are easy to operate the reaction may be adopted.

Furthermore, depending on the purpose, several types of fatty acids and lower alcohol esters thereof may be mixed and reacted.

Next, there is no limit to the amount of microbial alkaline lipase used, but for example, fatty acid or its lower alcohol ester/g-unit/θ~10oooo units, preferably 000
An example of the amount used is approximately 0 to too many units, and the amount may be determined taking into account that it depends on the reaction conditions and the type of microbial alkaline lipase used.

When using an enzyme immobilized on a carrier, the higher the specific activity is, the more preferable it is, and the specific activity is preferably from 1,000 to 1,000 per gram of carrier.
Approximately 300,000 units of microbial alkaline superse! Can you give an example of a supported carrier?

In addition, the amount of organic solvent used depends on the solvent used.

It depends on the type and concentration of the substrate, but as a guideline, it is desirable to add an organic solvent to the extent that the organic solvent in which the substrate is dissolved can move freely and come into contact with the enzyme to promote one reaction, and there are no particular restrictions. Not though.

For example, 1 to 9% (W/W) of the reaction system, preferably 1
0-9! % (W/W) for reaction. The seven features of the present invention are that it does not substantially add water (
, in the diglyceride production reaction carried out by microbial alkaline lipase in the presence or absence of an organic solvent (excluding primary alcohol solvents), which means that the water content of the reaction system is at an absolute θ. It's not a thing. It is impossible and unnecessary to actually create such a state. However, the water in the reaction system should be kept as low as possible in order to reach a high diglyceride production rate and to avoid the risk of re-decomposition ( Furthermore, since the activity of the enzyme can be maintained stably for a long time (maintained stably) and the degree of reuse of the enzyme can be increased, the present invention not only does not substantially add water (but also eliminates water by-produced by the reaction). The diglyceride production reaction is carried out using solid microbial alkaline lipase.

However, this does not mean that diglyceride will not be produced by allowing a small amount of water to coexist in the reaction system. In the present invention, the meaning of adding substantially no water means:
When using a substrate and microbial alkaline lipase in a reaction, the reaction is carried out without using any water, either as is or by dissolving or dispersing the substrate in an organic solvent. That is, the substances involved in the reaction all contain a certain amount of water, the amount of which is about θ and 0.7% for glycerin, about 0.02% for fatty acids or their lower alcohol esters, and about 0.02% for microbial alkalinity. Approximately with lipase /
Even in organic solvents, it is about 0.02~0.
Even if these are used as they are, the initial water content of the reaction system as a whole is in the range of about 0.2 to 7.0%, but in such a case, substantially no water is added. Furthermore, if water is produced as a by-product due to moisture absorption from the air or ester synthesis during the reaction, the water content will further increase; however, in this case as well, substantially no water is added.

And the water content of the reaction system is the substrate used for one reaction.

Enzymes, organic solvents, etc. should be used as dry as possible, and if there is water that enters from the air or is a by-product of a synthetic reaction, use a dehydrating agent such as zeolite, etc.
In addition to using silica gel, calcined plaster, and Glauber's salt to remove moisture, dry air or inert gas can be vented into the reaction tank, stirred, and exhausted outside the reaction tank, or the exhaust gas can be cooled and condensed to remove moisture. Water in the reaction system can be removed by removing the organic solvent and refluxing the organic solvent.

By reducing the water content of the reaction system to 0.7% or less by such means, for example, when the molar ratio of glycerin and oleic acid is 0.7:/, θ.

The ester synthesis rate can be increased to about 0-9%, and the concentration of diglyceride in the resulting glycerinoxylate can be increased to about 10-70%.

In the present invention, the diglyceride production reaction proceeds even at room temperature, so there is no particular need for heating.

In general, it is desirable to select an appropriate temperature by taking into consideration the boiling point of the organic solvent used and the action temperature of the enzyme; an example of this range is a temperature of 0-9000°C. , usually in the range of 20-6θ°C.

Moreover, the reaction time can also be selected as appropriate, for example, 7
An example is 0 minutes to 70 days.

Preferably/~? Be able to clearly indicate the opening time and reaction time.

In addition, if necessary, the ester production process can be tracked by measuring the rate of reduction of fatty acids in the reaction system, or by using techniques such as TLCr thin layer chromatography and Yatroscan analysis, to obtain the desired target product. The reaction time may be determined by confirming the formation of 0 After the reaction, the diglyceride produced in the absence of an organic solvent can be separated with an organic solvent. Diglyceride can be separated from the reaction system using silicic acid, alumina, gel for molecular sieves, etc. as necessary, distillation, solvent fractionation, urea addition, etc.
It can be separated and purified using high-performance liquid chromatography, centrifugal liquid-liquid partition chromatography, etc. [Effects of the Invention] According to the present invention, diglyceride production rate is high, which was not possible with the conventionally proposed enzymatic method. Diglycerides can be obtained with almost no residual fatty acids added. Furthermore, in the present invention, there is no need for a large excess of glycerin relative to the fatty acid or its lower alcohol ester as shown in the prior art, and a sufficiently high concentration of diester can be obtained even at a molar ratio of / to 0.7. The substrate concentration can also be varied over a wide range from a low concentration of several percent to a high concentration of several tens of percent. Furthermore, according to the present invention, glycerides with a particularly high diglyceride concentration can be obtained using a chemical method.

Furthermore, in the present invention, there is no need to use toxic reaction catalysts such as those used in chemical methods (because glyceride is produced under extremely mild reaction conditions, changes occur in the structure of glycerin and glyceride themselves). Therefore, glycerides consisting mainly of diglycerides with excellent properties and high safety can be obtained.

Furthermore, according to the present invention, it is possible to produce diglyceride having any fatty acid composition, so any fatty acid can be further added to the hydroxyl group portion where there is no acyl bond by chemical synthesis method or enzymatic method. By introducing an acyl group, it can be used as a raw material for producing a novel triglyceride with any fatty acid composition.
Furthermore, the diglyceride obtained by the present invention can be used as a base material for phospholipids such as phospholipids and β-type phospholipids.
It is also possible to use it as a raw material for synthesizing various glycerides.

Furthermore, the diglyceride obtained according to the present invention can be used as a substrate for measuring lipase activity that specifically acts on diglyceride, and is expected to be used in clinical tests.

〔Example〕

Examples of the present invention will be described below.

A dehydrating agent, Moregular Sieves (sold by Wako Tsumugi Co., Ltd.), was used so that the water content of the reaction system was 7% or less.

Example 1 Oleic acid 109-CB, f, j mmol), glycerin/,? ≦ノ(U/, 3 mmol 1. Tertiary butyl alcohol 100 ml 13. Lipase-PL Otsu7wa [Polysaccharide Sangyo C Co., Ltd.] manufactured, specific activity 100,000 u/j']!!.

Molecular Siegis 3A Koo! g- to j 00 rn
The mixture was placed in a 1-volume Erlenmeyer flask and subjected to a shaking reaction at 0° C. for 22 hours to prepare glycerin oleate. The ester synthesis rate of this reaction solution was 90%. The ester synthesis rate is determined by titrating the amount of fatty acids added to the reaction system that is consumed by the ester base with an alkaline solution, and is expressed as the percentage of reduction in fatty acids relative to the amount of fatty acids before 1 reaction. Thereafter, the mixture was centrifuged for 100oxy-/θ minutes to remove insoluble materials to obtain a supernatant. Furthermore, 100rnl of chloroform was added to the insoluble matter, and after washing the insoluble matter, centrifugation was performed to obtain a supernatant.O These supernatants were combined and concentrated in an evaporator to remove the solvent.
The obtained glycerin oleate was analyzed by TLC. That is, 10μ of a 7% chloroform solution of glycerin oleate
! A thin layer of silica gel (manufactured by Merck & Co.).

Silica Mol Otsu 0TL and play) No,! 7 pieces/.

20 x 20 Cm) and developed using 1 petroleum ether-ether-acetic acid (7 (7: 3o: /V/V'J) as a developing solvent. For detection of 0 spots, 50% sulfuric acid or 050% sulfuric acid using iodine was used. Or, when the spots were detected using iodine, other spots were detected besides oleic acid.
Glycerin-7,3-manufactured by Koshi Yakuhin Co., Ltd.] 0. In addition, the component composition ratio of the above-mentioned glycerin oleate was determined using Iatroscan (Iatroscan TH10, manufactured by Yatron Co., Ltd.). Namely, the glycerin oleate/
% chloroform solution/μ! and benzene-chloroform-acetic acid (!0:20:O,jV/Vl'
The mixture was developed in a 4-developing bath medium for about 10 crn, and then subjected to IATROScan to determine the weight ratio of the components from the peak area ratio. The composition ratio of the above glycerin oleate is 23% glycerin monoslate and glycerin diolate! ! % (glycerin-/, 3-diolate 4t!, glycerin-/, 2
-diolate 73%), glycerin triolate 9%,
It was 10% oleic acid. In addition, as a standard substance,
O using glycerin monooleate, glycerin-/, 2-diolate, glycerin-/, 3-diolate, glycerin triolate (all manufactured by Funakoshi Pharmaceutical Co., Ltd.) Example 2 Oleic acid 10g-(3j, 689 mol)-glycerin 2.413 fC21, mmol), diisobutyl ketone 7100 ml. Lipase-pLJ79 powder, ano powder, regular sieves 3A of; The ester synthesis rate of this reaction solution was 9 g%.

The component composition ratio of this glycerin oleate determined by Iatoroscan is: glycerin monooleate 30%, glycerin diolate ≦2% [glycerin-/, 3-diolate 53%, glycerin-/, 2-diolate 9%), glycerin Trio rate O%.

The 1st/in grading was 2%.

Also, instead of oleic acid/θ, stearic acid 10.
/No (3 nights, 689 mol) was carried out in the same manner as above to prepare glycerin stearate. The ester synthesis rate of this reaction solution was 92%. The component composition ratio of this glycerin stearate determined by IATROScan is glycerin monostearate co! %, glycerin distearate 70 ratio (glycerin-/, 3-distearate g♂
%.

glycerin-7, cordistearate co%], glycerin tristearate 3%-stearic acid-2%.

Furthermore, instead of 70% oleic acid, palmitic acid,
Glycerin palmitate was prepared in the same manner as above using /no [3 days, Tamrimol). The ester synthesis rate of this reaction solution was 9 g%.

The component composition ratio of this glycerin palmitate determined by Iatoroscan is 20% glycerin monopalmitate,
Glycerin dipalmitate O% (glycerin-/, 3
-dipalmitate 39%, glycerin-/, dipalmitate 27%), glycerin tripalmitate/2%
, Valmitic acid was the third group.

Example 3 Beef tallow 10iI-C//, 6 mmol], glycerin θ,
7ji PC♂, 789 mol), tertiary butyl alcohol 100rnlj, lipase-PL, 41;79 powder conomole regular sieves 3A, :toomtt
The mixture was placed in an Erlenmeyer flask and shaken at 0°C for 1 hour to prepare glycerin tallow fatty acid ester. From then on,
Glycerin beef tallow fatty acid ester 7 was prepared in the same manner as in Example 1.
I got 0 no.

The component composition ratio of this glycerin tallow fatty acid ester determined by Iatoroscan is: glycerin monoester, 20
%, glycerin diester 1.0%c glycerin/,
3-diester 37%, glycerin/2-diester 22%), glycerin triester 79%, fatty acid/
%Met.

In addition, in place of 2 g of Lipase-PL Otsu 79 powder, Lipase-PL2 J J powder [Polysaccharide Sangyo c stock], specific activity /, / 10,000 U / × 9- was used, and the same procedure as above was carried out.
Glycerin tallow fatty acid ester9. The? I got it.

The component composition ratio of this glycerin tallow fatty acid ester determined by Iatoroscan is: glycerin monoester, 23
%, glycerin diester≦O (glycerin-7,3
-diesterg θ%, glycerin/, diester 20%), glycerin triester/20%, and fatty acid/20%.

Furthermore, Lipase-PL≦79 powder U? Instead of Lipase-A L powder [Polysaccharide Sangyo C strain], specific activity /,! The same procedure as above was carried out using 20,000 u/g-]2f to obtain 9.9 g of glycerin beef tallow fatty acid ester.

The component composition ratio of this glycerin tallow fatty acid ester determined by Iatoroscan is: glycerin monoester 2st
l,, Glycerin diester B/% [Glycerin-/,
3-diester 36, glycerin/, 2-diester 2! %), glycerin triester 77.

And no fatty acids were detected.

Example 4 Palm oil 10yr//, 7 mmol), glycerin 0
．． 7! of〔? , commimol), tertiary butyl alcohol,
IT7700 m13. Lipase-PL 79 powder 25
F. Molecular Siegis 3A oil was placed in an Erlenmeyer flask having a capacity of 0.000 C and subjected to a shaking reaction at 0.degree. C. for an hour to prepare glycerin palm oil fatty acid ester. Thereafter, the same procedure as in Example 1 was carried out to obtain glycerin balm oil fatty acid ester and iP.

The component composition ratio of this glycerin balm oil fatty acid ester determined by Iatoroscan is: glycerin monoester 2
: 1%, glycerin diester Otsu θ% (glycerin-/
, 3-diester 37%, glycerin-/1.2-diester 3%), glycerin triester/? %Met. And no fatty acids were detected.

Example 5 Hardened beef tallow 10fC//, mmol), glycerin 0.
73PCF,/mmol), tertiary butyl alcohol 1
00m1V, Lipase-PT, Otsu7ri Powder 292 Molecular Sieves 3A20y- were placed in a t00mb Erlenmeyer flask and heated to 4t? A glycerin-hardened beef tallow fatty acid ester was prepared by shaking for a period of time. Thereafter, the same procedure as in Example 1 was carried out to obtain glycerin hardened beef tallow fatty acid esters /0 and /f.

The component composition ratio of this glycerin hardened beef tallow fatty acid ester determined by IATROScan is -glycerin monoester/
The subdivisions were glycerin diester otsuko (fl) (glycerin-/, 3-diester 32%, glycerin-/, cordierin 23%), and glycerin triester 20%. And fatty acids were not detected.

Example 6 Olive oil 10fC//, glymol], glycerin 0
．． 74 tons (!, O mmol), Lipase-PL otsu mulberry powder 29-, Molecular sieves 3A/θ9”t1
The mixture was placed in a 00rnl Erlenmeyer flask and subjected to shaking reaction at 0°C for <zr hours to prepare glycerin olive oil fatty acid ester. After reaction - tertiary butyl alcohol 10t)m
After that, the same procedure as in Example 1 was carried out to prepare glycerin olive oil fatty acid ester in 9. Evening i! I got -.

The component composition ratio of this glycerin olive oil fatty acid ester determined by Iatoroscan is - glycerin monoester 3stl, glycerin diester! !硲〔Glycerin-
7,3-diester (3%), glycerin/cordiester (aO), glycerin triester (z%), and fatty acid (%).

Claims (2)

[Claims] (1) Adding substantially all Production of diglyceride by an enzymatic method characterized by dehydrating it in the presence or absence of an organic solvent (excluding a primary alcohol solvent) without adding water and allowing microbial alkaline lipase to act. Method. (2) The method according to claim 1, characterized in that the reaction system is dehydrated to a water content of 0.1% or less as much as possible, and then microbial alkaline lipase is allowed to act.