JPH01309689A - Process for ester interchange of oil and fat using microbial cell - Google Patents

Abstract

PURPOSE:To improve the efficiency of ester-interchange reaction by reacting a glyceride with lipase-containing microbial cells while controlling the water- content of the cells. CONSTITUTION:Ester-interchange reaction of oil and fat is carried out using microbial cells by reacting a glyceride with lipasecontaining microbial cells (e.g., Rhizopus chinensis) while controlling the water-content of the cell to 0.1-20wt.%. The control of the water-content of the cell is preferably carried out by controlling the concentration of water in the reaction liquid.

Description

[Detailed Description of the Invention] [Industrial Application Field] The present invention relates to an enzymatic reaction method using microbial cells, and more specifically, the present invention relates to an enzymatic reaction method using a microbial cell, and more specifically, a reaction between triglycerides, which are constituents of oils and fats, which is carried out using a lipase enzyme as a catalyst. Concerning the transesterification reaction of

[Conventional technology]

Conventionally, the transesterification reaction of fats and oils has been carried out using alkali metals, alkali metal alkoxylades, alkali metal hydroxides, etc. as catalysts, but since this method lacks specificity in the exchange position, recently a method using lipase enzyme has been developed. Many cases have been reported. However, 1li, which exhibits activity only when dissolved in water or in the presence of water, in a water-immiscible substrate (reactant) such as triglyceride.
When using chlorine to act as a catalyst, there are several problems as described below.

(1) In order to increase the contact opportunities between the substrate and the enzyme, it is desirable to add the enzyme directly to the substrate, but in oils and organic solvents, the enzyme will rapidly disappear unless it is protected in some way. Live. Conventionally, many reported methods using lipase enzymes prevent deactivation by adsorbing the enzyme to a carrier such as an adsorbent, but deactivation occurs when the enzyme is desorbed.

(2) If the amount of water near the enzyme is too large, hydrolysis becomes dominant in the reaction and esterification does not proceed.

Conversely, if the water content is reduced too much, the transesterification reaction rate will decrease. In the conventional method, it is said that the water concentration in the reaction system at the initial stage of the reaction is in the range of o, o o s to 1.0% by weight, which is the suitable water content for the transesterification reaction. No adjustments have been made. Further, a method using a di- or trivalent lower alcohol instead of water has been proposed, but according to the experience of the present inventors, the reaction rate is low and it is impractical.

(3) When an enzyme is adsorbed on an adsorption carrier, the reactant diffuses to the enzyme on the carrier. In particular, the enzyme adsorbed in the pores of the carrier can substantially participate in the reaction. This tendency becomes stronger when a hydrophilic carrier is used.

In this way, when performing a transesterification reaction on an oil phase reactant using a lipase enzyme that exhibits activity in an aqueous phase as a catalyst, the enzyme must not be deactivated and the amount of water in the reaction system must be maintained at an appropriate level. It must be maintained in a microaqueous environment and provide sufficient contact opportunities for enzymes and reactants.

[Problem that the invention seeks to solve]

Conventional methods only take into account (2) above and only focus on the water concentration at the initial stage of the reaction.
According to the study results of the present inventors, the reaction rate is slow and it is impractical. Furthermore, since conventional methods utilize purified enzymes,
This method requires complicated enzyme separation and purification steps and immobilized enzyme production steps, which increases the production cost of the reaction catalyst and is disadvantageous from an industrial and economical point of view.

In addition, as mentioned above, the transesterification reaction using lipase converts triglyceride (TG) into diglyceride (DG).
, monoglyceride (MG), or glycerin and fatty acid (FA), and a reaction in which the hydrolyzate is resynthesized into triglyceride or the like as a result of a reversible reaction. Therefore, the presence of water is essential in the transesterification reaction, but when the amount of water added increases, a large amount of byproducts such as free fatty acids, monoglycerides, diglycerides, etc. are produced, reducing the yield of triglycerides, or Reduces the quality of product triglycerides. Also, if the amount of water in the reaction system is too small,
The catalytic activity of the enzyme cannot be fully expressed, and the reaction rate becomes low. Therefore, in order to carry out the transesterification reaction effectively, the reaction system must always be maintained at an appropriate microwater concentration.

Therefore, in order to make the transesterification reaction industrially possible, it is necessary to develop a method for producing inexpensive lipase and a method for constantly adjusting the water concentration near the enzyme to the optimal microwater concentration to obtain a high reaction rate and This was needed to suppress side reaction products and to effectively utilize expensive lipase enzymes without waste.

[Means for solving problems]

The present inventors solved the above-mentioned problems, and as a result of intensive study on the utilization form of lipase, which does not deactivate the enzyme for a long period of time, has a sufficient reaction rate, and can be used at an industrially low production cost. By culturing a microorganism capable of producing lipase, drying the microorganism with the lipase encapsulated in the microbial cell, or treating it with a polar solvent or a nonpolar solvent, and then applying it to a mixture of triglycerides as a lipase catalyst,
It has been found that the transesterification reaction can be carried out efficiently. Furthermore, it was discovered that in transesterification reactions using such microbial cells, a water adsorption equilibrium relationship is established between the reaction solution and the microbial cells, and this equilibrium relationship can be established by detecting the water concentration in the reaction solution. It was found that the water concentration of bacterial cells could be determined indirectly using the method. Therefore, it has been discovered that by adjusting the water concentration in the reaction solution to correspond to the desired water concentration inside the bacteria using a similar equilibrium relationship, the water concentration inside the bacteria can be adjusted to the desired value as a result, and the present invention has been made. I was able to complete it.

That is, the present invention reduces the water content of the lipase-containing bacterial cells to 0.
．． The content is a method for transesterifying fats and oils using microorganism cells, which is characterized by reacting with glyceride while controlling the amount to 1 to 20% by weight.

As the microorganism used in the present invention, any microorganism that has lipase-producing ability can be used, but examples of the Rhizopus genus include Rhizopus chinensis (Rh, chinensis), Rhizopus delemer (Rh, del), etc. 'emaar), Rhizopus jabonicus (Rh, japanicus), Rhizopus oligosporus (Rh, oligosporus)
us), Rhizopus niveus (Rh, n1veus)
, Rhizopus javupnicus (Rh, javani
As for the Mucor genus, for example, Mucor javanicus
icus); Aspergillus
Examples of the genus Aspergillus niger (Ius) include Aspergillus niger (
As-pergillus niger, etc.; as for the genus Geotrichum, Geotrichum candigum (Geotrichu+a
Candidum) etc.; Candida
a) As a genus, Candida cylindrasose (Ca
ndida cylindoracea); as for the genus Corynebacterium (Corynebacterium+), Corynebacterium
ebacteriu+sequ-1); Staphylococcus genus Staphylococcus aureus (Sequ-1);
ococcus aureus) etc., and all of them are easily available.

These microorganisms are usually cultured using known batch, semi-batch, continuous culture methods, etc., but according to the results of the study by the present inventors, the amino acid group that has an inhibitory effect on lipase production is found in the culture medium components. It is possible to enhance the lipase production ability of these microorganisms by using an organic nitrogen source with a low content of amino acids that have a large inhibitory effect as a main component. It has been found that the lipase-producing ability of these microorganisms can be increased by culturing while maintaining the amino acid concentration in the culture solution at a low concentration by feeding. Typical examples of these organic nitrogen sources include polypeptone, yeast extract, malt extract, yeast peptone, proextract, meat extract, corn staple liquor, and the like.

Furthermore, if 0.2 to 80% by weight of a lipid-related substance such as glyceride or fatty acid is added as an inducer to the medium at the beginning or during the culture, the lipase activity within the microbial cells is further enhanced. Such lipid-related substances include triglycerides such as olive oil, tea oil, and fish oil;
Typical examples include fatty acids such as oleic acid, linoleic acid, and linolenic acid, higher alcohols such as oleyl alcohol and linoleic alcohol, and esters such as methyl oleate and ethyl caprate.

Furthermore, a microorganism-retaining material, for example 1 to 2000μ, is added to the medium in advance.
By charging microorganisms with m porous particles and allowing microorganisms to proliferate within the particles or near the surface of the particles, the lipase activity within the microbial cells can be dramatically increased.

As such a microorganism-retaining material, any material that enables adsorption and proliferation of microorganisms due to its adhesive strength and adsorption power can be used. For example, the polymeric porous materials include polyolefin-based materials such as polyethylene or polypropylene; diene-based materials such as butadiene or isoprene;
Vinyl polymers such as polyvinyl chloride, acrylamide or borisnarene; condensation systems such as polyether, polyester, polycarbonate or polyamide; materials such as polyurethane, silicone and fluorine resins; and inorganic materials such as ceramics, glass, activated carbon and porous Quality metals and metal fiber processed materials can be used. In order to properly immobilize microorganisms on the holding material for any material, the porosity must be 10-99% and the pore size must be 1-2000%.
Porous material in the μm range or porosity of 10-99%
It is preferable to use a metal processing material that is.

Such a microorganism-retaining material can be appropriately selected depending on the species of microorganism, culture conditions, etc., and the shape may be, for example, spherical,
The block-like material can be processed into a sheet-like form and used. The dimensions vary depending on the type of microorganism, culture conditions, type of reactor, etc., but if it is spherical, it should be approximately 1 to 100 mm in diameter, and if it is block-shaped, it should be approximately 1 to 100 μL on each side. be done. Furthermore, immobilization of microorganisms on the above-mentioned holding material can be easily achieved using commonly known batch, half-batch, continuous culture methods, etc.

The amount of the microorganism-retaining material added is preferably 5 to 40% by volume as conditions for the growth and immobilization of microorganisms.

In the present invention, air, oxygen, or a mixture of both gases are used to aerate the culture tank, and any type of culture tank can be used, including a stirrer type, a non-stirrer type, and a bubble column type. In order to immobilize and proliferate microorganisms on the microorganism holding material, a non-stirrer type culture tank is more preferable in terms of operation and cost.

Furthermore, in the present invention, when culturing with a microorganism-retaining material, glycerides and fatty acids added as inducers of lipase in microbial cells as described above may be used as a microorganism-retaining material using a lipophilic polymeric material such as polyurethane. etc., these lipid-related substances are absorbed into the microorganism-retaining material. Therefore, if a large amount of lipid-related substances are absorbed into the microorganism-retaining material, the resistance to the movement of oxygen and substrates into the microorganism-retaining material will increase, which will only be a negative factor for growth within the microorganism-retaining material. As a result, the microorganisms cannot proliferate within the holding material, and lipase production is no longer promoted. Therefore, when using a microorganism-retaining material, the amount of lipid-related substances added is preferably 0.2 to 8
%, more preferably 0.2 to 2%.

Furthermore, in order to maintain stable growth within the microorganism-retaining material and to suppress the detachment of microorganisms from the microorganism-retaining material, the turbulence intensity in the culture solution and the concentration of the microorganism-retaining material in the culture solution are important factors. Become. Therefore, the turbulence intensity for maintaining stable growth of lipase-producing microorganisms in the microorganism holding material is preferably 102 to 107, more preferably 103 to 107, based on the stirring Reynolds number in a stirrer type culture tank. 10
It is operated under stirring conditions of 5.

No stirring P! In a machine-type bubble column, the problem of detachment of microorganisms from the microorganism-retaining material is not observed particularly within the range of the ventilation linear velocity at which the ventilation gas blow-through occurs. Therefore,
Usually 1 crn/sec, preferably 1.5 cm/
One mulberry is grown at an aeration linear velocity of sec or more. From the point of view of preventing leakage of lipase within the bacterial body, it is 1°2 to 2.8 c.
A range of m/sec is preferred.

The microorganisms obtained by culturing as described above, i.e., the immobilized microorganisms, can be used as viable cells in the reaction, but as mentioned above, if the water content is too high, the transesterification yield will be low. It is desirable to remove moisture from the bacterial cells. In addition, the lipase in dried bacterial cells is very stable when water is removed, and can be stored for a long period of time.

In principle, the method for removing water from microorganisms is to dry them at a temperature that does not deactivate the enzyme (below 80°C), but simply exposing the microorganisms to a single drop of water causes the cell tissue to shrink and become very hard. Contact between lipase in tissues and the outside world is cut off, making it difficult for lipase to exert its activity. Therefore, it is preferable to use a method that does not involve shrinkage of cell tissue to dry the bacterial cells. For this reason, the bacterial cells are immersed in a polar solvent and [
By replacing the moisture in the I-weave with a polar solvent and then evaporating the polar solvent, it is possible to suppress the shrinkage of the cell-weave and obtain dry bacterial cells. In this case, as a drying method, a known drying method such as vacuum or freeze drying, low temperature drying, hot air drying, etc. can be used. As the polar solvent used in the present invention,
Any substance that forms a homogeneous phase with water when mixed with water can be used; representative examples include ketones such as acetone, methyl ethyl ketone, and diethyl ketone;
Lower alcohols such as methanol, ethanol, isopropanol and n-butanol; diols such as ethylene glycol and propylene glycol; and triols such as glycerol. Among these, acetone and glycerol are preferred because they hardly reduce enzyme activity.

Furthermore, even without drying, by replacing such a polar solvent with a non-polar solvent, the moisture content within the bacterial cells can be reduced to a desired level.

As the nonpolar solvent used in the present invention, any solvent can be used as long as it can dissolve the oils and fats that are the reaction substrates. Typical examples include alkyl hydrocarbons such as hexane, heptane, octane, isooctane, nonane, and decane; aromatic hydrocarbons such as benzene, toluene, and xylene.

The microbial cells cultured in this manner are adjusted to have a water content of 0.1 to 20% by weight (hereinafter referred to as lipase-containing microbial cells), and subjected to transesterification.

Next, lipase-containing microbial cells or lipase-containing microbial cells immobilized on a microorganism holding material are suspended in the reaction solution, but they are easy to handle and have dramatically improved lipase activity. Therefore, from an industrial perspective, it is more convenient to use one immobilized on a microorganism-retaining material.

As mentioned above, if the amount of water near the enzyme is too large, the reaction will be dominated by hydrolysis and esterification will not proceed, so in order to carry out the transesterification reaction efficiently, the amount of water near the enzyme must be controlled to an appropriate value. It must be. As a result of extensive studies on this point, the present inventors have found that by controlling the water concentration in the reaction solution, the water concentration within the bacterial cells can be controlled based on the adsorption equilibrium relationship therewith.

A specific method for controlling moisture content will be described below.

It is said that it is difficult to directly determine the water content in the bacterial cells (enzyme catalyst) in a system where solid and liquid (bacterial cells - reaction liquid) coexist, such as the present reaction system. (enzyme catalyst)
We have found a method to indirectly determine the amount of water retained in In other words, we focused on the fact that there is a water adsorption equilibrium relationship between the microbial cell enzyme catalyst (enzyme catalyst) and the reaction solution (oil phase) in a microaqueous system, and performed the reaction using this equilibrium relationship determined in advance. This method determines the amount of water inside the bacterial cells by detecting the extremely small amount of water concentration in the liquid. Such an adsorption equilibrium relationship differs depending on the type and composition of the reaction liquid, the type of bacterial cells or the microorganism-retaining material on which the bacterial cells are immobilized, and each combination has its own unique equilibrium relationship. Such an equilibrium relationship can be easily obtained by following the usual method of determining an adsorption isotherm for liquid phase adsorption. For example, the water adsorption equilibrium relationship between the reaction liquid and the bacterial cells (enzyme catalyst) as shown in Figure 1 can be obtained, and by controlling the water concentration in the reaction liquid during the reaction, the water concentration inside the bacterial cells can be controlled. It becomes possible.

The effect of water concentration on the reaction rate varies depending on the type of microorganism holding material used for immobilization, the type of bacterial cells, the lipase activity of the bacterial cells, etc.

The optimum moisture concentration in such a system can be easily determined by a separately known method, and as a result of studies on various microorganisms, the optimum moisture concentration within the bacterial cells is preferably approximately 0.1 to 20% by weight. In order to adjust the water concentration inside the bacteria, it is necessary to adjust the moisture level in the reaction solution to a microwater level of about 5 to 11,000 pp, preferably 10 to 500 pp.
It is best to adjust it to ppm. More preferably 10-20
Within this range, hydrolysis as a side reaction is suppressed and the quality of the product is improved.

As a method for analyzing moisture, -C-wise, the gravimetric method in which only the moisture in the sample is evaporated and the moisture content of the sample is determined from the change in weight, or the Karl Fischer method that utilizes a dehydration reaction are well known.

In the Karl Fischer method, when a liquid sample is collected from a reactor, the solid phase is separated using an appropriate filter (for example, a porous filter made of sintered metal or resin), and the water concentration in the liquid phase is measured. Other methods include online moisture concentration meters based on various known principles, such as methods that project infrared light onto the sample and measure the amount of absorbed light, and methods that measure from changes in the electrical resistance of a part of the sensor. Method,
Alternatively, a moisture sensor based on a method such as measuring the capacitance of a liquid can be used. Ilt moisture in the reaction solution in the reactor using these sensors? W degree or reaction system (solid-liquid)
By measuring the average moisture concentration, it is possible to determine the amount of moisture retained in the bacterial cells (enzyme catalyst), which are in the same phase, using the moisture adsorption equilibrium relationship between solid and liquid. If the obtained moisture concentration is lower than the desired value, increase the moisture concentration in the reaction solution, and conversely, if it is higher, decrease it, so that the moisture and concentration in the catalyst can always be maintained at an appropriate level. .

As a method for increasing the water content in the reactor, a method of directly injecting water into the reactor or a method of dissolving water in the reaction liquid and then injecting the water into the reactor can be applied.

The reaction liquid to be injected may be a new reaction liquid or may be a reaction liquid extracted from the reactor with water dissolved therein. Further, the reaction solution may be one in which water is saturated and dissolved, or may be one in which water is dissolved to a certain extent without reaching saturation.

A method for saturated dissolving water in the reaction liquid can be achieved by bringing the reaction liquid into contact with water for a long period of time. In addition, it is possible to dissolve water to a certain extent by adjusting the contact time between water and the reaction solution, but it is also possible to dissolve water-soluble substances that do not mutually dissolve in the reaction solution, such as glycerin, ethylene glycol, diethylene glycol, and triethylene. The water concentration in the reaction solution can also be adjusted by bringing an aqueous solution of glycol, propylene glycol, butylene glycol, hexylene glycol, polyethylene glycol, etc. into contact with the reaction solution and utilizing the water distribution equilibrium between the reaction solution and the aqueous solution of a water-soluble substance. is possible.

Methods for reducing the water concentration in the reaction solution include directly injecting a new reaction solution from which water has been removed into the reactor, or removing water from the reaction solution extracted from the reactor and returning it to the reactor. is applicable.

A method for removing water from an organic solvent system such as a reaction solution can be achieved by bringing a water-absorbing material into contact with the reaction solution. Any water-absorbing material can be used as long as it does not dissolve in the reaction solution and does not react with the reaction substrate, such as glycerin, ethylene glycol, diethylene glycol, triethylene glycol, butylene glycol, propylene glycol, Water-soluble substances such as hexylene glycol and polyethylene glycol; molecular sieves, zeolites, silica gel,
Water-absorbing substances such as alumina, diatomaceous earth, activated carbon, kaolinite, perlite, cellulose powder, hydroxylapatite, and chitosan; monosaccharides such as glucose, galactose, ribose, and fructose; polysaccharides such as sucrose, trehalose, dextrin, glycogen, and starch Use salts that have water absorbing properties such as calcined gypsum, calcium carbonate, and calcium chloride; Salts that have deliquescent properties such as sodium hydroxide and calcium hydroxide; or metal salts that have lost crystallization water such as anhydrous sodium sulfate. be able to.

The moisture regulating device for increasing and decreasing the moisture content may be of any type, such as a stirred tank type or a packed bed type. A diaphragm type may be used in which the reaction solution and water or an aqueous solution of the above-mentioned water-soluble substance are flowed into and out of the tube, respectively.

The reaction method of the present invention may be a batch method or a continuous method, but since the degree of deterioration of enzyme activity is reduced in the present invention, it is particularly suitable for a continuous method over a long period of time. Further, the number of stages in the reactor may be one, but in order to increase the reaction yield, a multistage system may be used. Further, the reactor may be of any type such as a stirred tank type, a packed bed type, a fluidized bed type, or a combination thereof. In the case of the packed bed type, the supplied water is selectively absorbed by the bacterial cells near the reactor inlet, so in order to achieve an efficient reaction, the packed bed must be divided into smaller parts and the number of stages must be increased. It won't happen.

Examples of the reaction apparatus applied to the present invention will be specifically explained below with reference to FIGS. 2 to 5, but the present invention is not limited thereto.

FIG. 2 is an example of an apparatus that combines a reactor (1) consisting of a one-stage stirring tank and a stirring tank type water dissolving device (2).

In Figure 2, the water addition port (11) is connected to the water addition pump (7).
) is connected to the water melting device (2).The water concentration in the reaction solution is constantly measured by the water concentration sensor (4), and the measured value is compared with the set value in the processor (5). , the circulation pumps (3), (13) or the water addition pump (7) are turned ON and the flow rate of the injected water-dissolved reaction liquid to the reactor (1) is adjusted and maintained at the set value. .

Figure 3 shows a reactor (1) with a multi-stage fluidized bed, and each stage has a stirring tank-type water adjustment device (8).
This is an example of a device that combines the following. In FIG. 3, a water adjustment device (8) for bringing into contact with the reaction solution an aqueous solution of a water-soluble substance that does not dissolve in the reaction solution is connected to circulation pumps (3) and (13) in each stage of the reactor (1). connected via. In Fig. 3, the circulation pump (12) is constantly operated to flow the bacterial cells themselves or the bacterial cells immobilized on the microorganism holding material in the reactor (1). The moisture in the first stage, which is the lower stage, is supplemented by supplying water to the water dissolving device (2) from the water addition port (11) via the water addition pump (7), and further in the second and third stages. A water adjustment device (8) is provided through each circulation pump (3), and in the device (8), the aqueous glycerin solution and the reaction solution are brought into contact with each other by stirring gently to the extent that droplets do not form. The water concentration in the reaction solution is minutely adjusted using the water distribution equilibrium relationship between glycerin aqueous solutions.

Figure 4 shows the reactor shown in Figure 2, which consists of one stage of stirring tank (
This is an example in which a water removing device (14) consisting of a silica gel packed column is further combined with the stirring tank type water dissolving device (2) of 1). In the apparatus shown in FIG.
) is the filter (6) and circulation pump (3), (1
3) to the water dissolving device (2), and also to the water removing device (14) via the circulation pump (15).

As in the case of Fig. 2, the water concentration in the reaction solution is constantly measured by the water concentration sensor (4), and the water concentration is determined by comparing the measured value with a set value that has a certain range inside the pro sensor (5). is below the lower limit of the set value, the water addition pump (7),
Circulation pump (3).

Turn on (13) to check if there is water in the reaction solution. When the agricultural degree increases and exceeds the upper limit of the set value, the circulation pump (15) is turned on.
By removing water and reducing the water concentration in the reaction solution, the water concentration can be accurately maintained within a desired range.

Figure 5 shows the multi-stage fluidized bed in Figure 3 as a single stage, and similarly to Figure 4, a silica gel packed column is used with a water removal device (14
). Similarly to Fig. 3, the circulation pump (12) is constantly operated to flow the microbial cells themselves or the microbial cells immobilized on the microorganism holding material in the reactor (1), and the water content is adjusted in the same manner as shown in Fig. 4. By this method, the water concentration of the reaction solution, that is, the water concentration inside the bacterial cells, is controlled to a desired value.

If the moisture adjustment method shown in Figures 4 and 5 is used, the second
Compared to the method shown in Figures and Figure 3, it is possible to adjust the water concentration more minutely, so the progress of hydrolysis, which is a side reaction, can be more effectively suppressed, and the yield and quality of products due to transesterification can be improved. can be further improved.

As mentioned above, any type of reactor can be used for the transesterification reaction, including a stirrer-repellent type, a non-stirrer type, and a bubble column type, but when using microbial cells immobilized on a microbial holding material, From the viewpoint of suppressing detachment of bacterial cells, a non-stirrer type bubble column type is preferable, in which the flow of the reaction liquid is gentle and shearing force is small.

In particular, when a microorganism-retaining material is used, it is easy to separate, clean, etc., and therefore, it is also possible to use the apparatus used as the above-mentioned culture tank as a transesterification reactor as it is after culturing.

Glycerides used in the present invention include ordinary animal and vegetable oils or synthetic oils, such as olive oil, palm oil, shea butter, soybean oil, cottonseed oil, safflower oil, beef tallow, lard, fish oil, monkey fat, and mango oil. , cocum fat, triolein, tripalmitin, tristearin, diolein, civalmitin, distearin, monoolein, monobalmitin, monostearin, etc. Furthermore, the high melting point, intermediate melting point, and low melting point portions of these fats and oils obtained through a normal fractional crystallization process can also be used as reaction substrates.

In addition, in the present invention, the above reaction substances can be used directly, but if necessary, the substrates may be diluted with an organic solvent such as n-hexane, isooctane, acetone, ethanol, methanol, petroleum ether, or ethyl acetate. use

I can do both.

〔Example〕

Next, the present invention will be explained using Examples, but the present invention is not limited to these Examples. Note that % and parts mean % by weight and parts by weight, respectively, unless otherwise specified.

Example 1 Rhizopus delemma (Rh, delem) as a microorganism
ar[Fo 4697 ), glucose 1%, polypeptone 7%, NaNOs 0.1%, KIIzPO
A seed mother was prepared by preculturing at 30° C. for 24 hours in a medium consisting of 40.1% Mg5Oa.711□00.05%.

5j! Batch culture was carried out under the following conditions using a stirring tank jar fermenter (manufactured by Iwashiya Biochemical Co., Ltd., MBC-5, stirring blade diameter 8 cm).

Composition of medium used: Yeast extract 7%, NaN0+O, 1%
, K112PO40.1%, Mg5Oa・7HzOo,
05%, olive oil 6.5% pH: 5.6, stirring number: 40 Q rpm, ventilation l: Q, 5 vvm, temperature: 30°C After culturing for 40 hours, the bacterial cells were filtered out, washed twice with water, and then After washing twice with acetone, the cells were dried in a vacuum dryer for 48 hours to prepare dried bacterial cells. The water concentration in the dried bacterial body is 4
It was ~6%.

Five parts of the dried bacterial cells were mixed with palm oil medium melting point fraction (lV: 35,
Pop content in triglyceride: 62%) and 40 parts of soybean oil dissolved in 450 parts of hexane were added, and the second
Using the reaction apparatus shown in the figure, the water concentration in the reaction solution was adjusted to 5Q.
The transesterification reaction was carried out by stirring at 40° C. for 15 hours while controlling the ppm.

After the reaction, hexane was not separated and was subjected to solvent fractionation by a conventional method. That is, cut the high melting point fraction at 10°C, -
The low melting point fraction was canted at 15°C. The yield of the intermediate melting point fraction obtained was 45 parts after hexane removal.

This oil had a softening point of 32.8°C, and the content of monounsaturated disaturated glycerides in the oil was 90.2%.

Furthermore, chocolate was manufactured using this fat and oil, and the tempering properties and heat resistance were very good, and the raw material oil was clearly modified.

In addition, the same reaction was carried out with the water concentration of the reaction solution controlled at 800 ppm, and the solvent fractionation was performed in the same manner.
The yield of the intermediate melting point fraction obtained was 30 parts after hexane removal.

In addition, in liquid chromatography, Shimadzu Corporation (manufactured by Shimadzu Corporation)
(Product names: HGS-15 and 20) were used to measure the amount of diglyceride (hereinafter abbreviated as DG) in the total oil and fat after exchange. When the water concentration was soppm, the amount of diglyceride (DG) was 4.3%. In the case of 800 ppm, it was 30.2%, and with sooppm, the reaction yield was obviously poor and hydrolysis was progressing.

In all Examples including this Example, a moisture sensor manufactured by Panametrinox (trade name Nidistem I) was used as the moisture sensor.

Example 2 Mucorj javanicus as a microorganism
A seed mother was prepared in the same manner as in Example 1 using S. avanicus IFO4569).

Figure 6 shows the bubble column (C4r) used as a culture tank in Example 2.
culating Bed Fermentor) (
16) is a schematic cross-sectional view of FIG. As shown in Figure 6, this bubble column (16) has a height (H) of 400 mm, an inner diameter (D,
) is 150 dragons. In addition, FIG. 7 shows the bubble dispersion plate (17) provided in the bubble column (16) shown in FIG.
) is a plan view of The bubble dispersion plate (17) has a diameter (Dt)
is 150+u, and as shown in FIG. 7, a hole (18) with a diameter of 3 mm is provided, and air is vented in the direction shown by the arrow in FIG.

Using the bubble column (16) in Fig. 6, the ventilation amount is 2417w1.
Batch culture was performed at a temperature of 30° C. and a pH of 5.0. The medium used was the one used in Example 1 in which the yeast extract was replaced with meat extract.

Block-shaped polyurethane foam with 6 dragons on each side (manufactured by Prigistone 01, product name HR-40) as a microorganism retention material
was added to the medium used at a concentration of 1500 cells/l.

After culturing for 80 hours, the microbial cells immobilized on the microorganism holding material were filtered, washed with water and acetone in the same manner as in Example 1, and then washed with chitin three more times to remove the lipase-containing immobilized microbial cells. Prepared.

Using these immobilized bacterial cells, transesterification was carried out by controlling the water concentration in the reaction solution to 50 ppm and 800 ppm in the same manner as in Example 1. After 5 hours, almost the same reforming reaction results as in Example 1 were obtained.

Example 3 Palm oil medium melting point (AV: 0.1. l V:
35°5) 50 parts and shea butter medium melting point part (AV: 0.0
Aspergillus niger (＾spergillus n!gsr IFO4) prepared in the same manner as in Example 1 by dissolving 50 parts of 8°TV:50.2) in 300 parts of hexane.
Add 10 parts of lipase-containing bacterial cells of 343) and adjust the water concentration in the reaction solution to 20 to 30 pp in the reaction apparatus shown in Figure 4.
The mixture was stirred and reacted for 12 hours while controlling the temperature within the range of m. After solvent fractionation was carried out in the same manner as in Example 1, 85 parts of the medium melting point fraction after hexane was removed was obtained.

This oil has a softening point of 33.5°C, a content of monounsaturated disaturated glycerides in the oil and fat of 92.2%, and has good tempering properties.When chocolate was prototyped and subjected to a sensory test, it was found that it melts in the mouth. and heat resistance (fi).

Example 4 Monkey butter (AV:
0.065. IV: 37.6) Rh, n1veus immobilized on the microorganism holding material prepared in the same manner as Example 2.
IFO4759) lipase-containing microbial cells were added so that the amount of microbial cells to be added was the same as in Example 3, and the water concentration in the reaction solution was adjusted to 40 to 5 in the reaction apparatus shown in FIG.
Controlled to a range of 0 ppm, reacted for 3 hours,
Hard butter was obtained in the same manner as in Example 3. The yield of the obtained oil and fat was 47 parts after hexane was removed.

The softening point of this fat and oil was 31.9°C, the content of monounsaturated and disaturated glycerides in the fat and oil was 89.3%, and the tempering property was also good. When chocolate was prototyped and subjected to a sensory test, Example 3 Similarly, it had excellent melt-in-the-mouth properties and heat resistance.

Example 5 Cory-ncbact was prepared in the same manner as in Example 1 by dissolving 50 parts of triolein and 50 parts of tristearin in 1000 parts of hexane.
erium equi IFO3730), Staphylococcus aureus
aureus IFO3060), Candida cylindorace
a ATCC10571), Geotrichum candidum (IF)
Add 10 parts of lipase-containing microbial cells of O4597) to each of them, and while stirring at 40°C, adjust the moisture adjustment device of the reaction apparatus shown in Fig. 2 to determine the water distribution relationship with glycerin shown in Fig. 3. Change the water concentration to 50 instead of what you use.
The transesterification reaction was carried out for 5 hours while controlling the transesterification at ppm.

Analyze the reaction solution before and after the reaction using liquid chromatography,
Table 1 shows the composition of glycerides in the fats and oils and the stars of the DC produced.

Table 1 Indicates S nystearic acid and 0 nioleic acid.

The numerical values in the table indicate the amount of each glyceride in the total triglyceride and the amount of DC produced in the total 21 accents (%).

From Table 1, it can be seen that the triglyceride composition changes depending on each bacterial cell, and these bacterial cells serve as highly functional catalysts for carrying out the transesterification reaction. It can also be seen that by controlling the water concentration in the reaction solution, hydrolysis, which is a side reaction, is suppressed.

Example 6 Rhizopus oligosporus (Rh, ol- igosporus
Add 10 parts of lipase-containing microbial cells of IPO8631), and conduct transesterification by controlling the water concentration in the reaction solution within the range of 20 to 3 opps+ in the same reaction apparatus as in Example 3 while stirring at 40°C. The reaction was carried out.

During the reaction, the reaction solution was extracted as appropriate and the composition of glyceride in the oil and fat was analyzed by liquid chromatography.

Table 2 shows changes in triglyceride composition in each bacterial cell.

From Table 2, it can be seen that these bacterial cells act as catalysts for the transesterification reaction, and it can also be seen that the activity within the bacterial cells is dramatically increased by immobilization on the microorganism holding material. The amount of DC generated at 20 hours was 3.
2% and 4.0%, and side reactions were significantly suppressed.

Example 7 Similarly to Example 2, Rhizopus chinensis (Rh.

chinensis IFO4768), washed with water and acetone in the culture tank, and then aerated at room temperature to remove the bacteria immobilized on the microorganism holding material until the moisture content was 2 to 3.
%. Thereafter, the same reaction substrate as in Example 5 was charged, and a continuous transesterification reaction was carried out using the culture tank shown in FIG. 5 as a reactor and controlling the water concentration within a range of 15 to 20 ppm. The reaction substrate was supplied into the reactor (culture tank) using a feed pump and discharged from the reactor (culture tank) using a discharge pump. The average residence time of the reaction solution in the tank was 10 hours.

Table 3 shows changes over time in the triglyceride composition of the effluent and the amount of DG produced.

Table 3 shows that by controlling the water content inside the bacteria, the stability of lipase inside the bacteria can be further increased.
Approximately 90% activity remained after hours of continuous reaction.

In addition, side reactions were suppressed for a long time.

[Action/Effect]

As mentioned above, the enzyme reaction method of the present invention, which involves controlling the moisture content within the microbial cell within an appropriate range, stabilizes the lipase enzyme, prevents deactivation, and provides sufficient contact opportunities between the enzyme and the substrate. Not only does it provide a
The reaction can be maintained stably for a long period of time.

It also enables production of lipase catalysts at a lower cost than when using commercially available lipase enzymes.

Furthermore, when using bacterial cells immobilized on a microbial holding material, not only the lipase activity within the bacterial cells is dramatically improved, but also the operability of handling the bacterial cells in each step of the transesterification reaction is improved. This is extremely advantageous compared to conventional processes that utilize purified enzymes when carrying out transesterification reactions industrially.

4. Explanation of the Drawings Figure 1 is a graph showing an example of the water adsorption equilibrium relationship established between the bacterial cells and the reaction solution, and Figures 2 to 5 are used in the present invention, respectively. A schematic diagram showing an example of 2H, FIG. 6 is a schematic diagram showing an example of 2H. 4. 6. FIG. 7 is a schematic cross-sectional view of the bubble column used in Example 1, and FIG. 7 is a plan view of a bubble dispersion plate provided in the bubble column shown in FIG.

1...Reactor, 2...Water dissolving device 3
...Circulation pump, 4.Moisture concentration sensor 5.Processor, 6.Filter 7.
...Water addition pump, 8...Moisture adjustment device 9...
Reaction liquid inlet, lO...reaction liquid outlet 11...
Water addition port, 12...Circulation pump 13...Circulation pump, 14...Moisture removal device 15...Circulation pump, 16...Bubble column 17...Bubble portion 1
ijl board, 18...hole Fig. 1 + ooof- anti-a-o - Geki stock box i kainai main concentration (2-wood/y-h body) Fig. 2 5-3 Fig. 4 Fig. 5

Claims (1)

[Claims] 1. The moisture content of the lipase-containing bacterial cells is 0.1 to 20.
A method for transesterifying fats and oils using microbial cells, which is characterized by reacting with glyceride while controlling the weight percentage. 2. The transesterification method according to claim 1, wherein the water content of the bacterial cells is controlled by controlling the water concentration in the reaction solution. 3. The transesterification method according to claim 1 or 2, wherein the water concentration in the reaction solution is adjusted to 5 to 1000 ppm.