JPH0556943B2 - - Google Patents

Description

[Detailed description of the invention]

[0001]

TECHNICAL FIELD The present invention relates to dried bacterial cells used for transesterification of oils and fats. More specifically, when transesterifying oils and fats,
This invention relates to dried microbial cells used to increase the reaction rate and maintain the reaction stably for a long period of time.

【0002】

[Prior art and problems to be solved by the invention] Conventionally, transesterification reactions of oils and fats have been carried out using alkali metals, alkali metal alkoxylates, alkali metal hydroxides, etc. as catalysts, but this method lacks specificity in the exchange position. Therefore, a method using lipase enzyme has recently been adopted. In other words, when lipase acts on a mixture of fats and oils and fatty acids, transesterification occurs at specific positions depending on the specificity of the enzyme and the substrate, and various fatty acids are exchanged depending on the purpose (Japanese Patent Application Laid-Open No. 1983-1999). −
No. 104506, Special Publication No. 57-6480, No. 57-27159,
No. 57-28519). However, there are several problems when an enzyme that becomes active when dissolved in water or in the presence of water acts as a catalyst on a substrate (reactant) that is immiscible with water, such as oil or fat or fatty acid. .

(1) In order to increase the contact opportunities between the substrate and the enzyme, it is desirable to add the enzyme directly into the substrate, but in fats and oils or organic solvents, the enzyme is rapidly deactivated unless it is protected in some way. . The above-mentioned patent specifications attempt to prevent this by adsorbing the enzyme to a carrier such as an adsorbent, but if it is desorbed, it is likely to be deactivated.

(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 amount of water is reduced, transesterification will certainly proceed, but the reaction rate will be extremely slow and the enzyme will likely be deactivated. In JP-A-52-104506, the water content of the reaction system is 0.2 to 1.0% (weight %, the same applies hereinafter),
The specification of Japanese Patent Publication No. 57-27159 states that the water content is 0.005 to 0.18%, and the specification of Japanese Patent Publication No. 57-28519 states that a water content of 0.01 to 0.20% is suitable for transesterification. Also, in the specification of Japanese Patent Publication No. 57-6480, instead of water,
It has been shown that the use of trivalent lower alcohols allows transesterification without hydrolysis, 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, it is difficult for the reactant to diffuse to the enzyme on the carrier, and in particular, the enzyme adsorbed within the pores of the carrier cannot substantially participate in the reaction and cannot be effectively used. The number of working enzymes decreases. In particular, this tendency becomes stronger as a hydrophilic carrier is used.

[0006] As mentioned above, when carrying out the transesterification reaction of an oil-based reactant using a lipase enzyme that is active in the aqueous phase as a catalyst, the enzyme must not be deactivated, and the The water content of the enzyme must not be too high or low, and the chances of contact between the enzyme and the reactant must not be reduced. Just by paying attention to (2) above,
Little consideration has been given to (1) and (3).
However, problems (1) and (3) above are extremely important in systems such as transesterification systems, where the reactant and catalyst (enzyme) form a heterophasic system in an environment where enzymes are easily deactivated. This poses a major challenge.

【0007】

[Means for Solving the Problems] The purpose of the present invention is to solve the above-mentioned problems and to perform transesterification quickly without deactivating the enzyme for a long period of time. That is, the present invention relates to transesterification of oils and fats, in particular, by suspending and reacting dried bacterial cells containing lipase with a water content of 1 to 20% in a mixture of glyceride oils and fatty acids. This relates to dried bacterial cells used for transesterification to replace fatty acids with fatty acids.When culturing lipase-containing microorganisms, glycerides or fatty acids are used as lipase inducers during culture at a concentration of 1 to 80% in the culture solution. After adding the microorganisms at the initial stage or during the cultivation and washing the obtained microorganisms with a water-soluble solvent, the water content is 1 to 20%.
Concerning dried bacterial cells obtained by drying until dry.

[0008]

[Example] Since it is preferable that the moisture content in the dried bacterial cells is low from the viewpoint of preventing hydrolysis reactions, drying should be carried out so that the moisture content is 20% or less. When it exceeds 20%, hydrolysis takes precedence over transesterification, and the majority of the products are hydrolysates such as diglyceride, monoglyceride, and glycerin. Regarding the lower limit, it is better to keep it as low as possible, so there is no point in stipulating it. However, in general drying methods, the moisture content cannot be lowered below the characteristic moisture content of the dried material (equilibrium moisture content), and microorganisms can be dried under vacuum at room temperature. It is difficult to reduce the dry moisture content below 1%.

[0009] As the microorganism used in the present invention, any microorganism that produces lipase can be used, but Rhizopus (Rhizopus), Mucor (Mucor), Aspergillus (Aspergillus)
Microorganisms of the genus Candida, Geotrichum, etc. are suitable;
For example, Rhizopus delemer (Rhizopus
delemar), Rhizopus chinensis (Rhizopus
chinensis), Rhizopus pseudochinensis, Mucor javanicus, Mucor hiemalis, Aspergillus niger, Candida rugosa, Geotrichum candidum).

[0010] In order to make the above-mentioned microorganisms work efficiently as reaction catalysts, it is important to have a culture method that allows the body to contain a large amount of lipase, and a drying condition that makes it easier for the intracellular lipase to come into contact with fats and oils and fatty acids.

[0011] In the present invention, when culturing lipase-containing microorganisms, glyceride or fatty acid is added to the culture solution as a lipase inducer at the beginning or during the culture so that the concentration in the culture solution is 1 to 80%. By washing the obtained microorganisms with a water-soluble solvent and drying them to a moisture content of 1 to 20%, dried microorganisms that have the effect of rapidly and stably sustaining the transesterification reaction for a long period of time can be obtained. It will be done.

[0012] In such culture, mono-, di-, and triglycerides, fatty acids having 8 to 20 carbon atoms, and their esters are used as lipase inducers, but among these, normal culture temperatures (20 to 40
triolein (olive oil), which becomes liquid at
Diolein, monoolein, oleic acid and linoleic acid are preferred. As for the amount to be added, it is suitable that the concentration in the culture solution is 1 to 80%. When the amount of inducer is less than 1%, the amount of lipase contained in the body is small, and the rate of transesterification using such bacterial cells is very slow. When the amount exceeds 1%, the intracellular lipase content begins to increase rapidly, and reaches its maximum content at 5 to 10%. When the amount of inducer is further increased to 40 to 50% or more, the culture system forms a W/O emulsion, and the microorganisms multiply in the water droplets and accumulate lipase in the body. The intracellular lipase activity obtained by culturing in such a W/O emulsion system is still high and can be used sufficiently for transesterification. However, if it exceeds 80%, the amount of culture solution decreases and the bacterial cell yield also decreases, which is not practical.

[0013] As a method for removing water from the bacterial cells obtained as described above, in principle, it is sufficient to dry them at a temperature (below 40 to 60°C) that does not deactivate the enzyme, but it is possible to simply remove water. When evaporated, the cell tissue contracts and becomes extremely hard, cutting off contact between the lipase in the tissue and the outside world, making it unable to express its activity. Therefore, in order to dry the bacterial cells, it is necessary to use a method that does not involve shrinkage of the cell tissue. For this purpose, the cell tissue is contracted by immersing the bacterial cells in a water-soluble solvent such as acetone, or a lower alcohol such as methanol, ethanol, or isopropanol, replacing the tissue with the solvent, and then evaporating the solvent. It is possible to obtain dry bacterial cells by suppressing the In this case, vacuum drying is preferred as the drying method. Alternatively, if the use of a solvent is not preferred, freeze-drying may be used.

[0014] Furthermore, by soaking the cell tissue in a 5% or less glutaraldehyde aqueous solution to fix the cell tissue before immersing the cell tissue in the solvent, shrinkage of the cell tissue can be further suppressed, and a more preferable dry cell body can be prepared. It is possible to do so. If the concentration of the glutaraldehyde aqueous solution is higher than 5%, the degree of crosslinking will be too high and the transesterification reaction rate will be reduced, so it is preferably 5% or less.

[0015] The dried bacterial cells obtained by the above method usually have a moisture content of 1 to 5%, but in order to be used for the transesterification reaction, the moisture content must be 1 to 5% as described above. 20% is good. The moisture content can be easily adjusted by adjusting the drying time.

[0016] Using the dried bacterial cells of the present invention obtained in this manner, it is possible to carry out the transesterification reaction of fats and oils at a high rate and stably for a long period of time.

[0017] The fats and oils and fatty acids that can be used in transesterification using the dried bacterial cells of the present invention are as follows.

[0018] That is, the fats and oils are normal animal and vegetable oils or synthetic oils, and specifically include olive oil, palm oil, shea butter, soybean oil, cottonseed oil, beef tallow, lard, and fish fat.

[0019] In addition, fatty acids having 8 to 20 carbon atoms that exist in nature can be used, specifically stearic acid, palmitic acid, oleic acid,
Such as linoleic acid. Saturated fatty acids with a large number of carbon atoms have a high melting point of 60 to 80°C, and when using such fatty acids, hydrocarbons such as hexane, heptane, ethyl ether,
In addition to ethers such as propyl ether, esters such as methyl acetate and ethyl acetate, solvents such as benzene and acetone can be used, and dried bacterial cells can function in such solvent systems without being inactivated.

[0020] The amount of the dried bacterial cells of the present invention to be suspended in the reactant (mixture of glyceride oil and fatty acid) is determined by the amount of the reaction system (glyceride oil, fatty acid,
It is preferable to add dried bacterial cells so that the moisture content of the mixture (mixture of dried bacterial cells) is 0.1 to 10%. 0.1
If it is less than %, the amount of lipase in the reaction system will be small, the reaction rate will be low, and it will not be substantial. If the amount exceeds 10%, the reaction rate will increase accordingly, but the viscosity of the reaction system will increase, the mixing condition will deteriorate, and the reaction rate will not be proportional to the amount added, and the dried bacterial cells will be separated after the reaction. From the operational point of view, which can be difficult to perform, the amount of dried bacterial cells to be added is preferably such that the moisture content of the reaction system is 0.1 to 10%.
More preferably, the amount used is 1 to 5%, which is optimal in terms of reaction rate and operation.

[0021] In the conventional method of adsorbing enzymes onto a carrier, hydrolysis could not be prevented unless the water content of the reaction system was kept below 1%, whereas in the method using dried bacterial cells, the water content of the reaction system was reduced to 1% or less. It was confirmed that even when the amount exceeds 1%, the transesterification reaction can be carried out at a high reaction rate with almost no hydrolysis. In other words, although lipase in dried bacterial cells has a large apparent water content, it is presumed that the amount of water involved in the reaction is considerably smaller than the apparent water content. Although it is not clear in what form the water that is not involved in the reaction exists in the dried cells, it appears to contribute to enzyme activation and stabilization. This is because the method using dried bacterial cells has a much lower deactivation rate than conventional methods and can withstand long-term use. Originally, enzymes are biocatalysts that function well in mild environments and tend to be deactivated in extreme environments. Moreover, once it is deactivated, it is difficult to regenerate. In particular, in oil-water heterophase reactions such as transesterification reactions, enzymes are constantly placed in contact with the oil phase, which is considered to be a harsh environment, and it is precisely in this environment that the enzyme activity can be maintained for a long time. The important point is whether or not it can be sustained, and the dried bacterial cells of the present invention are satisfactory in this respect.

[0022] The advantages of the present invention can be summarized as follows.

[0023] () Since the dried intracellular lipase is under the protection of cell tissues and tissue water, its deactivation rate is low and it can be subjected to reactions for a long time.

[0024] () Perhaps because dry intracellular lipase or its vicinity is easily compatible with oil-based reactants, the amount of enzyme with the same number of units is 2 to 5 times lower than that of the carrier adsorption method.
It has twice the reaction speed.

() No decrease in activity is observed even when the reaction is carried out in a solvent such as hexane.

() Has strong resistance to changes in pH, temperature, etc.

[0027] Enzymes themselves have restrictions on pH and temperature in order to express their activity. For example, Rhizopus
The preferred environment for Delemer's lipase is a pH of 4 to 7 and a temperature of 30 to 40°C; in other environments, the enzyme is inactivated or its activity is significantly reduced. but,
Lipase in the form of dried bacterial cells exhibits stable activity in this environment, and even outside this environment, the activity does not decrease significantly and is persistent. The reason for this is probably due to the above-mentioned (), but such an advantage lies in the fact that the reaction rate can be increased by increasing the temperature (it can be raised to 50 to 60°C).

[0028] Dried intracellular lipase has the advantages mentioned above, but if a heat-resistant (thermophilic) strain is used as the lipase-producing strain, the reaction temperature can be lowered to 70°C.
It can be raised up to ℃. As such heat-resistant bacterial strains, strains of the genus Rhizopus, such as Rhizopus chinensis and Rhizopus pseudokinensis, are used. For example, a heat-resistant strain of Rhizopus chinensis can grow at temperatures of 50 to 60°C, and if the dried bacterial cells of the present invention are prepared using this strain, the transesterification reaction can be carried out at 70°C. Fatty acids such as stearic acid and palmitic acid have a melting point of 68 to 72°C, but if they can react at temperatures above 70°C, it is convenient because there is no need to use a solvent to dissolve the fatty acids. good.

[0029] Furthermore, if the dried bacterial cells of the present invention are prepared using a microorganism containing a lipase specific for the 1 and 3 positions, it is also possible to selectively transesterify only the 1 and 3 positions of glyceride. Examples of microorganisms that produce lipases specific for the 1st and 3rd positions include Rhizopus species such as Rhizopus deremer and Rhizopus chinensis, Mucor jabanicas, and Aspergillus niger.

[0030] Furthermore, when culturing microorganisms containing lipase, if porous particles with a diameter of 50 to 2000 μm are added to the culture medium in an amount of 5 to 30% of the volume of the culture medium, the microorganisms will enter the pores of the particles. It proliferates and begins to cover the particle surface. By treating the thus obtained immobilized microorganism with the drying method of the present invention, an immobilized microorganism that can be subjected to transesterification can be obtained. Continuous operation is possible. Incidentally, in continuous transesterification operations using immobilized microorganisms, the activity is stable for 1 to 2 weeks, and the activity remains over 60% even after 1 month.

[0031] Next, the present invention will be explained in more detail with reference to Examples, but the present invention is not limited to these Examples.

Example 1 Rhizopus delemer IFO4697 was cultured using a medium containing the components shown in Table 1 (olive oil was the inducer).
Aerated culture was carried out at pH 6.5 and temperature of 30°C for 50 hours.

【0033】

[Table 1] ■■■ Turtle shell [0009] ■■■

[0034] The obtained bacterial cells were washed twice with pure water, then immersed in a 50% acetone aqueous solution for 10 minutes, and further
After soaking in 100% acetone solution for 5 minutes, filter it.
It was then vacuum dried at 30°C for 2 hours. The moisture content of the dried bacterial cells thus obtained was approximately 5%. Enzyme activity was 2000 units/g dry cells.

[0035] The reaction system shown in Table 2 (the water content of the reaction system is
1.2%) was used to perform the transesterification reaction.

[0036]

[Table 2] ■■■ Turtle shell [0010] ■■■

[0037] Although the reaction was carried out with stirring at 40°C for 48 hours, the reaction had already been completed, and the products shown in Table 3 were obtained.

【0038】

[Table 3] ■■■ Turtle shell [0011] ■■■

[0039] The lipase of Rhizopus delemer is 1,
Since it is 3-position specific, oleic acid at the 1st and 3rd positions was replaced with stearic acid. 80% of olive oil is transesterified at the 1st or 1st and 3rd positions, and 5%
became diglyceride. When the amount of water near the enzyme is large, hydrolysis progresses and decomposes diglyceride and even monoglyceride, but in this example, the hydrolysis rate was suppressed to 5%.

[0040] Example 2 Heat-resistant strain of Rhizopus chinensis
Chinensis IFO4768 was dried into dried cells in the same manner as in Example 1, and transesterification was carried out in the same manner as in Example 1. However, the reaction temperature was changed to 40°C, 50°C, and 60°C, and the time required for the reaction to complete was compared. As a result, the reaction times were 45, 30, and 24 hours, respectively, and the reaction rate was nearly doubled by increasing the reaction temperature.

Example 3 Using the dried bacterial cells obtained in Example 1, the transesterification reaction was carried out in a continuous system (flow system), and the enzyme deactivation rate was investigated. The reactants shown in Table 2 and dried bacterial cells were charged in advance into the reaction tank, and then a certain amount of a mixture of the reaction substrates olive oil and stearic acid dissolved in hexane with the composition shown in Table 2 was added to the reaction tank. supplied. On the other hand, an amount of produced liquid equal to the amount supplied was extracted from the reaction tank. At this time, a filter was installed at the outlet to prevent dried bacterial cells from leaking. The amount supplied and the amount taken out were adjusted so that the average residence time in the reaction tank was 24 hours. The reaction temperature was 40°C.

[0042] The composition of the product solution was measured using this method, and the rate of enzyme deactivation was investigated from the change in reaction yield (transesterification rate) over time. The results are shown in Figure 1. After the reaction reached steady state, the steady state lasted for one week, after which the reaction rate began to gradually decrease and the enzyme activity was gradually lost, but even after one month, the steady state remained at 40%.
% or more. As a comparative example, a conventional method in which a commercially available enzyme Rhizopus lipase (manufactured by Seikagaku Kogyo Co., Ltd.) derived from Rhizopus deremer was adsorbed onto Celite was used. The results are shown in Figure 1, and the steady state only lasted for 2-3 days, and the enzyme activity decreased by 20% in 1 week.

Example 4 Rhizopus chinensis IFO4768 was grown in porous commercially available sponge particles (1 mm cubic, pore diameter 50-100 μm,
The cells were cultured for 50 hours in a medium containing the components shown in Table 1, in which the cells were suspended (empty rate: approximately 80%). Bacterial cells also proliferated within the particles and covered the surfaces of the particles. When the obtained particles were dried by the method of the present invention, the bacterial cells adhered to the particles and immobilized microorganisms were obtained. The immobilized microorganism thus obtained was added in an amount of 20% of the reaction system, and the enzyme deactivation rate was examined in the same manner as in Example 3. The results are shown in Figure 1. As shown in Figure 1, the steady state persisted further, lasting nearly two weeks, and the rate of deactivation slowed down.

Example 5 Rhizopus chinensis IFO4768 was cultured in the same manner as in Example 1, and dried bacterial cells were obtained using the same treatment method. Next, 2 g of dried bacterial cells were added to a reaction solution containing 10 g of low melting point part of shea fat, 20 g of palmitic acid, and 60 g of hexane, and transesterification reaction was carried out for 2 hours while stirring with a magnetic stirrer. Next, the bacterial strain was Aspergillus niger.
IFO4343, Mukor Ziyavanikas IFO 4569,
A transesterification reaction was carried out under the same reaction conditions using dried bacterial cells produced in place of Mucor heamaris IFO 8448 and Candeida rugosa ATCC 10571.

[0045] The reaction results were analyzed in the same manner as in Example 1, and 1-stearoyl-2,3-dioleoyl-glyceride (SOO) and 1-palmitoyl-2,3-dioleoyl-glyceride (POO) in triglyceride were analyzed. , 1,3-dipalmitoyl-2-oleoyl-glyceride (POP)
The composition is shown in Table 4.

【0046】

[Table 4] ■■■ Turtle shell [0012] ■■■

[0047]

Effects of the Invention The dried bacterial cells of the present invention enable efficient transesterification of fats and oils with a faster reaction rate and a stable reaction lasting for a long time.

[Brief explanation of drawings]

FIG. 1 is a graph showing changes in transesterification reaction rate over time.

Claims (1)

[Scope of Claims] 1. When culturing a microorganism containing lipase, glyceride or fatty acid is added to the culture solution as a lipase inducer so that the concentration in the culture solution is 1 to 80% by weight at the beginning or during the culture. Dried microorganisms are obtained by washing the microorganisms with a water-soluble solvent and drying the microorganisms to a water content of 1 to 20% by weight. 2. The dried bacterial cell according to claim 1, wherein the lipase inducer is triolein, diolein, monoolein, oleic acid, or linoleic acid. 3. The dried microbial cell according to claim 1, which is obtained by treating the cell surface by immersing the microorganism in an aqueous solution of 5% or less glutaraldehyde after completion of culturing, and then washing the cell surface with a water-soluble solvent. 4 Porous particles with a diameter of 50 to 2000 μm are placed in a medium in advance at 5 to 30% by weight of the medium volume, and microorganisms are cultured, the microorganisms are grown within the particles, the microorganisms are immobilized on the particles, and then dried. Dried bacterial cells according to claim 1.