JPH0716425B2 - Transesterification method for fats and oils - Google Patents

Description

TECHNICAL FIELD The present invention relates to a method for transesterification of fats and oils using a lipase (lipolytic enzyme). More specifically, a transesterification reaction between fats and oils and fatty acids or fatty acid esters, a transesterification reaction between fats and oils, a transesterification reaction between fats and oils and alcohols, using a lipase or an enzyme preparation containing lipase having sufficient ester synthesis activity It is about how to do etc.

The transesterification reaction of oils and fats is an important processing technology along with hydrogenation in the production of edible oils and fats such as margarine and shortening.

[Conventional technology]

The transesterification reaction of fats and oils has been conventionally performed by a chemical method. That is, it has been carried out by a method using an alcal substance such as an alkali metal, an alkali metal alcoholate or an alkali metal hydroxide, or various metal salts as a catalyst. However, in this method, regarding the arrangement of fatty acids in oils and fats, rearrangement occurs according to the law of indiscriminate distribution, so that no specificity is observed in the position of the arrangement of fatty acids bound to the obtained oils and fats. . That is, conventional chemical methods are non-selective with respect to the position of bound fatty acids in glycerides. This property is normal margarine,
In the case of producing an edible processed oil / fat such as shortening, it may bring about an effect of improving physical properties of the oil / fat.

However, such conventional non-selective methods cannot be used for the purpose of producing fats and oils having a unique glyceride composition. The unique glyceride composition as used herein is a case where most glycerides have a symmetrical structure, such as natural cacao butter. Recently, in the case of intending to produce such fats and oils, a method for regioselectively carrying out the transesterification reaction of fats and oils has been developed instead of the conventional non-selective chemical method.

That is, it is intended to carry out regioselective transesterification of fats and oils using lipase, which is an enzyme that originally hydrolyzes fats and oils (JP-A-52-104506). According to this method, the presence of water in the reaction system is an essential condition for activating the lipase. Although this water content is as small as 0.2 to 1.0%, due to the inherent property of lipase, hydrolysis of oils and fats inevitably occurs, and the yield of exchanged fats due to by-product diglyceride cannot be avoided.

In addition, the water content is reduced to reduce the production of by-products.
Attempts have been made to reduce the amount of water to 0.1% or less for the reaction, but the reduction of the water content substantially lowers the reaction rate, which is not practical in practice (Japanese Patent Laid-Open No. 55-71797).

Further, as a method for increasing the transesterification rate, there is also a proposal of a method in which the reaction step is divided into two steps of decomposition and synthesis (JP-A-60-19495, JP-A-60-203196), There are difficulties in controlling the reaction, especially in the decomposition process.
In the method, it is interesting to focus on diglyceride,
It is technically difficult to selectively produce diglyceride in the decomposition step, and while decomposition to monoglyceride and glycerin occurs, a large amount of undecomposed triglyceride remains. In addition, 1, resulting from the non-enzymatic transfer of diglycerides,
The presence of 3-diglyceride inevitably reduces the yield of the desired transesterification product in the second synthetic step. Further, the second-stage synthetic reaction rate is not sufficient as compared with the usual transesterification rate. From this point of view, it is inevitable that the process operation becomes complicated.

As described above, the transesterification reaction of fats and oils with lipase has a characteristic and advantageous point as compared with the above-mentioned chemical method, but on the other hand, there are many problems to be solved,
In order to carry out industrially, it is necessary to solve these.

[Problems to be solved by the invention]

From an industrial economic perspective, the price of the enzyme needed to catalyze these reactions is still very high,
In industrialization of the reaction, it is premised that the amount of the enzyme used is reduced or recovered and repeatedly used. Under such circumstances, an attempt to reduce the amount of the enzyme causes a substantial decrease in the reaction rate, resulting in enormous reaction equipment and a decrease in production efficiency. Deterioration is unavoidable, and the amount of transesterification product that can be produced from a given weight of enzyme is naturally limited.

As described above, in the conventional technique, the reaction speed in a substantial aspect,
And, it is not recognized that the enzyme has both durability in terms of economy.

[Means for solving problems]

In such an actual situation, the present inventors diligently studied a method of suppressing the hydrolysis of fats and oils, and efficiently and economically performing only transesterification, and in order to solve the above problems, a lipase or a lipase was contained. As a result of various studies on the method of transesterification using the enzyme preparation (hereinafter, abbreviated as lipase agent) and the characteristics of lipase, an effective method of using the lipase agent was found, and the present invention was completed.

That is, the present invention, when performing the transesterification reaction of fats and oils and fatty acids or fatty acid esters using a lipase or enzyme preparation containing lipase, transesterification reaction of fats and oils, or transesterification reaction of fats and alcohols,
The transesterification reaction is performed by adjusting the concentration of lipase or an enzyme preparation containing lipase so that the decomposition activity is 500 to 20,000 units / reactive substrate weight (g) and the synthetic activity is 0.05 unit or more / reactive substrate weight (g). The present invention relates to a method for transesterification of fats and oils, which is characterized in that it is carried out.

That is, the present invention relates to a method for performing a transesterification reaction of fats and oils with fatty acids or fatty acid esters, or a transesterification reaction of fats and oils with each other, or a transesterification reaction of fats and oils with alcohols in the presence of a high-concentration lipase agent. Is.

It has been revealed by pioneering research by Iwai, Tsujisaka, etc. that lipase catalyzes not only hydrolysis but also a synthetic reaction which is a reverse reaction (M. Iwai, Y. Tsujisaka, J. Fukumoto, J.
Gen. Appl. Microbiol., 10, 13 (1964)). The present inventors analyzed the transesterification reaction of oils and fats based on this experimental fact from the viewpoint of enzyme chemistry and reaction engineering. As a result, the complex of diglyceride and enzyme was involved in the reaction, and the transesterification rate was It has been found that is expressed by the following equation.

V = k [E · DG] [FA] where k is the overall reaction rate constant, [FA] is the fatty acid concentration, and [E]
・ DG] represents the concentration of diglyceride / enzyme complex. k greatly depends on the water content and the enzyme concentration in the reaction system, but an increase in the water content only promotes an increase in free diglyceride, and not only a substantial increase in the transesterification reaction rate is not observed. However, it is necessary to involve a complicated dehydration process. On the other hand, the increase of the enzyme concentration suppresses the increase of free diglyceride in the reaction system, and enables the increase of the transesterification reaction rate as is clear from the above rate equation. Even more surprisingly, the mechanism is not clear by increasing the enzyme concentration in the reaction system, but it is considered that the mutual stabilizing action of the enzymes is expressed, and the decrease of the transesterification activity due to the deterioration of the enzyme over time is suppressed. It became clear that it could be greatly improved.

Furthermore, the present inventors have focused on the fact that the transesterification rates of various enzymes are different, and as a result of developing and testing a measuring method for the enzyme activity of synthesizing triglyceride from diglyceride,
It was found that those having a significantly higher triglyceride synthesis activity than the decomposition activity had a higher transesterification rate.

The present inventors have completed the present invention as a result of further detailed research based on these findings.

According to the method of the present invention, it is possible to significantly reduce the time by one-step reaction without requiring a complicated reaction step, and without causing a decrease in the yield of the target component due to decomposition of the substrate by water, and a further lipase agent. It is possible to suppress a decrease in transesterification activity of (lipase or an enzyme preparation containing lipase) and increase the number of times of collecting and using the lipase agent.

As the lipase agent used in the present invention, commercially available lipase may be used as it is, and any of the activated lipase preparations described in JP-A-60-251891 or an immobilized lipase preparation retained on various carriers as necessary is used. However, it is preferable to use one having a ratio of synthetic activity to decomposition activity of 1 × 10 ⁻⁴ or more. Further, the presence of an excessive amount of lipase agent is not preferable because the workability is impaired due to an increase in the slurry concentration of the reaction system. An appropriate enzyme concentration is 0.05 unit or more as synthetic activity / reactive substrate weight (g) and 500-20,000 unit as reactive activity / reactive substrate weight (g).
1,000 to 10,000 units / reaction substrate weight (g) is preferable.

In order to activate the lipase agent in the transesterification reaction, the water content in the reaction system should be 0.00 based on 1 part by weight of the reaction substrate.
It is desirable that the amount is 5 to 0.2 parts by weight, preferably 0.01 to 0.1 parts by weight.

Regarding the degradability of lipase agents, the method of Fukumoto et al. (J. Gen.
According to Appl. Microbiol., 98, 353 (1963), add a predetermined amount of enzyme (lipase agent) to 5 ml of olive oil emulsion and 4 ml of 0.1M phosphate buffer and measure the amount of fatty acid produced when reacting at 37 ° C for 30 minutes. One unit of oleic acid corresponding to 1 μmol / min was used. In addition, the synthetic activity means 0.9 g of high-purity diglyceride obtained by decomposing and purifying olive oil according to the method described in Reference Example 1 and Reference Example 2 and olein.
Dissolve 1 g and 4 ml of n-hexane to obtain 6 as decomposition activity.
The amount of triglyceride produced when reacting at 40 ° C for 1 hour with the addition of 00 unit of enzyme was 1 μmol / triolein.
The one equivalent to Hr was set as 1 unit.

Further, as the lipase for the lipase agent used in the present invention,
Microorganisms such as Rhizopus genus, Aspergillus genus, Mucor genus, Geotrichum genus having fatty acid specificity, and Candida genus having no position specificity, which have excellent position specificity Examples include lipases of animal origin and lipases of animal origin such as pancreatic lipase.

If necessary, a known carrier can be used together with the lipase. As the carrier, Celite, diatomaceous earth, kaolinite, silica gel, perlite, glass fiber, molecular sieve, activated carbon, inorganic carriers insoluble in transesterification reaction system such as calcium carbonate, and cellulose powder, ion exchange resin, chitosan, etc. Any organic polymer such as the above organic polymers can be used as long as it does not adversely affect the lipase activity. Further, the carrier may have various shapes such as powder, granules, fibers and sponge, and any of them may be used.

Examples of the fats and oils used in the present invention include general vegetable fats and oils, animal fats and oils or processed fats and oils, or mixed fats and oils thereof. Examples of these include soybean oil, cottonseed oil, rapeseed oil, corn oil, safflower oil, sunflower oil, coconut oil, beef tallow, lard, fish oil and the like. Further, in the case of producing a cocoa butter substitute fat by a transesterification reaction, fats and oils containing a large amount of oleic acid at the 2-position of the glyceride, such as palm oil, oleic oil, high oleic sunflower oil, high oleic acid succinate, etc. Flower oil, camellia oil, azalea oil, monkey butter, shea butter, illipe butter, kokum butter, mola butter, fulwalla butter, borneo tallow butter, mango kernel oil, or fractionated oils or fats thereof can be used.

Transesterification is performed by using fats and oils, fatty acids and fatty acid esters,
It is performed by reacting fats and oils or fats and alcohols. As the fatty acid, a straight chain having 2 to 24 carbon atoms and usually existing in nature, for example, a saturated fatty acid such as acetic acid, caprylic acid, palmitic acid, and stearic acid, or an unsaturated acid such as oleic acid, linoleic acid, and eicosapentaenoic acid is used. Saturated fatty acids and the like can also be used.

As the fatty acid ester, an esterified product of the above fatty acid (linear fatty acid having 2 to 24 carbon atoms) and linear saturated monohydric alcohol having 1 to 6 carbon atoms is used. For example, methyl palmitate, ethyl palmitate, methyl stearate, ethyl stearate and the like can be used.

As the alcohol, a linear aliphatic monohydric alcohol having 1 to 24 carbon atoms can be used.

The transesterification reaction of the present invention can be carried out in a solvent inert to lipase, if necessary. Preferred solvents include n-hexane, petroleum ether, petroleum benzine and the like. A suitable amount of the solvent used is, for example, about 1 to 10 parts by weight with respect to 1 part by weight of the fatty acid. The reaction temperature in the present invention is 20 to 70 ° C. as in the usual enzyme reaction.
Can be done at. It is also effective in reducing the by-products that the excess water in the reaction system is blown out of the reaction system by blowing an inert gas such as dry nitrogen, if necessary.

From the reaction mixture that has completed the transesterification reaction, fatty acid,
Easy to use small amount of monoglyceride, partial ester such as diglyceride, unreacted alcohol, etc. by liquid-liquid extraction, alkali neutralization, vacuum or molecular distillation, etc. using conventional separation and purification means alone or in appropriate combination. It is possible to obtain a purified transesterification product.

〔The invention's effect〕

The method of the present invention is for fully exhibiting the synthetic activity of lipase, and effectively utilizes that the synthetic activity of lipase is proportional to the enzyme concentration. When the enzyme concentration is sufficiently high with respect to the reaction substrate as in the present invention, even if a large amount of water that promotes hydrolysis, which is a conventional side reaction, is present in the reaction system, any by-products will increase. A noticeable increase in the reaction rate is seen without looking. In addition, not only the initial velocity but also the time required to reach the equilibrium of the transesterification reaction (final velocity) is short-circuited more than expected.

Furthermore, the greatest effect of the present invention is that by sufficiently increasing the concentration of the lipase agent in the reaction system, the mutual stabilizing effect of the enzymes is expressed, and as a result, the decrease in the enzyme activity over time decreases, and after the reaction, This is the point that the recovered lipase agent can be effectively reused, the productivity per enzyme weight can be remarkably improved and the economical efficiency can be improved in the practice on an industrial scale.

Furthermore, the present invention can effectively produce an expensive cocoa substitute fat from, for example, inexpensive palm oil by using a regioselective lipase agent.

〔Example〕

Hereinafter, the present invention will be described in more detail with reference to Reference Examples, Examples, Comparative Examples, and the like. All percentages and parts in these examples are by weight.

Reference Example 1 (Preparation of 1,2-diglyceride) Olive oil 500 g and commercially available enzyme 1 [Olipase 4S (intracellular enzyme of Rhizopus japonicas origin) manufactured by Osaka Bacterial Research Institute; decomposition activity 1,500 unit / g] 20 g and water 1,500 Olive oil hydrolyzate was obtained by mixing with ml and stirring at 40 ° C. for 3 hours.

This hydrolyzate was repeatedly extracted three times with 500 ml of diethyl ether, dehydrated and distilled off by a conventional method to obtain a mixture of triglyceride, diglyceride and fatty acid. Iatroscan TH-10 synchrography method (M. Tanaka et.al., Lipids vol 15 (10), 87
2 (1980), etc.) to analyze triglycerides, 1,2-diglycerides, 1,3-diglycerides, fatty acids and the like. At this time, use a silica gel rod treated with 0.3% boric acid,
Chloroform: acetone = 96: 4 was used as the developing solvent. The analysis results at this time are shown in Table 1.

Take 50 g of the oil obtained here and add silica gel (Merck Sili
Ca gel 60, 200-400 mesh) 200 g of column chromatography was used to separate n-hexane: diethyl ether = 9: 1 and 8: 2 (1,000 ml each) to obtain crude diglyceride in the subsequent section. The composition of the obtained crude diglyceride is shown in Table 2.

The crude diglyceride was purified by column chromatography using Florisil which had been previously treated with boric acid. The composition of the obtained 1,2-diglyceride is shown in Table-3.

Reference Example 2 (Measurement of synthetic activity) The diglyceride (high-purity diglyceride in Table 3) obtained in Reference Example 1 was mixed with oleic acid so that the ratio of diglyceride: fatty acid was 9: 1, and the mixture was mixed. 1g to 4ml n-
600 units of each of the commercially available enzyme 1, the commercially available enzyme 2 or the prepared enzyme was added to the product dissolved in hexane as a degrading activity, and the amount of triglyceride produced when reacted at 40 ° C. was quantified by an iatroscan. For each enzyme, the amount of production (μmol) of triglyceride produced after reaction for 1 hour was determined, and 1 μmol / Hr as the activity of the synthetic reaction, which is the reverse reaction of decomposition, was defined as 1 unit of synthetic activity.

The respective synthetic activities and activity ratios (ratio of synthetic activity to decomposition activity) are shown in Table 4.

The commercially available lipase 1 in Table 4 is the lipase 4S (intracellular enzyme derived from Rhizopus japonicas) manufactured by Osaka Bacterial Research Institute, and the commercially available lipase 2 is Talipase manufactured by Tanabe Seiyaku Co., Ltd. Enzyme), and the prepared enzyme is a lipase agent prepared by treating commercially available lipase 2 as shown in Example 1 below.

Example 1 Medium melting point portion (iodine value 32.5) obtained by solvent fractionation of palm oil 10
250 parts of 0 parts and commercially available stearic acid (Lunack S-90, 95% stearic acid purity, manufactured by Kao Corporation) at 40 ° C.
Part of n-hexane. According to the method described in Japanese Patent Application Laid-Open No. 60-251891 related to the application by the present applicant, a commercially available lipase 2 [Talipase (Tanabe Seiyaku Co., Ltd., bacterium external enzyme derived from Rhizopus delemer)], decomposition activity 4800 unit / g, was added to the solution.
Synthetic activity 0.42 unit / g] was added and 50 parts of the prepared enzyme (decomposition activity 2,500 unit / g, synthetic activity 1.20 unit / g, ratio of synthetic activity to decomposition activity 4.8 × 10 ⁻⁴ ) was added for 24 hours. The reaction was carried out. The decomposition activity at this time is equivalent to 1,250 units / weight of oil / fat (g) (hereinafter, based on the same standard) (0.60 unit / weight of oil / fat (g) as synthetic activity). The water content in the reaction system at this time was 0.023 parts per 1 part of oil and fat.

After the reaction was completed, the product was recovered, and the triglyceride fraction was separated by silica gel column chromatography (developing solvent, n-hexane: ethyl ether = 90: 10). The fractionated triglyceride fraction was analyzed for its alkyl group composition by gas chromatography as a methyl ester according to the standard oil and fat analysis test method.

Based on the amount of stearic acid incorporated in the triglyceride by the reaction, the progress of the transesterification reaction was calculated by calculating the reaction rate when the equilibrium value represented by the following formula was 100%. The reaction rate at this time was 96.3%, showing the result that the reaction was sufficiently performed.

Reaction rate% (after t hours) = 100 × (St−So) / (S∞−So) In the above formula, St; Stearic acid content in oil / fat at time t So; Stearic acid in raw oil / fat before reaction Content S ∞; 1,3 means the content of stearic acid at random equilibrium.

Example 2 In Example 1, the enzyme after the completion of the reaction was recovered,
The raw materials described inside were newly added and the reaction was repeated. The reaction time at this time was constant at 24 hours for each time.

No decrease in activity was observed up to the 10th repetition (reaction rate 100%). When the action was further repeated, the reaction rate became 83% at the 14th time.

Example 3 Palm oil mid-melting point (iodine value: 31.5, diglyceride content: 1.6)
%) 100 parts and commercially available stearic acid (Lunac S-90, stearic acid purity 95%, manufactured by Kao Corporation) 90 parts at 40 ° C.
It was dissolved in 0 part of n-hexane. Commercially available revertase 1 (Olipase 4S manufactured by Osaka Bacterial Research Institute, decomposition activity 1,500u) was added to the solution.
40 parts of nit / g, synthetic activity 1.40 unit / g, ratio of synthetic activity to decomposition activity 9.333 × 10 ⁻⁴ ) were added and reacted for 20 hours. The synthetic activity at this time corresponded to 0.56 unit / weight of fat (g) (as decomposition activity 600 unit / weight of fat (g)). The water content in the reaction system at this time was 0.0214 parts to 1 part of oil and fat.

When the reaction rate was determined from the stearic acid content of the oil and fat obtained by carrying out the same treatment as in Example 1 after recovering the product after reacting for 20 hours, a reaction equilibrium value of 100% was reached.
A further portion was removed by Florisil column chromatography (developing solvent, n-hexane: diethyl ether = 5: 5) to remove the fatty acid, and the diglyceride content produced as a by-product was analyzed by reversed-phase high performance liquid chromatography (stationary phase = Hitachi Gel 3057). ,
As a result of analysis by Hitachi Chemical Co., Ltd. ODS silica, eluent = acetone: acetonitrile = 5: 5), the increase in diglyceride was only 4.7%.

Example 4 In the same manner as in Example 3, the amount of commercially available lipase 1 used was 80 parts (synthetic activity 1.02 unit / weight of fat (g)), 160 parts (synthetic activity 2.04 unit / weight of fat (g)), 320 parts ( Synthetic activity 4.08un
The same reaction was performed as it / fat weight (g)),
When 0.2 unit or more / fat weight (g), which is sufficient as the synthetic activity unit, was used, it was confirmed that the reaction reached equilibrium within a shorter time than expected from the proportional relationship of the enzyme concentration.

Example 5 The enzyme after completion of the reaction in Example 3 was recovered,
The raw materials described inside were newly added and the reaction was repeated. The reaction time at this time was constant at 20 hours for each time.

No decrease in activity was observed up to the 11th repetition (reaction rate 100%). After further repeated use, the reaction rate became 85% at the 16th time.

Example 6 When the amount of commercial lipase 1 used in Example 4 was set to 160 parts, the enzyme was recovered in the same manner as in Example 5 and repeatedly reacted with the new raw material. The reaction time at this time was constant at 4 hours.

No decrease in activity was observed up to the 80th repetition (reaction rate 100%). Repeatedly, 120
The reaction rate was 85% at the first time.

Comparative Example 1 The same operation as in Example 1 was performed except that the amount of the prepared enzyme was changed to 10 parts. The decomposition activity at this time is 250 unit / weight of fat (g) (as synthetic activity, 0.12 unit / weight of fat (g))
Met. The water content in the reaction system at this time was 0.0048 parts to 1 part of oil and fat. The reaction rate after reacting for 24 hours is 33.6.
It was only%. When the reaction was continued for 72 hours, the reaction rate became 95.8%, but when the enzyme was recovered and repeatedly reacted for 72 hours as in Example 5, the reaction rate dropped to 65% at the third time. .

Comparative Example 2 The same operation as in Example 3 was performed except that the addition amount of the commercially available lipase 1 was changed to 10 parts in Example 3. The synthetic activity at this time is 0.14 unit / weight of fat (g) (150un as decomposition activity)
Equivalent to it / fat weight (g)). The water content in the reaction system was 0.55% with respect to the fat and oil. The reaction time was 96 hours, and the reaction rate was 99.7%.

The enzyme was recovered in the same manner as in Example 5 and repeatedly reacted with a new raw material. The reaction time at this time was constant at 96 hours. A rapid decrease in the reaction rate was observed at the fourth repetition (reaction rate 55%).

As a result, in a system having a sufficiently high enzyme concentration of 500 units or more per weight of fat (g) as a decomposition activity, the activity decrease during repeated use of the enzyme is reduced, and the productivity of transesterified product per unit weight of the enzyme is improved. Was confirmed. In addition, the reaction reached equilibrium in a short time in a system in which a lipase and / or a lipase agent with a sufficiently high synthetic activity ratio was used in a sufficient amount of 0.2 unit or more / fat weight (g) as synthetic activity with respect to the oil or fat as a substrate. It was approved to do.

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Claims (4)

[Claims] 1. A lipase for performing a transesterification reaction between a fat and oil and a fatty acid or a fatty acid ester, a transesterification reaction between fats and oils, or a transesterification reaction between a fat and oils and alcohols using a lipase or an enzyme preparation containing a lipase. Alternatively, the enzyme preparation containing lipase is subjected to transesterification by adjusting the concentration such that the decomposition activity is 500 to 20,000 unit / reactive substrate weight (g) and the synthetic activity is 0.05 unit or more / reactive substrate weight (g). Characterized by that
Transesterification method for fats and oils. 2. The ratio of synthetic activity to degrading activity is 1 × 10 ^-4.
The method for transesterifying oils and fats according to claim 1, which is as described above. 3. The amount of water in the reaction system for activating the lipase or the lipase-containing enzyme drug is 0.005 to 0.2 part by weight per 1 part by weight of the reaction substrate.
Item 4. The method for transesterification of fats and oils according to Item 2. 4. The method for transesterification of fats and oils according to claim 1, 2 or 3, wherein lipase or an enzyme drug containing lipase is repeatedly used after the transesterification reaction.