Method for reduction of re-esterification lypase activity and method for re-esterification

FIELD: medicine.

SUBSTANCE: what is presented is a method for reduction of re-esterification activity of lipase prepared of Thermomyces sp. and immobilised on a carrier, or a lypase powder mixture which contains a filter excipient and lypase prepared of Thermomyces sp. immobilised on the carrier. The lipase powder mixture is ground to average particle size 1 mcm to 300 mcm. The method is implemented by washing said lypase or lypase powder mixture in triacylglycerol. What is also presented is a method for re-esterification. It involves conducting a re-esterification reaction with the use of said lypase or lypase powder mixture. Then lypase or lypase powder mixture is separated from the reaction system and washed in triacylglycerol for reduction of re-esterification activity. One more re-esterification reaction is conducted with the use of reduced lypase or lypase powder mixture.

EFFECT: method provides effective reduction of low re-esterification activity of lypase or lypase powder mixture to be re-used in the re-esterification reaction.

10 cl, 3 dwg, 1 tbl, 3 ex

 

The technical field to which the invention relates

The present invention relates to methods for recovery of lipase activity, such as the ability of a specific immobilized lipase or lipase powder mixture to form various reactions of esterification or interesterification. The present invention also relates to the reactions of esterification or ways interesterification of oils and fats using immobilized lipase or lipase powder mixture.

The level of technology

Lipase is widely used in esterification reactions between various carboxylic acids such as fatty acids, and alcohols, such as monatomic and polyatomic alcohols; or in the interesterification reaction of esters of carboxylic acids. Including the interesterification reaction is an important technology for modification of animal and vegetable fats and for the production of esters of various fatty acids, esters, sugars and steroids. When lipase, which is hydrolases fats and oils, is used as the catalyst for these reactions, the interesterification reaction can be conducted under mild conditions from room temperature to about 70°C. Thus, in comparison with standard chemical reactions, lipase not only inhibits side reactions and reduces energy costs is about and has a high level of security, because lipase as the catalyst is a natural product. Next, the target compounds can be effectively carried out due to their positional and substrate specificity. However, although lipase powder is used directly in the interesterification reaction, the lipase activity is manifested not completely. In addition, it is difficult to evenly dispersing the lipase, which is mainly soluble in water, in a wet substrate is oil-based, and it is also difficult to collect her. Normally, therefore, the lipase immobilized in certain media, such as anion-exchange resin (Patent document 1), finalarray resin (Patent document 2), a hydrophobic carrier (Patent document 3), cation-exchange resin (Patent document 4) and chelating resin (Patent document 5), and is used in the esterification reaction or transesterification.

However, since the immobilization of lipase on the carrier of its lipase activity is reduced, various technologies have been developed with the use of lipase powder.

In particular, a method is proposed, which includes the stage of dispersion lipase powder in the raw substrate containing ether(s) in the presence or absence of an inactive organic solvent, where the interesterification reaction from 90% of the particles of the dispersed lipase powder have a size of from 1 to 100 μm; and the ATEM conduct of the interesterification reaction (Patent document 6). Further it is also proposed to use the enzyme powder obtained by drying the enzyme suspension containing phospholipid(s) and fat soluble(e) vitamin(s) (Patent document 7).

At the same time as lipase which is an enzyme that is expensive, after completion of the reaction it is collected and re-use, and throw away when there is a significant reduction in lipase activity. However, if reduced lipase activity can be restored, the practicality of using lipases will increase significantly. Therefore, from the point of view of industrial production, creating an efficient method of recovery of lipase activity is of great practical interest.

Patent document 1: JP-A60-98984

Patent document 2: JP-A61-202688

Patent document 3: JP-A2-138986

Patent document 4: JP-A3-61485

Patent document 5: JP-A1-262795

Patent document 6: JP-B2668187

Patent document 7: JP-A2000-106873

The invention

The purpose of this invention is to provide methods for recovering reduced lipase activity.

Another aim of the present invention is to provide methods of esterification or interesterification, each of which includes a step of using immobilizovannoi lipase or lipase powder mixtures, each of which has a Voss is set out lipase activity.

The present invention was made on the basis of detection of the fact that the original lipase activity of immobilized lipase or lipase powder mixture, which contains the crushed product of the immobilized lipase and an auxiliary filtering material can be recovered by washing the above lipase or lipase powder mixtures with reduced activity of triacylglycerides.

In particular, the present invention provides a method of restoring lipase activity, which includes stages of use of the lipase derived fromThermomyces sp.and immobilized on a carrier, or lipase powder mixture, which contains an auxiliary filtering material and a lipase derived fromThermomyces sp.and immobilized on the carrier, ground to an average particle size of from 1 μm or more to less than 300 μm in the reaction of esterification or interesterification; and washing the specified lipase or lipase powder mixture with triacylglycerides.

The present invention also provides a method of esterification or interesterification, which includes stages of use of the lipase derived fromThermomyces sp.and immobilized on a carrier or lipase powder mixture, which contains an auxiliary filtering material and a lipase derived fromThermomyces sp.and immobilized on the ositelu, crushed to an average particle size of from 1 μm or more to less than 300 μm in the reaction of esterification or interesterification; Department of lipase or lipase powder mixture from the reaction system and washing their triacylglycerides to restore their lipase activity; and then carrying out the esterification reaction or transesterification using the obtained immobilized lipase or lipase powder mixture.

Brief description of drawings

Figure 1 illustrates the reduction preterition activity in time when using lipase powder mixture in the interesterification reaction (Example 1(3)).

Figure 2 illustrates that, in accordance with the present invention, when laundering lipase powder mixture, preterition activity which has been reduced, there is a restoration of its preterition activity (Example 1(4)).

Figure 3 illustrates that, in accordance with the present invention, when laundering immobilized lipase, preterition activity which has been reduced, there is a restoration of its preterition activity (Example 3(3-2)).

The best option of carrying out the invention

The lipase used in the present invention, was obtained from Thermomyces sp. and immobilized on a carrier, preferably a carrier of silicon dioxide. At present the eat the invention can be applied directly specified lipase or use specified lipase, which is crushed to an average particle size of from 1 μm or more to less than 300 microns. In particular, preferably, the lipase is immobilized on a carrier of silica with a particle size of from about 300 to 1000 μm, was crushed to an average particle size of from 1 μm to 300 μm. This immobilized lipase can be obtained, for example, from the company Novozymes A/S, which produces the product Lipozyme TL-IM.

When grinding such immobilized lipase is preferable to use the standard mill and grind it to an average particle size of from 1 μm or more to less than 300 μm, preferably from 1 μm to 200 μm, preferably from 1 μm to 100 μm and particularly preferably from 20 μm to 100 μm. Examples of mills include mortar, rod drum mill mill mill, millstone (Mycolloider, Masscolloider), coffee mill, electric mill, pin mill, impact mill (hammer mill or ball mill), roller mill, electric mill, a homogenizer and an ultrasonic mill.

When the lipase used in this invention is the product of the above-mentioned pulverization, it is preferable to use in combination with an auxiliary filtering material. Examples of auxiliary filtering material include inorganic filter material is ialy, such as celite and the organic auxiliary filtering material, such as fiber, for example, cellulose, and their milled products. Among them, preferred organic auxiliary filter materials, especially preferred organic polymeric auxiliary filter materials are particularly preferred cellulose and similar materials. Preferred examples thereof include brand: KC Flock, produced by Nippon paper Chemicals Co., Ltd. Preferably, the filter material was in powder form and had an average particle size of from 10 to 90 microns.

The preferred mass ratio of the above-described product of grinding of the lipase to the auxiliary filtering material is from 1/10 to 10/1, and particularly preferably from 1/7 to 2/1.

Although the above-described immobilized lipase or lipase powder mixture used in the present invention, can be used directly in the esterification or interesterification of oils and fats, they can be cleaned during their interaction with triglycerides of fatty acids with long chain and triglycerides of fatty acids with medium chain length; after which they can be separated. At the same time, it is possible to increase lipase activity.

Because it uses a triglyceride of fatty acids with long the th circuit and the triglyceride fatty acids with medium chain length for washing the immobilized lipase or lipase powder mixture, it is preferable to use those listed in the following section.

Preferably the use of a triglyceride of fatty acids with long chain triglycerides of fatty acids with medium chain length in a weight ratio of from 95:5 to 50:50, and it is preferable to add from 2 to 100 mass of triglyceride on the total weight of the lipase.

The reaction of esterification using lipase or lipase powder mixture, in the preferred embodiment is a process comprising a stage of esterification of fats and oils in the presence of immobilized lipase or lipase powder mixture; then the collection of immobilized lipase or lipase powder mixture and reuse.

In particular, since lipase activity and suitability for the esterification reaction and a transesterification of the above lipase powder mixture is recovered enough for recycling and reuse in the reaction, it is possible to appropriately use the mixture in the modification of fats and oils in industrial scale by interesterification.

However, when repeating the processing and the use of such lipase powder mixture or immobilized lipase in the reaction of esterification or interesterification, lipase activity, such as the ability to esterification and interesterification, reduced depending on the frequency of use.

N the present invention makes it possible to increase lipase activity of this immobilized lipase or lipase powder mixture, lipase activity of each of which is reduced, when washing in special conditions, and for lipase powder mixture for a long time remain elevated lipase activity and reusability.

In the present invention immobilized lipase or lipase powder mixture, each of which has a reduced lipase activity, and may include those where the original lipase activity even slightly reduced. However, from the viewpoint of industrial use, it is preferable to choose such lipase, the initial activity (100%) reduced to level from 70% to 50%.

Meanwhile, it is preferable that triacylglycerol used for washing the immobilized lipase or lipase powder was in a liquid state at room temperature. Particularly preferable to use a mixture of triglycerides of fatty acids with long chain triglycerides of fatty acids with medium chain length, each of which is used for purification of lipase powder mixture.

If you are using a triglyceride of fatty acids with long-chain, preferably the residue of a fatty acid such triglyceride contains from 14 to 24 carbon atoms, also most preferably, the vegetable oil is selected from the group consisting of canola oil, soybean oil, podzone the aqueous oil safflower oil and corn oil.

For the triglyceride fatty acids with medium chain length preferably the residue of a fatty acid such triglyceride contains from 6 to 12 carbon atoms. Such triglycerides of fatty acids can be obtained by a commonly known method or use of the product available on the market. Examples of brand name products available on the market include ODO, produced by The Nisshin OilliO Group, Ltd.

Preferably the use of a triglyceride of fatty acids with long chain triglycerides of fatty acids with medium chain length, mass ratio of from 95:5 to 50:50 and it is better to add from 2 to 100 mass. triglycerides on the total weight of lipase, most preferably from 5 to 50 mass. triglycerides on the total weight of the lipase.

In particular, triacylglycerol used for washing, and preferably is crude oil for interesterification.

Preferably, the immobilized lipase or lipase powder mixture was filtered so that the above-mentioned lipase or lipase lipase in the mixture could sufficiently to contact with the above described triacylglycerides. In particular, it is preferable to perform washing with mixing and dispersing immobilized lipase or lipase powder mixture used in the esterification reaction or the transesterification in t is acylglycerides; and separate them from triacylglyceride.

Communication, more specifically stirring, preferably at a temperature of from 10°to 45 ° C, particularly preferably at room temperature; preferably from 2 hours or more, more preferably 10 hours or more, particularly preferably from 12 to 48 hours. If desired, it can be 48 hours or more. The choice of mixer for mixing is not particularly limited, and it is preferable to use a propeller stirrer, magnetic stirrer, three-phase motor or similar device.

Thus, the immobilized lipase or lipase powder mixture in sufficient contact with triacylglycerides; filtered in the usual way to separate the immobilized lipase or lipase powder mixture from triacylglyceride; and then reused in the esterification reaction or transesterification.

To date lipase, lipase activity, which was reduced as a result of her participation in various reactions were thrown out. However, according to the method of the present invention, since the lipase activity can be restored, duration of use, the lipase may be increased and the cost of the products produced using the lipase can be reduced. Thus, from the point of view of industrial Prim is the link of the present invention has many advantages.

The following examples disclose the present invention.

Example 1

(1) 1 kg Lypozyme TL-IM firm Novozymes A/S with an average particle size of 800 μm was ground using a pin mill (fine impact mill 100 UPZ) the company Hosokawa Micron Corporation at 17600 Rev/min particle Size of the powdered lipase was measured using the analyzer of the distribution of particle size LA-500 company Horiba, Ltd., and the average size of these particles was equal to 13.8 μm. For the preparation of lipase powder mixture to lipase powder as an auxiliary filter material was added to 1 kg of cellulose powder company Nippon Paper Chemicals Co., Ltd. with an average particle size of 30 microns.

(2) 90 g of bleached canola oil and 10 g ODO (medium chain triglyceride fatty acids) of the company Nisshin OilliO Group, Ltd. was added to 5 g of the thus obtained lipase mixture and stirred 24 hours at room temperature. After that, the mixture was filtered to select lipase mixture. Then use the following method was measured preterition activity of this lipase mixture, and its relative activity was equal to 714, if you take activity Lypozyme TL-IM before chopping for 100.

Method for measuring lipase activity

Lipase mixture was added to the oil, which triolein, tricaprylin was in the ratio of 1:1 (by volume), and reaction was carried out at 60°C. For PR is the procession of time as the sample was selected 10 µl reaction mixture, was diluted in 1.5 ml of hexane and then the solution, which was filtered lipase mixture was taken as a sample for gas chromatography (GC). The solution was analyzed by gas chromatography (column DB-1ht), and the reaction rate was calculated by the following formula. The GC conditions: initial column temperature 150°C, temperature rise of 15°C/min and final temperature of 370°C.

The reaction rate (%)={square S/(area of C24+square S)}×100, where C24 is tricaprylin; S is tricaprylin, where one fatty acid is replaced by oleic; and area is the area of each of them. On the basis of the reaction rate at each point in time using analytical software (Origin ver. 6.1) was calculated reaction rate constant k.

The lipase activity was presented through the relative activity, assuming k for Lipozyme TL-IM is equal to 100.

(3) about 1 wt.% lipase mixture obtained above in (2)was added to 85 g of bleached canola oil company Nisshin OilliO Group, Ltd. and 15 g ODO company Nisshin OilliO Group, Ltd., and for the carrying out of the interesterification reaction was stirred 19 hours at 60°C. after the lapse of time expected rate of interesterification and confirmed the completion of reaction. For the interesterification reaction using gas chromatography was analyzed composition of triglycerides, also was calculated proportional to the ratio exceed the existing substance of the interesterification reaction in the measured sample.

After the lipase reaction mixture was filtered and collected, and the collected lipase mixture was re-used in the interesterification reaction. Further reaction was carried out several times. Change the reaction rate, represented as relative velocity, shown in figure 1.

According to the results of figure 1, it became clear that when the total reaction time is 82 hours, lipase activity lipase mixture is reduced to about 60%.

(4) Lipase mixture described in (3), lipase activity, which decreased to about 60%, was filtered and collected. Collected in this way lipase mixture was added to 18 g of bleached canola oil company Nisshin OilliO Group, Ltd. and 2 g ODO company Nisshin OilliO Group, Ltd. and stirred with a magnetic stirrer for 24 hours at room temperature. After lipase mixture was collected by filtration, was repeated interesterification reaction, as indicated in (3). Change the reaction rate, represented as relative velocity, shown in figure 2.

According to the results of figure 2 it became clear that the lipase activity is restored to the initial activity of 100% at the washing with stirring lipase mixture with reduced lipase activity and that this lipase mixture may be recycled and used many times.

Example 2

(2-1) 90 g of bleached canola oil and 10 g ODO company Nisshin OilliO Group, Ltd. was added to 5 g of lipase mixture obtained in (1) of example 1, and stirred for 2 hours at 60°C. Then, in order to collect lipase mixture, the mixture was filtered. Preterition activity of this lipase mixture was measured in the same way as in example 1, and it was equal to 557.

(2-2) 1.2 wt.% lipase mixture obtained above in (2-1)was added to 100 g of soybean oil and 25 g of fully hydrogenated soybean oil company Yokozeki Fat & Oil Corporation and was stirred for 120 hours at 70°C. Then lipase mixture was collected by filtration. Lipase activity in the part of the collected lipase mixture was measured in the same way as in example 1 (2-2A). Preassembled lipase mixture was dispersed in acetone and filtered. Its the precipitate on the filter was again collected and dispersed in 50 g butter, mixed in a ratio of bleached canola oil: ODO company Nisshin OilliO Group, Ltd.=9:1 (by weight). Then the mixture was filtered to flush and replacement, and was collected lipase mixture. Preterition activity of this lipase mixture was measured in the same way as in example 1(2-2b). Each activity was recorded in relative units in the table 1.

Table 1
The relative lipase activity per mass of the lipase preparation
Before chopping (TL-IM)100
(2-1)557
(2-2a)11
(2-2b)200

Example 3

5 wt.% Lypozyme TL-IM(immobilized lipase) company Novozymes A/S was added to 85 g of bleached canola oil company Nisshin OilliO Group, Ltd. and 15 g ODO company Nisshin OilliO Group, Ltd. and was stirred 19 hours at 60°C for the reaction of transesterification. Over time, calculated the rate of interesterification and confirmed the completion of reaction. For the interesterification reaction using gas chromatography was analyzed composition of triglycerides, also was calculated proportional to the ratio of the reacting substances of the interesterification reaction in the measured sample.

After the reaction described above, the immobilized lipase was filtered and collected, and the collected immobilized lipase was re-used in the interesterification reaction.

Further reaction was carried out several times. Change the reaction rate, presented in the form of the relative velocity, is shown in F. the D.3 (3-1).

The above-described immobilized lipase, lipase activity, which decreased to about 60%, was filtered and collected. Collected thus immobilized lipase was added to 18 g of bleached canola oil company Nisshin OilliO Group, Ltd. and 2 g ODO company Nisshin OilliO Group, Ltd. and mixed with a magnetic stirrer for 24 hours at room temperature. Then by filtering the immobilized lipase was collected, and as shown in (3-1)was repeated interesterification reaction. The change in reaction rate with time, represented as the relative velocity, shown in figure 3 (3-2).

1. Method of recovering preterition activity of lipase derived from Thermomyces sp. and immobilized on a carrier, or lipase powder mixture, which contains an auxiliary filtering material and a lipase derived from Thermomyces sp. and immobilized on the carrier, ground to an average particle size of from 1 μm to 300 μm, by washing the specified lipase or lipase powder mixture triacylglycerides.

2. The method according to claim 1, where triacylglycerol is in a liquid state at room temperature.

3. The method according to claim 1, in which triacylglycerol includes a triglyceride(s) fatty acids with medium chain length.

4. The method according to any one of claims 1 to 3, where triacylglycerol is a crude oil is eletrification.

5. The method according to any one of claims 1 to 3, where washing is carried out with the help of the stages of mixing and dispersion of immobilized lipase or lipase powder mixture used in the interesterification reaction, triacylglyceride; and separating the lipase or lipase powder mixture from triacylglyceride.

6. The method according to any one of claims 1 to 3, where the carrier is silicon dioxide.

7. The method according to any one of claims 1 to 3, where the average particle size of the crushed carrier with immobilized lipase is from 1 to 200 microns.

8. The method according to any one of claims 1 to 3, where the auxiliary filter material is cellulose.

9. The method according to any one of claims 1 to 3, where an auxiliary filtering material is in powder form, the average particle size is from 10 to 90 microns.

10. The way interesterification, which includes stages of use of the lipase derived from Thermomyces sp. and immobilized on a carrier, or lipase powder mixture, which contains an auxiliary filtering material and a lipase derived from Thermomyces sp. and immobilized on the carrier, ground to an average particle size of from 1 μm to 300 μm, the interesterification reaction; separating the lipase or lipase powder mixture from the reaction system and washed her triacylglycerides to restore its preterition activity; and then carrying out the reaction p is reesterification using the obtained lipase or lipase powder mixture.



 

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EFFECT: simple and safe procedure for production of lubricating additive to diesel fuel corresponding to mixture of ethyl ethers of fat acids.

2 cl

FIELD: power industry.

SUBSTANCE: hydrocarbon fuel obtaining method involves contacting of glycerides of fatty acids with C1-C5 alcohol in presence of solid double cyanide of metals as catalyst at temperature of within 150-200°C during 2-6 hours, cooling of the above reaction mixture to temperature within 20-35°C, filtration of reaction mixture for separation of catalyst with further removal of unreacted alcohol from the obtained filtrate by vacuum distillation so that hydrocarbon fuel is obtained; at that, one metal of catalyst is Zn2+, and the second one is Fe ion.

EFFECT: high output of hydrocarbon fuels.

11 cl, 9 ex, 2 tbl

FIELD: chemistry.

SUBSTANCE: present invention relates to a reesterification catalyst and its preparation method. The invention describes a reesterification catalyst of general formula: Zn3M2(CN)n(ROH)·xZnCl2·yH2O, where R is tertiary butyl and M is a transition metal ion selected from Fe, Co and Cr; x lies between 0 and 0.5, y lies between 3 and 5 and n equals 10 or 12. Described is a method of preparing the catalyst, involving the following steps: a) dissolving ZnCb in a mixture of water and tertiary butanol, b) adding the said solution obtained from step a) to an aqueous solution of K4Fe(CN)6 while stirring, c) adding a ternary block copolymer of poly(ethylene glycol)-block-poly(propylene glycol)-block-poly(ethylene glycol) (EO20-PO70-EO20; molecular weight of approximately 5800), dissolved in a mixture of tert-butanol and water, to the above mentioned mixture obtained at step (b) while stirring and at temperature 25°C-70°C, d) filtering the reaction mixture obtained at step (c) to obtain a solid product ad then washing with distilled water and drying at temperature 20-50°C and e) activating the said dried solid product at temperature 150-200°C to obtain the desired reesterification catalyst.

EFFECT: ensuring high catalyst activity even in moderate conditions during reesterification of glycerides, esters of fatty acids and cyclic carbonates during reaction with alcohols; leaching of metal ions from the solid catalyst is not observed.

12 cl, 2 tbl, 12 ex

FIELD: food industry.

SUBSTANCE: invention relates to compositions of breast milk fat substitutes, methods of their production, compositions of fat bases and methods of their production; baby formula containing specified substitutes. Composition of fat base according to invention includes mixture of triglycerides of vegetable origin, characterised by the fact that less than 50% of remains of fatty acids bound in sn-2 position are saturated; and/or amount of remains of saturated fatty acids bound in sn-2 position of glycerin frame makes less than approximately 43.5% of overall amount of remains of saturated fatty acids, 45-65% parts of unsaturated fatty acids in sn-1 and sn-3 positions make parts of oleic acid and/or 7-15% parts of unsaturated fatty acids in sn-1 and sn-3 positions make parts of linoleic acid. Composition of breast milk fat substitute according to invention includes mixture of at least 25% or at least 30% of specified composition of fat base according to the invention and up to 75% or accordingly up to 70% of at least one vegetable oil, in which specified vegetable oil is randomised. Baby formula according to invention includes composition of fat base or composition of breast milk fat substitute.

EFFECT: compositions of fat base make it possible to optimally imitate breast milk fat and are suitable for use in various baby formulas, and methods of production provide for low consumption of fat bases in process of their production.

28 cl, 17 tbl

FIELD: process engineering.

SUBSTANCE: invention relates to oil-and-fat industry. Method and system for fermentative treatment of initial material containing lipides comprises brining initial material in contact with process admixture, passing initial material at, in fact, constant flow rate through treatment system that includes several reactors with ferments and stationary layer connected in series. Reactors with stationary layer may be serviced individually while initial material flow rate being, in fact, constant in cutting one reactor off for servicing purposes. Process admixture is, in fact, dehydrated silicon dioxide with pore size exceeding 150 angstrom. Said admixture may be placed in one or several reactors above layer of ferment, or be placed in pretreatment system that includes one or several reactors.

EFFECT: increased fermentative activity of treatment.

32 cl, 6 dwg, 20 tbl, 6 ex

FIELD: chemistry.

SUBSTANCE: gum is treated with one or more enzymes having PLA activity at temperature of about 40-60°C and pH of about 3-7 for not more than 4 hours, which leads to formation of lysophospholipids and free fatty acids. Gum is treated with one or more enzymes having PLC activity at temperature of about 40-80°C and pH of about 8 or lower for not more than 30 minutes to form diacylglycerols and phosphates.

EFFECT: diacylglycerols and free fatty acids formed from independent reactions are combined in the presence of not less than one of said enzymes to form novel triacylglycerol molecules.

22 cl, 8 dwg, 9 tbl, 16 ex

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