HK1115414B - Lipase powder composition - Google Patents

Description

Lipase powder compositions

Technical Field

The present invention relates to a lipase powder composition which can be suitably used in various esterification reactions, transesterification reactions, and the like, and a method for transesterification of fats and oils using the lipase powder composition.

Background

Lipases are widely used in esterification reactions of various carboxylic acids such as fatty acids with alcohols such as monohydric alcohols and polyhydric alcohols, transesterification reactions between a plurality of carboxylic acid esters, and the like. Among them, transesterification is an important technique as a method for modifying animal and vegetable oils and fats and for producing esters of various fatty acids, sugar esters and steroids. When lipase as a fat or oil hydrolase is used as a catalyst for these reactions, the transesterification reaction can be carried out under mild conditions of room temperature to about 70 ℃ and, as compared with conventional chemical reactions, the lipase as a catalyst is a natural product and therefore is highly safe, while suppressing side reactions and reducing energy consumption. In addition, the target substance can be efficiently produced according to its substrate specificity or position specificity. However, even when lipase powder is used as it is in the transesterification reaction, the activity thereof is not sufficiently exhibited, and it is difficult to uniformly disperse the lipase which is originally water-soluble in the oily raw material, and the recovery thereof is difficult. Therefore, conventionally, lipases have been generally used in esterification reactions, transesterification reactions, and the like by immobilizing them on some carriers, for example, anion exchange resins (patent document 1), phenol adsorption resins (patent document 2), hydrophobic carriers (patent document 3), cation exchange resins (patent document 4), chelate resins (patent document 5), and the like.

However, when a lipase is immobilized on a carrier, the lipase activity is reduced, and thus various techniques using lipase powder have been developed.

Specifically, the following scheme is proposed: a method of dispersing a lipase powder in an ester-containing raw material in the presence or absence of an inactive organic solvent to perform transesterification so that 90% or more of the particles of the dispersed lipase powder are maintained in a particle diameter range of 1 to 100 μm during the transesterification (patent document 6). Further, it has been proposed to use enzyme powder obtained by drying an enzyme solution containing phospholipids and fat-soluble vitamins (patent document 7).

Patent document 1: japanese laid-open patent publication No. 60-98984

Patent document 2: japanese laid-open patent publication No. 61-202688

Patent document 3: japanese laid-open patent publication No. 2-138986

Patent document 4: japanese laid-open patent publication No. 3-61485

Patent document 5: japanese laid-open patent publication No. 1-262795

Patent document 6: japanese patent No. 2668187

Patent document 7: japanese patent laid-open publication No. 2000-106873

Disclosure of Invention

The purpose of the present invention is to provide a lipase powder composition having improved lipase activity and improved handling properties.

It is another object of the present invention to provide a method for transesterification of fats and oils using the lipase powder composition.

It is also an object of the present invention to provide an esterification process using the lipase powder composition.

The present invention has been made based on the finding that the above-mentioned problems can be solved by using a pulverized product of a specific immobilized lipase together with a filter aid.

That is, the present invention provides a lipase powder composition characterized by containing a pulverized product having an average particle size of 1 μm or more and less than 300 μm of a lipase derived from the genus Thermomyces immobilized on a silica carrier and a filter aid.

The present invention also provides a method for transesterification of fats and oils, characterized in that the transesterification of fats and oils is carried out in the presence of a filtration aid and a ground product having an average particle diameter of 1 μm or more and less than 300 μm of a lipase derived from the genus Thermomyces immobilized on a silica carrier, and then the ground product and the filtration aid are recovered and recycled.

The present invention also provides an esterification method characterized by carrying out an esterification reaction in the presence of a pulverized product having an average particle diameter of 1 μm or more and less than 300 μm of a lipase derived from a thermophilic fungus immobilized on a silica carrier and a filter aid, and then recovering the pulverized product and the filter aid and recycling them.

Drawings

FIG. 1 shows the change in the ester exchange rate in the case of using the lipase powder composition of the present invention (example 4) and immobilized lipase before pulverization (Lipozyme TL-IM).

Detailed Description

The lipase used in the present invention is a lipase derived from Thermomycesp, and the lipase immobilized on a silica carrier is pulverized to have an average particle size of 1 μm or more and less than 300 μm. Here, the average particle size of the lipase immobilized on the silica carrier is preferably about 300 to 1000. mu.m. Such immobilized lipase can be, for example, Lipozyme TL-IM obtained from Novozymes A/S.

The immobilized lipase is pulverized by a common pulverizer into particles having an average particle size of 1 μm or more and less than 300. mu.m, preferably 1 to 200. mu.m, more preferably 1 to 100. mu.m, and particularly preferably 20 to 100. mu.m. Examples of the pulverizer include a mortar, a shearing friction pulverizer, a cutting pulverizer, a grindstone (mycolloid, mascholeide), a coffee mill, a power mill, a pin mill, an impact pulverizer (hammer mill, ball mill), a roll pulverizer, a gas flow pulverizer, a homogenizer, and an ultrasonic pulverizer.

On the other hand, examples of the filter aid used in the present invention include inorganic filter aids such as fluoromagnesian; and organic filter aids such as fibers of cellulose or pulverized products thereof. Among these, organic filter aids, particularly organic polymer filter aids are preferable, and among them, cellulose and the like are preferable, and examples thereof include products sold under the trade name KC Flock by NIPPON PAPER chemicultco. The filter aid is also preferably powdery, and preferably has an average particle diameter of 10 to 90 μm.

The mass ratio of the lipase crushed product to the filter aid is preferably 1/10-10/1, and particularly preferably 1/7-2/1.

The lipase powder composition of the present invention must contain the above-mentioned lipase powder and a filter aid.

The lipase powder composition of the present invention can be used for transesterification and esterification of fats and oils as it is, but can be purified and at the same time, the lipase activity can be improved by collecting the lipase powder composition after contacting it with a long-chain fatty acid triglyceride and a medium-chain fatty acid triglyceride.

The long-chain fatty acid triglyceride used herein is preferably a triglyceride having 14 to 24 carbon atoms in a constituent fatty acid, and particularly preferably a vegetable oil selected from the group consisting of rapeseed oil, soybean oil, sunflower oil, safflower oil and corn oil.

The medium-chain fatty acid triglyceride is preferably a triglyceride having 6 to 12 carbon atoms constituting a fatty acid. Such fatty acid triglycerides can be produced by a known production method, and commercially available products can be used. As a commercial product, for example, a product sold by riqing orlistat group ltd under the product name of ODO is available.

Preferably, the long-chain fatty acid triglyceride and the medium-chain fatty acid triglyceride are used in a mass ratio of 95: 5 to 50: 50, and the lipase is preferably contacted with the triglycerides in an amount of 2 to 100 times by mass based on the total mass of the lipase.

As the esterification reaction using the lipase powder composition of the present invention, it is desirable to carry out an esterification reaction of fats and oils in the presence of the lipase powder composition, and then recover and recycle the lipase powder composition.

It is also preferable that the esterification reaction of fats and oils is carried out in the presence of a pulverized product having a particle size of 1 μm or more and less than 300 μm of a lipase derived from a thermophilic fungus immobilized on a silica carrier and a filter aid, and then the pulverized product and the filter aid are collected and recycled to esterify the fats and oils.

According to the present invention, the lipase activity can be improved, the ease of use (handling) in the esterification reaction and the transesterification reaction can be improved, and the lipase can be recycled and used for these reactions many times, and therefore, the lipase can be suitably used for the modification of fats and oils by transesterification of fats and oils on an industrial scale. Further, according to the method of the present invention, an oil or fat having an improved flavor can be provided.

The present invention will be described in more detail with reference to examples.

Examples

Example 1

5g of Lipozyme TL-IM by Novozymes A/S was pulverized using L-type MYCOLLOIDER manufactured by Special industries. The particle size of the lipase after pulverization was measured using a particle size distribution meter LA-500 manufactured by horiba Ltd, and the average particle size was 66.4. mu.m. To the powder was added 5g of cellulose powder (NIPPON PAPERE CHEMICALS CO., LTD: 30 μm in average particle diameter) as a filter aid to prepare a lipase powder composition. The transesterification activity of the lipase composition was measured by the following method and is shown in table 1 as a relative value.

Method for measuring lipase activity

To an oil obtained by mixing triolein and tricaprylin in a ratio of 1: 1(w), a lipase composition was added and allowed to react at 60 ℃. 10. mu.l of the lipase composition was sampled with time, diluted with 1.5ml of hexane, and the obtained solution was used as a sample for Gas Chromatography (GC). The reaction rate was determined by the following formula by GC (column: DB-1ht) analysis. GC conditions were column temperature: the temperature was initially 150 deg.C, increased by 15 deg.C/min, and finally 370 deg.C.

Reaction rate (%) { C34area/(C24area + C34area) } × 100

In the formula, C24 represents tricaprylin, C34 represents a substance in which one fatty acid of tricaprylin is replaced with oleic acid, and area is their peak area. The reaction rate constant k was determined from the reaction rate at each time by analytical software (Origin ver.6.1).

The lipase activity was expressed as relative activity when the k value of Lipozyme TL-IM was set to 100.

Example 2

5g of Lipozyme TL-IM manufactured by Novozymes A/S was pulverized using a mortar. The particle size of the lipase after pulverization was measured using a particle size distribution meter LA-500 manufactured by horiba Ltd, and the average particle size was 52.1. mu.m. To this powder was added 2.5g of cellulose powder (NIPPON paperemicals co., LTD) as a filter aid to prepare a lipase powder composition. The transesterification activity of the lipase composition was measured in the same manner as in example 1, and the relative values are shown in table 1.

Example 3

50g of rapeseed oil was added to 5g of Lipozyme TL-IM manufactured by Novozymes A/S, and the mixture was pulverized at 12500rpm for 3 minutes under ice-cooling using a homogenizer (Multipro395) manufactured by DREMEL. The particle size of the lipase after pulverization was measured using a particle size distribution meter LA-500 manufactured by horiba Ltd, and the average particle size was 91.5. mu.m. To this was added 5g of cellulose powder (NIPPON PAPERE CHEMICAL S co., LTD) as a filter aid, and rapeseed oil was filtered under reduced pressure to obtain a lipase composition. The transesterification activity of the lipase composition was measured in the same manner as in example 1, and the relative values are shown in table 1.

TABLE 1

	Average particle diameter (μm)	Relative transesterification Activity with respect to Lipase preparation Mass
			(TL-IM) before comminution example 1 example 2 example 3	80066.452.191.5	100201304202

Example 4

To 5g of the lipase composition obtained in example 1, 90g of rapeseed oil and 10g of ODO (Nisshin Oillio group Co., Ltd.) were added, and the mixture was stirred at 25 ℃ for 5 hours, followed by filtration to recover the lipase composition.

Subsequently, 0.5% of the lipase composition pretreated by the above method was added to 150g of ODO (Nisshin Oillio Co., Ltd.) and 850g of rapeseed oil (Nisshin Oillio Co., Ltd.), and the mixture was stirred at 50 ℃ for 15 hours to conduct a transesterification reaction. The transesterification rate was determined with time, and the progress of the reaction was confirmed. The glyceride composition was analyzed by a gas chromatograph, and the ratio of the transesterification reaction product in the measurement sample was calculated to determine the transesterification ratio. The change in the ester exchange ratio between this example and lipozyme TL-IM (before pulverization) is shown in FIG. 1.

After the reaction, the lipase composition was filtered and recovered. The obtained oil was deacidified, decolored and deodorized by a conventional method to purify the ester-exchanged oil. The flavor of the obtained transesterified oil was evaluated by a trained panel. As a result, the same transesterification method as that used for the lipase composition which had not been pretreated with rapeseed oil and ODO was used to obtain a transesterified oil, which exhibited a significantly better flavor.

Further, the recovered enzyme was used to carry out the transesterification reaction, and as a result, recycling was carried out 10 times without any problem.

Example 5

1Kg of Lipozyme TL-IM manufactured by Novozymes A/S was pulverized at 17600rpm using a pin pulverizer (precision impact pulverizer 100UPZ) manufactured by Hosokawa Micron. The particle size of the lipase after pulverization was measured using a particle size distribution meter LA-500 manufactured by horiba Ltd, and the average particle size was 13.8. mu.m. To this powder was added 1Kg of cellulose powder (NIPPONPAERE CHEMICALS CO., LTD: 30 μm in average particle diameter) as a filter aid to prepare a lipase powder composition.

Example 6

To 5g of the lipase composition obtained in example 5, 90g of decolorized rapeseed oil and 10g of ODO (produced by Olympic corporation, Nisshin) were added, and the mixture was stirred at 60 ℃ for 2 hours, followed by filtration to recover the lipase composition. The transesterification activity of the lipase composition was measured in the same manner as in example 1, and the relative values are shown in table 2.

Example 7

To 5g of the lipase composition obtained in example 5, 90g of decolorized rapeseed oil and 10g of ODO (manufactured by Olympic corporation, Nisshin) were added, and the mixture was stirred at room temperature for 24 hours, followed by filtration to recover the lipase composition. The transesterification activity of the lipase composition was measured in the same manner as in example 1, and the relative values are shown in table 2.

Example 8

50g of decolorized rapeseed oil and 50g of ODO (produced by Oillio Rivietyol Co., Ltd., Nisshin) were added to 5g of the lipase composition obtained in example 5, and the mixture was stirred at room temperature for 24 hours, followed by filtration to recover the lipase composition. The transesterification activity of the lipase composition was measured in the same manner as in example 1, and the relative values are shown in table 2.

TABLE 2

	Relative transesterification Activity with respect to Lipase preparation Mass
		(TL-IM) example 5 example 6 example 7 example 8 before comminution	100474557714600

Claims (4)

1. A lipase powder composition characterized by containing a lipase powder having an average particle diameter of 13.8 μm derived from Thermomyces and a cellulose powder having an average particle diameter of 30 μm as a filter aid, which are immobilized on a silica carrier, the mass ratio of the lipase powder to the cellulose powder being 1: 1.

2. The composition of claim 1, which is ester-exchangeable or esterified.

3. A process for transesterification of fats and oils, which comprises conducting the transesterification of fats and oils in the presence of the lipase powder composition as defined in claim 1 or 2, and subsequently recovering and recycling the lipase powder composition.

4. An esterification method characterized in that an esterification reaction is carried out in the presence of lipase powder having an average particle diameter of 13.8 μm derived from the genus Thermomyces immobilized on a silica carrier and cellulose powder having an average particle diameter of 30 μm as a filter aid, and then the pulverized product and the filter aid are recovered and recycled, the mass ratio of the lipase powder to the cellulose powder being 1: 1.