JP2007068426A - Lipase powder preparation, method for producing the same and use of the same - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a lipase powder preparation improved in lipase activity and stability, and to provide a method for producing the same. <P>SOLUTION: The lipase powder preparation consists of granules comprising a lipase and bean protein. Another version of this preparation comprises the above two components and fat-and-oil. The method for producing the lipase powder preparation comprises evaporating an aqueous solution with the lipase and the bean protein dissolved/dispersed therein to dryness. <P>COPYRIGHT: (C)2007,JPO&INPIT

Description

  The present invention provides a lipase powder preparation that can be suitably used for various esterification reactions, transesterification reactions, and the like, a lipase powder preparation containing a fatty acid ester and / or a fatty acid, a production method thereof, and a lipase powder preparation thereof. It relates to a transesterification method to be used.

Lipases are widely used in esterification reactions of various carboxylic acids such as fatty acids with alcohols such as monoalcohols and polyhydric alcohols, and transesterification reactions between a plurality of carboxylic acid esters. Among these, the transesterification reaction is an important technique as a method for producing various fatty acid esters, sugar esters and sterol esters, including the modification of animal and vegetable fats and oils. When lipase, which is an oil and fat hydrolase, is used as a catalyst for these reactions, a transesterification reaction can be carried out under mild conditions of room temperature to about 70 ° C., which suppresses side reactions compared to conventional chemical reactions. In addition to reducing the cost of energy and energy, the lipase as a catalyst is a natural product, so safety is high. Moreover, the target product can be produced efficiently due to its substrate specificity and position specificity. However, even if the powder lipase is used as it is in the transesterification reaction, the activity is not sufficiently exhibited, and it is difficult to uniformly disperse the originally water-soluble lipase in the oily raw material, and the recovery thereof is also difficult. .
For this reason, conventionally lipase is used as some kind of carrier, for example, anion exchange resin (Patent Document 1), phenol adsorption resin (Patent Document 2), hydrophobic carrier (Patent Document 3), cation exchange resin (Patent Document 4), chelate. It is common to fix to resin (patent document 5) etc. and to use for esterification or transesterification. Further, an emulsion in which an aqueous phase in which a lipase and a substance acting as a carrier for lipase are dissolved is dispersed in a hydrophobic phase is produced, and water is removed from the emulsion, whereby the aqueous phase is coated with a lipase. A method for producing immobilized lipase particles to be changed into particles has been proposed (Patent Document 6).

Thus, conventionally, lipase was immobilized and used for transesterification, but such immobilized lipase is not only accompanied by a loss of the original lipase activity due to the immobilization treatment, but also when a porous support is used. The pores were clogged with raw materials and products, resulting in a decrease in the transesterification rate. Furthermore, in the transesterification reaction using the conventional immobilized lipase, since the water held by the carrier is brought into the reaction system, it is not possible to avoid side reactions such as the formation of diglycerides and monoglycerides in the transesterification reaction of fats and oils. It was difficult.
In view of such a situation, various techniques using powder lipase have been developed. For example, in the presence or absence of an inert organic solvent, the powder lipase is dispersed in the raw material containing the ester so that 90% or more of the dispersed lipase powder particles are maintained at a particle size in the range of 1 to 100 μm during the transesterification reaction. And a method for carrying out transesterification has been proposed (Patent Document 7). It has also been proposed to use an enzyme powder obtained by drying an enzyme solution containing phospholipids and fat-soluble vitamins (Patent Document 8).
However, there is a need for a powder lipase with further improved lipase activity and stability.

JP-A-60-98984 JP-A-61-202688 Japanese Patent Laid-Open No. 2-138986 Japanese Patent Laid-Open No. 3-61485 Japanese Patent Laid-Open No. 1-262795 Japanese Patent No. 3403202 Japanese Patent No. 2668187 JP 2000-106873 A

An object of the present invention is to provide a lipase powder formulation having improved lipase activity and stability.
Another object of the present invention is to provide a lipase powder preparation further containing a fatty acid ester and / or a fatty acid.
Another object of the present invention is to provide a method for producing these lipase powder formulations.
Another object of the present invention is to provide a transesterification method and an esterification method using these lipase powder formulations.

Since the present invention was made on the basis of the knowledge that when a granulated product containing lipase and legume protein is used as a lipase powder preparation for esterification and / or transesterification, the lipase activity and stability are significantly improved. is there.
That is, the present invention provides a lipase powder preparation characterized by being a granulated product containing lipase and legume protein.
The present invention further provides a lipase powder formulation containing a fatty acid ester and / or a fatty acid.
The present invention also provides a method for producing a lipase powder preparation characterized by drying an aqueous solution in which lipase and legume protein are dissolved and dispersed.
The present invention also provides a method for producing a lipase powder formulation further comprising a step of bringing a fatty acid ester and / or a fatty acid into contact with an aqueous solution in which lipase and legume protein are dissolved and dispersed.
The present invention also provides a lipase powder formulation that can be used for transesterification or esterification.
The present invention also provides a transesterification product and / or an esterification product obtained using a transesterification and / or esterification lipase.

ADVANTAGE OF THE INVENTION According to this invention, the lipase powder formulation which carried out transesterification reaction or esterification reaction efficiently, can collect | recover after a reaction and can be reused, and the enzyme activity and stability improved significantly can be obtained. .
In addition, according to the present invention, a lipase that can be used safely and inexpensively to produce a food or food additive for people who cannot take animal-derived protein or fat for religious reasons or health reasons. A powder formulation can be obtained.

Examples of the lipase used in the present invention include lipoprotein lipase, monoacyl glycero lipase, diacyl glycero lipase, triacyl glycero lipase, galacto lipase, and phospho lipase. Of these, triacylglycerolipase is preferred.
The microorganisms that produce these lipases are not particularly limited to bacteria, yeasts, filamentous fungi, actinomycetes, etc., but include Pseudomonas sp., Alcaligenes sp., Asrobacter sp. sp.), Staphylococcus sp., Torulopsis sp., Escherichia sp., Mycotorula sp., Propionibacterum sp., Chromo Bacteria (Chromobacterum sp.), Xanthomonas sp., Lactobacillus sp., Clostridium sp., Candida sp., Geotrichum sp .), Sacchromycopsis sp., Nocardia sp., Fusarium sp., Asper Aspergillus sp., Penicillium sp., Mucor sp., Rhizopus sp., Phycomyces sp., Puccinia sp., Bacillus (Bacillus sp.), Streptomyces genus (Streptmycese sp.), Thermomyces genus (Thermomyces sp.) And the like.
Of these, 1,3-specific lipases are preferred in the present invention, and in particular, 1,3-specific lipases derived from the genera Rhizomucor, Alkagenes, Rhizopus, and Thermomyces are preferred. Rhizomucor miehei, Alcaligenes sp. From the genus Algegenes, Rhizopus delemar, Rhizopus oryzae, and the Rhizopus oryzae, and the Rhizopus oryzae, the Rhizopus oryzae-specific Rhizomuthus genus L. preferable. Conventionally, Rhizomucor miehei may be treated as a genus Mucor (Mucor sp.).
The lipase used in the present invention includes those obtained by drying a lipase-containing aqueous solution containing a lipase medium component and the like.

Examples of the lipase derived from Rhizopus oryzae that can be used in the present invention include Amano Enzyme's product: Lipase F-AP15. This is a powder lipase.
Examples of lipases derived from Rhizopus delemar include Amano Enzyme's product: Lipase D. This is a powder lipase. Note that this lipase D is currently derived from Rhizopus oryzae, but conventionally it was derived from Rhizopus delemar.
Examples of lipases derived from Alcaligenes sp. Include products from the famous sugar industry: Lipase QLM. This is a powder lipase.
Examples of the lipase derived from Rhizomucor miehei include Novozymes' product: Palatase 20000L. Patalase is marketed in a form in which lipase is dissolved and dispersed in an aqueous solution, and the aqueous solution is filtered using an ultrafiltration membrane to remove low molecular components to form an aqueous lipase-containing solution. It can be obtained by spraying using spray drying or freeze drying.
Examples of the lipase derived from Thermomyces lanuginosus include Novozymes' product: Lipozyme TL 100L. The powder lipase can be obtained in the same manner as described above.

Examples of the legume protein used in the present invention include legume proteins such as pea, broad bean, soybean, azuki bean, green beans and cowpea. Of these, pea protein, broad bean protein and soybean protein are preferable, and pea protein and broad bean protein are particularly preferable.
As the bean protein used in the present invention, a protein extracted by a conventional method or a commercially available one can be used. Examples of commercially available products include soy protein and pea protein. In particular, soy protein is classified according to protein content. In order to further improve the activation and stability of the enzyme, the protein content in the legume protein is preferably 60 to 95% by mass, more preferably 70 to 90% by mass.
Examples of the method for measuring the protein content include the Kjeldahl method, the UV method, the Raleigh method, the BCA method, and the like, and the Dumas (combustion) method that can be easily measured in a small amount, and can be obtained by measuring the nitrogen content.
The mass of the legume protein relative to the lipase is preferably 0.1 to 200 times, more preferably 1.0 to 100 times, and most preferably 3.0 to 50 times.

Examples of a method for extracting protein from beans include a method in which beans are crushed and then extracted with water or an alkaline aqueous solution, and the protein is precipitated from the extract with an acid.
More specifically, the beans of the raw material are soaked in water, the absorbed beans are crushed, a sodium hydroxide aqueous solution is added to make a 0.1 N alkaline aqueous solution, and the protein is solubilized in water from the crushed beans. Extract. Thereafter, for example, a dilute hydrochloric acid aqueous solution is added to the solution to form a weakly acidic aqueous solution having a pH of about 3 to 6, and the solubilized protein can be precipitated and precipitated. The acid to be used may be either an inorganic acid or an organic acid, but when hydrochloric acid or the like which is a strong acid is used, it is preferably diluted or used in small amounts in the extract. Examples of the organic acid include citric acid and acetic acid.
In addition, as a method for obtaining protein from soybean, there is also a method for obtaining protein by washing defatted soybean with an acidic aqueous solution or alcohol. Since soybeans have a higher fat content than other beans, it is preferable to extract and separate proteins using defatted soybeans by the method described above.
The bean protein thus precipitated can be separated, for example, by centrifugation at 1,000 to 3,000 × g, and the separated bean protein can be separated by, for example, vacuum drying such as freeze drying, drying by spray drying, or the like. It can be dried to a powder.
In order to prevent deterioration of the obtained protein, when performing protein extraction and drying, do not let the protein come into contact with high concentration strong acid / strong alkaline aqueous solution for a long time or leave it at high temperature for a long time. It is important to.
A protease inhibitor or the like may or may not be added.

The lipase powder preparation of the present invention preferably has a water content of 10% by mass or less, particularly preferably 1 to 8% by mass.
The particle size of the lipase powder formulation of the present invention can be set arbitrarily, but 90% by mass or more of the lipase powder formulation preferably has a particle size of 1 to 100 μm. The average particle size is preferably 10 to 80 μm. The shape of the lipase powder preparation is preferably spherical.
The particle size of the lipase powder preparation can be measured using, for example, a particle size distribution measuring device (LA-500) manufactured by HORIBA.
The lipase powder preparation of the present invention is produced by drying an aqueous solution in which lipase and legume protein are dissolved and dispersed by any one drying method selected from spray drying, freeze drying, and solvent precipitation / drying. be able to.
Here, the aqueous solution in which lipase and legume protein are dissolved / dispersed may be obtained by dissolving / dispersing powder lipase and legume protein in water, mixing powder lipase in an aqueous solution in which legume protein is dissolved / dispersed, or explained later. The lipase-containing aqueous solution can be obtained by mixing legume protein.

In the process of drying the aqueous solution in which lipase and legume protein are dissolved and dispersed, the particles of lipase and / or legume protein aggregate to form a granulated product containing lipase and legume protein. This granulated product may contain a medium component of lipase.
The lipase powder preparation thus prepared can be used for transesterification or esterification as it is.
The amount of water in the aqueous solution in which lipase and legume protein are dissolved and dispersed adjusts the mass of water with respect to the total mass of lipase and legume protein. Specifically, the mass of water relative to the total mass of lipase and legume protein is preferably 0.5 to 1,000 times, more preferably 1.0 to 500 times, and 3.0 to 100 times is most preferable.
In particular, when producing a lipase powder formulation by spray drying, the mass of water relative to the total mass of lipase and legume protein is preferably 2.0 to 1,000 times from the characteristics of the apparatus, 500 times is more preferable, and 3.0 to 100 times is most preferable. If the lipase-containing aqueous solution is used as a raw material and the lipase content in the lipase-containing aqueous solution is unknown, the lipase-containing aqueous solution is pulverized by freeze drying or other reduced-pressure drying to determine the lipase content, and the lipase mass is calculated. can do.

Here, the lipase-containing aqueous solution includes a lipase culture solution from which bacterial cells have been removed, a purified culture solution, a lipase obtained by dissolving and dispersing the lipase in water again, and a commercially available powder lipase dissolved and dispersed in water again. And commercially available liquid lipase. Further, those obtained by removing low molecular components such as salts in order to further increase the lipase activity are more preferable, and those obtained by removing low molecular components such as sugars in order to further improve the powder properties.
Examples of the lipase culture solution include aqueous solutions containing soy flour, peptone, corn staple liquor, K ₂ HPO ₄ , (NH ₄ ) ₂ SO ₄ , MgSO ₄ .7H ₂ O and the like. These concentrations include 0.1 to 20% by weight of soybean flour, preferably 1.0 to 10% by weight, 0.1 to 30% by weight of peptone, preferably 0.5 to 10% by weight, corn staple liquor 0.1 to 30% by weight, preferably from 0.5 to 10 mass%, K ₂ HPO ₄ 0.01 to 20 wt%, preferably from 0.1 to 5 mass%. Further, (NH ₄ ) ₂ SO ₄ is 0.01 to 20% by mass, preferably 0.05 to 5% by mass, and MgSO ₄ · 7H ₂ O is 0.01 to 20% by mass, preferably 0.05 to 5%. % By mass. The culture conditions are as follows: culture temperature is 10 to 40 ° C., preferably 20 to 35 ° C., aeration rate is 0.1 to 2.0 VVM, preferably 0.1 to 1.5 VVM, and stirring speed is 100 to 800 rpm, preferably It is good to control 200-400 rpm and pH to 3.0-10.0, Preferably it is 4.0-9.5.

Separation of the cells is preferably performed by centrifugation, membrane filtration or the like. Moreover, removal of low molecular components such as salts and sugars can be performed by UF membrane treatment. Specifically, by performing a UF membrane treatment, concentrating the aqueous solution containing lipase to a volume of ½ volume, and repeating the operation of adding the same amount of phosphate buffer as the concentrated solution 1 to 5 times, A lipase-containing aqueous solution from which low molecular components have been removed can be obtained.
Centrifugation is preferably 200 to 20,000 × g, and membrane filtration is preferably controlled to 3.0 kg / m ² or less with an MF membrane, filter press or the like. In the case of intracellular enzymes, it is preferable to crush the cells with a homogenizer, Waring blender, ultrasonic crushing, French press, ball mill or the like, and remove cell residues by centrifugation, membrane filtration or the like. The stirring rotation speed of the homogenizer is 500 to 30,000 rpm, preferably 1,000 to 15,000 rpm, and the rotation speed of the Waring blender is 500 to 10,000 rpm, preferably 1,000 to 5,000 rpm. The stirring time is 0.5 to 10 minutes, preferably 1 to 5 minutes. The ultrasonic crushing may be performed under conditions of 1 to 50 kHz, preferably 10 to 20 kHz. The ball mill preferably uses glass spheres having a diameter of about 0.1 to 0.5 mm.
In the middle step before the drying step, the lipase-containing aqueous solution may be concentrated. The concentration method is not particularly limited, but evaporator, flash evaporator, UF membrane concentration, MF membrane concentration, salting out with inorganic salts, precipitation method with solvent, adsorption method with ion-exchange cellulose, water absorption with water-absorbing gel Law. Preferably, UF membrane concentration and an evaporator are used. The UF membrane concentration module is preferably a flat membrane or hollow fiber membrane having a molecular weight cut-off of 3,000 to 100,000, preferably 6,000 to 50,000, and the material is preferably polyacrylonitrile or polysulfone.

Next, spray drying, freeze drying, or solvent precipitation / drying, which is a method of drying an aqueous solution in which lipase and legume protein are dissolved and dispersed, will be described.
Spray drying is preferably performed using a spray dryer such as a nozzle countercurrent type, a disk countercurrent type, a nozzle cocurrent type, and a disk cocurrent type. The disk co-current type is preferable, the atomizer speed is 4,000 to 20,000 rpm, the heating is preferably controlled at an inlet temperature of 100 to 200 ° C., and an outlet temperature of 40 to 100 ° C. and spray dried.
Freeze-drying (freeze-drying) is preferably performed by, for example, a lab-size small-scale freeze-dryer or a shelf-type freeze-dryer. Furthermore, it can also be prepared by drying under reduced pressure.
In solvent precipitation / drying, an aqueous solution in which lipase and legume protein are dissolved / dispersed is gradually added to the solvent used to produce a precipitate, and the resulting precipitate is centrifuged using a centrifuge. After collecting the precipitate, it is dried under reduced pressure. In order to prevent denaturation / deterioration of the lipase powder formulation, the series of operations is preferably performed under a low temperature condition of room temperature or lower.
Examples of the solvent used in the solvent precipitation include water-soluble solvents or hydrophilic solvents such as ethanol, acetone, methanol, isopropyl alcohol, and hexane, and a mixed solvent thereof can also be used. Of these, ethanol or acetone is preferably used in order to further enhance the activity of the lipase powder preparation.
The amount of the solvent to be used is not particularly limited, but it is preferable to use a solvent having a volume of 1 to 100 times the volume of an aqueous solution in which lipase and legume protein are dissolved and dispersed, and a solvent having a volume of 2 to 10 times. It is more preferable to use.
Moreover, after solvent precipitation, the precipitate can be obtained by filtration after standing, but can also be obtained by mild centrifugation at about 1,000 to 3,000 × g. The obtained precipitate can be dried, for example, by drying under reduced pressure.

Next, the lipase powder formulation containing a fatty acid ester and / or a fatty acid will be described. The lipase powder preparation containing the fatty acid ester and / or fatty acid of the present invention may be powder or oily.
The lipase powder preparation containing the fatty acid ester and / or fatty acid of the present invention can be obtained by drying after the step of bringing the fatty acid ester and / or fatty acid into contact with an aqueous solution in which lipase and legume protein are dissolved and dispersed. it can. By making such fatty acid ester and / or fatty acid contact, lipase activity and stability can be further improved.
Examples of the fatty acid ester used include fatty acid esters of monoalcohol or polyhydric alcohol and fatty acid. The fatty acid ester of the polyhydric alcohol may be a partial ester or a full ester.
Here, examples of the monoalcohol include sterols such as alkyl monoalcohol and phytosterol. The alkyl moiety constituting the alkyl monoalcohol is preferably a medium-chain alkyl having 6 to 12 carbon atoms or a long-chain alkyl having 13 to 22 carbon atoms, which may be saturated or unsaturated, and may be linear or branched Good. As phytosterol, for example, sitosterol, stigmasterol, campesterol, fucostol, spinasterol, brush castrol and the like are preferable. Examples of the polyhydric alcohol include glycerin, glycerin condensates such as diglycerin and decaglycerin, glycols such as propylene glycol, and sorbitol.
The constituent fatty acid of the fatty acid ester to be used and the fatty acid to be used are not particularly limited, but are preferably oil-derived fatty acids. For example, a medium chain fatty acid such as hexanoic acid, octanoic acid, decanoic acid, undecanoic acid, etc. having a carbon number of 6-12, oleic acid, linoleic acid, linolenic acid, ricinoleic acid, erucic acid, etc. Examples include chain unsaturated fatty acids. In addition, long-chain saturated fatty acids such as tetradecanoic acid, hexadecanoic acid, octadecanoic acid, eicosanoic acid, and docosanoic acid are also included.

As fatty acid ester used for this invention, 1 type, or 2 or more types chosen from diglyceride and monoglyceride which use fats and oils and fatty acid derived from fats and oils as a structural component are preferable. Moreover, the mixture of partial ester and a fatty acid obtained by hydrolyzing a part of fatty acid ester can also be used.
In addition, it is preferable to select the same fatty acid ester and fatty acid used for the lipase powder preparation as the raw materials used for transesterification and esterification performed using the lipase powder preparation.
Here, the fats and oils used as fatty acid esters in the present invention are not particularly limited, but when a lipase powder preparation is produced by performing a hydrolysis reaction and an esterification reaction, liquid fats and oils may be used at the reaction temperature. preferable.
As fats and oils, for example, rapeseed oil, sunflower oil, olive oil, corn oil, coconut oil, sesame oil, safflower oil, soybean oil, oils of these hyolein varieties, cottonseed oil, rice oil, linseed oil, palm oil, palm oil fractionated oil Plant oils such as palm kernel oil, camellia oil, cacao butter, shea butter, monkey butter, lippe butter, triolein (trioleic acid glyceride), tricaprylin (trioctanoic acid glyceride), triacetin (triacetic acid glyceride) , One or a mixture of two or more fats and oils such as triglycerides (synthetic fats and oils) such as tributyrin (tributyric acid glyceride), animal fats such as fish oil, beef tallow and lard. Among these, vegetable oils and fats are preferable.

Next, the manufacturing method of the lipase powder formulation containing a fatty acid ester and / or a fatty acid is demonstrated.
When using fatty acid ester, or fatty acid ester and fatty acid as raw materials, add fatty acid ester, or fatty acid ester and fatty acid to an aqueous solution in which lipase and legume protein are dissolved and dispersed, and contact with them, using a stirrer or three-one motor. After being hydrolyzed and / or emulsified / dispersed by stirring, the lipase powder formulation can be produced by drying by one drying method selected from spray drying, freeze drying or solvent precipitation / drying. Here, drying can also be performed by dehydration accompanied by an esterification reaction. That is, after hydrolyzing and / or emulsifying / dispersing, an esterification reaction is subsequently performed while dehydrating, and if necessary, an oil such as an unreacted product is filtered to produce a lipase powder formulation. .
Here, the hydrolysis will be described.
The hydrolysis reaction performed by adding a fatty acid ester or a fatty acid ester and a fatty acid to an aqueous solution in which lipase and legume protein are dissolved and dispersed is not particularly limited, but can be performed under normal pressure.
The temperature of the hydrolysis reaction is preferably 20 to 50 ° C, particularly preferably 30 to 45 ° C. When oil and fat is used as a raw material, the timing for terminating the hydrolysis can determine the degree of progress of hydrolysis by the G degree, and the degree varies depending on the reaction time, the amount of water, the stirring conditions, and the like. The hydrolysis reaction may be terminated when the target G degree is reached, and is preferably performed until the G degree becomes 2.9 to 0, more preferably in the range of 2.5 to 0. preferable. Here, the G degree of 0 indicates a state in which the G degree is completely hydrolyzed to fatty acid and glycerin.

Here, the G degree means that the reaction solution collected during the hydrolysis reaction and trimethylsilylated (TMS converted) is subjected to GC (gas chromatography) analysis, and each glyceride (glycerin (GLY), monoglyceride (MG), The peak area% of diglyceride (DG) and triglyceride (TG) was determined. Using the obtained numerical values, perform molar conversion from each glyceride converted to TMS, and again determine the abundance ratio of each component (GLY, MG, DG, TG) as a percentage of glyceride, and apply that value to the following equation: Can be calculated.
G degree = (MG + 2 × DG + 3 × TG) / 100
In addition, when using a partial ester of a fatty acid as the fatty acid ester, after adding the fatty acid ester or the fatty acid ester and the fatty acid to a solution in which hydrolysis treatment is not performed, that is, an aqueous solution in which lipase and legume protein are dissolved and dispersed. The lipase powder preparation can also be produced by stirring and immediately drying by one drying method selected from spray drying, freeze drying or solvent precipitation / drying.
When using only fatty acids, add the fatty acid to the aqueous solution in which lipase and legume protein are dissolved and dispersed, and after stirring and emulsifying and dispersing uniformly with a stirrer or three-one motor, spray drying, freeze drying or A lipase powder formulation can also be produced by drying by one drying method selected from solvent precipitation and drying.

The added amount of fatty acid ester and / or fatty acid used in the production of the lipase powder preparation is preferably 0.1 to 500 times the mass of the total mass of lipase and legume protein, and is 0.2 to 100 times greater. The mass is more preferable, and the mass of 0.3 to 50 times is most preferable.
However, when manufacturing a lipase powder formulation using spray drying, the added amount of fatty acid ester and / or fatty acid used is 0.1 to 10 times the mass of the total mass of lipase and legume protein. The mass is more preferably 0.2 to 10 times, and most preferably 0.3 to 10 times.
When spray drying is used, if the amount of fatty acid ester and / or fatty acid is increased, the evaporation of water becomes incomplete, or the resulting lipase powder formulation becomes difficult to recover due to excess fatty acid ester and / or fatty acid. This is because the problem occurs.
The upper limit of the amount of fatty acid ester and / or fatty acid to be used can be increased by improving the spray drying apparatus or changing the recovery form. However, if it contains more fatty acid ester and / or fatty acid than necessary, filtration, etc. A process is required.
When a lipase powder preparation containing a fatty acid ester and / or fatty acid is produced using solvent precipitation, the amount of the solvent used is an aqueous solution in which the fatty acid ester and / or fatty acid, and the lipase and legume protein are dissolved and dispersed. It is preferable to use a solvent having a volume of 1 to 100 times, and more preferably a solvent having a volume of 2 to 10 times the total mass value of the total.
When the filter aid described later is added before the solvent precipitation, the solvent is used with the total mass added to the filter aid.

Next, drying by dehydration accompanied by an esterification reaction will be described. That is, after hydrolyzing and / or emulsifying / dispersing, an esterification reaction is subsequently performed while dehydrating, and if necessary, an oil such as an unreacted product is filtered to produce a lipase powder formulation. .
The esterification reaction performed after hydrolysis is preferably performed in an environment that reaches a reduced pressure of about 10 hPa from normal pressure while dehydrating.
Although the reaction temperature of esterification reaction is not specifically limited, 20-80 degreeC is preferable and 30-60 degreeC is more preferable. This is because within this temperature range, the esterification reaction proceeds more rapidly, and the lipase inactivation is less likely to occur.
The timing for terminating the esterification reaction can also determine the degree of progress of esterification by the degree G, and the degree varies depending on the reaction temperature and the lipase concentration. The degree G of the esterification reaction solution can be calculated by the method described above. Then, when the target G degree is reached, the esterification reaction is terminated.
Here, after the esterification reaction, if the triglyceride content is 100% based on the total of each glyceride, the G degree of the esterification reaction solution is 3.0.
The esterification reaction performed in the present invention is preferably performed so that the G degree value after the reaction becomes a value increased by 0.2 or more from the G degree value after hydrolysis.
And it is preferable that G degree after esterification reaction is 1.0-2.99.

Here, there is a portion that overlaps with the preferred range of hydrolysis described above. For example, when the degree of G after hydrolysis is 0.5, esterification to about 1.0 by esterification reaction or This is because when the G degree after hydrolysis is 1.5, esterification may be performed up to about 2.0 by an esterification reaction.
After the esterification reaction is completed, excess oil such as unreacted substances can be removed by filtration as necessary to obtain the lipase powder preparation containing the fatty acid ester and / or fatty acid of the present invention.
The mass of the fatty acid ester and / or fatty acid (hereinafter also referred to as oil-containing component) contained in the lipase powder preparation obtained by the drying is 0.01 to 50 times the total mass of the lipase and legume protein. Is preferable, and it is more preferably 0.1 to 10 times.
Moreover, when including the filter aid demonstrated later, it is preferable that it is 0.01-50 times with respect to the total mass of lipase, legume protein, and filter aid, and it is more preferable that it is 0.1-10 times. .
Further, as will be described later, even when the lipase powder preparation produced by dehydration accompanied by an esterification reaction is washed using the raw materials used for transesterification or esterification, the fatty acid ester contained in the lipase powder preparation and / or Or the preferable mass of a fatty acid is the same as this.

In this invention, the process of adding a filter aid can further be included.
When drying is performed by dehydration accompanied by an esterification reaction, a filter aid can be added before the esterification reaction, during the esterification reaction, or after the esterification reaction. It is preferable to add a filter aid because the filtration treatment can be performed smoothly after the esterification reaction.
When adding a filter aid before or during the esterification reaction, oils and fats may be further added. This is because the fluidity of the reaction solution is improved by adding the fats and oils in this manner when the viscosity increases and the stirring becomes worse due to the addition.
Examples of filter aids that can be used include silica gel, celite, cellulose, starch, dextrin, activated carbon, activated clay, kaolin, bentonite, talc, and sand. Of these, silica gel, celite, and cellulose are preferable. The particle size of the filter aid may be arbitrary, but is preferably 1 to 100 μm, particularly preferably 5 to 50 μm.
The filter aid that can be used before, during, or during the esterification reaction is preferably added in an amount of 1 to 500% by mass, and in an amount of 10 to 200% by mass, based on the total mass of lipase and legume protein. Further preferred. This is because if the amount is within this range, the burden during filtration is reduced, and filtration pretreatment such as large-scale filtration equipment and advanced centrifugation is not required.

Moreover, a filter aid can also be contained in the lipase powder formulation obtained by a drying method other than dehydration involving the esterification reaction of the present invention. When drying by spray drying or freeze drying to obtain a lipase powder formulation, a filter aid may be added either before or after drying.
When drying by a method of drying after solvent precipitation, it is preferable to add a filter aid to the lipase powder formulation obtained by drying.
The amount of the filter aid to be contained in the obtained lipase powder preparation can be 1 to 500% by mass, more preferably 10 to 200% by mass, based on the total mass of lipase and legume protein.
Moreover, the lipase powder formulation obtained by the manufacturing method demonstrated previously can also be refine | purified by the method including the process of wash | cleaning with a fatty acid ester and / or a fatty acid.
Washing can be performed by adding a fatty acid ester and / or a fatty acid to the lipase powder preparation obtained by the production method described above, heating as necessary, stirring in a liquid state, and then filtering.
Examples of the filtration include natural filtration, suction filtration, and pressure filtration.
Here, the fatty acid ester and / or fatty acid used for washing is preferably the same as the fatty acid ester and / or fatty acid used as a raw material when performing a transesterification reaction using the lipase powder preparation of the present invention. . This is because it is possible to avoid contamination of impurities in the obtained transesterification reaction product.
In addition, when drying by dehydration accompanied by esterification reaction to obtain a lipase powder formulation, such washing can be performed before or after the filtration step of the unesterified product and fatty acid, but after the filtration step. Is preferred.

Next, the transesterification product or the manufacturing method of an esterification product obtained by transesterification or esterification reaction using the lipase powder formulation of this invention is demonstrated.
The transesterification reaction performed using the lipase powder preparation of the present invention is an ester exchange reaction between one or more selected from fatty acid esters, fatty acids, and alcohols and fatty acid esters. Examples include transesterification, transesterification of fats and oils and fatty acid esters, and transesterification of alcoholysis and acidolysis.
The esterification reaction performed using the lipase powder preparation of the present invention is an esterification reaction between a fatty acid partial ester and a fatty acid, or an esterification reaction between a monohydric or polyhydric alcohol and a fatty acid. For example, glycerin And esterification reaction of fatty acid with fatty acid.
More specifically, as a transesterification reaction between fats and oils, for example, rapeseed oil, which is a triglyceride of a long chain fatty acid, and trioctanoic acid glyceride, which is a triglyceride of a plant-derived medium chain fatty acid, can be transesterified, A triglyceride mixed with a long chain and a medium chain can be produced.
In addition, as a transesterification reaction using acidolysis by fats and oils and fatty acids, it is possible to produce structured fats and oils that greatly utilize the 1,3-specific lipase possessed by lipases. A specific fatty acid is left at the second position of the glycerin skeleton, and the fatty acid at the first and third positions is replaced with the target fatty acid. The obtained product can be used for fats and oils used for chocolate and the like, and can also be used for fats and oils having a specific nutritional effect.

The conditions for the transesterification reaction and esterification reaction using the lipase powder preparation of the present invention are not particularly limited, and can be carried out by a conventional method.
Generally, it is carried out under normal pressure or reduced pressure while avoiding the mixing of water that causes hydrolysis. The reaction temperature depends on the raw materials used and the freezing point of the mixture in which the raw materials are mixed, but is preferably about 20 to 80 ° C, and more preferably 40 to 60 ° C if not limited by the freezing point.
Moreover, as addition amount to the reaction raw material of a lipase powder formulation, 0.05-10 mass% is preferable, and 0.05-5 mass% is more preferable. The optimum amount is determined by the reaction temperature, the set reaction time, the activity of the resulting lipase powder formulation, and the like. After completion of the reaction, the lipase powder preparation is removed by filtration, centrifugation, etc., and can be used repeatedly (evaluation of stability) until the activity is reduced to an impossible production.
Therefore, it is generally desirable that expensive lipase can impart high activity and high stability to a lipase powder formulation at the same time in as little amount as possible.
The transesterified product or esterified product thus obtained is not particularly limited, but is preferably a transesterified fat or esterified fat used in the food field, and also for religious reasons or health reasons. More preferably, it is a transesterified oil or esterified oil / fat derived from vegetable oil that can be used to produce food or food additives for people who cannot take animal-derived protein or oil / fat.
Next, the present invention will be described in detail with reference to production examples and examples.

<Production Example 1: Extraction of blue pea protein>
100 g of dried blue peas were immersed in 200 g of water overnight to obtain 210 g of beans containing water (water content 110 g). The water 190g was added here and it grind | pulverized with the mixer. The obtained bean pulverized solution was stirred with a stirrer in a cold water bath at 15 ° C., and 200 g of an aqueous solution containing 2 g of NaOH was added little by little to prepare an aqueous solution of about 0.1 N sodium hydroxide. The protein was solubilized by allowing it to stand for about 30 minutes, and this was centrifuged with a centrifuge (manufactured by Beckman: GS-6KR) at 3000 rpm, 15 ° C. for 15 minutes to separate insoluble components. did. An aqueous citric acid solution was dropped into the obtained aqueous phase to adjust the pH to 5 to 6, and proteins were precipitated. Centrifugation was performed with a centrifuge under conditions of 3000 rpm, 15 ° C., and 15 minutes, and the precipitate was collected. The precipitate was dried by freeze drying and pulverized in a mortar to obtain a powdered blue pea extract protein. The yield was about 13% by weight, based on the weight of the dry blue pea used. Moreover, when the protein content of this extract was measured by the Dumas method, the nitrogen content was about 12%, which corresponds to about 78% as the protein content.

<Production Example 2: Extraction of red pea protein>
The same operation as in Production Example 1 was performed to obtain a powdered red pea extract protein. The yield was about 16% by mass. Further, when the protein content of this extract was measured by the Dumas method, the nitrogen content was 12%, which corresponds to about 76% as the protein content.
<Production Example 3: Extraction of blue broad bean protein>
The same operation as in Production Example 1 was performed to obtain a powdered blue broad bean extracted protein. The yield was about 20% by mass. Further, when the protein content of this extract was measured by the Dumas method, the nitrogen content was 13%, corresponding to a protein content of about 83%.
<Production Example 4: Extraction of Otafuksora Bean Protein>
The same operation as in Production Example 1 was carried out to obtain powdered Otafuxolame extract protein. The yield was about 17% by mass. Further, when the protein content of this extract was measured by the Dumas method, the nitrogen content was 13%, corresponding to a protein content of about 83%.
<Production Example 5: PP-CS>
When the protein content of a commercially available yellow pea protein (trade name: PP-CS, Organo Danisco Food Techno Co., Ltd.) was measured by the Dumas method, the nitrogen content was 13%, and the protein content was about 83%. It corresponds to.
<Production Example 6: Soy protein>
When the protein content of commercially available soybean protein (trade name: Solpy 5000, Nissin Oilio Group Co., Ltd.) was measured by the Dumas method, the nitrogen content was 14%, which corresponds to about 90% as the protein content. To do.

<Example 1>
Powder lipase (trade name: Lipase D: Hydrolytic activity 19 million U / g, Amano Enzyme Co., Ltd., the same shall apply hereinafter) and water 12 ml (based on the total mass of powder lipase and legume protein) 5.7 times, the same shall apply hereinafter) was added and dissolved and dispersed with stirring to obtain a lipase-containing aqueous solution.
To this lipase-containing aqueous solution, 2.0 g of yellow pea protein (PP-CS) described in Production Example 5 (20 times the mass of powdered lipase, the same shall apply hereinafter) is added, stirred, dissolved and dispersed. It was.
50 g of high oleic sunflower oil (trade name: olein rich, Showa Sangyo Co., Ltd.) (23.8 times the total mass of powdered lipase and legume protein, hereinafter the same) was added as the fat. While maintaining the temperature in the reaction vessel at 40 ° C. using a water bath, the mixture was stirred for 2 hours to hydrolyze the fats and oils. The G degree at this time was 1.8.
While maintaining this solution at 40 ° C., 2 g of powdered cellulose (trade name: KC Flock (average particle diameter of about 10 μm), Nippon Paper Chemicals Co., Ltd.) (95% by mass based on the total mass of powdered lipase and legume protein) The mixture was stirred for 20 hours under reduced pressure using a vacuum pump to remove excess water, followed by a dehydration esterification reaction. The final degree of vacuum was about 25 hPa. The G degree of the obtained solution was 2.4.
By filtering the total amount of the obtained solution using a quantitative filter paper (Advantech Toyo Co., Ltd.), excess oils and fats, esterified unreacted substances and fatty acids were removed. Furthermore, the oil and fat of the target transesterification reaction (fat obtained by mixing trioctanoic acid glyceride: high oleic sunflower oil at a mass ratio of 1: 1 and drying with molecular sieves) was washed and filtered with about 10 g, and purified lipase powder formulation (1) 7.3 g was obtained. When it was assumed that 100% of the total mass of the solid content including the filter aid was recovered, the oil content was 0.8 times.

<Example 2>
The raw materials and operations were the same except that the yellow pea protein used in Example 1 was replaced with the blue pea extract protein obtained in Production Example 1. The degree of G after hydrolysis was 1.9. The G degree of the obtained solution was 2.5. 6.5 g of lipase powder formulation (2) obtained by washing and filtration was obtained. The oil content was 0.6 times.
<Example 3>
The raw materials and operations were the same except that the yellow pea protein used in Example 1 was replaced with the red pea extract protein obtained in Production Example 2. The degree of G after hydrolysis was 2.0. The G degree of the obtained solution was 2.5. 6.4 g of lipase powder formulation (3) obtained by washing and filtration was obtained. The oil content was 0.6 times.
<Example 4>
The raw materials and operations were the same except that the yellow pea protein used in Example 1 was replaced with the blue broad bean extract protein obtained in Production Example 3. The degree of G after hydrolysis was 1.8. The G degree of the obtained solution was 2.5. 7.1 g of lipase powder formulation (4) obtained by washing and filtration was obtained. The oil content was 0.7 times.
<Example 5>
The raw materials and operations were the same except that the yellow pea protein used in Example 1 was replaced with the Otafuxola bean extract protein obtained in Production Example 4. The degree of G after hydrolysis was 1.9. The G degree of the obtained solution was 2.5. 6.9 g of lipase powder formulation (5) obtained by washing and filtration was obtained. The oil content was 0.7 times.

<Example 6>
The raw material and operation were the same except that the yellow pea protein used in Example 1 was replaced with the soy protein described in Production Example 6, water was 20 ml (9.5 times), and high oleic sunflower oil was 18 g (8.6 times). Went to. The degree of G after hydrolysis was 1.6. The G degree of the obtained solution was 1.9. 10.2 g of lipase powder formulation (6) obtained by washing and filtration was obtained. The oil content was 1.5 times.
<Example 7>
The raw materials and operations were the same except that the high oleic sunflower oil used in Example 1 was replaced with rapeseed oil (trade name: canola oil, Nissin Oilio Group Co., Ltd.), and a powdered cellulose having an average particle size of about 28 μm. went. The degree of G after hydrolysis was 1.9. The G degree of the obtained solution was 2.7. 5.8 g of lipase powder formulation (7) obtained by washing and filtration was obtained. The oil content was 0.4 times.
<Example 8>
The powdered cellulose used in Example 1 was replaced with Celite (trade name: RocaHelp, Mitsui Mining Co., Ltd.), and high oleic sunflower oil was added as 12 g (5.7 times) and added as a filter aid before the esterification reaction. The raw materials and operations were performed in the same manner except that the reaction was once returned to normal pressure and added 1 hour before the end of the reaction. The degree of G after hydrolysis was 1.0. The G degree of the obtained solution was 2.1. 8.9 g of lipase powder formulation (14) obtained by washing and filtration was obtained. The oil content was 1.2 times.

<Example 9>
The raw materials and operations were the same except that the yellow pea protein used in Example 1 was 4 g (40 times), water was 24 g (5.9 times), and no filter aid was added. The degree of G after hydrolysis was 1.9. The G degree of the obtained solution was 2.1. 7.8 g of lipase powder formulation (9) obtained by washing and filtration was obtained. The oil content was 0.9 times.
<Example 10>
The raw materials and operations were the same except that 1 g of yellow pea protein used in Example 1 (10 times) and 8 g of water (7.3 times) were used. The degree of G after hydrolysis was 1.8. The G degree of the obtained solution was 2.4. 4.7 g of lipase powder formulation (10) obtained by washing and filtration was obtained. The oil content was 0.5 times.
<Comparative Example 1>
The raw materials and operations were the same except that the yellow pea protein used in Example 1 was not used and water was changed to 4 g (40 times). The degree of G after hydrolysis was 2.4. The G degree of the obtained solution was 2.6. 2.8 g of a protein-free lipase powder formulation (1) obtained by washing and filtration was obtained. The oil content was 0.3 times.

<Example 11>
To a reaction vessel equipped with a stirrer, 0.1 g of lipase D and 9 ml of water (5.6 times) were added and dissolved and dispersed while stirring to obtain a lipase-containing aqueous solution.
To this lipase-containing aqueous solution, 1.5 g (15 times) of yellow pea protein (PP-CS) described in Production Example 5 was added, stirred, and dissolved and dispersed.
Here, 1.0 g (0.6 times) of high oleic sunflower oil was added as fat. While maintaining the temperature in the reaction vessel at 40 ° C. using a water bath, the mixture was stirred for 1 hour to hydrolyze the fats and oils. The G degree at this time was 0.8.
While maintaining this solution at 40 ° C., 1 g (60% by mass) of powdered cellulose was added and stirred, and then dried using a spray dryer (SD-1000 type: manufactured by Tokyo Rika Kikai Co., Ltd.) at an inlet temperature of 130 ° C. Spraying was performed under the conditions of an air amount of 0.7 to 1.1 m ³ / min and a spraying pressure of 11 to 12 kPa to obtain 1.7 g of a lipase powder formulation (11). In this condition, since the amount of added oil is small, the oil content becomes 0.4 times when converted at the time of preparation.
<Example 12>
2 g (20 times) of yellow pea protein used in Example 11, 11 g (5.2 times) of water, 1.6 g (0.8 times) of high oleic sunflower oil, 1.2 g (60% by mass) of powdered cellulose The raw materials and operations other than those described above were the same. The degree of G after hydrolysis was 1.0. 2.6 g of lipase powder formulation (12) was obtained. The oil content is 0.5 times when converted at the time of preparation.

<Example 13>
The high oleic sunflower oil used as the fat in Example 12 was replaced with a mixture of partial glycerides and fatty acids. As partial glycerides, 0.6 g of fatty acid monoglycerides derived from safflower oil (trade name: Sunsoft 8090, Taiyo Kagaku Co., Ltd.) and as fatty acids oleic acid (reagent names: oleic acid, Tokyo Chemical Industry Co., Ltd.) ) 1.0 g was mixed and used. Also, at this time, the work is performed at room temperature of about 25 ° C., and yellow pea protein and powdered cellulose are simultaneously added to the lipase-containing aqueous solution while stirring, and finally a mixture of partial glycerides and fatty acids is added. Spray drying was performed quickly except for the operation. As a result, 2.4 g of a lipase powder formulation (13) was obtained. The oil content is 0.5 times when converted at the time of preparation.
<Example 14>
The raw materials and operations were the same except that the mixture of partial glyceride and fatty acid used in Example 13 was replaced with oleic acid alone. Thereby, 2.8 g of a lipase powder formulation (14) was obtained. The oil content is 0.5 times when converted at the time of preparation.
<Example 15>
The raw materials and operations were the same except that the yellow pea protein used in Example 12 was replaced with the red pea extract protein. The degree of G after hydrolysis was 1.6. As a result, 2.3 g of a lipase powder formulation (15) was obtained. The oil content is 0.5 times when converted at the time of preparation.

<Example 16>
The raw materials and operations were the same except that the high oleic sunflower oil used in Example 12 was changed to 0.8 g (0.4 times). The degree G after hydrolysis was 0.7. Thereby, 2.1 g of lipase powder formulation (16) was obtained. The oil content is 0.2 times when converted at the time of preparation.
<Example 17>
The raw materials and operations were the same except that the yellow pea protein used in Example 12 was 4 g (40 times), water was 22 g (5.4 times), and high oleic sunflower oil was 3.0 g (0.7 times). It was. The degree of G after hydrolysis was 1.9. As a result, 5.2 g of a lipase powder formulation (17) was obtained. The oil content is 0.6 times when converted at the time of preparation.
<Comparative example 2>
The raw materials and operations were the same except that the yellow pea protein used in Example 12 was not used and powdered cellulose was changed to 3.2 g (320 mass%). The degree G after hydrolysis was 1.4. As a result, 2.2 g of protein-free lipase powder formulation (2) was obtained. The oil content is 0.5 times when converted at the time of preparation.

<Example 18>
In a reaction vessel equipped with a stirrer, 0.1 g of lipase D and 10 ml (5.6 times) of water were added and dissolved and dispersed while stirring to obtain a lipase-containing aqueous solution.
To this lipase-containing aqueous solution, 1.7 g (17 times) of yellow pea protein (PP-CS) described in Production Example 5 was added, stirred, and dissolved and dispersed.
Here, 10 g (5.6 times) of high oleic sunflower oil was added as an oil. While maintaining the temperature in the reaction vessel at 40 ° C. using a water bath, the mixture was stirred for 1 hour to hydrolyze the fats and oils. The G degree at this time was 1.4.
This solution was gradually added to about 180 ml (8.3 times) of ethanol previously cooled to 0 ° C. or lower to obtain a precipitate. The obtained precipitate was centrifuged with a centrifuge (manufactured by Beckman: GS-6KR) under conditions of 3000 rpm, 15 ° C. and 15 minutes to collect the precipitate, and then the freeze dryer (Tokyo Science Instruments Co., Ltd.). ): FDU-830) for 16-20 hours under reduced pressure at room temperature to obtain 2.0 g of lipase powder formulation (18). In addition, in addition to the low recovery rate in solvent precipitation, the oil content is unclear because most of the oil is dissolved in the solvent.
<Example 19>
The high oleic sunflower oil used as the fat in Example 18 was replaced with partial glycerides. As partial glyceride, 10 g of monoglyceride of fatty acid derived from safflower oil (trade name: Sunsoft 8090, Taiyo Kagaku Co., Ltd.) was used. At this time, work at room temperature of about 25 ° C., add yellow pea protein to the lipase-containing aqueous solution while stirring, and finally add a partial glyceride. went. The amount of ethanol at this time was 100 ml (4.6 times). As a result, 1.9 g of lipase powder formulation (19) was obtained.

<Example 20>
The raw materials and operations were the same except that the yellow pea protein used in Example 18 was replaced with the red pea extract protein. The amount of ethanol at this time was 100 ml (4.6 times). The degree of G after hydrolysis was 2.1. Thereby, 3.5 g of lipase powder formulation (20) was obtained.
<Example 21>
The high oleic sunflower oil used as the fat in Example 18 was hydrolyzed from the fat and oil and replaced with a blended oil (G = about 1) adjusted to the hydrolyzate composition when the G degree was about 1. The blended oil (G = about 1) was adjusted by calculating the molecular weight from the GC analysis value to determine the composition mass of each blended component, and mixing each component. More specifically, glycerin (reagent name: glycerin, Tokyo Kasei Kogyo Co., Ltd.) 2.4 g, fatty acid-derived monoglyceride 10.0 g, diglyceride oil 9.9 g, high oleic sunflower oil 11.4 g, olein 66.3 g of acid was mixed to make 100 g of blended oil (G = about 1). The yellow pea protein was 3.0 g (30 times), water was 25 ml (8.1 times), and the blended oil (G = about 1) was 25 g (8.1 times). At this time, the operation is performed at room temperature of about 25 ° C., yellow pea protein is added to the lipase-containing aqueous solution while stirring, and finally the blended oil (G = about 1) is added. A quick solvent precipitation was performed. The amount of ethanol at this time was 250 ml (4.7 times). Thereby, 3.5 g of lipase powder formulation (21) was obtained.

<Example 22>
The blended oil (G = about 1) used in Example 21 was replaced with a blended oil (G = about 2) prepared by performing the same calculation so that the G degree was about 2. Specifically, 0.5 g of glycerin, 5.6 g of monoglyceride of fatty acid derived from safflower oil, 33.7 g of diglyceride oil, 25.9 g of high oleic sunflower oil, 34.4 g of oleic acid, and 100 g of mixed oil (G = About 2). Further, yellow pea protein was 0.5 g (5 times), water was 1 ml (1.7 times), and blended oil (G = about 2) was 10 g (16.7 times). At this time, the operation is performed at room temperature of about 25 ° C., yellow pea protein is added to the lipase-containing aqueous solution while stirring, and finally the blended oil (G = about 2) is added. A quick solvent precipitation was performed. The amount of ethanol at this time was 55 ml (4.7 times). As a result, 0.9 g of a lipase powder formulation (22) was obtained.
<Comparative Example 3>
The raw materials and operations were the same except that the yellow pea protein used in Example 18 was not used and 1.7 g (170% by mass) of powdered cellulose was used. The degree G after hydrolysis was 1.5. As a result, 2.1 g of a protein-free lipase powder formulation (3) was obtained.

<Example 23: Evaluation of transesterification activity ability>
The transesterification reaction was performed using the lipase powder formulations obtained in Examples 1 to 22 and Comparative Examples 1 to 3, and the transesterification activity was examined.
As a raw material for the transesterification reaction, a fat and oil (hereinafter referred to as a transesterification raw material fat and oil) obtained by mixing trioctanoic acid glyceride: high oleic sunflower oil at a mass ratio of 1: 1 and drying with molecular sieves was used.
First, the transesterification reaction of fats and oils was carried out by charging the transesterification raw material fats and fats and various lipase powder preparations in a reaction vessel and reacting them at 60 ° C. Note that the lipase powder formulation was added to the transesterified raw oil and fat with the addition amount defined as follows for each treatment method. In the case of the lipase powder formulations of Examples 1 to 10 and Comparative Example 1 obtained by dehydration accompanied by an esterification reaction, 0.05% by mass was used in terms of lipase D (19 million U / g). The conversion method of lipase D is as follows. Assuming that the lipase D used is 100% contained in the obtained lipase powder formulation, for example, when 0.1 g of lipase D (19 million U / g) is used and the yield of the lipase powder formulation is 10 g The lipase D content in the lipase powder formulation is 1% by mass. When 10 g of the transesterified raw material fat is used, 0.5 g of this lipase powder formulation is added. The setting of the addition amount is adjusted in order to perform the measurement in a short time, and when used for production, a smaller amount may be added.

Moreover, in the case of the lipase powder formulation of Examples 11-17 obtained by spray drying and the comparative example 2, 2 mass% of this lipase powder formulation was added with respect to transesterification raw material fats and oils. The lipase D content at that time was determined by calculation from the content of lipase D in all raw materials except water. For example, when 0.1 g of lipase D, 2 g of yellow pea protein, 1.2 g of powdered cellulose, and 1.6 g of high oleic sunflower oil are used, the lipase D content in the lipase powder formulation is 2.1% by mass. . In this case, 0.042 mass% of lipase D was added. In the case of a lipase powder preparation obtained by freeze drying, the same measurement is performed.
In the case of the lipase powder formulations of Examples 18 to 22 and Comparative Example 3 obtained by the solvent precipitation method, 2% by mass of this lipase powder formulation was added to the transesterified raw material fat. The lipase D content at that time was calculated in the same manner as in the case of dehydration with an esterification reaction. For example, when 0.1 g of lipase D (19 million U / g) is used and the yield is 2 g, the lipase D content is 5.0% by mass, and lipase D is added by 0.1% by mass.

Next, the production rate of the monooleic acid substitution product produced by the transesterification reaction was determined from the following formula, and the reaction rate constant K serving as an index of the transesterification activity was calculated.

Production rate of monooleic acid substitution product (%) =
{Monoleoleic acid substituted product / (trioctanoic acid glyceride + monooleic acid substituted product)} × 100

Specifically, among the values of area% of the peak from trioctanoic acid glyceride to trioleic acid glyceride obtained by GC analysis, area% of trioctanoic acid glyceride and dioctanoic acid monooleic acid glyceride (monooleic acid substitution product) The production rate of monooleic acid substitution product was calculated from the above formula using
The GC analysis was performed under the conditions of column temperature: initial 50 ° C., temperature increase 15 ° C./min., And final 370 ° C.
Included in various lipase powder formulations by inputting the value of the production rate (%) of monooleic acid substitution product in several reaction times from the start of the reaction to the end of the reaction into the analysis software (Origin Ver.6.1) The reaction rate constant K (reaction rate constant per lipase) based on the lipase obtained was calculated as K1. The reaction time for this activity measurement is about 1 hour.
The reaction rate constants K obtained by the above measuring methods are shown in Tables 1 to 3 separately for each drying method.

<Measurement method of active sustainability>
The transesterification reaction was performed using the lipase powder formulations obtained in Examples 1 to 22 and Comparative Examples 1 to 3, and the transesterification activity sustaining performance was examined as an index for evaluating stability.
The calculation of the reaction rate constant K (reaction rate constant per lipase) based on the lipase contained in various lipase powder formulations was carried out in the same manner as the measurement of the transesterification activity.
Moreover, the reaction method at the time of investigating activity sustainability was performed by a batch type, and it performed 4 batches by making 23 hours into 1 batch. The time for filtration / recovery of the lipase powder formulation between batches was not added, and 1 hour of activity measurement performed after 4 batches was added to 93 hours. The reaction rate constant K was determined on the assumption that the lipase powder formulation was not reduced by filtration between batches.
As an index of the transesterification activity sustainability, the half-life (h) was determined from the transesterification activity (K1) at the start of the reaction and the transesterification activity (K2) at the reaction time of 93 hours using the following formula (I). Furthermore, the following formula (II) is obtained from the transesterification activity (K1) at the start of the reaction and the half-life (h) as an index considering both the transesterification activity at the start of the reaction and the transesterification activity sustaining performance. It was. Specifically, the area obtained from the time from K1 until reaching the half-life was defined as a half-value area and obtained by calculation. This makes it possible to compare high activity and short half-life with low activity and long half-life.

The transesterification activity sustaining performance of various lipase powder formulations when the half-value areas of Comparative Examples 1 to 3 (protein-free) of each treatment method were each 100 was also expressed as a half-value area relative value.

(I) = 93 × (K1) / 2 / {(K1) − (K2)}

(II) = 1.5 × (K1) × (I) / 2

Tables 1 to 3 show K2, (I), and half-value area relative values obtained by the above measurement and calculation methods for each of the various drying methods.

  As is clear from the results in Table 1, in the case of Comparative Example 1 in which the formulation was formed by dehydration accompanied by an esterification reaction without containing a bean protein, the transesterification ability was improved to some extent, but the half-life was 50 hours. It is short and not suitable for manufacturing. On the other hand, in the lipase powder formulation containing the pulse protein of the present invention, the reaction rate constant K per lipase was about 2 to 3 times that of Comparative Example 1, and the half-life was also long. Furthermore, the half-value area relative value in consideration of both the high activity and the sustained activity performance was about 4 to 50 times, indicating that the obtained lipase powder formulation was very excellent.

As is clear from the results in Table 2, in the case of Comparative Example 2 which was formulated by spray drying without containing legume protein, the transesterification ability was improved to some extent, but the half-life was as short as 49 hours. Not suitable. On the other hand, in the lipase powder formulation containing the pulse protein of the present invention, the reaction rate constant K per lipase was about 1 to 3 times that of Comparative Example 2, and the half-life was also long. Furthermore, the half-value area relative value in consideration of both high activity and active sustainability was about 4 to 50 times, indicating that the obtained lipase powder formulation was very excellent.

As is clear from the results in Table 3, in the case of Comparative Example 3 which was formulated by solvent precipitation and drying without containing legume protein, the transesterification ability was improved to some extent, but the half-life was as short as 60 hours. Not suitable for. On the other hand, in the lipase powder formulation containing the pulse protein of the present invention, the reaction rate constant K per lipase was about 1 to 2 times that of Comparative Example 3. In addition, the half-life was long. Furthermore, the half-value area relative value in consideration of both high activity and active sustainability was about 2 to 130 times, indicating that the resulting lipase powder formulation was very excellent. Even if the reaction rate constant K per lipase is the same as or lower than that of Comparative Example 3, it is clear that it is superior in actual production due to its high activity sustaining performance.
Moreover, as can be seen from the results in Tables 1 to 3, the initial activity value per lipase of the lipase powder preparation obtained was different depending on the drying method. The highest initial activity value per lipase was produced by drying by dehydration accompanied by an esterification reaction, and the next highest value was produced by drying by spray drying.

Claims (14)

  A lipase powder preparation characterized by being a granulated product containing lipase and legume protein.   The lipase powder formulation according to claim 1, wherein the legume protein is a protein of one or more beans selected from the group consisting of pea, broad bean and soybean.   The lipase powder preparation according to claim 1 or 2, wherein the legume protein is obtained by solubilizing and extracting a legume protein with water or an alkaline aqueous solution and precipitating the protein with an acid from the extract.   Furthermore, the lipase powder formulation of any one of Claims 1-3 containing a fatty acid ester and / or a fatty acid.   The lipase powder formulation of Claim 4 whose said fatty acid ester is fats and oils.   A method for producing a lipase powder preparation, comprising drying an aqueous solution in which lipase and legume protein are dissolved and dispersed.   The production method according to claim 6, further comprising a step of bringing a fatty acid ester and / or a fatty acid into contact with an aqueous solution in which lipase and legume protein are dissolved and dispersed.   The manufacturing method according to claim 7, wherein the fatty acid ester is hydrolyzed in the step of contacting the fatty acid ester and / or the fatty acid.   The manufacturing method according to any one of claims 6 to 8, wherein the drying is any one selected from spray drying, freeze drying, and solvent precipitation / drying.   The production method according to claim 7 or 8, wherein the drying is dehydration accompanied by an esterification reaction.   The manufacturing method according to claim 10, further comprising a step of filtering after the esterification reaction.   Furthermore, the manufacturing method of any one of Claims 6-11 including the process of adding a filter aid.   The manufacturing method of any one of Claims 6-12 including the process of wash | cleaning the obtained lipase powder formulation with a fatty acid ester and / or a fatty acid further.   Using the lipase powder preparation obtained by the production method according to any one of claims 6 to 13, performing an ester exchange reaction or an esterification reaction on one or more selected from fatty acid esters, fatty acids, and alcohols. A process for producing a transesterified product or an esterified product.