JPS6261582A - Production of lipase formulation - Google Patents

Abstract

PURPOSE:To obtain a lipase formulation having improved decomposition ratio of fat or oil or ester interchange ratio as compared with that of a conventional well-known production process, by adding a water-insoluble carrier to a culture fluid of a microorganism containing lipase and drying the resultant mixture solution. CONSTITUTION:A microorganism capable of producing lipase, e.g. microorganism of the genus Rhizopus or Aspergillus, is cultivated by a conventional method, i.e. solid cultivation method using bran or liquid cultivation method is employed. 100-200pts.vol. water-insoluble carrier, e.g. zeolite or diatomaceous earth, is added to 100pts.wt. resultant culture fluid, and the resultant mixture is stirred and dried at <=50 deg.C to afford the aimed lipase formulation. The drying method in this case is particularly preferably freeze-drying.

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial Application Field] The present invention relates to a powder formulation of a microbial culture solution containing lipase (lipid-degrading enzyme), that is, a method for producing a lipase formulation.

Attempts are being made to use enzymes to obtain fatty acids from or modify vegetable or animal fats and oils that exist in nature.

In the production of fatty acids by decomposing fats and oils, lipases are being applied instead of the high-temperature, high-pressure decomposition of fats and oils, which consumes a lot of energy. Regarding the modification of fats and oils, in recent years there have been many proposals regarding the production of fats and oils for chocolate using the transesterification reaction of fats and oils by lipase. As described above, how to apply lipase to fats and oils is one of the most important factors in making advanced use of fats and oils.

[Conventional technology and problems]

Since lipase is water-soluble, it is dissolved in water and used for the reaction reaction, but after the reaction is completed, lipase cannot be separated and recovered from the lipase solution containing the reaction product, so lipase conversion is fixed for industrial use. Things have been thought out. As the immobilized Northern Δ method, methods have been reported in which lipase is immobilized on a carrier by covalent bonding, ionic bonding, physical adsorption, etc., and techniques such as enveloping immobilization of lipase, microencapsulation, and the like.

On the other hand, in the fermentation production of lipase using microorganisms,
Efforts are being made to search for microorganisms that produce distinctive lipases by adding gold additives during the microorganism cultivation stage to increase the productivity of lipases.

However, since the production of lipase and the production of immobilized lipase are considered separately, no consideration is given to the fact that lipase is produced by microorganisms and has characteristics in nine states. Furthermore, there is a method in which the lipase, which has been made into a powder, is redissolved in water or the like and then fixed.

This may lead to deactivation of lipase.

[Means for solving problems]

Under these circumstances, the present inventors investigated the characteristics of lipase contained in the culture solution obtained by culturing lipase-producing microorganisms, namely, fat-degrading activity and fat-and-oil transesterification activity. As a result of intensive research with the aim of preventing this from occurring, we have discovered a new method for producing a lipase preparation that retains the properties of lipase, and have completed the present invention.

That is, the present invention maintains the original characteristics of the lipase enzyme, which is characterized by adding a water-insoluble carrier to the lipase-containing culture solution obtained by culturing lipase-producing microorganisms and drying it. The present invention relates to a method for producing a lipase preparation.

In the decomposition reaction of nine fats and oils using the lipase preparation obtained in the present invention, a superior fat and oil decomposition rate and fat and oil transesterification activity can be obtained compared to lipase preparations obtained by conventional formulations.

Regarding the method for producing a lipase preparation according to the present invention, detailed production conditions are as follows. First, a known technique may be used for culturing the microorganisms, such as a solid culture method using 1 ton of bran or a liquid culture method. In the solid-state culture method, after culturing, a culture solution is obtained by extraction with a gold water medium. The culture medium II solution can be concentrated by ultrafiltration or salting out/dialysis, if necessary.

Rhizopus (Rhi) is a microorganism that produces lipase gold.
zopus) @, Aspergillus (Aspθrgi
It is preferable to use fungi of the genus G.llug, genus Muoor, and genus Geotrichum, or yeast of the genus Candida.

The carrier may be a water-insoluble carrier such as celite, cane earth, kaolinite, perlite, silica gel, activated carbon, or cellulose powder, and any carrier that does not adversely affect the enzyme activity can be used.

These water-insoluble carriers are usually used alone, but two or more types may be used in combination.

The ninth specific conditions for producing the lipase preparation of the present invention are as follows. That is, microorganisms ~ 200! A lipase preparation is obtained by adding a certain amount and stirring, followed by drying at 50° C. or lower. In this case, as for the drying method, freeze drying is preferred, but methods such as normal pressure drying or reduced pressure drying may also be used. The lipase preparation thus obtained can be subjected to a fat/oil decomposition reaction or a fat/oil transesterification reaction.

〔Effect of the invention〕

As is clear from the following examples, the method of producing a lipase preparation while retaining the properties of lipase according to the present invention is as follows:
Compared to the conventional production method of lipase preparations, the decomposition rate,
Since the transesterification rate is improved, it is clear that the present invention is superior as a method for obtaining lipase preparations.

〔Example〕

EXAMPLES Hereinafter, the present invention will be explained in more detail using Examples and Comparative Examples. All percentages in the examples refer to weight Example 1 Rhizopus dele r - (Rhizopus dele
Bactosoyto (mar)
ne; peptone for culture medium, DIFCOLABORA, USA
TORI! 3 companies) 5.0%, glucose 2.0%,
Liquid medium consisting of 0.1% sodium nitrate (NaN03), 0.1% potassium dihydrogen phosphate (KH2PO4) and 0.05% magnesium sulfate (Mg804)1.
5 days at 26°C with aeration and agitation, 11.1 g of a culture solution containing lipase was obtained, which was completely desalted by ultrafiltration and concentrated to 200 ml. Next, this concentrated liquid 10
After adding 20 parts by weight of Celite to 0 parts by volume and stirring, the entire process was freeze-dried to obtain 24 parts by weight of a lipase preparation.

Lipase preparation II obtained above (3000 units/g)
A decomposition reaction was carried out by stirring 50 g of beef tallow and 2501 g of water at -40°C for 24 hours.

After the reaction is complete, separate the oil layer from the decomposed product and extract the free fat in the oil layer! was titrated with alkali to determine the acid value (AV), and the ratio of acid value to saponification value (SV) of beef tallow (AV/SV) was used to calculate the decomposition ``4 (%).The results are shown in Table 1. Indicated.

Comparative Example 1 A lipase preparation was obtained in the same manner as in Example 1 except that 20 parts by weight of lactose was used in place of Celite. Using this lipase preparation, a decomposition reaction was carried out under the same conditions as in Example 1 (60 units/9 beef tallow).
4 (%) 1 Ratio determined using the same analytical method as Example 1. Table 1 shows the results.

Table 1 Example 2 2 IJpase formulation obtained in the same manner as Example 1.
, palm oil medium melting point (iodine number [IV] = 54°, diglyceride content 1%) 50 g, stearic acid 5 (M+, ion-exchanged water 0,075.9 and n- to #san 20014@
After the completion of the reaction, fatty acids are distilled off from the product and the stearic acid content in the resulting oil is determined by a conventional method. Friend to calculate the rate (%). The content (%) of diglyceride, a reaction by-product, was determined by high-performance liquid chromatography. the result? It is shown in Table 2.

Comparative Example 2 Lipase preparation obtained by the same method as Comparative Example 1 = 8,9
, Celite 59, M point m in palm oil < Iv=34,
Diglyceride content 1%) 509, stearic acid 503,
Ion exchange water 0.0759 and n-hexano 200d'
The enzymatic reaction (ester exchange reaction) was carried out by stirring in a closed container at 40° C. for 7 days. After the reaction was completed in the same manner as in Example 2, the reaction rate and diglyceride content were determined, and the results are shown in Table 2.

As is clear from Table 2, Comparative Example 2 has no transesterification activity at all, whereas Example 2 has high transesterification activity.

Table 2

Claims (1)

[Scope of Claims] 1. A method for producing a lipase preparation, which comprises adding a water-insoluble carrier to a lipase-containing culture solution obtained by culturing lipase-producing microorganisms, and then drying the culture solution. 2. The microorganism that produces lipase is Rhizopus.
pus) genus, Aspergillus
Genus, Mucor, Geotrichum
trichum genus or Candida
The method for producing a lipase preparation according to claim 1, which is a microorganism of the genus ). 3. The lipase according to claim 1, wherein the water-insoluble carrier is one or more carriers selected from the group consisting of celite, diatomaceous earth, kaolinite, perlite, silica gel, activated carbon, and cellulose powder. Method of manufacturing the formulation.