WO1998018952A1

WO1998018952A1 - Process for producing fats containing highly unsaturated fatty acids containing selectively concentrated docosahexaenoic acid

Info

Publication number: WO1998018952A1
Application number: PCT/JP1997/003949
Authority: WO
Inventors: Yuji Okita; Yukie Imai; Nobuyoshi Shimizu
Original assignee: Nippon Suisan Kaisha, Ltd.
Priority date: 1996-10-30
Filing date: 1997-10-30
Publication date: 1998-05-07

Abstract

A process for producing fats containing highly unsaturated fatty acids containing selectively concentrated docosahexaenoic acid by hydrolyzing fats containing highly unsaturated fatty acids with lipases having an elevated capability of selectively concentrating docosahexaenoic acid. These lipases are at least two lipases including monoglyceride lipase and/or diglyceride lipase originating in microorganisms belonging to the genera Penicillium and Candida, Penicillium and Mucor, or Penicillium and Rhizopus. This process makes it possible to produce fats containing highly unsaturated fatty acids with an elevated ratio of DHA to EPA.

Description

Description Process for selectively producing fats and oils containing highly unsaturated fatty acids in which docosahexanoic acid is concentrated

The present invention relates to a method for producing a highly unsaturated fatty acid-containing oil or fat in which docosahexanoic acid is selectively concentrated.

In the present invention, polyunsaturated fatty acids (hereinafter sometimes abbreviated as “PUFA”) are eicosapentaenoic acid (hereinafter abbreviated as “EPA”) and docosahexaenoic acid (hereinafter abbreviated as “DHA”). ) Means fatty acids having at least 18 carbon atoms and at least 3 double bonds per molecule. Background art

It is known that fish oils are rich in PUFAs such as EPA and DHA which have various physiological actions. Approximately 10 to 40% of PUFA is contained in the constituent fatty acids of fish oil. However, these PUFAs are structurally vulnerable to heat and the like, and are susceptible to oxidation. Since enzymes such as lipase act at normal temperature and normal pressure, they are considered to be particularly effective in modifying oils and fats containing a large amount of PUFA, such as fish oil. Already, there have been many reports on the method of enriching PUFAs using lipases that have low activity on PUFAs.For example, fish oil was purified using lipase derived from Candida cvl indracea. A method of hydrolyzing (Japanese Patent Publication No. 4-16619) is known.

Recently, EPA antagonized arachidonic acid, which is essential for infant growth, T JP97 / 03 9

2 Phenomenon such as suppression of length has become known, and the importance of lipases, which have the ability to selectively concentrate only DHA, among other PUFAs, is growing in importance. Such rivases include Candi da Lugosa (ς.

rugosa) -derived lipase (Japanese Published Patent Application No. Hei 11-hei 3-106694). However, when only one kind of lipase is used in this way, the produced fats and oils are governed by the properties of the lipase. For example, these lipases are not satisfactory in terms of PUFA recovery, since they have low activity on PUFA but do not act at all _(in addition, the lipase method of η: ί- The decomposition reaction reached an equilibrium state, and there was also a problem that the concentration of PUF II did not proceed any further.

An object of the present invention is to produce a highly unsaturated fatty acid-containing fat or oil having an increased ratio of DHA to EPA. The present inventors have started to search for rivases that achieve both selective enrichment ability and high recovery rate with respect to the above-mentioned problems, and through experiments in which multiple types of lipases obtained from various biological species are combined to act on a substrate. However, they have found that an increase in the DHA selective enrichment ability of a reaction system, which could not be achieved with a single lipase, can be achieved by using a specific combination of lipase groups, and thus completed the present invention.

The present invention provides a highly unsaturated fatty acid in which docosahexaenoic acid is selectively concentrated by performing a hydrolysis reaction of a polyunsaturated fatty acid-containing oil or fat with a lipase group having an enhanced ability to selectively concentrate docosahexaenoic acid. This is a method for producing fats and oils. / JP97 / 03949

Fatty acids other than PUFA in the constituent fatty acids of fish oils are hydrolyzed by lipase, which hardly or only slightly hydrolyzes the ester bond between PUFA and glycerin in the fatty acids of fish oil. The ester bond between glycerin and glycerin is easily hydrolyzed, and PUFA is concentrated in the glyceride part. In addition to the lipase hydrolysis reaction system, which does not act on triglyceride but acts on diglyceride and monoglyceride to carry out the hydrolysis reaction. The concentrated PUFA hydrolyzes EPA rather than DHA, and as a result, DHA is more concentrated in the glyceride than EPA. The intrinsic properties of the rivase, that is, the property of hardly or only slightly hydrolyzing the ester bond between PUFA and glycerin in the constituent fatty acids of fish oil are hardly affected. The system exhibits a new property in that the added rivase hydrolyzes EPA in preference to DHA. The lipase group that has enhanced the selective enrichment ability of docosahexaenoic acid is two or more lipases including one or more monodari lipase and / or diglyceride lipase and one or more other lipases. Here, the term “monoglyceride lipase and / or diglyceride driver” means a lipase which does not act on triglyceride but acts on diglyceride and monoglyceride to cause a hydrolysis reaction.

By adding monoglyceride lipase and Z or diglyceride rivase to the lyase hydrolysis system and performing the hydrolysis reaction, the DHA selective enrichment ability of the reaction system is increased.

The present invention relates to a method for producing a highly unsaturated fatty acid-containing fat or oil using a monoglyceride lipase. And / or hydrolysis by two or more rivases, including one or more diglyceride lipases and one or more other lipases, to selectively concentrate docosahexanoic acid. This is a method for producing saturated fatty acid-containing fats and oils. The above monoglyceride lipase and / or diglyceride lipase is preferably derived from a microorganism (Penicilliun microorganism. The present invention relates to a method for preparing a highly unsaturated fatty acid-containing fat or oil,

Penicillium) By selectively conducting the hydrolysis reaction with two or more lipases including one or more of mono-glyceride lipase and Z or diglyceride rivase derived from microorganisms and one or more other lipases, docosahexane is selectively obtained. This is a method for producing fats and oils containing highly unsaturated fatty acids in which the acid is concentrated. The above microorganism belonging to the genus Penicillium is used as a microorganism of the genus Penicillium.

Penicl 1 ium cameraberti i).

The present invention relates to a highly unsaturated fatty acid-containing fat or oil, comprising: a monoglyceride drino derived from Penicllium camemberti. —Selective enrichment of docosahexaenoic acid by hydrolysis with two or more lipases, including at least one lipase and / or at least one diglyceride rivase This is a method for producing fats and oils containing highly unsaturated fatty acids. Among the above lipase groups, lipases other than monoglyceride lipase and / or diglyceride lipase are known genus Candida (

Candida), Mucor 厲, Rhizopus 厲, As ぺ Noreginoles 厲 (Aspergi 1 lus) _x Shoot “Monas 厲 (Pseudomonas), Agnore Power Ligenes 厲 (Mcaligenes), Penicllium, Pengull Acuobacterium (Achromobacter) Tric Geotrichum F Fusarium, Huraicuia, Serratia, Chromobacterium, and / Or lipase derived from microorganisms of Staphylococcus (Staphylococcus) microorganisms, which are described in "Enzymes in Food Processing" Third Edition p.206-208, ACADEMIC PRESS. , INC., Llarcourt Brace & Company ".

The present invention relates to a method for producing a polybutadiene containing polyunsaturated fatty acid,

Penicillium) By selectively performing a hydrolysis reaction with at least one of mono- and diglyceride rivases derived from microorganisms, and at least one lipase derived from microorganisms belonging to the genus listed above. This is a method for producing a highly unsaturated fatty acid-containing oil in which docosahexaenoic acid is concentrated. A combination of two or more lipases, including one or more of the above-mentioned monoglyceride lipase and / or diglyceride lipase and one or more other lipases, is a combination of Penicillium and Candida or Penicillium. Rhium and Mucor II, or Nissiliium and Rhizops II. The present invention relates to a method for producing a monoglyceride derived from a microorganism containing a polyunsaturated fatty acid, which is derived from microorganisms of Pseudomonas and Candida, Pseudomonas and Mucor, or Pseudomonas and Rhizopus. Hydrolysis by two or more lipases, including one or more lipases and / or one or more other lipases, selectively converts docosahexaenoic acid. Contains concentrated polyunsaturated fatty acids This is a method for producing an oil and fat.

Hereinafter, the present invention will be described in detail. What kind of fats and oils used in the present invention contain PUFA such as arachidonic acid (C20: 4w6), EPA (C20: 5ω3) and DHA (C22: 6w3). May be something. It is known that such fats and oils containing PUFA are produced by fish such as sardines and tuna, marine animals such as crustaceans, and certain microorganisms. For example, tuna oil is known to contain DHA in a large amount of 22.0%, compared to 14.6% of valmic acid and 17.2% of oleic acid. The monoglyceride lipase and Z or diglyceride lipase used in the present invention have little or no action on triglyceride, and may be any that act on monoglyceride and / or diglyceride. Examples include those derived from Penicllium microorganisms. These lipases are all commercially available.

For example, as a microorganism of the genus Penicillium, Penicllium camemberti is exemplified as a preferred example. It is also possible to use commercially available and easily available rivase derived from P. nigerium mannberti [trade name: Lipase 6, Amano Pharmaceutical Co., Ltd.]. -The lipase used in the present invention, which is used in combination with one or more of the above-mentioned monoglyceride lipase and Z or diglyceride lipase, includes Candida, Mucor, Rhizopus, Aspergillus, Pseudomonas; Panorama, Alcal igenes, Peniclium, Peniclium Terminology (Achromobacter), genus Geotrichum, Fusarium, Humicula, Serratia, Chromobacterium ), Lipases derived from Staphylococcus microorganisms, etc. These lipases can be appropriately selected from these known lipases according to the purpose. Commercially available enzymes include lipase derived from Candida cylindrache [trade name: Lipase OF, Meito Sangyo Co., Ltd.], and Mucor My High.

Livase derived from (Mucor miehei) (trade name: Lipposim, Novo Nordeisk) can be used. These lipases may be immobilized if necessary. Examples of the two or more lipases containing one or more of the above-mentioned monoglyceride rivase and Z or diglyceride rivase and one or more other rivases used in the present invention include, for example, Penicillium and Candida. Or a combination of a genera of the genus Pseudomonas and the genus Mukoru, or a genus of the genus Pseudomonas and Rhizopus.

These two or more rivases may be added all at once from the start of the reaction, or may be added one or several at a time. The amount of lipase to be added may be any amount as long as the above purpose can be achieved.For example, 0.1 to 1000 units per 1 g of fat or oil, usually about 1 to 2000 units are used. I do. The hydrolysis reaction in the present invention must be carried out in the presence of a sufficient amount of water or a buffer, for example, 1 to 100% (% by weight) with respect to fish oil, preferably 10 to 10%. Use around 0%. The hydrolysis reaction proceeds by static or stirring at a temperature at which the lipase used is not inactivated. The fat is hydrolyzed using two or more of the above specific lipases, and the glycerin-fatty acid produced by the hydrolysis reaction is obtained by a known method, such as alcohol deoxidation, steam distillation, solvent extraction, ion exchange resin, etc. It can be removed by the following method. In this way, the glyceride portion is fractionated from the hydrolyzed oil, and an oil or fat achieving the above object can be obtained.

The progress of the hydrolysis reaction can be grasped by measuring the acid value of the reaction mixture. That is, the hydrolysis rate is given by the following formula [1]:

Hydrolysis rate (%) = (acid value Z saponification value of base oil) X 100 I: 1] Also, the recovery rate of each fatty acid is based on the corresponding fatty acid amount in the base oil and the corresponding fatty acid amount in the lipase-treated oil. It is determined from the ratio of For example, the DI-IA recovery rate is obtained from the ratio of the amount of DHA in the feedstock to the amount of DHA ift in rivase-treated Shanchu. That is, the following equation [2]:

DHA in lipase-treated oil (%) x {100 hydrolysis rate (%)} DHA recovery rate (%) = ~ [2] DHA in feedstock oil (%)

Can be calculated by BEST MODE FOR CARRYING OUT THE INVENTION

Hereinafter, the present invention will be described in more detail with reference to Examples, but the present invention is not limited to these Examples. Example 1

Tuna oil (D HA 21.97% _s 7.60% EPA, D HA / EPA 2.89, saponification value 185) 2 m1, Mucor Meiha in 2 m1 of distilled water Lipase derived from lipase (trade name: Lipozyme, Novo Nordisk) 400 units per unit, lipase derived from Penicillium Power Mmberberry [trade name: Reno. —Ze G, Amano Pharmaceutical Co., Ltd.] 80 units were added and stirred at 35 ° C. for 16 hours.

The hydrolysis rate was 43.4%. The glyceride fraction obtained by deacidifying and dehydrating the hydrolyzate is DHA 35.72%, EPA 7.27%, DHA ZE PA 4.92, and DHA is selected for EPA. Concentrated. At this time, the DHA recovery rate was as high as 92.6%. Example 2

The test was performed again under the same conditions as in Example 1. The hydrolysis rate was 46.6%, and the resulting glyceride fraction was DHA 37.14%, EPA 7.01%, and DHA / EPA 5.30. It was selectively concentrated. At this time, the DHA recovery rate was as high as 90.23%. Example 3

300,000 units of tuna oil and 300,000 ml of distilled water in the same manner as in Example 1 were used for 600,000 units of lipase derived from Mucor Mihai, and 120,000 units of lipase derived from Penicillium Power Lumberti. The unit was added and stirred at 350 for 16 hours (800 rpm).

The hydrolysis rate was 50.6%. The glyceride fraction obtained by deacidifying and dehydrating the hydrolyzate is DHA 39.58%, EPA 7.48%, DHA In ZE PA 5.29, D HA was selectively enriched for EPA. At this time, the DHA recovery rate was as high as 88.94%. Comparative Example 1

The same as in Example 1, 2 ml of tuna oil and 2 ml of distilled water were added with 400 units of rivase derived from Mucor Mihai, and 35 ° C without addition of lipase derived from Penicillium Power Mamberti For 16 hours. The hydrolysis rate was 25.1%, and the hydrolysis reaction did not proceed much as compared with Example 1. The glyceride fraction obtained by deoxidizing and dehydrating the hydrolyzate showed a high DHA recovery rate of 97.17%, but the concentration capacity was DHA 28.5 2%, EPA 7.74%, DHA / EPA 3.69 resulted in less concentrated DHA.

Comparative Example 2

To 2 ml of tuna oil and 2 ml of distilled water the same as in Example 1, 80 units of rivase derived from P. nimbium umberti was added, and the revase derived from Mucor Mihai was not added. The mixture was stirred for 6 hours. The hydrolysis rate was 0.2%, and the hydrolysis reaction hardly proceeded. The glyceride fraction obtained by deacidification and dehydration of the hydrolyzate was DHA2 2.18%, EPA7.35%, DHA / EPA3.02, and DHA was hardly concentrated. The value was almost the same as that of the raw material mug oil. Thus, two or more lipases, including one or more monoglyceride lipases and / or one or more other lipases, have little effect when used alone. It can be seen that it is not possible to demonstrate 1 ^

However, by using these rivases together, it is possible to increase the concentration of DHA and increase the ratio of DHA to EPA without significantly reducing the high recovery of lipozyme for DHA ₍

Example 4

Lipase derived from Candida Silicone Drache [Trade name: Lipase OF, Meito Sangyo Co., Ltd.] in 2 m 1 of tuna oil and 2 m 1 of distilled water as in Example 1 200 units, Penicillium power 40 uM of mannose-derived lyase was added, and the mixture was stirred at 35 ° C for 16 hours. The hydrolysis rate was 52.6% (the glyceride fraction obtained by deacidifying and dehydrating the hydrolyzate was DHA 40.86%, EPA 8.84%, DHA / EPA 4.6) In step 2, DHA was selectively enriched for EPA.

To 2 ml of tuna oil and 2 ml of distilled water as in Example 1, 400 units of lipase derived from Candida silyl dorache and 400 units of lipase derived from Penicillium power manberti were added. The mixture was stirred at C for 20 hours. The hydrolysis rate was 74.0%. The glyceride fraction obtained by deacidifying and dehydrating the hydrolyzate is DHA48.10%, EPA5.50%, and DHA / EPA8.75. Was selectively concentrated. Comparative Example 3

To 2 ml of tuna oil and 2 ml of distilled water, the same as in Example 1, 200 units of lipase derived from Candida silicon dorache was added, and at 35 ° C without addition of lipase derived from Penicillium kerman Berti. Stirred for 16 hours. The hydrolysis rate was 34.1%, and the hydrolysis reaction did not progress as compared with Example 4. won. The glyceride fraction obtained by deacidifying and dehydrating the hydrolyzate was DHA 31.82%, EPA 9.08%, DHA / EPA 3.51, and although DHA was concentrated, As compared with Example 4, the value was low, and the selective enrichment of DHA with respect to EPA was low. Comparative Example 4

To 2 ml of the same tuna oil and 2 ml of distilled water as in Example 1, 1600 units of lipase derived from Candida Silicone Drachet were added, and 3 g of lipase derived from Penicillium Powerummberti were added. Stirred at 5 ° C for 20 hours. The hydrolysis rate was 62.7%. Compared with Example 5, the hydrolysis reaction had not progressed. The glyceride fraction obtained by deacidifying and dehydrating the hydrolyzate was DHA 44.74%, EPA 5.78%, and DHA / EPA 7.78. The results showed a low value in comparison, and the selective enrichment of DHA for EPA was also low. Thus, even if the lipase combined with the monoglyceride lipase and / or the diglyceride lipase is changed from mucor myhai lipase to another lipase, these two or more lipases are added simultaneously. It can be seen that the effect is exhibited only when used in the water splitting reaction. Example 6

Ripase derived from Rhizopus oryzae (trade name: Lipase F—AP15, Amano Pharmaceutical Co., Ltd.) 400 units, Benishirium power in 2 ml of tuna oil and 2 ml of distilled water as in Example 1 80 units of lipase derived from Nmberti were added, and the mixture was stirred at 35 ° C for 16 hours. The hydrolysis rate was 51.8%. The glyceride fraction obtained by deacidifying and dehydrating the hydrolyzate is 丄 0

D HA 4 1.13% Ε Ρ A 7.02% D Η A / Ρ Ρ A 5.86.

DHA was selectively enriched over EPA.

Comparative Example 5

To 2 ml of tuna oil and 2 ml of distilled water as in Example 1, 400 liters of lipase derived from Rhizobium spores was added, and 35 ° C without addition of lipase derived from P. nigerium manmberti. For 16 hours. The hydrolysis rate was 32.7%, and the hydrolysis reaction did not proceed as compared with Example 6. The glyceride fraction obtained by deoxidizing and dehydrating the hydrolyzate was DHA 32.08% EPA 7.05% DHA / EPA 4.55, and DHA was concentrated. Compared to 6, the value was low, and the selective enrichment of DHA for EPA was also low. Example 7

With respect to the DHA concentrated oil obtained in Example 6, the rivase reaction was further repeated. That is, 2 ml of the glyceride fraction obtained in Example 6 and 2 ml of distilled water were added to 400 ml of lysose olase-derived lipase and 80 liters of lipase derived from sillium mannberti. Then, the mixture was stirred at 35 C for 16 hours. The hydrolysis rate was 35.6%. The hydrolyzate was deacidified. The glyceride fraction obtained by dehydration was DHA 52.36% EPA 5.81% DHA / EPA9 · 011. Compared with Example 6, the selective enrichment of DHA with respect to EPA was further improved.

Thus, lipases with much lower DHA-selective enrichment compared to lipases from Candida silin dorache can be combined with monoglyceride lipase and Z or diglyceride lipase. ^ _Λ

It can be seen that the selective concentration of DHA is remarkably improved by 14 and that an oil or fat containing 50% or more of DHA can be produced practically. Industrial applicability

A highly unsaturated fatty acid-containing fat or oil with an increased ratio of DHA to EPA can be produced.

Claims

Claim range ffl

1. A highly unsaturated fatty acid-containing fat or oil containing docosahexaenoic acid selectively concentrated by subjecting a highly unsaturated fatty acid-containing fat or oil to a hydrolysis reaction with a group of lipases having an enhanced ability to selectively concentrate docosahexaenoic acid. How to manufacture.

2. The method according to claim 1, wherein the lipase group is two or more lipases containing at least one monoglyceride lipase and at least one Z or diglyceride lipase and at least one other lipase. A method for producing fats and oils containing highly unsaturated fatty acids in which oxaenic acid is concentrated.

3. The method according to claim 2, wherein the monoglyceride lipase and Z or diglyceride lipase are derived from a Penicillium microorganism.

4. The microorganism of the genus Nissan is

4. The method for producing a highly unsaturated fatty acid-containing fat or oil in which docosahexaenoic acid is selectively concentrated according to claim 3, which is (Penicllium camembertii).

5. Among the above lipases, lipases other than monoglyceride lipase and Z or diglyceride lipase are Candida, Mucor II, EzozoDUS, and Eszoreginoles.厲 (Aspergillus), Pseudomonas, Panaure genus (Alcaligenes), Penicillium (ni-cl ^ ium), Achromobacter, Geotrichum, Lipase derived from any of the microorganisms of the genus Fusarium, Humicula, Serratia, Chromobacterium, and Staphylococcus. 5. The method for producing a highly unsaturated fatty acid-containing cross-linked butterfat according to any one of claims 1 to 4, wherein docosahexaenoic acid is concentrated.

6. Two or more lipases, including one or more of the above-mentioned monoglyceride lipases and / or diglyceride lipases, and one or more other rivases, are of the genus Penicillium and Candida or Penicillium lipase. A method for producing a highly unsaturated fatty acid-containing fat or oil enriched with docosahexaenoic acid according to any one of claims 2 to 5, which is derived from microorganisms of the genus Muco and Mucor, or Benicilum and Rhizopus. .