Microorganisms that extracellularly secrete lipid particles encapsulating lipids

Abstract

There are provided microorganisms having a property of producing a lipid containing unsaturated fatty acids as constituent fatty acids and extracellularly secreting the produced lipid encapsulated in lipid particles, methods of screening said microorganisms, as well as methods of efficiently producing a fatty acid-containing lipid using said microorganisms. Furthermore, there are provided lipid particles encapsulating a lipid containing unsaturated fatty acids, and foods, cosmetics, and animal feeds comprising said lipid particles added thereto. Artificially treated microorganisms or microorganisms collected from nature are grown on a solid medium, and microbial strains that form lipid particles at the periphery of the colonies and/or microbial strains that, when cultured in a transparent liquid medium, make the culture liquid cloudy are selected. The microorganisms obtained are cultured, lipid-containing lipid particles secreted in the culture liquid, are separated from the culture liquid, and the lipid is separated and purified.

Claims

1. A composition comprising isolated lipid particles extracellularly secreted by a mutated Mortierella alpina, wherein the isolated lipid particles encapsulate an unsaturated fatty acid-containing lipid, wherein the isolated lipid particles comprise 20-60% sugar, 0-30% protein, and 30-80% lipid, wherein the mutated M. alpina grows into colonies covered with the lipid particles on a solid medium, or produces a cloudy culture liquid in a transparent liquid medium, and wherein the isolated lipid particles are produced by M. alpina SAM 2241 or M. alpina SAM 2242.

2. The composition of claim 1, wherein the unsaturated fatty acid-containing lipid contains an unsaturated fatty acid having 18 or more carbons and two or more double bonds.

3. The composition of claim 1, wherein the microorganism is cultured in a liquid medium, and wherein the isolated lipid particles are obtained from a culture liquid.

4. The composition of claim 1, wherein the isolated lipid particles can be uniformly dispersed in water or a hydrophilic substance.

5. The composition of claim 1, wherein the isolated lipid particles stably retain the unsaturated fatty acid-containing lipid thereby preventing oxidation of the unsaturated fatty acid.

6. The composition of claim 1, wherein the isolated lipid particles are isolated by centrifugation and/or filtration.

7. The composition of claim 1, wherein the isolated lipid particles have an average diameter of 0.2 to 10 μm.

8. The composition of claim 1, wherein the unsaturated fatty acid-containing lipid comprises 50% or more triglyceride.

Description

EXAMPLES

(1) The present invention will now be explained in more details with reference to specific examples. It should be noted, however, that the present invention will not limited by these examples in anyway.

Example 1

Obtaining a Microbial Strain That Extracellularly Secretes Lipid Particles by Mutation of Mortierella alpina IFO8568

(2) Mortierella alpina IFO8568 was inoculated into a large slant bottle containing 300 ml of Czapek agar medium (0.2% NaNo.sub.3, 0.1% K.sub.2HPO.sub.4, 0.05% MgSO.sub.4.Math.7H.sub.2O, 0.05% KCl, 0.01% FeSO.sub.4.Math.7H.sub.2O, 3% sucrose, 2% agar, pH 6.0), and was cultured at 28° C. for 2 weeks.

(3) After culturing, 50 ml of sterile water to which had been added 2 drops of Tween 80 was added to the large slant bottle, which was shaken sufficiently, and then filtered with 4 ply gauze. This procedure was repeated twice, and the filtrate was centrifuged at 8000× g for 10 minutes. Spores thus obtained were suspended. into 50 mM Tris/maleate buffer solution (pH 7.5) to 1×10.sup.6/ml to prepare a spore solution.

(4) To 1.0 ml of the spore solution thus obtained, 0.5 ml of 100 mM Tris/maleate buffer solution (pH 7.5) was added, and 500 μl of the NTG solution (5 mg of N-methyl-N′-nitro-N-nitrosoguanidine per ml of deionized water) was added, which was subjected to mutation treatment by incubating at 28° C. for 15 minutes. After adding 3 ml of 10% Na.sub.2S.sub.2O.sub.3, the reaction mixture was centrifuged at 5500× g for 10 minutes, and the precipitate (spores subjected to mutation treatment) was washed with 3 ml of sterile water and centrifuged at 5500× g for 10 minutes, to which sterile water was added to prepare a NTG-treated spore suspension.

(5) The NTG-treated spore suspension was diluted to about 10.sup.−3 to 10.sup.−4 and then plated on a GY agar plate (1% glucose, 0.5% yeast extract, 0.005% Triton X-100, 1.5% agar, pH 6.0). After incubating at 28° C., those that developed colonies were examined for morphology with a result that microbial strains having a growth morphology distinctly different from that of the parent strain were obtained. The entire colonies of the highly unsaturated fatty acid-producing microbial strains including the parent strain were covered with mycelia as the strain accumulate the produced lipids in the cell, whereas the mutants obtained were covered with lipid particles.

(6) Subsequently, 4 ml of a transparent liquid medium (4% glucose, 1% yeast extract, pH 6.0) was dispensed into a test tube and sterilized at 120° C. for 20 minutes. Then a platinum loopful of the microbial strain obtained above was inoculated thereinto and cultured under shaking at 28° C. for 2 days, which made the medium cloudy. Even after allowing the medium to stand for over 10 minutes, no lipid layers were observed on the surface of the medium. Thus, the lipid was possibly secreted as lipid particles.

(7) The lipid particles that covered the colonies obtained in the culturing on the GY agar plate were analyzed for lipid by thin layer chromatography (TLC). To a previously activated plate (Merck 5554, 200×200×0.25 mm, silica gel 60F-254, aluminium sheet), the sample and the control (phospholipids, triglycerides, fatty acids) were plated, which was developed with n-hexane:diethylether:acetic acid=80:20:2 (V/V/V) using phosphomolybdic acid (10% phosphomolybdic acid in ethanol) and primulin (0.01% primulin in 80% acetone) as a color developer. For primulin, bands were examined under UV light of a long wavelength (366 nm). As a result, the majority of the lipids in the lipid particles observed outside of the cell were found to be triglycerides.

(8) Thus, about 3000 colonies yielded mutants Mortierella alpina SAM2241 FERM BP-7272 and SAM2242 that extracellularly secrete lipid particles encapsulating mainly triglycerides having highly unsaturated fatty acids as constituent fatty acids.

Example 2

Fatty Acid Analysis of an Extracellularly Secreted Lipid When Mortierella alpina SAM2241 That Extracellularly Secretes Lipid Particles was Cultured on Various Media

(9) Four ml each of medium A, B, C, D, E, and F was distributed in a test tube, and was sterilized at 120° C. for 20 minutes. A platinum loopful of Mortierella alpina SAM2241 (FERM BP-7272) obtained in Example 1 was inoculated into the medium, and cultured under shaking at 28° C. for 2 days and then at 12° C. for 7 days. After culturing, the cells and the filtrate were separated by filtration. The filtrate obtained was placed in a screw-capped test tube (16.5 mmφ), and lyophilized. To this were added 1 ml of methylene chloride and 2 ml of anhydrous methanol-hydrochloric acid (10%), which was methylesterified by treating at 50° C. for 3 hours. Four ml of n-hexane and 1 ml of water were added to this, and then extracted twice. The solvent after extraction was evaporated using a centrifuge evaporator (40° C., 1 hour), and the fatty acid methylesters thus obtained were analyzed by capillary gas chromatography. At the time of adding the methylester, 0.2 mg/ml n-heptadecanoic acid (17:0) was added as an internal standard, and fatty acids were quantitated based on the ratio of surface area of GLC.

(10) TABLE-US-00001 Medium A Glucose 1.0% K.sub.2HPO.sub.4 0.3 MgSO.sub.4•7H.sub.2O 0.02 Polypeptone 1.5 NaCl 0.2 Yeast extract 0.1 pH 7.0 Medium B Polypeptone 1.0% Meat extract 0.5 Yeast extract 0.1 NaCl 0.5 pH 7.0 Medium C Glucose 5.0% Polypeptone 0.5 KH.sub.2PO.sub.4 0.2 K.sub.2HPO.sub.4 0.1 MgSO.sub.4•7H.sub.2O 0.02 Yeast extract 0.1 pH 6.5 Medium D Glucose 2.0% Yeast extract 1.0 Bactopeptone 1.0 Medium E Glucose 2.0% (MRS medium) Meat extract 1.0 Yeast extract 0.5 Casein trypsin digest 1.0 K.sub.2HPO.sub.4 0.2 Sodium acetate 0.5 Diammonium citrate 0.2 MgSO.sub.4•7H.sub.2O 0.02 MnSO.sub.4•7H.sub.2O 0.02 Tween 80 0.1 Medium F Glucose 1.0% Yeast extract 0.5 pH 6.5

(11) The result is shown in Table 1.

(12) TABLE-US-00002 TABLE 1 The composition of extracellularly secreted fatty acids in the lipid obtained on various media 16:0 18:0 18:1 (n − 9) 18:2 (n − 6) 18:3 (n − 6) DGLA AA Others Medium A 14 8 20 11 4 4 31 8 Medium B 15 6 28 16 9 — 26 — Medium C 17 14 13 10 3 2 37 4 Medium D 14 9 23 9 5 2 31 7 Medium E 16 3 30 11 7 2 27 4 Medium F 15 5 23 10 5 3 32 7 16:0, palmitic acid; 18:0, stearic acid; 18:1 (n − 9), oleic acid; 18:2 (n − 6), linoleic acid; 18:3 (n − 6), γ-linoleic acid; DGLA, dihomo-γ-linoleic acid; AA, arachidonic acid

(13) In any of the media, a lipid containing unsaturated fatty acids were observed to be extracellularly secreted. Furthermore, the total amount of the extracellularly secreted lipid obtained for medium A to F and the amount of arachidonic acid were found to be positively correlated with glucose concentration. Thus, the total amount of lipid per test tube was 0.18 and 0.6 mg at a glucose concentration of 1%, 0.53 and 0.96 mg at a glucose concentration of 2%, and 2.11 mg at a glucose concentration of 5%, and the amount of arachidonic acid per test tube was 0.05 and 0.15 mg at a glucose concentration of 1%, 0.11 and 0.19 mg at a glucose concentration of 2%, and 0.62 mg at a glucose concentration of 5%.

(14) A result with similar tendency was obtained for the mutant SAM2242.

Example 3

The Amount of Arachidonic Acid Produced by Aerated Agitating Culture Using a 10 L Jar Fermentor of Mortierella alpina SAM2241 that Extracellularly Secretes Lipid Particles

(15) Five liters of a medium (A: pH 5.0, B: pH 6.0, C: pH 7.0) containing 2% glucose, 1.5% soy flour, 0.3% KH.sub.2PO.sub.4, 0.1% Na.sub.2SO.sub.4, 0.05% MgCl.sub.2.Math.6H.sub.2O, 0.05% CaCl.sub.2.Math.2H.sub.2O, and 0.2% soybean oil was placed in a 10 L jar fermentor, and sterilized at 120° C. for 30 minutes. Mortierella alpina SAM2241 (FERM BP-7272) obtained in Example 1 was inoculated therein, and were subjected to aerated agitating culture at an aeration rate of 1.0 vvm and a culture temperature of 24° C. for 10 days, 2.0% glucose was added on day 1 of culturing, 1.5% glucose on day 2, 1.0% glucose on days 3 and 4, and 0.5% glucose on days 5 and 6.

(16) Sampling was carried out every day. The culture liquid was separated by filtration. into the cells and the filtrate (extracellularly secreted lipid particles are dispersed therein). The cells were dried at 105° C. for 2 hours, and 20 mg of the dried cells was placed in a screw-capped test tube (16.5 mmφ)and was subjected to methylesterification as in Example 2. The filtrate (1 ml) was placed in a screw-capped test tube (16.5 mmφ), was lyophilized, and then was subjected to methylesterification as in Example 2. The fatty acid esters thus obtained were analyzed by capillary gas chromatography. Table 2 shows the amount produced of arachidonic acid and its content on day 9 of culturing.

(17) TABLE-US-00003 TABLE 2 The amount produced of arachidonic acid and its content on day 9 of culturing Extracellular arachidonic Outside of the acid In the cell cell percentage (%) Medium A pH 5.0 6.4 g/L(33.2%) 0.14 g/L(37.4%) 2.1 Medium B pH 6.0 5.8 g/L(32.4%) 0.24 g/L(34.5%) 4.0 Medium C pH 7.0 3.6 g/L(29.7%) 0.43 g/L(30.8%) 10.7

(18) Figures in parentheses indicate the ratio of arachidonic acid relative to the total fatty acids.

(19) With increased pH of the medium, the extracellular secretion of arachidonic acid-containing lipids was prompted.

Example 4

Lipid Analysis of Lipid Particles Secreted by Mortierella alpina SAM2241 That Extracellularly Secretes Lipid Particles

(20) The culture filtrates on day 9 of culturing in medium A, B, and C obtained in Example 3 were treated with. chloroform/methanol/water (1:2:0.8) by the Blight-Dyer method and the total lipids were extracted from the extracellularly secreted lipid particles. The total lipids obtained contained neutral lipids (triglycerides) and polar lipids (phospholipids). The total extracted lipids were charged into the Sep-pak Silica cartridge (manufactured by Waters) and eluted to obtain the neutral lipid fraction with chloroform and the polar lipid fraction with methanol. After evaporating the solvent, methylesterification was carried out as in Example 2, and fatty acid methylesters obtained were analyzed by capillary one chromatography. As a percentage of the total fatty acids to which triglycerides and phospholipids bind, the percentages of triglyceride and phospholipids were calculated. As a result, the percentage of the triglycerides in the total lipids for medium A, B, and C were 95.3%, 97.7%, and 96.2%, respectively.

Example 5

Continuous Culture of Mortierella alpina SAM2241 That Extracellularly Secrets Lipid Particles in a 10 L Bioreactor

(21) To a 10 L bioreactor having two built-in ceramic filters, 5 L of a medium containing 2% glucose and 2% yeast extract with pH adjusted to 7 was prepared, to which a precultured microbial strain of Mortierella alpina SAM2241 (FERM BP-7272) obtained in Example 1 was inoculated and subjected to aerated agitating culture. On the next day, a glucose solution was added through a ceramic filter to increase the glucose concentration in the medium by 3%. On day 2 also, the glucose solution was added through the ceramic filter to increase the glucose concentration in the medium by 3%.

(22) On day 3 and after, a 5% glucose solution and a 0.05% yeast extract solution were continuously passed through a ceramic filter at a speed of about 1000 ml/day. And the culture liquid was continuously extracted through a ceramic filter at about 600 ml/day (the amount of is adjusted to remain constant). In order to prevent the clogging of the filter with the cells, feeding of the glucose and yeast extract solutions and extracting of the culture liquid were alternately carried out as appropriate. Due to evaporation of water vapor by aeration, the amount of liquid in the jar remained almost constant. The feeding speed of glucose was adjusted by the glucose concentration to he extracted.

(23) As a result, a medium (culture liquid) that contained about 1 g/L arachidonic acid-containing triglycerides was able to be continuously extracted.

Example 6

Microbial Transformation of the Fat and Oil Added to the Medium and its Migration into Lipid Particles by Mortierella alpina SAM2241 That Extracellularly Secretes Lipid

(24) To 2 ml of a medium (pH 6.0) containing 1% glucose and 1% yeast extract, 2% linseed oil or fish oil was added, which was then put into a 10 ml Erlenmeyer flask and sterilized at 120° C. for 20 minutes. One platinum loopful of Mortierella alpina SAM2241 (FERM BP-7272) obtained in Example 1 was inoculated into the medium, and cultured using a reciprocating shaker (150 rpm) at 28° C. for 8 days. The filtrate was recovered by filtration, was lyophilized, and then the extracellularly secreted lipid was subjected to methylesterification as in Example 2, and the fatty acid methylester was analyzed by capillary gas chromatography.

(25) When linseed oil was added to the medium, a major fatty acid of linseed oil, 9,12,15-octadecatrienoic acid (α-linolenic acid) served as a substrate of the fatty acid biosynthetic enzymes of the mutant, and was converted to 6,9,12,15-octadecatetraenoic acid (stearidonic acid), 8,11,14,17-eicosatetraenoic acid, and 5,8,11,14,17-eicosapentaenoic acid, and the lipid in the lipid particles contained 2.4, 3.3, and 8.1% of 6,9,12,15-octadecatetraenoic acid (stearic acid), 8,11,14,17-eicosatetraenoic acid and 5,8,11,14,17-eicosapentaenoic acid, respectively, confirming that the converted fatty acids are extracellularly secreted as the constituent fatty acids of triglycerides. When fish oil was added to the medium, the fact that 5,8,11,14,17-eicosapentaenoic acid and 4,7,10,13,16,19-docosahexaenoic acid of fish oil are incorporated into the microbial strain and are extracellularly secreted as constituent fatty acids of triglycerides was confirmed because the lipid of the extracellularly secreted lipid particles contained 8.1 and 12.2% of 5,8,11,14,17-eicosapentaenoic acid and 4,7,10,13,16,19-docosahexaenoic acid, respectively.

Example 7

Component Analysis of Lipid Particles Extracellularly Secreted by Mortierella alpina SAM2241

(26) In order to analyze components of extracellularly secreted lipid particles, a spore suspension of Mortierella alpina SAM2241 obtained in Example 1 was plated to the Gtr agar plate (1% glucose, 0.5% yeast extract, 0.005% Triton X-100, 1.5% agar, pH 6.0), and cultured at 28° C. for 4 days. As shown in Example 1, the entire colonies were covered with lipid particles. Thus, the small particles were collected into a screw-capped test tube (16.5 mmφ). Chloroform (2 ml) and KCl solution (2 ml) were added thereto, shaken and extracted. Lipids migrated into the chloroform layer and sugars and proteins migrated into the KCl layer, and these were analyzed for the components according to a standard method and were found to comprise sugars: 38.1%, proteins: 18.2%, lipids: 43.7%.

Example 8

Preparation of Formula Using Lipid Particles

(27) The culture filtrate obtained in Example 3 was separated using a centrifuge (TOMY RL-101) at 1500× g and washed with sterile water to prepare lipid particles fit for consumption. The lipid particles (0.92 g) were added to 100 g of powdered milk to prepare a formula containing lipid particles. The composition of arachidonic acid in the formula obtained way 0.5% of the total fatty acids, which was similar to that of the mother's milk.

(28) When the formula was dissolved in water, its dispersion in water was good and uniformly dispersed without any separation of oils.

Example 9

Preparation of Capsules

(29) Water was added to 100 parts per weight of gelatin and 35 parts per weight of food additive glycerin, which was dissolved at 50 to 60° C. to prepare a gelatin coating with a viscosity of 20000 cps. Then, from the lipid particles separated by centrifugation from the culture filtrate obtained in Example 3, lipids were extracted and purified according to a standard method. Then, 97% the refinded oil and 3% vitamin E oil were mixed to prepare a content. Using these, capsule molding and drying were carried out according to a standard method so that soft capsules containing 180 mg content per capsule were produced.

Example 10

Preparation of Lipid Particles-Containing Beverages

(30) The lipid particles (10 g) fit for consumption obtained in the method shown in Example 8 were added to 10 L of orange juice to prepare juice containing lipid particles.

(31) Reference to the microorganisms deposited under the Patent Cooperation Treaty, Rule 13-2, and the name of the Depository Authority

(32) Depository Authority:

(33) Name: the National Institute of Bioscience and Human Technology, Agency of Industrial Science and Technology Address: 1-3, Higashi 1-chome, Tsukuba city, Ibaraki Pref., Japan

(34) Organism (1)

(35) Name: Mortierella elongata SAM0219

(36) Accession number: FERM BP-1239

(37) Date deposited: Mar. 19, 1986

(38) Organism (2)

(39) Name: Mortierella alpina SAM2241

(40) Accession number: FERM BP-7272

(41) Date deposited: Aug. 11, 2000