JP2002502612A - Mortierella - Google Patents

Abstract

  (57) [Summary] The use of morphologically compact varieties reduces the adherence of Mortierella to the surface during culture.

Description

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to Mortierella.

[0002] Mortierella is a genus of filamentous fungus in which at least some microorganisms of Mortierella have residues of polyunsaturated fatty acids, especially arachidonic acid, and other polysaccharides that can be metabolized by humans to the fatty acids. There is industrial interest in producing lipids containing residues of unsaturated fatty acids (eg, gamma-linolenic acid). It is believed that such lipids and / or such acids derived therefrom are beneficial to human health when used as nutrient supplements. Such lipids and / or acids may be added to infant formulas to make them more human milk.
la) (artificial milk for human infants). It is possible to eat these acids, lipids or the microorganisms that produce them themselves, and their polyunsaturated fatty acids are then converted to the normal process of digestion (normal process).
sses).

[0003] The present inventors have found that during the course of fermentation, Mortierella becomes plant surface (plant).
surfactants). According to microscopic examination,
Generally, Mortierella filaments have a very high tendency to tangle,
It is generally not possible to distinguish one individual from another to quantify morphological characteristics. However, untangled ends are tangled mass
Microorganisms that protrude and that are desirable to inspect at their ends
sm) is more highly branched than the corresponding known microorganisms.

[0004] The following measures can show that the desired microorganisms have a more compact morphology (corresponding to a greater degree of branching) than known variants. It is not known for what reason and in what manner the desired microorganisms hardly adhere to the solid surface.

[0005] To identify the desired strain of Mortierella, some strains may be grown in liquid and plated cultures under comparative conditions. The nutrient medium for the plates may be different from the nutrient medium used in the liquid culture. The area covered after such plating for a period of time is substantially smaller for the desired microorganisms than for other microorganisms of the same liquid culture growth rate. Typically, the area covered by the desired microorganism is less than half, and preferably less than one-fourth of the area covered by a known strain of Mortierella of the same growth rate in liquid media.

The morphology of Mortierella has a morphological index (morph) expressed by M = Kr / μmax.
and may more generally be quantified as an index. In this equation, Kr is an average expanding radius per hour (average) on a flat plate.
radial expansion, unit: a [mu] m), mu] max in a liquid culture and the same temperature using a liquid culture medium having the following composition log _e 2 multiplication time (doubling time) (i.e., unconstrained culture conditions Divided by the time required to double the dry cell weight below (ie, its maximum growth rate).

The concentration (gl ⁻¹ ) (NH ₄ ) ₂ SO ₄ 3.0 MgSO ₄ . 7H ₂ O _{1.0 K 2 SO 4 1.3 H 3} PO 4 0.75ml yeast autolysate (Bio Springer) 3.0 Glucose 45.0 trace component solution 1.0 (composition MnSO ₄ .4H ₂ O 40gl ^-1 ZnSO _{^{4 .7H 2 O 50gl -1 CuSO 4}} .5H 2 O 5gl -1 biotin _{^{50mgl -1 H 2 sO 4 5mll -1}} ) the composition is the initial pH 6.8, which addition of NaOH as required By doing so, the decline is prevented. The average value of Kr is based on measurements taken over a 24-hour period on an equal number of plates of the following composition over a period of 4 days. (A) 1.9 (g / l) K ₂ HPO ₄ 1.6 (g / l) NaH ₂ PO ₄ 1.8 (g / l) (NH ₄ ) ₂ SO ₄ 0.2 (g / l) MgSO ₄ . 7H ₂ O (added after sterilization by filtration after high-pressure steam sterilization) 0.9 (MG / L) FeCl ₃ 1.0 (ml / l) Trace component (720 mg / l Ca, 5 mg / l Cu, 23 mg / l) PH adjusted to pH 7.2 with 2M HCl) In addition to the above, glucose was added until the final concentration was 45 g / l, and yeast extract was added until the final concentration was 5 g / l. Agar was added until the final concentration was 15 g / l. (B) Potato extract 4.0 g / liter Glucose 20.0 g / liter Agar 15.0 g / liter pH 5.6 ± 0.2 (c) Malt extract 30.0 g / liter Mycological peptone 5.0 g Agar 15.0 g / l pH 5.4 ± 0.2 (d) 1.9 g / l K ₂ HPO ₄ 1.6 g / l NaH ₂ PO ₄ 0.1 g / l (NH ₄ ) ₂ SO ₄ 0.1 g / l 2 g / l MgSO ₄ . 7H ₂ O (added by filtration sterilization after high-pressure steam sterilization) 0.9 mg / l FeCl ₃ 1.0 ml / l Trace components (containing 720 mg / l Ca, 5 mg / l Cu, 23 mg / l Zn, 0.1 g / l yeast extract 45 g / l glucose 15 g / l agar The value of M is M ≦ 4 × 10 ³ μm and preferably M ≦ 3.5 × 10 ³ μm. preferable. If the colonies on the plate are almost circular, measuring two vertical radii may be sufficient, but more measurements are needed if the colonies on the plate deviate from a circle. Might be.

The present invention comprises a novel strain of Mortierella having a morphological index M as defined above of 4 × 10 ³ μm or less, preferably 3.5 × 10 ³ μm or less. Suitable strains of Mortierella may be, for example, those listed below: a
lpina (Mortierella alpina), M.P. bainieri (Mortierella bainieri), M.E. elongata (Mortierella elongata); e
xigua (Mortierella exigua), M.E. hygrophila (Mortierella hygrofia); globalpina (Mortierella globalpina); minutesima (Mortierella minutima), M.P.
verticillata (Mortierella Vertilata); polyc
epuala (Mortierella polysepuala) and M. zyzchae (Mortiera Zizkae). These are eicosapentaenoic acid, γ-linolenic acid (GL
A) or preferably arachidonic acid and / or their derivatives, in particular lipids containing their residues, can be produced depending on the process conditions.

The present invention further provides a method for selecting a novel Mortierella strain. One such method involves culturing the strain on an agar plate under the same conditions and selecting a strain with a small colony area. Strains showing little growth observed as low density in the colony area should be ignored. Strains so selected are most likely to have the desired morphological index. Suitable strains can be produced by culturing Mortierella strains under nutrient-restricted (particularly nitrogen-limited) conditions for a long time, and then selecting individuals with appropriate values of colony area and / or morphology. Such individuals can then be suitably cultured under conditions of nutrient restriction, and the selection may be repeated if necessary.

[0010] The present invention provides for the selection of a dense form of microorganisms from a culture thereof, preferably after continuous culture, until substantial attachment of Mortierella to the fermentor surface occurs, and the subsequent fermentation of said more dense microorganisms. Fermenter, comprising performing
or) the method of culturing Mortierella in This procedure may be repeated as many times as necessary until a microorganism with low adherence to the surface is obtained.

[0011] "Low adhesion to surfaces"
) "Means that the rate of removal of the turbulent microorganisms encountered in the fermentation from the surface under normal conditions is sufficient to prevent build-up on such surfaces. Means

[0012] The present invention also provides a method for producing lipids, which comprises the step of culturing Mortierella as described above. In batch culture, it is preferred that the medium is first inoculated with a microorganism that has been grown to a desired content of the microorganism of interest and then further cultured after one or more nutrients have been consumed. And recover the biomass therefrom. One or two
The supply of more than one species of nutrient may be supplemented during the cultivation ("feed batch (" fed b
atch ")"). In continuous culture, new nutrients are added and products are removed without stopping the culture. Such additions and removals may be continuous or intermittent. The new variety Mortierella is convenient for batch or fed-batch operations, but is particularly suitable for continuous culture. In each embodiment, the new Mortierella forms a more homogenous mixture and thus a better mix (mixi
ng), thereby generally providing faster and / or greater biomass formation.

[0013] Continuous culture is performed by a known method, for example, an air lift fermenter (air lift f).
ermentor (the fermenter has no moving parts, but the groove is formed by a known variety (var)
may be packed) or in a stirred vessel.

The microorganism according to the invention typically has a pH of 45 to 10, preferably of pH 6
-8. For lipid production, the pH is preferably adjusted so that the initial value is between 5 and 6 and the final value is between 6.5 and 7.5. The temperature is 5
The temperature is -40C, preferably 20-30C. The microorganism may be cultured on a solid medium, but it is preferable to use a liquid medium. The carbon source in the liquid is a solubilized
bles), for example glucose (fructose), fructose (fructose), sucrose (sucrose), maltose (maltose) or soluble starch, or poorly soluble carbohydrates, fatty acids or lipids and / or (metabolizes hydrocarbons for growth) In the case of microorganisms), it may contain a hydrocarbon. The nitrogen source may be complex, for example, proteins, peptides and / or amino acids, such as yeast extract, meat extract, corn steep liquor, or may be simple. For example, nitrate and / or ammonium ions (ammonium ions are supplied, for example, as sulfate or ammonia gas), or the nitrogen source may be a mixture of multiple and single sources. Good. The source of phosphate is preferably an inorganic substance. Trace components and vitamins may be included. The concentration of the carbon source (w / w based on the total medium) is preferably 0.1% or more, and may be 30% or more, but is preferably 1-15%, for example, 2-10%. The concentration of the nitrogen source (w / w based on the whole medium), calculated as equivalent nitrogen atoms (equivalent N), is, for example, 0.01 to 1%, especially 0.1 to 0.4%. As nutrients are depleted, additional nutrients may be added. Multiple carbon sources may be used. In order to increase the yield of lipids, it is preferred to manipulate the phase of culture after consumption of nutrients other than carbon material, in particular nitrogen or phosphate.

The cultivation is carried out under aerobic conditions, preferably in the presence of dissolved oxygen (which may be concentrated) supplied as a gas. Preferably, the culture is agitated, for example, by stirring or gas flow. Preferably, an airlift fermenter (maintaining liquid circulation with gas injection of oxygen, air, etc.) is used.

The cultured cells are preferably made of an organic solvent or the like (for example, hexane, ester, or alcohol, but may be a combination thereof, or may be used continuously. Good). If necessary, it may be extracted with a halogenated hydrocarbon such as chloroform, but in order to avoid environmental problems, it is preferable to use hydrocarbons, alcohols or esters.

[0017]

【Example】

Mortierella Alpina ATCC3
2222 was grown in continuous culture under aerobic conditions at pH 6.8 and 26 ° C., with dissolved oxygen tension ODT maintained at about 20% of air saturation. SD4 medium supplemented with excess glucose as the primary carbon source (see table below) so that the glucose concentration is maintained continuously at 5-20 g / l throughout the fermentation period and ammonium sulfate is the limiting substrate. ). The medium was added to make the dilution ratio equal to 0.05 h- ¹ .

[0018] Every day, a culture sample was collected from the fermenter, grown on an S2GYE agar plate (see Appendix 2), and the colonies obtained were carefully examined for colony mutation. 620 hours after inoculation in the continuous culture fermenter, colony mutation was observed. These colonies occupied a smaller area than the parent strain. Furthermore, colony mutants had more branches.

When the culture was further continued for 165 hours, 88% of the existing strains became colony mutants. Pure culture of the new colony mutants was performed on S2GYE plates at 28 ° C. After 7 days of growth, the spores on the mycelium were aseptically transferred to sterile saline to prepare a spore suspension. Then, a 10 μl drop of the spore solution was transferred to the center of the agar plate containing the desired medium.

The plates were then incubated at 28 ° C. and the growth rate of the colonies was measured daily by measuring the diameter of the colonies on each plate (measuring two perpendicular angles). This was performed in the following four media. (A) S2GYE agar (B) Potato dextrose agar (C) Malt extract (D) S2 Low N (see Appendix 2). A similar process was performed on the original stock.

The radial growth rate for each day was calculated and the average value for the entire growth period was determined (see Appendix 3). The data show that the radius growth rate of the new stock is significantly lower than that of the original stock.

The new microorganism was cultured in liquid medium at 26 ° C. under aerobic conditions (Appendix 4
See). Adopt batch fermentation, start pH 6.8, sterile 2M Na
OH was added to prevent the culture pH from dropping. To control pH rise
No means were provided. The maximum growth rate of microorganisms is CO_TwoThe release rate (CER) was determined by measuring. Similarly, cultivate the original microorganism under the same conditions, and
The growth rate was determined. As a result, these growth rates are similar (the original stock is 0.06 h ^-1 0.07h new shares^-1), It was shown that the new strain had lower adhesiveness. Culture
The samples were analyzed after 6 days of growth. 2: 1 dichloromethane and methanol
The oil content of this biomass was determined by solvent extraction of the v / v mixture,
This is extracted by forming a tellurium and analyzing by gas liquid chromatography.
The arachidonic acid content of the oil was determined. The results are shown in the table below.

[0023]

[Table 1]

Using the above data, it is possible to calculate the morphological index (M) for each strain.

[0025]

(Equation 1)

Appendix 1 SD4 Medium Component Concentration (gl ^-1 ) (NH ₄ ) ₂ SO ₄ 3.2 MgSO ₄ .7H ₂ O 0.5 K ₂ SO ₄ 1.0 H ₃ PO ₄ 1.0 Yeast Autolysis things (Bio Springer) 0.5 trace element solution 1.0 ^* trace element solution ^* component concentration _{^{(gl -1) MgSO 4 · 4H}} 2 O 40 ZnSO 4 · 7H 2 O 50 CuSO 4 · 5H 2 O 5 biotin 50 mg H 5 ml of ₂ SO ₄ .

Appendix 2 Formulation of Seed2 Seed2 medium 1.9 (g / l) K ₂ HPO ₄ 1.6 (g / l) NaH ₂ PO ₄ 1.8 (g / l) (NH ₄ ) ₂ SO _{40 .2 (g / l) MgSO 4} · 7H 2 O ( after autoclaving, sterile filtered and added) 0.9 (mg / l) FeCl 3 1.0 of (ml / l) Fisons trace elements (720 mg / l Ca , 5mg / l
Of Cu, 23 mg / l Zn and pH 7.2 with 2M HCl). Medium (A) S2GYE is obtained by adding glucose to a Seed2 medium to a final concentration of 45 g / l. Yeast extract (Oxoid L21) was added to a final concentration of 5 g / l, and agar (Oxoid Bacteriological L1) was added.
1) was added to a final concentration of 15 g / l. Medium (B) Potato dextrose was obtained from Oxoid (CM139). It is prepared as follows. Formulation gm / liter potato extract 4.0 glucose 20.0 agar 15.0 pH 5.6 ± 0.2 Medium (C) Malt extract agar was obtained from Oxoid (CM59). It is prepared as follows. Formulation gm / liter Malt extract 30.0 Fungal peptone 5.0 Agar 15.0 pH 5.4 ± 0.2 Medium (D) S2Low N is a seed in which the level of ammonium sulfate has been changed to 0.1 gl ^−1.
d2 medium. Yeast extract (Oxoid L21) was added to a final concentration of 0.1 g / l, glucose was added to a final concentration of 45 g / l, and further to a final concentration of 15 g / l.

Appendix 3 Comparison of Radial Colony Expansion Rate (Kr) of New and Original Mortierella Strains on Different Media A-D

[0029]

[Table 2]

[0030] The new strain was established on January 8, 1998 by the Commonwealth Agricultural Administration and the International Institute for Mycology (Comm.
onwealth Agricultural Bureau, International Mycological Institute), Ferr
y Lane, Kew, Surrey TW9 3AF, United Kingdom (Currently UK Center (Egham), B
akeham Lane, Egham, Surrey, TW209TY) and deposited under IMI 378077.

Appendix 4 Concentration (gl ⁻¹ ) (NH ₄ ) ₂ SO ₄ 3.0 MgSO ₄ . _{7H 2 O 1.0 K 2 SO 4} 1.3 H 3 PO 4 0.75ml yeast autolysate (BioSpringer) 3.0 Glucose 45.0 Trace element solution 1.0 (composition MnSO ₄ .4H ₂ O 40gl ^{- _{1 ZnSO 4 .7H 2 O 50gl -1}} CuSO 4 .5H 2 O 5gl -1 biotin _{^{50mg l -1 H 2 SO 4 5ml}} l -1)

CABI Biosciences A division of CAB International UK Center (Egham), Bakeham Lane, Egham, Surrey, TW20 9TY Phone: ++ 44 (0) 1784 470111 Fax: ++ 44 (0) 1784 470909 Dear Sir Patent Proceedings Microorganism: Mortierella alpina IMI CC Number: 378077 (Mortierella alpina) Strain Number: 26C The above microorganism, which was accepted on January 12, 1998, is included in the application that you signed on January 8, 1998. Subject to the terms and conditions set forth, it will be deposited on January 12, 1998 as a deposit in patent proceedings, a copy of which you must possess. Sincerely yours, Secretary-General International Mycological Institute Egham, UK Genetic Resources Collection Date February 6, 1997

CABI Biosciences Division of CAB International UK Center (Egham), Bakeham Lane, Egham, Surrey, TW20 9TY Phone: ++ 44 (0) 1784 470111 Fax: ++ 44 (0) 1784 470909 Dear Sir Patent Proceedings Microorganism: Mortierella alpina IMI CC Number: 378077 (Mortierella alpina) Strain number: 26C A test was conducted on January 27, 1998 for the survival of the above microorganisms. On this day, the microorganism was alive. Conditions under which the survival test was performed (only if the test is negative) The IMI Genetic Resources Collection, an international depositary authority recognized by the Convention,
A certificate will be issued in accordance with the Budapest Treaty's deposit of microorganisms in the patent procedure. Sincerely yours, Secretary-General International Mycological Institute Egham, UK Genetic Resources Collection Date February 6, 1997

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) C12R 1: 645) C12R 1: 645) (81) Designated countries EP (AT, BE, CH, CY, DE, DK, ES, FI, FR, GB, GR, IE, IT, LU, MC, NL, PT, SE), OA (BF, BJ, CF, CG, CI, CM, GA, GN, GW, ML, MR, NE, SN, TD, TG), AP (GH, GM, KE, LS, MW, SD, SZ, UG, ZW), EA (AM, AZ, BY, KG, KZ, MD, RU, TJ) , TM), AL, AM, AT, AU, AZ, BA, BB, BG, BR, BY, CA, CH, CN, CU, CZ, DE, DK, EE, ES, FI, GB , GE, GH, GM, HR, HU, ID, IL, IN, IS, JP, KE, KG, KP, KR, KZ, LC, LK, LR, LS, LT, LU, LV, MD, MG, MG, MK, MN, MW, MX, NO, NZ, PL, PT, RO, RU, SD, SE, SG, SI, SK, SL, TJ, TM, TR, TT, UA, UG, US, UZ, VN , YU, ZW

Claims (11)

[Claims] Claims 1. Preferably, after culturing is continued until Mortierella substantially adheres to the surface of the fermenter, a dense form of organism is selected from the culture, and A method for culturing Mortierella in a fermentor, comprising fermenting Mortierella. 2. The method according to claim 1, comprising repeating the above steps until an organism having low adherence to the surface is obtained. 3. A variant of Mortierella characterized by a morphological index (M) of at most 4 × 10 ⁻³ μm. 4. Growing a large number of strains of Mortierella in both liquid and plate cultures and selecting strains that cover a lower area of the plate in liquid culture compared to strains of Mortierella of similar growth rate. Methods for selecting Mortierella varieties having low adhesion to solid surfaces, including. 5. The Mortierella strain is cultivated in a liquid medium under nutrient-limited conditions, and the resulting individual is cultivated in plate culture and carried out under conditions comparable to reculturing said organism. Selecting at least one organism having a smaller colony area compared to another plate culture and optionally repeating this process until an organism with low adherence to the surface is obtained, producing a Mortierella varieties Method. 6. A method for producing a lipid or a lipid-containing food, comprising a liquid cultivation step of Mortierella, wherein Mortierella is as defined in claim 3 or selected as described in claim 4. Or the method of producing as described in claim 5. 7. The method according to claim 1, wherein the culture of Mortierella is continuous. 8. The method of claim 1, 2, 5 or 6, wherein the Mortierella is Mortierella Alpina, or the method of claim 3, or
A Mortierella selected as described in claim 4 or produced as described in claim 5. 9. The Mortierella is cultured in a liquid medium containing soluble carbohydrates at a pH of 6 to 8 and a temperature of 5 to 40 ° C. in the presence of dissolved oxygen.
Or the method described in 8. 10. A method according to claim 3 or a choice as described in claim 5,
Alternatively, a method for recovering lipids from Mortierella produced by the method according to any one of claims 1, 2, 6, 7, 8 and 9, preferably by solvent extraction with an organic solvent. 11. Mortierella having the properties of the strain deposited at the International Mycological Institute under the accession number IMI 378077, and a variant thereof;
Mutants and derivatives.