KR20010040732A - Mortierella - Google Patents

Abstract

The present invention relates to the reduction of motiella adhesion to the surface in culture with the use of dense strains on morphology.

Description

Mortiella {MORTIERELLA}

The present invention relates to motiella.

Mortiella, a genus of filamentous fungi, is of industrial interest because at least some members produce lipids containing residues of polyunsaturated fatty acids, particularly arachidonic acid and other polyunsaturated fatty acids, such as gamma linolenic acid, which can be metabolized by humans. It is a target. These lipids and / or acids derived therefrom are believed to be useful for human health when used as nutritional supplements. It has been proposed to add these lipids and / or acids to baby foods (baby artificial milk) to make them closer to breast milk. Acids, lipids or organics themselves can be eaten and then their polyunsaturated fatty acids may be made available by human digestion processes.

We found a tendency for motiella to adhere to plant surfaces in culture. Thus, a built-in stirrer with fungal biomass can be made relatively inefficient and conduits blocked.

We have now found a kind of motiella ("intentional organic") that causes substantially less of this problem.

Microscopic examination shows that Mortiella hyphae generally tend to be very entangled, making it impossible to quantify morphological properties in general. However, since the non-tangled ends protrude from the agglomerated mass, their examination shows that the intended organics are more branched than the known organics.

The following procedure shows that the intended organic matter is more dense than the known strains, resulting in a denser form. It is not known why or how the intended organics adhere to the solid surface less easily.

Many strains may be cultured under comparable conditions in liquid and plate cultures to identify preferred strains of Motiella. Plate nutrition media are different from those used in liquid culture. The area covered after plate culture for a period of time is substantially less for these preferred organics than for other organics with similar liquid culture growth rates. Generally, the area covered by the preferred organics is half, preferably less than one quarter, of that covered by known Motiella strains having similar growth rates in liquid culture.

The morphology can be more generally quantified as the morphology index M-Kr / μ max, where Kr is the average radius expansion (μm) per hour on the plate and μ max is the next composition at initial pH 6.8 that was not dropped by adding NaOH if necessary. Log _e 2 divided by twice the time in liquid incubation at the same temperature using a liquid culture medium of

The mean value of Kr is based on measurements taken at 24 hour intervals over 4 days on equal plates of the following composition:

(a) 1.9 (g / l) K ₂ HPO ₄

1.6 (g / l) NaH ₂ PO ₄

1.8 (g / l) (Na ₄ ) ₂ SO ₄

0.2 (g / l) MgSO ₄ .7H ₂ O (added by autoclaving and sterilized by filtration)

0.9 (mg / l) FeCl ₃

PH is 7.2 with trace element 2M HCl containing 1.0 (ml / l) 720 mg / l Ca, 5 mg / l Cu, 23 mg / l Zn

Add glucus at a final concentration of 45 g / l, yeast extract at a final concentration of 5 g / l and agar at a final concentration of 15 g / l.

The value of M is 4 × ^{10 3} μm, preferably 3.5 × 10 ³ μm. If the colonies on the plate were nearly circular, the radial expansion would be sufficient for two orthogonal measurements, but more deviations would be needed if leaving the circle.

The present invention includes novel Mortiella strains whose morphology index M is 4x10 ³ μm or less, preferably 3.5x10 ³ μm or less, as described above.

Suitable Mortiella strains are, for example, M. elongata, M. excigua, M. hygrophila, M. globalpina, M. minutisma, M. vertequilata, M. polykepuala and M. It could be Zizkai. They may produce lipids containing eicosapentainoic acid, gamma linolenic acid (GLA) or preferably arachidonic acid and / or derivatives thereof, in particular residues thereof, depending on the process conditions.

The present invention also provides a method for selecting novel Mortiella strains. One such method involves culturing strains on agar plates under similar conditions to select those with small colony regions. Strains that show little growth that are observed to be low density in the colony region should be ignored. The strains thus selected are most likely to have the desired morphology index. Mortiella strains may be cultured under nutritional constraints (especially nitrogen restriction) and then selected to show appropriate colony region values and / or morphologies to produce the appropriate strains. Each of these can then be appropriately cultured under nutrient limits and the selection can be repeated if necessary.

The present invention preferably continues the culture until the motiella is substantially adhered to the surface of the incubator, and then culturing the motiella in the incubator comprising the step of selecting the organic material of the dense morphology and culturing the dense organic material It includes a method to make. The procedure may be repeated as desired until an organic material with low surface tack is obtained.

"Low surface tack" means that the rate at which organics are removed from the surface under the usual harsh conditions encountered in culture is sufficient to prevent the formation of deposits on the surface.

The present invention also provides a method of producing lipids comprising the step of culturing motiella as described above.

In batch cultivation, the organic material to be grown first with the desired content of organic material is placed in a medium, preferably, after consuming one or more nutrients, the culture is again incubated, and the biomass is collected. It may be possible to continue to fill one or more nutrient sources in culture (“pad batches”). In continuous culture, new nutrient (s) are added and the product is isolated without discontinuing the culture. Such additions and separations may be continuous or hepatic. The novel Mortiella variant is advantageous in batch or pad batch operation but is particularly suitable for continuous culture. In each method the novel mortella is a more homogeneous mixture, generally producing faster and / or larger myomass.

Known methods may for example be continuous incubation in an air lift incubator (no flow parts but the conduit may be entangled with known varieties) or a stirred vessel.

The organics of the present invention will generally be able to grow at a pH of 4-10, preferably 6-8. In lipid preparation the pH is preferably adjusted to increase from an initial value of 5-6 to a final value of 6.5-7.5. The temperature may be 5-40, preferably 20-30 ° C. The organics may be incubated on a solid medium but preferably a liquid medium is used. Carbon sources in liquids include, for example, soluble substances such as glucose, fructose, sucrose, maltose or soluble starch or low density carbohydrates, fatty acids or lipids and / or hydrocarbons (for organic matter that metabolize hydrocarbons during growth). You can do it. Nitrogen sources include, for example, proteins, peptides and / or amino acids such as yeast extracts, meat extracts, complexes such as corn soaks or for example ammonium ions supplied with nitrates and / or eg sulfates or gaseous ammonia. It may be the same simple or a mixture of complex and simple source. The phosphate source is suitably inorganic. Trace elements and vitamins may be included. The concentration of the carbon source (w / w over the entire medium) is preferably at least 0.1%, possibly at least 30%, but preferably 1-15%, for example 2-10%. The concentration of the nitrogen source (w / w over the whole medium) is, for example, 0.01-1, in particular 0.1-0.4% when calculated as equivalent N. If nutrients are deficient, additional nutrients may be added. Multiple carbon sources may be available. In order to increase the yield of lipids, the post-consumption culture phase of nutrients, in particular nitrogen or phosphate, preferably excluding carbonaceous material is operated.

Air culture is suitably in the presence of dissolved oxygen supplied to the air. The culture is suitably stirred, for example by agitation or by gas flow. Suitably an air lift incubator (eg maintain liquid circulation by injection of gas such as oxygen or air) is used.

The cultured cells are suitably extracted using, for example, organic solvents such as hexane, ester or alcohol, possibly together or continuously. If desired, it may be possible to extract with halogenated hydrocarbons, for example chloroform, but it is preferable to use hydrocarbons or alcohols or esters as additives to avoid environmental problems.

Mortierella Alpina ATCC 32222 was grown by continuous incubation at pH6.8, 26 ° C. under aerobic conditions in which dissolved oxygen tension (DOT) was maintained at air saturation of about 20% or more. The culture was added to excess glucose as the first carbon source so that this glucose was maintained at a fixed concentration of 5-20 g / l in the culture as a whole during fermentation and ammonium sulfate was the limiting substrate (see table below) on SD4 medium. Has grown. The medium was added at a rate equal to the dilution rate of 0.05 h ⁻¹ .

Samples of the culture, grown on S2GYE agar plates (see Appendix 2), were removed from the fermentor daily, and the colonies obtained were carefully measured for colony variants. After inoculation of the continuous culture fermenter for 520 hours, colony variants were observed. The colonies of this colony variant were found to occupy smaller areas than the original parent organism. Moreover, colony variants were more branched.

For an additional 165 hours of continuous culture, colony variant species appeared to constitute 88% of the population present.

Pure cultures of novel colony variants were grown on S2GYE dishes at 28 ° C. After 7 days of growth, the spore suspension was made by aseptic transfer of mycelium spores into sterile saline solution. 10 μl drops of the spore solution were then transferred to the center of the agar containing the required medium.

The dishes were then incubated at 28 ° C. and the rate of expansion of the colonies was measured daily by measuring the diameter of the colonies on each dish at two vertical positions. This was done with four different types of media.

(A) = S2GYE agar

(B) = potato textloss agar

(C) = molten extract

(D) = S2 LOW N

(See Appendix 2). The same methods were repeated with the original strain.

The daily radial growth rate was calculated and the average rate was measured during the growth period (see Appendix 3). From the data, it can be seen that the radial expansion rate of the new strain is considerably slower than the reflex expansion rate of the original strain.

The new organisms were incubated in liquid medium (see Appendix 4) at 26 ° C. under aerobic conditions. Batch fermentation was used at an initiation pH of 6.8, which was a culture pH that was prevented from falling by adding sterilized 2 moles of NaOH. There was no adjustment to prevent an increase in pH. The maximum growth rate of the organism was confirmed by measuring the CO ₂ development rate (CER). Similarly, the original organisms were incubated under the same conditions, and their maximum growth rate was also measured by the same means. The results will demonstrate that the growth rates were similar, (0.06h ^-1, 0.07h ^-1 with respect to a novel strain with respect to the original strain), a novel strain that has a less adhesiveness. Samples of the cultures were analyzed after 6 days of growth. The oil content of the biomaterial was measured by solvent extraction using a mixture of dichloromethyl and methanol of 2: 1 v / v, and the content of arachidonic acid contained in the extracted oil was measured by producing methyl ester, and gas chromatography Analyzed. The results are shown in the table below.

Using this data, one can calculate the value of the morphology index (M) for each strain.

Appendix 1

Appendix 2

Seed 2 treatment

Seed 2 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 ₄

0.2 (g / l) MgSO ₄ 7H ₂ O (added to sterilized filter after autolysis)

0.9 (mg / l) FCl ₃

1.0 (ml / l) fisons trace elements containing 720 mg / l Ca, 5 mg / l Cu, 23 mg / l Zn with pH 7.2 with 2M HCl

Medium (A)

S2GYE was seed 2 medium with glucose added at a final concentration of 45 g / l. Yeast extract (oxoid L21) was added at a final concentration of 5 g / l and agar (oxoid bacteriologic L11) was added at a final concentration of 15 g / l.

Medium (B)

Potato Textose agar was obtained from Oxoid (CM139). Its configuration is as follows:

Chemical formula gm / L Potato Extract 4.0 Glycos 20.0 Agar 15.0

pH 5.6 ± 0.2

Medium (C)

Molten extract agar was obtained from oxoid (CM59). Its configuration is as follows:

Chemical formula gm / L Molten extract 30.0 Fungus peptone 5.0 Agar 15.0

pH 5.4 ± 0.2

Medium (D)

S 2 Low N was seed 2 with ammonium sulphate varying to 0.1 gl ⁻¹ . Yeast extract (oxoid L21) was added at a final concentration of 0.1 g / l, glucose was added at a final concentration of 45 g / l and again at a final concentration of 15 g / l.

Appendix 3

Comparison of Spinal Colony Expansion Rate (Kr) for Original and New Strains of Mortierella on Various Media A-D

The new strain was deposited on January 8, 1998 with accession number IMI 378077 to the Department of Surgery, YW9 3AF, Ferry Lane, Institute for International Fungiology, Health and Agriculture, UK (currently TW20 9TY, Surrey, Egham, Bakeham Lane). UK Center (Egham).

Appendix 4

Claims (11)

Preferably, the method comprises culturing the mortarella in a incubator comprising continuing to incubate until substantial adhesion of the motiella to the surface of the fermentor is followed by selecting organics of the dense morphology from the culture and subsequently culturing the denser organics. The method of claim 1 comprising repeating the procedure until an organic with low surface tack is obtained. Mortiella variant characterized by a morphology index (M) of ⁴ × 10 ³ μm or less. Growing a plurality of Mortiella strains on both liquid and plate cultures and selecting those that cover less plate area when compared to Mortiella strains with similar growth rates in liquid cultures; How to Screen Mortiella Variants. Culturing the Mortiella strains in a liquid medium under nutrient limitation conditions, culturing each of the so obtained in plate culture, and selecting one or more organics with a small colony area compared to other plate cultures performed under comparable conditions. Re-culturing the organic material and optionally repeating the process until an organic material having low surface tack is obtained. A method of making a lipid or lipid comprising food comprising the step of liquid culturing of motiella as defined in claim 3 and selected according to claim 4 or prepared according to claim 5. The method according to claim 1, 2 or 6, wherein the motiella is continuously cultured. 7. The method of claim 1, 2, 5 or 6, wherein the motiella defined in claim 3 and selected in claim 4 or prepared in claim 5 is motiella alpina. The motiella according to claim 1, 2, 6, 7 or 8, wherein the liquid medium comprises soluble carbohydrates in the presence of dissolved oxygen at a temperature of 5-40 ° C. and at a pH of 6-8. How to incubate. Preferably from motiella defined in claim 3 and selected according to claim 5 and prepared by the method of any one of claims 1, 2, 6, 7, 8 or 9. A method for recovering lipids by solvent extraction with an organic solvent. Mortiella and its variants, mutations and derivatives having the characteristics of the strain deposited with the International Mycology Laboratory under accession no. IMI 378077.