The strain of the fungus mortierella alpina peyronel bs-2 for the production of polyunsaturated higher fatty acids and their derivatives

 

(57) Abstract:

The invention relates to industrial Microbiology. A new strain of Mortierella alpina Peyronel BS-2 (VKM F-3625D) produces lipids with a high content of polyunsaturated higher fatty acids (PLURK), in particular arachidonic (20 : 4, W - 6) acid up to 60 - 65%. The strain also produces eykozapentaenovuyu (20 : 5, W - 3) and docosahexaenoic (22 : 6 W - 3) acid. The mixture PLURK used in the production of various drugs used in agriculture, veterinary science, medicine, cosmetics and food industry. High level production of arachidonic acid provides the prospective use of the strain. table 2.

The invention relates to agricultural, medical and food Microbiology, in particular the strain-producer of polyunsaturated higher fatty acids, which are used for the production of various drugs used in agriculture, veterinary science, medicine, cosmetics and food industry.

Known strains of fungi of the genus Mortierella: Mortierella alpina, Mortierella bainiari, Mortierella elongata, Mortierella exigua, Mortierella minutissima, Mortierella verticilata, Mortierella hygrophila and Mortierella polycephala to obtain arachidonic acid (EP, 0223960 A2, CL mm Mortierella hygrophila BKM F-1854, producing 10-45 (Rel. %)) arachidonic acid from the amount of fatty acids (RU, patent N 2058078, class A 01 N 63/04, 1996).

The disadvantage of these methods using specific strains is that, despite the relatively large amounts of arachidonic acid, it is unclear is whether these strains to produce other higher polyunsaturated fatty acids (PLURK) depending on the cultivation conditions, because in some cases it is necessary to change the ratio between PLURK, preserving the maximum amount of arachidonic acid, that is unclear is whether these strains to be universal in terms of production.

A known strain of the fungus Mortierella alpina Peyronel BS-1 (VKM F-3605D), which is the producer of polyunsaturated higher fatty acids. The strain produces up to 30-50% of arachidonic acid from the sum of all higher fatty acids (RU, patent N 2075934, class A 01 N 63/04, A 01 C 1/8, 1996). Availability and description of the strain given in this patent (the parent strain, prototype).

A known strain does not allow arachidonic and other PLURK in even larger quantities.

The aim of the invention is to obtain a strain that produces an even more significant amount of arachidonic acid culturing conditions.

A new strain of the fungus Mortierella alpina Peyronel BS-2 (VKM F-3625D) for the production of polyunsaturated higher fatty acids and their derivatives.

A new strain obtained by selection monoporosa isolates after exposure to ultraviolet radiation on the spore suspension Mortierella alpina Peyronel BS-1 (VKM F-3605D), with subsequent analysis of the content of arachidonic acid (a marker characteristic) in the isolates by GLC. Author number of the new strain BS-2. Is stored in the all-Russian collection of microorganisms under international Depository number VKM F-3625D. At this time, the strain is production.

Strain Mortierella alpina Peyronel BS-2 (VKM F-D) has the following cultural-morphological features in its cultivation in liquid media.

When the cultivation of the strain in flasks in liquid media occurs, as a rule, mycelial growth. Sometimes the fungus grows in the form of loose balls with a diameter of from 2 to 6 mm Mycelium is yellowish-cream color and has an odor similar to the smell of fish oil. At the beginning of the cultivation hyphae loosely branching, wide 5-9 μm, a length of 50-100 μm. In the process of cultivation is observed the formation of fat cells, filled with the drops of fat. Fat cells have the non-is C in flasks (750 ml) with 100 ml of medium on a shaker at 220 rpm for 5-7 days.

Standard growing conditions given below as examples.

Example 1. Environment 1, the following composition (g/l):

glucose - 80

peptone - 10

yeast extract Difco - 5

KH2PO4- 2

MgSO47H2O - 0,5

Example 2. Environment 2 (g/l):

millet - 40

(NH4)2SO4- 1,0

KH2PO4- 1,0

MgSO47H2O - 0,5

KCl - 0,5

CaCl2- 0,15

Water - 1000 ml

Millet boiled in a large volume of water for 1 hour, to make salt, sterilized at 115oC 30 minutes

Example 3. Environment 3 (g/l):

glucose - 80,0

yeast extract Difco - 1,0

(NH4)2SO4- 3,0

MgSO47H2O - 0,5

KCl - 0,5

KH2PO4- 2,0

CaCl2- 0,15

FeSO47H2O - 40 mg/l

ZnSO47H2O - 30 mg/l

CuSO45H2O - 10 mg/l

MnSO45H2About 5 mg/l

CoCl20.5 mg/l

The strain has the following cultural morphological traits in dense environments.

Example 4. Cultural morphological traits on wort agar.

The composition of the medium - wort nikolenka 6-7oB, agar 2%, pH 7.

Sterilization at 115oC for 30 minutes.

The fungus is cultivated on h is white, fluffy, grow small blades in the wrong sockets, with a linear growth rate of 6-7 mm per day; with the advanced air vegetative mycelium, often pubescent, 0.5-0.8 cm height; weakly paronomasia. Sporangiola straight, subulate, 60-150 microns in length; the top 2-4 microns in diameter, at the base of 6-10 microns with stem cell; simple, single and depart frequently from the substrate. Sporangia rounded, with knobby protrusions, at base slightly flattened, 10-20 µm in diameter, mnogopudovye are formed on the longer sporangiola; and odnostoronie elliptical-ovate, sometimes slightly angular, 10-14 µm in diameter, occur on shorter sporangiola. Sporangiospore elliptic-cylindrical 4-5,5 1,5-2,5 µm, usually with 1-2 drops of fat.

Example 5. Cultural morphological traits on glucose-potato agar.

The composition of the medium - broth potato - 100 ml, glucose - 10 g, agar 20 g, tap water up to 1000 ml Sterilize at 115oC for 30 minutes.

The fungus is cultivated on Petri dishes (diameter 9 cm) in the dark at 28oC 7 days.

Temistocle colonies on glucose-potato agar 35-40 mm in diameter, white, fluffy, grow small blades in the form of napredno-fluffy, 0,5-0,8 cm tall; weakly paronomasia. The structure and dimensions of sparganosis, sporangium and sporangiospore similar to that shown in example 4.

Example 6. Cultural and morphological characteristics at the hungry agar.

The composition of the medium - agar - 20 g, water of water and 100 ml. Sterilization - 115oC for 30 minutes.

The fungus is cultivated on Petri dishes (diameter 9 cm) in the dark at 28oC 7 days.

Temistocle colony on short agar 12-15 mm in diameter, white, fluffy with a few hypha glomeruli, grow small blades in the wrong socket with a linear speed of 1.5-2 mm per day; with the advanced air vegetative mycelium, flocculent-fluffy 0,4-0,6 cm tall; well paronomasia. The structure and dimensions of sparganosis, sporangium and sporangiospore analogous to example 4.

Example 7. Cultural morphological traits on the environment of čapek.

The composition of the medium (g/l) - sucrose - 30,0, NaNO3- 2,0, K2HPO4of-1.0, KCl And 0.5, MgSO47H2O - 0,5, FeSO47H2O - 0,01, agar - 20, distilled water - 1000 ml

The fungus is cultivated on Petri dishes (diameter 9 cm) in the dark at 28oC 7 days.

Temistocle colony on the environment of the čapek IDA wrong socket with a linear speed of 2-2,8 mm per day; with advanced air vegetative mycelium, flocculent-fluffy 0,5-0,6 cm tall; weakly paronomasia. The structure and dimensions of sparganosis, sporangium and sporangiospore similar to that shown in example 4.

Physiological and biochemical characteristics of strain Mortierella alpina Peyronel BS-2 (VKM F-D):

Strain the saprophyte aerobe, is growing in the range of pO2from 10 to 100% of saturation with air, does not grow under anaerobic conditions.

Able to grow in the range of from 5 to 32oC. the Optimum temperature for growth 24-28oC. Increases in the range of pH from 5.4 to 8.0, the optimum pH value of 6.0 to 7.2.

Disposes of starch, glucose, fructose, lactose, maltose, mannose, dextrin, galactose, glycogen, ammonia nitrogen, nitrate, amine nitrogen (gelatin, yeast extract, meat hydrolysates, fish meal, casein).

The technical result of the invention to provide a strain that produces a relatively greater amount of arachidonic acid in the fatty acid composition (up to 62%), as well as producing acid C20:5- C22:6depending on cultivation conditions.

Comparative producing ability of parent and new strains on the yield of fatty acids are given mcelearney biomass strains of Mortierella alpina Peyronel BS-1 and BS-2 when grown in different environments (table. 1).

Strain differs from its parent by a higher content of arachidonic acid (C 20:4, -6) from the amount of fatty acids: up to 62% vs. 34%. In addition, the strain BS-2 allows you to grow biomass to 25 g/l vs. 14 g/l in the case of strain BS-1.

The rise is higher unsaturated and long higher fatty acids such as eicosapentaenoic (20:5, -3) and docosahexaenoic (C 22:6, -3) can be achieved by changing the temperature of the cultivation of the strain, namely by reducing to 12oC in the last day (example 9).

Example 9. The content of higher fatty acids (Rel.%) in the sample extract obtained from mycelial biomass of the fungus Mortierella alpina BC-2, after culturing for 12oC (cultivation on the environment 1) (PL. 2).

Isolated from the biomass of strain the extract is used directly in the form of mono-, di-, triglycerides and other lipids or after saponification and chemical modification, for example, in the form of salts, esters, amides and other derivatives of polyunsaturated higher fatty acids, for the manufacture of various drugs used in agriculture, veterinary science, medicine, cosmetics and food industry.

The strain of the fungus Mortierella alpina Peyronel B is

 

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2 dwg, 12 ex

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