SU540578A3 - The method of obtaining protein-containing substances - Google Patents

Description

(54) METHOD FOR OBTAINING PROTEIN SUBSTANCE

From the type of gram kiearum, the Schwabe strain deposited in the Mycological Institute of the British Commonwealth for the number of Sh1 145425, its variants and mutants are used.

In addition to the form of graminearuro use

strains for N 1M1 154209, 1M1 154211, Ш1 154212, Ш1 154213, Ш1 154210.

Strain Fusarium aminearum: fflBa6e 1M1 145425 non-pathogenic relative to wheat and has the following morphological properties.

/ Wednesday. Potato agar with sucrose Czapek-Docks (modified agaroxoind). 250 g of potatoes are washed, cut into small pieces, placed in a press brew for 15 minutes under a pressure of 10.3411-10 n / m. The broth is filtered through two layers of muslin. 2% glucose and 2% agar are added to the cloudy filtrate, after which the medium is autoclaved and dispersed.

Growth conditions. A few weeks at 25 ° C. Growth rate 4.0 cm for 3 days, 3.0 cm for 3 days.

The nature of growth. Flaky, common colonies with white aerial mycelium. The substrate on potato agar with sucrose is a grayish-pink q slices from raspberry to yellow. Tendency to straw-yellow color on agar Chapek-Doksa. Sometimes a dark red pigment is formed (especially with aging).

After one to two weeks, aerial mycelium is prone to burish staining (brown) color and flattening. Colonies become mucous when sporodokhii are formed and color from pink to brown on KSA (potato agar with sucrose) and orange-pink on the analytical grade (Capes Doxa agar).

Exudate is not formed, and the formation of pigment corresponds to the staining of the mycelium.

Conidia Microorganisms do not form microconidia. Microconidia are formed from separate lateral sterigms or branched conidiophores with short sterigmas. In older cultures, conidiophores aggregate to form a sporodoha (especially on the analytical grade). Conidi are different: from sickle-shaped to curved dorso-ventral partitions from 3 to 5, in younger cultures usually up to 5.

Spore sizes range from 25-50x2.5 to 4.0 microns.

The support cell is often provided with a leg (especially in long spores with 5 partitions). Swollen cells are present in the mycelium and sometimes chlamydospores are included, separate or chains.

The following is an example of the production of variants (or isolates) of the Fusarium draminearum Schwabe strain 1M1 145425 and their morphological features.

The isolate obtained on a continuous culture of Fusarium graminearum Schwabe 1M1 154-209 to 1M1 154213 is taken from the plates of malt agar inoculated with a broth from fermenJeJ) a in which Fusarium graminearum Schwab is grown

154245 on glucose in a saline medium at 30 ° C. A bunch of uninterrupted culture conditions with the limitation of the gloroid at a dilution rate of 0, 1-0.15 hour7.

Samples are taken from the fermenter at intervals of about 100 hours to determine variants. Isolates Ш1 154209-154213 are taken from the plates prepared from the sample at the 00 o'clock. These isolates represent the main types of morphological variation obtained during the fermentation process. In the conditions given in the example, variations appear on the plates after one hour. From this time on, they are formed temporarily, and after 1Gul of 1C1, they slowly change in percent of each species.

When this is used in the fermenter culture medium composition,%:

Solution 1.

Glucose3.0

Ammonium sulfate 0.25

Monopotassium phosphate

ILIKN2P040,3

Against OB shenivatel-polypropylene glycol 2000, sterilized at pH 3.0 for 30 min at 10.341 MO n / m 0.01 Solution 2.

MnSO44H200,0005

FeS047H200,0005

ZnSO4 7H2O0,0005

CoCl26H2O0,0001

CuS045H200,0001

Na,., 00,0001.

Sterilized for 15 min at 10.3411 n / m Solution 3.

Biotin0,10 or 20 mkg / l

Choline 0.1 or 2 mg / l

Methionine O or 600 mg / l.

Sterilized separately by filtration. All solutions are septicated into a tank placed in a 8.5 L chemostat under the following growth conditions.

Temperature 30 ° С, aeration 1 v / m, stirring at 800 rpm on a 6-blade turbine agitator in a fermenter with baffle.

The fermenter is maintained at pH 5.0 by the automatic addition of sterile ammonia. The composition of solution 3 is changed at various time intervals to determine the maximum by various additives, for example, biotin or biotin, scholin.

Growth rate 0.1 -0.15 hour Five isolates have the following morphological properties.

Wednesday. Potato agar with sucrose

Agar Čapek-Doke (oxoid). 250 g of potatoes are washed, crushed, placed in a press billet under a pressure of 10.3411-10 N / m for 15 minutes.

Broth is passed through a double layer of muslin.

2% glucose is added to the cloudy filtrate along with 2% agar, after which the medium is autoclaved and dispersed.

Growth conditions; 25 ° C. °

The nature of growth. Fusarium graminearum Schwab NMMl 154209.

Colonies are morphologically similar to the original A3 / 5, except that the diameter of the colony is less than and equal to 1.5 cm after 3 days at 30 ° on the analytical grade. Flaky aerial mycelium is different in colonies: x, but less than No. IMi 154211. Older cultures show a loss of mycelium.

On the reverse side are yellow-brown to gray-pink sectors with some pigmentation. Fusarium graminearum Schwabe No. 1M1 154211.

Intensive white with flaky pubescence aerial mycelium. The diameter of the colonies is smaller than the original A3 / 5.2 cm after 3 days at 30 ° ha of the analytical grade. On the reverse side, the segments from orange {gray to grayish-pink ...

 Fusarium graminearum Schwabe No. 1M1 154212. HaFusarium gramiinearum IMl 154211 is microscopically similar, but the other side is lighter (from salmon-pink to transparent).

Fusarium graminearum Schwabe NMM1 154213. Small dome-shaped colonies. Mycelium is short, matted, with a tendency to form spirals. Many structures have a pinkish appearance at the beginning of the strip. Pink coloring on the periphery. The diameter of the colony in, 4 cm after 3 days at 30 ° on the analytical grade.

Fusarium graminearum Schwabe No. 1M1 154210.

The species is very unstable and continuously varies to the type 1M1 154213. Similar to 1M1 154209, the colonies have a slightly larger diameter (2.4 cm after 3 days or more). View 1M1 154210 more resistant, than 1M1 154213.

Conidia . Fusarium graminearum Schwabe No. 1 Ml 154209.

Abundant microconidia are formed in a manner similar to the initial AZ / 5.

In older cultures, sporodokhii are formed. Individual macroconidia are similar to the original AZ / 5 in shape and size, 25-50; 3.0-5.0 µm, B-old cultures form a lot of sporodokhii.

In the mycelium of this species, chlamydospores are abundant, intercameral and terminal. They are spherical 10-12 microns. Sometimes a single microconidium cell forms chlamydospore.

Fusarium graminearum Schwabe 1M1 154211.

Less microconidia than 1M1 154209, smaller, simpler, 1 partition, 25-35 microns long. Forms a little sporodohii.

Abundant chlamydospores, inercally, separate and in chains. Fusarium graminearum Schwabe 1 Ml 154212.

It is similar to IMl 154211, but it has more macroconidiums, in number as much as in the species W1.154209.

Fusarium iJraminearum Schwabe 1 Ml 154213.

Macroconidium is small, there are types of ciiopofloxi-in. Up to the packs of chlamydospores. Many chlamydospores in the mycelium. Smaller macroconidia; than the original, 30-35 4 microns with 2 partitions. , Fusarium grarrtnearum Schwabe Ш1 154210.

 Looks like the look of W1 154209, with abundant macroconidia and chlamydospores. Typical macroconidia, 35-45 4 microns.

The incubation temperature is maintained within 25-34 ° C (preferably 30 ° C), inoculation by a preliminary stage of germination 2-10% inoculum, usually from 5-10%.

the pH of the substrate during the incubation process is maintained in the range of 3.5-6.7, contributing to maximum growth.

The period of growth under serial cultivation under these conditions is usually 20 to 48 hours.

With both periodic and continuous cultivation, aeration and mixing should be carried out, ensuring sufficient dissolved oxygen content and overcoming its deficiency, which limits growth.

In the substrate environment there should be a sufficient amount of the main nutrient sources of nitrogen, sulfur, phosphorus.

Vitamins, such as biotin, are necessary for maximum growth rate.

A nontoxic antispirator is added to the substrate to control the foaming during the enzyme process.

The resulting mycelium is separated by washing, filtering and drying. By reducing the moisture content to 50% by filtration under pressure, the subsequent drying is not carried out in strict temperature conditions. The method of drying should not reduce the nutritional value of the mycelium; therefore, drying is carried out with an air stream at 75 ° C or by flooding (sublimation).

The fungal mycelium obtained by the proposed method binds water and can be used as a thickener or gelling agent.

Without being an isolate, it retains vitamins and other nutrients, such as lipids and some carbohydrates.

Fungal mycelium has satisfactory exudation properties and can be used in the production of protein-enriched bread and snacks.

Due to the fibrous structure of the fungal mycelium, it is possible to bake, fry, cook and bake it and other food products that are comparable in appearance and acceptability to ordinary food products.

Example Prepare 10 liters of culture medium and sterilize in a fermenter while mixing the composition.

Molasses (sugar cane) to

l% weight. sugar amount

Ammonium sulfate 1 2% Na, H, PO, 0.25% Sterilized for 30 min at 10.3411 U and / m, CAC0.5 wt.% / Vol. Sterilized for 3 hours. The components of the medium are aseptically and cooled to 30 ° C. An inoculum equivalent to 5-10% by volume of a culture medium grown on glucose medium (corn liquid) or on another appropriate material in a shaker flask is inoculated with a suspension of an organism spores containing Fusarium draminearuro Schwabe IMl 145425 strain. It is grown for 18-24 hours at 30 ° C in a rotary shaker and introduced aseptically into the fermenter. Fermenter incubated at 30 ° C; mixing at 800 rpm using a turbine-disc clutch with 6 blades and a baffle, with aeration through a medium of sterile air 1 rpm. After 35 hours, the grown mycelium is recovered, centrifuged, washed with water and stored in a belt dryer with hot air at 75 ° C. The dried product has the following common nitrogen Ash Lipids 52 calculated as total NPUop AZ Example 2. Prepare 10 liters of culture medium and sterilize in a fermenter with a capacity of 14 liters (NEW-Bunsvik, Microferm). Data on the composition of the culture medium are given in Table 1. The indicated culture medium is sterilized by heating for 15 minutes at 10.3411 10 n / m, after which sterilized vitamins or amino acids are added to it. Solutions are added to the fermenter aseptically. The inoculum is grown and applied as in Example 1, but the final concentration in the fermenter is adjusted to 0.5 g of air-dry mycelium per liter. Growing conditions: AOR temperature is 1 rpm, agitator speed is adjusted to maintain dissolved oxygen in the culture medium by 25% higher than the saturation concentration measured by npH6qpoM of New Brunswick Inc. A sterile antifoaming agent — propylene glycol 2000 is added to suppress foaming, and the pH is maintained in the range of 6.0-6.3 by the addition of a sterile solution of potassium hydroxide. When growing a culture of microorganisms without adding sterilized vitamins or amino acids, their growth rate is very slow, and when the latter are added with a final biotin concentration of 50 µm / l in a culture medium, the growth rate is 0.2 hour. When sterilized vitamins or amino acids are added to the culture medium with a final biotin concentration of 50 μg / l, choline chloride 30 mg / l and methionine 300 mg / l, the growth rate is 0.25 hour. PRI me R 3. Prepare 100 L of culture medium and sterilize in a 130 L stainless steel fermenter. ComponentsFinal concentration1-1% Glucose4.0 Kutsuruz hardening liquid (50% solid) Ammonium sulfate °, pH is adjusted to 5.0 with sterile ammonia. Biotin is sterilized by filtration, added aseptically, providing a final concentration of 40 µg / l, the fermenter is inoculated with 10 l of culture grown in a tank for 18 hours at 30 on medium containing,%: 2 glucose, 0.4 tryptone (oxo), 0 , 1 yeast extract (oxoid) 0.15 ammonium sulfate, 1 monopotassium phosphate, 0.1 caustic soda, 0.025 magnesium sulphate, 0.001 ferrous sulphate, 0.001 zinc sulphate, 0.0005 manganese sulphate, 0.001 - copper sulphate, 0.05 anti-foaming agent -propylene glycol 2000, sterilized for 45 minutes at 10, 34110-10 n / m inoculated with spenziey spores Fusarium graminearum Schwabe strain 1M1 145425. growth conditions: temperature 30, aeration is controlled in accordance with neobhodamoy dissolved oxygen concentration at 10% vptse nasptseni concentration in the culture medium. Polypropylene glycol 2000 is added to suppress pricing, maintaining a pH of about 5.0 by adding sterile ammonia. Samples of the mycelium after the cultivation of content is calculated on the basis of dry weight: total nitrogen - 8.0–8.6%; alpha amino acid nitrogen - 6.4-6.6%. The initial growth rate in the complex medium obtained from the final medium and inoculum is about 0.3 hour.

Below are examples 5 and 6, the implementation of the proposed method in the unsuccessful cultivation,

Example 4 A culture medium of the following composition was prepared:

final concentration,%

T-aciuop 1

 3.0

Glucose 0.25

Ammonium Sulphate Monopotassium Phosphate

orKNO 0.3

Magnesium sulfate 0.025

Polypropylene glycol 2000 0.01 is sterilized at pH 3.0 for 30 minutes at 10, 3411 n / m. Solution 2

MnSO44H2O0,0005

FeS04-7HiO0,0005

0.0005 0.0001 0.0001 0.0001

Stronger for 15 minutes. Solution 3.

 and / or amino acids are sterilized by filtration, as described in JTO below.

All solutions are aseptically applied.

Conditions for growing in a 8.5 liter chemostat are as follows.

Temperature 30 ° C, aeration 1 rpm, mixing at 800 rpm using a generous turbine-disk memeshalki in a fermenter with a baffle plate.

The strain of FusarJum qraminearum Schwabe IM 145425 is used as a microorganism, pH 5.0 is maintained automatically by the addition of sterile ammonia.

Data on the growing conditions of the microorganism are given in Table. 2

Table 2

Solution 320 0.17-0.19 0.5 Solution 3 20600 0.2-0.21 0.5

Example 5. Preparing a culture medium of arrest.%;

Starch legumes (processed by alpha-amylase) 0.3 carbohydrates

Corn locking liquid1,33

Ammonium sulfate 0.25

Monopotassium phosphate 0.15

EXAMPLE 6 The culture medium and conditions are the same as in Example 5.

The pH is adjusted to 5.0 throughout the entire process at 26-34 ° C. The optimum temperature is 30-32 ° C.

Example 7. Duplicate rocking flasks with an elongation of 1 l are filled with 200 ml of medium of the following composition, final concentration,%:

Magnesium sulfate 0.025

Polypropylene glycol 2000 (volume / weight) 0.025 Sterilized for 30 min at 10.3411 10 n / m and pH 4.0.

The medium is introduced into a 8.5 l chemostat under the conditions of example 4 at a pH of 3.5-6.0 and a growth rate of 0.1

Data on the growth conditions of the microorganism are given in Table 3,

Table 3

Solution 1. Glucose (sterilization separately at pH 3.0 for 10 min at 6.8941; 10 n / m) Solution 2. Ammonium sulfate MoH (starch phosphate MgS0.7H20

FfeS04 (H4) 2-0 ° 6jH20

MnS04-4H2O 7.26.35465 7.7-8.6 6.1-6.3 .5978

0.0001

CuS04-5H2O

SOS-bN About 0.0001

0,0015

CaCl2-2H20 0.000001

Na2Mo04-2H20

0.2

NaOH

Sterilization was carried out at 10.3411 "10 n / m for 15 minutes at a final pH of 6.0.

 Solution 3. Vitamin stertisated by filtration. The solutions are aseptically applied, providing a final volume of 200 ml, then the flasks are inoculated with washed spores of strain Fusarium graminearum Schwabe III 154209 to a concentration of 8-.

Growth conditions, temperature 30 °, 140 rpm on an orbital rocking chair with a swing arc of 508 mm-.

After one hour, growth intervals are measured by determining the optical density of the sample at, 600 microns.

When growing the microorganism strain Fusariumgraminearum Schwabe IMi 154209 on a medium not including solution 3, its growth rate is very slow.

When solution 3 is turned on in the medium and the final concentration of biotin in it is 50 μg / l, the growth rate is 22.

With the inclusion of solution 3 in the medium and the final concentration of biotin in it 50 µg / l and choline chloride 50 mg / l, the growth rate is 0.27 hours.

With the growth of microorganisms of Fusarium graminearum Schwabe IMl 154211, IMl 54212, IMl 154213, IMl 154210 and IMl 145425 strains on the substrate described in Example 7, without including the solution 3 sterilized by filtration of vitamins, their growth rate is very slow.

irradiation of these strains on the same medium with the inclusion of a solution of 3 sterilized vitamins with a final concentration of biotin in a culture of 50 mcc / l, a growth rate of 0.21-0.22, and with the addition of bioten 50 mcc / l and choline chloride {50 mg / l) - 0.27 hours.

 the invention

L A method for producing a protein-containing substance by treating fungi of the genus Fusarium under aerobic conditions in an aqueous nutrient medium containing a carbon source in the form of carbohydrate, a nitrogen source, mineral salts and growth stimulants, followed by separation of the resulting product from the culture fluid, lt h But in order to improve the quality of the semi-precious product, from the genus pusaFusarium use the form of graminearum.

2. A method according to Claim 1, characterized in that Schwabe strain is used from the graminearum species. for №1М1 145425.

3. The method according to claim 1.2, is different and so .... That, from the graminearum type, use is made of the number SCH1 154209, Ш1154211.1М1 154212.1М1 154213, 1М1 154210.

4. A method according to claim 1-3, characterized in that the growth is carried out at a pH of predominantly equal 3.5-7.

5. A method according to claim 1-4, characterized in that biotin is used as a growth promoter.

6. A method according to claim 1-4, characterized in that hopin is used as a growth promoter.

7. A method according to claim 1-6, characterized in that the method is carried out with a dilution factor of the medium preferably equal to 0.1 hour.