RU2796923C1 - Ogataea parapolymorpha vkpm y-5081 yeast strain - producer of protein biomass - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: invention relates to a strain of Ogataea parapolymorpha BF20-17Y methylotrophic yeast deposited in the Bioresource Center of the All-Russian Collection of Industrial Microorganisms (BRC VKPM) of the National Research Center Kurchatov Institute — GosNIIgenetika under registration number VKPM Y-5081, as a producer of protein biomass.

EFFECT: invention allows to expand the arsenal of means for the production of microbial protein mass, creating a new highly productive strain of methanol-oxidizing yeast for obtaining microbial protein mass with utilization in the process of methanol cultivation; the activity (productivity) of the strain is up to 6.0 kg×m³/hour.

1 cl, 3 ex

Description

The invention relates to the microbiological industry and can be used to obtain protein biomass, which is used as a source of protein in protein-vitamin complexes intended for balancing feed for aquaculture, agricultural and domestic animals.

Various types of microorganisms are used as producers of complete protein and vitamins.

Currently, methods are being developed for obtaining high-protein feeds by their industrial biosynthesis with the help of lower autotrophic organisms - yeast and bacteria. Microorganisms convert simple, complex and synthetic substances (cellulose, simple sugars, ammonium salts, alcohol, acetic acid, acetaldehyde, carbon, paraffin, oil, natural gases, etc.) into valuable feed proteins. Yeast is grown on various raw materials - straw, corn cobs, sunflower husks, cotton husks, sulfite lye, wood hydrolysates, starch waste, reeds, wood waste, etc.

Known yeast strain Saccharomyces cerevisiae VKPM Y-3585, with amylase activity, which is deposited in the All-Russian Collection of Industrial Microorganisms (VKPM) and can be used in the production of feed protein [RF patent RU 2478701, IPC: C12N1/16; publ. April 10, 2013]. The method of microbiological production of fodder yeast is carried out on waste grain raw materials.

Known yeast strain Hansenula sp. VSB-943 - producer of fodder biomass, characterized by high productivity when cultivated on molasses and hydrolyzate [application RU 93039024 A, IPC: C12N1/16; publ. 01/10/1997]. The method of microbiological production of fodder yeast is carried out on waste grain raw materials.

A known strain of yeast Endomycopsis fibuligera VSB-12 is a producer of protein biomass, a decomposer of agricultural waste, characterized by high productivity when cultivated on a carbohydrate and hydrocarbon substrate [RF patent RU2092549, IPC: C12N1/16, A63K 1/00, C12R 21/00, C12R 1/ 645; publ. 10.10.1997].

Known yeast strains of the genus Candida used as producer cultures for the production of fodder biomass. Yeast strain Candida scottii VKPM U-546 feed protein producer [author's certificate SU1378373, IPC: C12N1/22, C12R 1/72; publ. 10.10.1995]. The yeast strain Candida tropicalis VKPM V-1987 is a producer of protein-enriched biomass [RF patent RU2031115, IPC: C12N1/16, C12R 1/74; publ. March 20, 1995]. Yeast strain Metschnikowia pulcherrima VKPM - Y-3147 - feed protein producer [RF patent RU2370529, IPC: C12N1/16, publ. October 20, 2009]. Yeast strain Metschnikowia pulcherrima VKPM - Y-3152 - feed protein producer [RF patent RU2370530, IPC: C12N1/16, publ. October 20, 2009]. The yeast strain Candida ethanolica BKM U-2752 is a biomass producer [RF patent RU2061751, IPC: C12N1/16, C12R 1/72, publ. 06/10/1996].

The yeast strain Candida utilis VKMP U-263 is a producer of protein biomass. When cultivating on ethanol, the accumulation of biomass is at the level of 10.2-13.2 g/l, the biomass yield from a given standard is 71.8-77% with a protein content of 58.1-63.9% [RF patent RU1817471, IPC: C12N1/ 16, publ. December 12, 1990].

The disadvantage of these strains is that according to their systematic position they belong to imperfect yeast-like conditionally pathogenic microorganisms, which reduces their consumer value.

Known strain of yeast Metschnikowia pulcherrima VKPM - Y-3146 - producer of feed protein. The strain is grown on a nutrient medium from the green mass of Sakhalin mountaineer [RF patent RU2370531, IPC: C12N1/16, publ. October 20, 2009].

Known strain of yeastHansenulasp. VKPM Y-2133 producer of fodder biomass, characterized by high productivity when cultivated on molasses and wood hydrolyzate [RF patent RU2077573, IPC: C12N1/16, C12R 1/78, publ. July 27, 1993].

The disadvantage of all of the above strains is the limitation of the nutrient media used to obtain protein biomass. None of these strains uses methanol as a source of hydrocarbons.

Methanol is a hazardous organic waste, its disposal without harm to the environment is one of the problems of the industry. The technical problem, the solution of which is provided by the implementation of the invention, is the creation of a new highly productive strain of methanol-oxidizing yeast for the production of fodder biomass.

The technical result consists in expanding the arsenal of means for the production of microbial protein mass, creating a new highly productive strain of methanol-oxidizing yeast for obtaining microbial protein mass with utilization in the process of methanol cultivation. The technical result also consists in increasing the resistance of the strain to changing cultivation conditions without reducing productivity when the temperature regime changes from 37°C to 42°C and pH values from 3.0 to 5.8.

To achieve this technical result, it is proposed to use a strain of methylotrophic yeast Ogataea parapolymorpha BF20-17Y, deposited at the Bioresource Center All-Russian Collection of Industrial Microorganisms (BRC VKPM) of the National Research Center "Kurchatov Institute" - GosNIIgenetika under the registration number VKPM Y-5081, as a producer of protein biomass.

The strain Ogataea parapolymorpha BF20-17Y was isolated from soddy-podzolic soil (Moscow region, Russia). A sample of soddy-podzolic soil was used to obtain an enrichment culture of methanol-oxidizing yeast. After incubation in a liquid mineral medium with methanol as the sole carbon source, the culture was subcultured using the serial dilution method. After several passages, the culture was sown on an agar mineral medium to obtain isolated colonies of a pure culture of methylotrophic yeast. The thus obtained strain Ogataea parapolymorpha BF20-17Y was selected during long-term (more than half a year) passaging on liquid and agar media with methanol.

The bacterium strain Ogataea parapolymorpha VKPM Y-5081 was identified by cultural-morphological, physiological-biochemical properties and genetic characteristics and has the following features.

The systematic position was confirmed by the results of the analysis of the ITS region of the rRNA gene, as well as the decoding of the genome. The work was carried out by SciBear OU (Tartu mnt 67/1-13b, Kesklinna linnaosa, Tallinn, 10115 Harju maakond, Reg. No. 16221849). The resulting sequences were deposited with the NCBI as OM992321.

Genetic features of the strain:

a) there are no mutations, deletions, inversions;

b) phage-resistant;

c) no plasmids were found;

d) no prophages were found.

Information about the safety of using the strain:

a) the strain is not genetically modified and does not contain genes from other organisms; transferred resistance genes; genetic changes associated with the use of genetic engineering techniques;

b) the strain is not zoopathogenic and phytopathogenic; does not pose a risk for any other reason.

The shape and size of cells in a one-day culture on a synthetic medium with methanol: yeast cells are oval, separate, multiple budding is characteristic. The cell size is 1.5-4.6×1.9-5.3 µm. On a synthetic agar medium with methanol, it forms round, smooth-edged, shiny, cream-colored colonies that form on days 5-7, reaching 1-2 mm. On Sabouraud agar they form creamy, shiny, round colonies with a smooth edge, reaching 1-5 mm.

In addition to methanol assimilate

1) alcohols: ethanol, mannitol;

2) sugars: glucose, sucrose, maltose, inulin, xylose;

Does not absorb potassium nitrate, absorbs ammonium sulfate.

Yeast also needs phosphorus and sulfur.

Metals: Magnesium, Iron, Calcium and Zinc are also essential for good yeast growth.

Needs autolysate or vitamins for growth: biotin and thiamine.

It uses ammonium salts as a source of nitrogen, worse than nitrates.

The culture is facultatively anaerobic.

Able to grow at a temperature of 37-42°C (optimum 38°C) and pH 3.0-5.8 (optimum 4.0-5.5).

The activity (productivity) of the strain is up to 6.0 kg×m ³ /hour. A method for determining the activity of a strain indicating the method: on a liquid mineral medium with methanol (1% vol.) and pH values (4.0) at a temperature of 40 ° C with the addition of yeast autolysate in an amount of 0.3%.

Method, conditions and composition of media for long-term storage of the strain: in a lyophilized state; cryopreservation at -80°C as a cryoprotectant, a 30% solution of glycerin is used.

The proposed strain is characterized by:

- sustainable productive growth on media of different composition, in a wide range of pH and temperature;

- the ability to maintain activity for a long time in a lyophilized state;

- lack of virulence, toxigenicity, toxicity and harmlessness.

The cultivation is carried out mainly in a continuous way.

Method, conditions and composition of media for propagation of the strain:

Option 1

The composition of the mineral medium for growing culture in flasks (g/l): (NH4) ₂ SO ₄ - 3.3; MgSO ₄ × 7H ₂ O - 0.7; NaCl - 0.5; K ₂ HPO ₄ -1.0; KH ₂ PO ₄ - 0.13; yeast autolysate - 3.0. Trace elements (mg/l): MnSO ₄ × 5H ₂ O - 0.2; FeSO ₄ ×7H ₂ O - 1; H ₃ BO ₃ - 0.2; Na ₂ MoO ₄ × 2H ₂ O - 0.2; KI - 0.2. Methanol 10 ml/l, pH 5 (adjusted with a 70% solution of H ₃ PO ₄ ), cultivation temperature 38-40°C.

Option 2

Optimal conditions and composition of the fermentation medium (10 g/l methanol):

_H3PO4 ( ₇₀ %) 15.86 ml _{K2SO4 _} 9.53 g MgSO ₄ × 7H ₂ O 7.8 g CaCl ₂ × 2H ₂ O 0.6 g KOH (85%) 2.6 g Biotin 20 mcg Thiamine 200 mcg FeSO ₄ × 7H ₂ O 650 mg CuSO ₄ × 5 H ₂ O 60 mg MnSO ₄ × 2 H ₂ O 30 mg ZnSO ₄ × 7 H ₂ O 200 mg.

Example 1

Cultivation of the Ogataea parapolymorpha BF20-17Y strain is carried out in batch mode in heat-resistant glass flasks with a volume of 0.7 l at a fill factor of 0.1-0.2 in a thermostatically controlled shaker-incubator at 38°C. Cultivation is carried out for 24 hours. At the end of cultivation, the concentration of solids is 1.2 g/l. The resulting culture is used as seed material for the subsequent cultivation of yeast in an automated fermentation complex with a volume of 30 l (working volume 20 l).

Example 2

The culture of Ogataea parapolymorpha BF20-17Y was grown continuously in a fermenter with a magnetic stirrer with a volume of 5 l (working volume - 3.5 l) with a constant supply of a mineral medium containing the following components (10 g/l methanol):

_H3PO4 ( ₇₀ %) 15.86 ml _{K2SO4 _} 9.53 g MgSO ₄ × 7H ₂ O 7.8 g CaCl ₂ × 2H ₂ O 0.6 g KOH (85%) 2.6 g Biotin 20 mcg Thiamine 200 mcg FeSO ₄ × 7H ₂ O 650 mg CuSO ₄ × 5H ₂ O 60 mg MnSO ₄ × 2H ₂ O 30 mg ZnSO ₄ × 7H ₂ O 200 mg.

The cultivation process was carried out under non-sterile conditions, by varying the cultivation conditions: changing the temperature regime from 38°C to 42°C and pH from 4.0 to 5.5. The methanol feed rate was 100 ml/h. With a flow rate coefficient of 0.25 h ^-1 . The pH value was adjusted with 1.5% ammonia water solution or 70% phosphoric acid solution. The process temperature was controlled by supplying cooling water to the apparatus heat exchanger. The air flow rate per 1 liter of the culture medium was 60 l/h.

As a result of applying such a cultivation regime for a long time, using water from different sources, the culture productivity remained stable, the biomass concentration in the fermenter was 13-16 g/l with a crude protein content in the biomass of 42-45%.

Example 3

The culture of Ogataea parapolymorpha BF20-17Y was grown continuously in a fermenter with a magnetic stirrer with a volume of 30 l (working volume - 20 l) with a continuous supply of mineral salt solutions (as in the previous example). The growing process was carried out at a temperature of 38°C and a pH of 4.0. The pH value was maintained with a 10% ammonia water solution. The fermenter was supplied with air in the amount of 360 l/h. The flow rate coefficient was 0.25 h ^-1 . The culture fluid at the outlet of the fermenter was separated from the cells in a non-sterile mode and returned to the fermentation process instead of water. The share of accompanying microflora with this method of cultivation was unchanged and did not exceed 20% of the total number of cells. The concentration of biomass at the outlet of the fermenter was 15-19 g/L. The content of crude protein in biomass is 42-46%. The data obtained indicate that the cultivation of the Ogataea parapolymorpha BF20-17Y strain in a mineral medium of the specified composition with a continuous flow and in a wide range of cultivation conditions (temperature, pH) makes it possible to obtain a stable biomass yield comparable to the prototype.

Claims (1)

The strain of methylotrophic yeast Ogataea parapolymorpha VKPM Y-5081 is a producer of protein biomass.