Method of obtaining fungal protein biomass

FIELD: chemistry.

SUBSTANCE: invention relates to biotechnology. Claimed is method of obtaining food fungal biomass with high protein content. Multicycle deep cultivation of Fusarium sambucinum All-Russian collection of industrial microorganisms F-1161 on liquid nutritional medium, containing sources of carbon, nitrogen, mineral salts, separation and drying of wet fungus biomass are carried out. Cultivation is performed at pH from 3.5 to 7.0 under conditions of air aeration from 0.5 to 2.0 l/l/min. Temperature mode in each cycle of fermentation is supported from the beginning of the cycle to the point of switch at the level from 26 to 30°C, and further to the end of the cycle at the level from 22 to 25°C. Point of switch is determined by accumulation of biomass to concentration from 45 to 60% from maximally achievable in fermentation apparatus, or point of switch is determined by concentration of dissolved oxygen by its reduction to the value from 20 to 40% of saturation ( calculated per atmospheric air pressure).

EFFECT: invention makes it possible to obtain the largest accumulation of biomass with high protein content with specified quantity of nucleic acids.

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The technical field

The invention relates to biotechnology and pharmacology, and relates to a method of production of edible protein by microbial synthesis. The most effective present invention can be used in food and medical industries.

The level of technology

Traditional sources of fungal protein in the human diet are the fruiting bodies of higher strains of edible mushrooms of the genera Agaricus, Flammulina, Lentinula, Morchella, Panus, Pholiota, Pleurotus, Volvariella, etc. obtained by the method of solid-phase cultivation. The application of industrial methods of obtaining fungal biomass by microbial synthesis contributes not only to the intensification of the process of cultivation of strains studied higher edible mushrooms, but also extends the range of potential producers.

A method of obtaining protein biomass of the strain of Pleurotus ostreatus VKPM F - 697 method of submerged cultivation in conditions of aeration in a nutrient medium with the subsequent stages of separation and drying of biomass [EN 2092559, SR 21/00, C12N 1/14, C12N 1/14, C12R1:645, publ. 10.10.1997]. The fermentation process is carried out in a pH range from 6.0 to 7.5 and a temperature of 26 to 28°C in conditions of aeration (0.5 l air/l of medium per minute) at different nutrient media with the addition of from 0.1 to 3% surfactant (bone, fat or vegetable oil - sunflower, corn) about what Yamu. Seed pre "zakolerovat" at a temperature of from 4 to 6°C for 4 to 24 h, which, according to the authors, allows to reduce significantly the duration of fermentation due to the receipt of multiple maxima in the dynamics of crop growth. The separation of the biomass is carried out by filtering the culture fluid. The fermentation process lasts from 24 to 48 hours the Yield of dry biomass ranges from 13 to 23 g/l, the mass fraction of protein is from 20 to 50% by AFM. The disadvantage of this method is the long way of seed preparation method "Sahelian", as well as the use of an additional component of the nutrient medium - surfactant (bone, fat or vegetable oil - sunflower, corn), which increases the process of biomass production, as well as the periodicity of the process that is technologically not effective.

A method of obtaining protein biomass of the strain of Pleurotus ostreatus WKMR-720 [EN 2126835, SR 21/00, 12N 1/14, 12R 1:645, publ. 27.02.1999] in deep conditions, based on detachable-topping-up method. The duration of one cycle is 30 to 35 hours, the volume of selected cultural liquid ranges from 40 to 50% of the total. The duration of the process varies from 2.5 to 3 months. Seed pre "zakolerovat" at a temperature of from 4 to 12°C for 4 to 8 hours The fermentation process is carried out in a pH range from 6.0 to 7.5 and a temperature of 26 to 28°C in conditions of aeration (0.5 l/l/min) at different nutrient media with the addition of 0.1-1% surfactant by volume. The yield of dry biomass is from 13 to 20 g/l, the mass fraction of protein is from 20 to 50% by AFM. The disadvantage of this method is the relatively low productivity of the strain on the biomass and the use of expensive surfactants.

Known methods for producing a protein biomass strains of Pleurotus ostreatus 2-204 VKPM F-811 [EN 2189395, SR 21/00, C12N 1/14, C12N 1/14, C12R1:645, publ. 20.09.2002] and strain Panus tigrinus 3-204 VKPM F-810 [EN 2186851, SR 21/00, C12N 1/14, C12N 1/14, C12R1:645, publ. 10.08.2002] in deep conditions detachable-topping-up method with the subsequent stages of separation of the biomass by filtration of the culture liquid and drying of the biomass at a temperature of 60°C. the Duration of one cycle is 60 h with weaning culture liquid in an amount of from 40 to 50% of the total. The fermentation process is carried out in a pH range from 5.8 to 6.2 and a temperature of 26 to 28°C and 32 to 34°C for strains of Pleurotus ostreatus and Panus tigrinus, respectively, under conditions of aeration (from 0.8 to 1.0 l/l/min) and stirring in a nutrient medium containing dry or native whey from 80 to 100 g/l (NH4)2SO4- 0,250 g/l (NH4)2HPO4- 0.125 g/l At the initial stage for 18 to 20 h of cultivation is of Tambov as antifoam use sterile sunflower refined oil at a concentration of 0.05 vol.% to the working volume of the apparatus. The yield of dry biomass ranges from 17 to 25 g/l, the mass fraction of crude protein for strains of Pleurotus ostreatus and Panus tigrinus from 28 to 40% and from 36 to 40% on AFM, respectively. The disadvantage of these methods is the length of the process of cultivation of strains, as well as a relatively small value of the mass fraction of crude protein in the biomass.

A method of obtaining protein biomass of food and fodder strains of Penicillium nonatum and Penicillium chrysogenum [US 3865951; C12j 13/06, A23j 3/00, publ. 11.02.1975] by the method of deep cultivation with subsequent stages of separation of the biomass from the culture fluid, rinsing, biomass, filtering, drying. As substrates for the cultivation of strains-producers assume the use of various plant materials such as feed wheat, hydrolyzed potato, molasses, bagasse and/or citrus waste. Cultivation is expected to be implemented in the temperature range from 25°to 35 ° C, preferably about 30°C in the pH range from 4.0 to 7.0, while maintaining the pH value at the level corresponding to the maximum growth rate. The amount of inoculum on the stage of fermentation is 5 to 10% by volume, preferably 10%. It is advisable to make in the nutritional environment of vitamins (e.g., Biotin) to ensure the maximum growth rate of the culture, as well as non-toxic PE is arasites. The duration of the periodic process of cultivation in the conditions of aeration and mixing ranges from 20 to 48 hours, in the case of the use of lactose as a carbon substrate time of cultivation can be increased to 72 hours. The process is limited by the main substrate. Mass fraction of crude protein in the resulting biomass ranges from 43 to 47% (total nitrogen by Kjeldahl from for 6.81 to 7,53%, the conversion factor -6,25). The disadvantage of this method is the relatively low content of proteins in the resulting biomass.

A method of obtaining protein biomass of the strain of Neurospora sitophila [US4938972, A23L 1/00; publ. 03.07.1990; Moo-Young, 1993. Fermentation of cellulosic materials to mycoprotein foods/ Murray Moo-Young, Yusuf Chisti, Dagmar Vlach // Biotech Adv. - 1993. - Vol.11, - pp.469-479] by the method of deep cultivation for different crops waste subjected prior to alkaline hydrolysis. The cultivation is carried out in a pH range from 5.5 to 7.5 and a temperature of from 20 to 40°C., preferably at 26°C, under conditions of aeration (0.5 to 1.0 l/l/min) and stirring. The required content of dissolved oxygen in the medium is about 50%, a level lower than 30% leads to a sharp decrease in productivity. The amount of inoculum is from 3 to 10% of the working volume of the fermenter. The process is carried out batch and continuous way. The most effective is active for this process are devices with relatively low intensity of mixing (arletty bioreactor), than machines with mechanical stirring. The end of the process is determined by the achievement of the culture of the stationary phase of growth. Mass fraction of crude protein in the biomass ranges from 30 to 60% by AFM. The disadvantage of this method is the low productivity albumen from 2.2 to 2.6 g/l

A method of obtaining food mycelial biomass strains of the genus Polyporus. Polyporus squamosus and Polyporus brumalis [US 4212947, C12N 1/14, C12R 1/645, publ. 10.07.1980] by the method of deep cultivation with subsequent stages of selection of the biomass by filtration (filter press or vacuum filter or separation and spray drying the biomass. Cultivating implement detachable-topping-up method at a temperature of from 24 to 28°C, pH from 6.0 to 7.0, aeration from 0.6 to 1.0 l/l/min in a nutrient medium of the following composition, wt.%: molasses from 4 to 5%, NH4NO3to 0.2%KN2RHO4- 0,12%, vegetable oil - 0,04% antifoam. The concentration of inoculum is 10 vol.%. The duration of the first cycle is 24 hours, subsequent cycles from 5 to 7 p.m weaning culture liquid in the amount of 50% of the total. The duration of the process is from 1 to 5 days. The biomass yield is 9 g/l AFM. Mass fraction of crude protein from 50 to 60%. The disadvantage of this method is negligible accumulation of biomass, which leads toáanáincrease number of production cycles and high costs of water.

A method of obtaining food mycelial biomass of fungi of the genus Fusarium: strains of Fusarium species to frost, Fusarium oxyspomm and Fusarium solani [US 4501765, A23J 3/00, publ. 26.02.1985; US 4555485, SR 21/00, C12R 1/77, publ. 26.11.1985] by the method of deep cultivation with subsequent stages of separation of the biomass from the culture fluid, rinsing, filtering, denuclearization biomass drying. As substrates for the cultivation of strains-producers assume the use of various carbon-containing raw materials (starch, starch-containing raw materials and products of their hydrolysis, sucrose, charonosaurus raw materials and their hydrolysates (invert sugar, etc.), hydrolyzed potato, molasses, glucose, maltose, hydrolysed starch beans or cassava). The cultivation is carried out in the temperature range from 25 to 34°C, preferably 30°C, number of seed fermenting from 5 to 10% by volume, in the range of pH from 3.5 to 7.0, preferably to maintain at a constant level corresponding to the maximum growth rate of the culture. Preferably the introduction of a nutrient medium with vitamins (e.g., Biotin) to ensure the maximum growth rate of the culture, as well as non-toxic antifoam. The process is carried out periodic [US 4501765, A23J 3/00, publ. 26.02.1985] and continuous [US 4501765, A23J 3/00, publ. 26.02.1985; US 4555485, SR 21/00, C12R 1/77, op is nl. 26.11.1985] way. The duration of the periodic process of cultivation in the conditions of aeration and mixing ranges from 20 to 48 hours. The process is limited by the main substrate. Mass fraction of crude protein in the resulting biomass is from 45 to 61% (total nitrogen by Kjeldahl - from 7.2 to 9.9%, the conversion factor and 6.25). The process of reducing the content of RNA in the biomass is carried out by exposure to solvents (lower alcohols containing not more than 3 carbon atoms) at a concentration of from 40 to 100% within 1.5 to 40 minutes at a temperature of from 45 to 60°C [US 4501765, A23J 3/00, publ. 26.02.1985] or by abrupt heating the biomass to a temperature above 68°C, preferably from 72 to 74°C, for 30 to 45 min [US 5739030, C12N 1/14, publ. 14.04.1998]. The method allows to reduce the amount of RNA to less than 2%, but losses of biomass is from 30%to 33%. The disadvantage of this method is the increased content in the obtained biomass RNA (from 7 to 12%), which significantly reduces permissible for human consumption amount of biomass (not more than 2 g per day) and requires a stage of denuclearization that, in turn, leads to the loss of biomass (from 30 to 33% AFM).

Thus, at present no known way to produce food of fungal biomass by the method of deep cultivation, providing at high speed R is a hundred accumulation of protein rich biomass with a high concentration of valuable unsaturated fatty acids and low concentrations of nucleic acids (from 3.0 to 4.1% AFM), allowing the use of this biomass for human food.

Disclosure of inventions

The aim of the invention is to develop a method of obtaining food

the fungi biomass with high protein content (from 53 to 63% AFM) and low amounts of nucleic acids (from 3.1 to 4.1% AFM) by the method of deep cultivation with high growth rate.

The method of obtaining dietary protein biomass is characterized by the fact that, as a producer strain using strain Fusarium sambucinum Fuck. var. ossicolum (Berk. et Curt.) D-104 deposited in PMBC number F-1161, the cultivation process is carried out in-depth conditions in the pH range from 3.5 to 7.0 in conditions of aeration (0.5 to 2.0 l/l/min with a linear speed of the mixing device 1 to 4.9 m/s). As a carbon source use a variety of carbon-containing raw materials (products of various types of grain (wheat, rye, triticale, potatoes, sugar beet and cane, the juice of the roots and green parts of Jerusalem artichoke, turnip, red beet, and other species and their hydrolysates) in an amount of from 2.0 to 6.0 wt.%.

Temperature of the cultivation process in each cycle fermentation support from the beginning of the cycle to the switching point at the level of 26 to 30°C, then till the end of the cycle - at the level of 22 to 25°C, and the switching point is determined by the biomass accumulation prior to the concentrations from 45 to 60% of the maximum achievable in this unit (maximum achievable concentration of biomass depends on the mass transfer characteristics of the fermenter, where is the cultivation of the microorganism, and its specific value is determined experimentally on the basis of trial downloads).

In another embodiment of the method, the switching point is determined by the concentration of dissolved oxygen after its reduction to a value of from 20 to 40% of saturation (in terms of atmospheric air pressure).

The process is cyclical. In the first cycle, the amount of inoculum is from 1 to 5 vol.%. In subsequent cycles weaning cultural liquid of the apparatus ranges from 95 to 99%vol.

Used a strain of Fusarium sambucinum Fuck. var. ossicolum (Berk. et Curt.) D-104 deposited in the collection PMBC number F-1161. The strain is characterized by a high growth rate (from 0.28 to 0.32 h-1) in liquid submerged culture and accumulates biomass with high protein content from 53 to 63%, the full amino acid composition, with a high number of valuable unsaturated fatty acids (from 83 to 87% of the sum of fatty acids) and the content of nucleic acids from 4 to 5% ACM.

The invention contributes to the intensification of the process of production of edible biomass of the fungus with a high protein content and high amount of valuable unsaturated fatty acids and helps to ensure a low content of nucleic acids in biomass (from 3.1 to 4.1% AFM) without the use of additional on roostaei and time-consuming operations.

The invention is illustrated by examples.

Example 1

Culture of a strain of Fusarium sambucinum D-104 was kept mowed wort agar in test tubes at a temperature of +4°C with periodic re-seeding every 6 months. Growing inoculum was performed in aseptic conditions at a temperature of 26 to 28°C in katalozhnyh flasks of 250 ml containing 100 ml of nutrient medium of the following composition: wheat starch 20 g/l, ammonium nitrate - 2.8 g/l, potassium phosphate one-deputizing - 1.2 g/l, corn steep condensed - 10 g/l, tap water - the rest, pH of 5.8 by reseeding culture with a solid nutrient medium (jamb) at the rate of 1 the casing 1 catalogno flask - the first passage. The duration of the growing seed: the first passage 96 h, the second 48 h, the third and subsequent 24 hours

Deep cultivation was carried out in periodic mode in a fermentation apparatus 30 litres displacement of 20L, mode rn-strirovaniya when the value of 4.0, a gauge pressure of 0.4 ATM, aeration under sterile conditions 1.0 l/l/min with mechanical stirring at 400 rpm with a stirrer turbine type on nutrient medium of the following composition: wheat starch - 45,0 g/l; ammonium nitrate - 4.5 g/l; potassium phosphate one-deputizing - 1.8 g/l; corn steep condensed - 10.0 g/l, propanol B-400 - 0.4 g/L. Starch was subjected to pre is sustained fashion hydrolysis by enzyme preparation, aminocoumarin in the calculation 0,024% by volume of nutrient medium (4.8 g/l). Wednesday was inoculable mycelial suspension (second and subsequent passages) in the amount of 3% by volume. Pre-defined maximum achievable in this environment for producer strain, the concentration of biomass at the end of the fermentation cycle, which amounted to 2 to 5 g/l of the AFM. Temperature of the cultivation process was supported from the beginning of the cycle to the switching point at the level of 26 to 30°C, then till the end of the cycle - at the level of 22 to 25°C, and the switching point was determined by the accumulation of biomass and the concentration of 11.0 g/l AFM, which accounted for about 45% of the maximum achievable concentration in this unit. Dehydration culture fluid was performed on a suction filter, drying wet biomass were carried out in a fluidized bed dryer.

The chemical composition of biomass, % ACM:

Total protein - 58,0;

Lipids - 7,8;

Nucleic acid - 3,1.

Example 2

The cultivation of the producer strain was carried out similar to that presented in example 1, however, the switching point was determined by the accumulation of biomass and the concentration of 15.0 g/l of the AFM, which corresponded to 60% of the maximum achievable concentration in this unit. Dehydration culture fluid was performed on a suction filter, drying wet biomass were carried out in a fluidized bed dryer.

The chemical composition of biomass, % ACM:

General the th protein 62,1;

Lipids - 6,5;

Nucleic acid - 3,7.

Example 3

The cultivation of the producer strain was carried out similar to that presented in example 1, however, the switching point was determined by the concentration of dissolved oxygen after its reduction to a value of 20% of saturation (in terms of atmospheric air pressure). Dehydration culture fluid was performed on drum vacuum filter, drying wet biomass were carried out in a fluidized bed dryer.

The chemical composition of biomass, % ACM:

Total protein - 63,0;

Lipids - 6,4;

Nucleic acid - 4,0.

Example 4

The cultivation of the producer strain was carried out similar to that presented in example 1, however, the switching point was determined by the concentration of dissolved oxygen after its reduction to a value of 40% of saturation (in terms of atmospheric air pressure). Dehydration culture fluid was performed on drum vacuum filter, drying wet biomass were carried out in a fluidized bed dryer.

The chemical composition of biomass, % ACM:

Total protein - 60,8;

Lipids - 7,0;

Nucleic acid - 3,3.

Thus, the use of this method allows to obtain high quality mushroom protein biomass food destination with a low content of nucleic acids (from 3.1 to 4.1% AFM)that does not require the additional duty to regulate the stage of denuclearization biomass and therefore, avoids undesirable fission products NC. The method can be successfully applied in the food industry and pharmacology for the production of edible mushroom protein biomass.

1. The method of obtaining food fungal biomass with high protein content, providing cyclical deep cultivation of the producer strain in a liquid nutrient medium containing sources of carbon, nitrogen, mineral salts, separating and drying the wet biomass of the fungus, characterized in that as a producer of biomass use strain of the fungus Fusarium sambucinum VKPM F-1161, the cultivation process is carried out at a pH of from 3.5 to 7.0 in conditions of aeration of 0.5 to 2.0 l/l/min, temperature of the cultivation process in each cycle fermentation support from the beginning of the cycle until the switch point level from 26 to 30°C, then till the end of the cycle - at the level of 22 to 25°C, and the switching point is determined by the accumulation of biomass to a concentration of from 45 to 60% of the maximum achievable in a fermentation apparatus or the switching point is determined by the concentration of dissolved oxygen after its reduction to a value of from 20 to 40% of saturation (in terms of atmospheric air pressure).



 

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26 cl, 25 dwg, 3 tbl, 4 ex

FIELD: biotechnologies.

SUBSTANCE: invention proposes a method for obtaining recombinant core protein of hepatitis E virus (rtHEV-ORF2) and recombinant vaccine for prophylaxis of hepatitis E virus. Core protein is obtained by cultivation of recombinant yeast strain Hansenula polymorpha "КБТ"-11/pHEV-001, which contains DNA sequence integrated into genom of yeast cell and coding the fragment of amino-acid sequence from position 86 to 607 of core protein of hepatitis E virus of genotype 3 (rtHEV-ORF2) under control of promoter of MOX gene. The method allows obtaining immunogenic antigene of hepatitis E virus, which has properties of natural protein. Based on the obtained antigene there created is recombinant vaccine for prophylaxis of hepatitis E virus. Vaccine includes effective amount of rtHEV-ORF2 protein, adjuvant and a physically acceptable diluter.

EFFECT: vaccine is immunodominant and non-toxic and has no by-effects.

3 cl, 5 ex

FIELD: biotechnologies.

SUBSTANCE: renatured membrane protein obtaining method is proposed. The above method involves production of a homogeneous solution containing a denatured membrane protein, a detergent mixture, phospholipide or phospholipide mixture and apolipoprotein or its equivalent with: further removal of detergents and formation of lipid-protein nanodiscs (LPND) containing renatured protein.

EFFECT: method provides production of renatured membrane proteins with high yield, which are built into LPND, which can be used at development of new medical products, biocatalysts, biosensors and biophotonic devices.

8 dwg, 2 ex

FIELD: biotechnologies.

SUBSTANCE: invention proposes a production method of recombinant protein through its hybrid precursor substance with natural decomposition site with enteropeptidase. The result is achieved by replacement in natural decomposition site with enteropeptidase Asp-Asp-Asp-Asp-Lys of amino-acid residue of lysine (Lys) with amino-acid residue of arginine (Arg) and further decomposition of hybrid precursor substance with light catalytic subunit of enteropeptidase of a human being or a bull.

EFFECT: improving quality and yield of target product under conditions when hybrid protein detects additional sites of decomposition with enteropeptidase.

3 tbl, 3 dwg, 4 ex

FIELD: chemistry.

SUBSTANCE: present inventions relate to protein engineering, plant molecular biology and pest control, as well as a hybrid insecticide protein and use thereof. Described is a hybrid insecticide protein which includes from the N-end to the C-end an N-end portion of Cry3A protein which is fused with the C-end portion of Cry1Ab protein, wherein the position of the crossover of the Cry3A protein and the Cry1Ab protein is located in a conservative block 2, in a conservative block 3 or in a conservative block 4 and has anti-western corn rootworm activity. Also disclosed are nucleic acid molecules which code the novel proteins, methods of producing proteins, methods for use thereof, as well as transgenic plants and seeds thereof which contain such proteins.

EFFECT: inventions enable to obtain cheap means of controlling Diabrotica worms.

39 cl, 8 dwg, 9 tbl, 46 ex

FIELD: biotechnology.

SUBSTANCE: method is characterised in that the DNA of the structure RNAb indicated on Figure 1, which encodes the fused protein of three parts, where N-terminal position is green fluorescent protein GFP, central - peptide of 73 amino acid residues with the amino acid sequence of SRKKCNFATTPICEYDGNMVSGYKKVMATIDSFQAFNTSYIHYTDEQIEW KDPDGMLKDHLNILVTKDIDFDT, and C-terminal - light chain of double-stranded protein Kunitz-type inhibitor from potato tubers (PKPI-BI), are introduced into cells of E. coli. The cells transformed by this construction are cultured, the biomass is lysed, the insoluble fraction of the lysate is separated by centrifugation. The product of expression in the form of inclusion bodies is solubilised with the denaturant. Chromatography is carried out under denaturing conditions. The resulting product is used for detection of specific antibodies in serum of patients with hemorrhagic fever with renal syndrome.

EFFECT: invention enables to obtain the recombinant antigen G2 of Hantavirus Dobrava with increased yield.

6 dwg, 1 ex

FIELD: biotechnologies.

SUBSTANCE: method is proposed to produce a polypeptide, including cell cultivation, which intensely expresses a bicarbonate carrier and has a transferred DNA, which codes the desired polypeptide.

EFFECT: invention makes it possible for the cell to produce the specified polypeptide and the appropriate cell.

12 cl, 15 dwg, 6 ex

FIELD: biotechnologies.

SUBSTANCE: mutant strain is obtained by impact on Glarea lozoyensis ATCC 20957 strain by nitrosoguanidine and it is deposited in CGMCC with number CGMCC 2933.

EFFECT: fungi strain has stable genetic and productive properties, produces low quantity of impurities during fermentation and is acceptable for commercial production of antibiotic.

6 cl, 2 dwg, 2 tbl, 3 ex

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