Method of producing microorganism growth stimulator

FIELD: chemistry.

SUBSTANCE: method of producing microorganism growth stimulators involves growing Fusarium sambucinum MKF-2001-3 or Fusarium sambucinum BKM F-3051D yeast strains on a medium which contains a source of carbon 3-4%, nitrogen 0.2-0.3%, phosphorus - 0.2-0.3% and trace elements - 0.07-0.08% in sterile conditions at temperature of 26-30°C with stirring and aeration for 40-48 hours. The obtained yeast suspension or culture fluid is titrated with hydrochloric acid to pH=4.0-4.5, heated for 1.0-1.5 hours to 115°C, held for 2 hours, cooled for 1.0-1.5 hours to room temperature.

EFFECT: method provides intense growth of microorganisms by stimulating biological activity of their growth factors which results in more complete recycling of the substrate and increases output of the end product.

10 ex

 

1. The technical field

The invention relates to the field of biotechnology, in particular to Microbiology, and can be used to obtain nutrient media of biologically active substances.

2. The level of technology

A known method of growing yeast under conditions of aeration on mineral nutrient medium containing sources of carbon, nitrogen, phosphorus and trace elements using as a growth promoter of the yeast one or more a-aminocarbonyl acids, mixed with individual amino acids such as arginine, leucine and lysine, as well as the b vitamins, especially pyridoxine and Pantothenic acid (French patent No. 1538957, CL SS, 1969).

The disadvantage of this method is the high cost of the original products.

A known method of growing yeast using as a growth stimulator of crotonates and bucillamine (Japan patent No. 5417026, CL SS 11/08, 1979).

However, the effect of these stimulants tested only in periodic modes of cultivation of yeast by shaking the flasks at low concentrations of biomass. Another disadvantage of this method is the considerable expense of crotonates and bucillamine, which is 0.1 mg per 1 liter of nutrient medium.

A method of obtaining an extract of fodder yeast, in which fodder yeast is mixed in with the Oh, boil with constant stirring and filtered (EN 2084171, M CL6: C12N 1/00, 1/14, 20.07.1997,).

The disadvantage of this method is the length of the filtration process, the presence of suspended particles and high pigmentation of the extract.

The closest analogue of the proposed method by the features of the invention and the achieved effect, taken as a prototype, is a method of obtaining growth stimulants microorganisms of fodder yeast, in which fodder yeast is mixed with water, followed by boiling, add the extract, sodium phosphate disubstituted, mix until the salt is completely dissolved, alkalinized to a pH of 7.2 to 8.3, and then add calcium chloride, alkalinized to a pH of 6.7 to 7.1, boil for one hour, the filtrate concentrated and dried (RU # 2227158, M CL7: C12N 1/00, 1/14, 22.07.2002 year).

The disadvantage of the prototype is limited performance and a significant duration of the process.

3. Disclosure of inventions

The objective of the invention is to intensify the growth of the microorganisms used in the biotechnological production of probiotics, the production of enzymes in the production of feed protein products, etc.

This object is achieved in that in the method of producing growth of microorganisms cultivated fungi of the genus Fusarium sambusinum strains MKF-201-3 or BKMF-3051D on the environment, contains a source of carbon - 3-4%, nitrogen - 0,2-0,3%, phosphorus - 0,2-0,3% and trace elements - 0,07-0,08% in sterile conditions at a temperature of 26-30°C With stirring and aeration for 40-48 hours, the suspension of the fungus and/or culture fluid petitrenaud hydrochloric acid to pH=4.0 to 4.5, heated for 1.0 to 1.5 hours up to 115°C, incubated for 2 hours, cooled for 1.0 to 1.5 hours to room temperature, the thus obtained suspension and/or culture fluid is growth of microorganisms.

4. The implementation of the invention

The claimed method of obtaining growth of microorganisms is as follows: cultivated mushrooms of the genus Fusarium sambusinum stamov MKF-2001-3 or BKMF-3051D on a medium containing a carbon source 3-4%, nitrogen - 0,2-0,3%, phosphorus - 0,2-0,3% and trace elements - 0,07-0,08% in sterile conditions at a temperature of 26-30°C With stirring and aeration for 40-48 hours.

While the bulk of these technological operations carried out at the following granularity of processes and parameters.

The suspension of the fungus petitrenaud hydrochloric acid to pH=4.0 to 4.5, heated for 1.0 to 1.5 hours up to 115°C, incubated for 2 hours, cooled for 1.0 to 1.5 hours to room temperature, make the stimulator in the amount of 0.002 to 0.8% by volume of the nutrient medium.

The culture fluid is separated by filtration is tons of biomass, petitrenaud hydrochloric acid to pH=4.0 to 4.5, heated for 1.0 to 1.5 hours up to 115°C, incubated for 2 hours, cooled for 1.0 to 1.5 hours to room temperature, make the stimulant in an amount of 0.1 to 8.0% by volume of the nutrient medium.

Examples of implementation of the method.

Example 1. Prepare a nutrient medium, which is 37.5 g of bran and 87.5 g of rye bran fill in 875 ml of water. The water ratio: solid and liquid fraction: 1:7.

Culture medium was subjected to heat treatment in a boiling water bath for 4 hours at pH of 6.7. After heat treatment, the mixture was cooled to 35°C, using 10% sulfuric acid brought the pH to 5.0 and added salt (NH4)2SO45 g/l and KN2RHO41.5 g/l In the resulting environment was made enzyme - glucosamines. Fermentors was carried out for 5 hours, after which the resulting fermentolizat analyzed (on the sugar content - RV), was poured into the flasks; in the amount of 200 ml in each flask.

Flasks with culture medium were sterilized for 40 min at 0.5 ATM. After sterilization of the nutrient medium in the flask was cooled to 30°C. and added into the nutrient medium of the biostimulator.

The yeast Saccharomyces cerevisiae diastaticus PMBC-u-1218 were grown in flasks on a shaker for 24 hours at a temperature of 32°C and the stirring speed environment 240 rpm After growing yeast biomass production in flasks biostimulation concentration of sugars (PB) decreased from 6 g/l to 0.4 g/l, in the control flasks she was within 0.6-0.7 g/l In the experimental flasks amount of yeast biomass production was 3.5 g DIA, in control - 2.9 g/l, i.e. 20,67% less. The content of crude protein in the target product was 42.8%, whereas in control - 41,4%.

Example 2. Yeast-like fungus Trichosporon cutaneum BWA-775 was grown in the same culture medium as in example 1, in flasks on a shaker for 36 hours, using the biostimulator in an amount of 0.1% by volume. In the end determined the amount of locally grown biomass, the degree of assimilation of sugars (RS) and the content of crude protein in the biomass of the fungus.

In the experimental flasks amount of accumulated biomass was in the range of 4.0 to 4.2 g DIA, and in the control- 3,7-3,8 (+9,33%). The content of crude protein in the test samples of the biomass was 43.2 per cent, and in the control- 41,8% (+3,35%).

Example 3. Conducted testing of the biostimulator for growing yeast on fermentolizate of SINOSURE.

The cultivation of the yeast Saccharomyces cerevisiae (S. cerevisiae diastaticus PMBC-u-1218) was performed in the device V-30 litres (useful volume 20 l) fermentolizate of SINOSURE.

Fermentolizat and the culture fluid was prepared according to the mode specified in example 1. In the control cultivation of yeast was carried out without addition of stimulator. Growing yeast culture was carried out under the following parameters:

t environment - 30-32°C

pH - 4,5-5,0

the intensity of paramesh is of 800 rpm and aeration - 5-8 l/l environment.

The duration of the cultivation process was determined to achieve the amount of residual sugars RV = 0,4. In controlling this period was 16 hours, the experience is 12 hours. The process of cultivation of yeast decreased by about 25%. DIA in experience was more than in control, 4,34%.

At the end of the fermentation process the yeast suspension was admitted to plasmas (t - 80°C, 1 hour); plasmolysis was dried in the spray dryer.

The test results were gathered samples of the protein product. Control the content of crude protein was 41.2%, protein by Barnstein - 30,8%. In an experimental batch of the content of crude protein was 44.5%, protein by Barnstein of 33.8%. Protein content increased by 8%, protein at 9,74%. As you can see, the use of biostimulant allows you to accelerate the bioconversion of sugars and improve the quality of the target product.

Example 4. The cultivation of Baker's yeast Saccharomyces cerevisiae (strain VNII PP) was performed in the fermenter BIOR-01 volume 100 l working volume was 50 l

The apparatus was filled with 40 l of tap water, making the components of the nutrient medium according to the following recipe (g/l):

Sugar20,0
Yeast extract5,0
MgSO40,3
(NH4)2SO45,0
K2HPO42,0
Laprol0.5 ml
The biostimulant4 l (8%)
Water waterup to 50 l

pH before planting to 5.8.

In the control of the biostimulator in the environment has not been made. The cultivation was conducted at 30°C under continuous stirring and aeration. The process is conducted until reaching the stationary phase of growth. Biomass accumulation was in control 1150 g, experience -1400, the Amount of biomass grown on 21,74%.

Example 5. Growing cells Bacillu.licheniformis B-8130, producer probiotic preparations were carried out in the fermentor BIOR-01 volume 100 l working volume was 50 l

The apparatus was filled with 40 l of tap water, making the components of the nutrient medium according to the following recipe (g/l):

Glucose25,0
Peptone8,0
Yeast extract3,0
MgSO40,2
CaCl20,1
K2HPO41,0
KN2PO40,5
Laprol, ml0,5
The biostimulant, l2,5 (5%)
Distilled water, l50

pH before planting - 7,0-7,2.

In the control of the biostimulator in the environment has not been made. The cultivation was conducted at 37°C with continuous stirring and aeration. The process led to improving pH to 8.6-8.8 and the appearance of free spores.

In the control apparatus of the duration of cultivation - 48-72 h, education dispute 10-20%. In the experimental apparatus, the length of growing - 26-28 h, the formation of spores in 95-100% of the cells.

Example 6. Growing cells Bacillu.subtilis HA-13, component bookreport of biocompost was carried out in the fermentor BIOR-01 volume 100 l working volume was 50 l

The apparatus was filled with 40 l of tap water, making the components of the nutrient medium according to the recipe (g/l) of example 5, the biostimulator in a volume of 2.5 l (5%).

In the control of the biostimulator in a nutrient medium was not made. The cultivation was conducted at 37°C at ararauna stirring and aeration to improve pH to 8.6-8.8 and the appearance of free spores.

In the control apparatus of the duration of cultivation - 48-52 h, education dispute 20-30%. In the experimental apparatus, the length of growing - 25-27 h, the formation of spores in 95-100% of the cells.

Example 7. Mycelial fungus Penicilliun verruculosum were grown on a nutrient medium containing sources of Priroda, nitrogen and mineral salts, before sterilization in an environment add 5-8% of the biostimulator, and then were cultured for 120 h on a rocking chair at a temperature of 29°C and stirring, in the process of fermentation, samples were taken and in the filtrate of the culture fluid was determined activity karbohidrat (cellulases and xylanases). Products (activity) of the enzyme in the presence of the biostimulator was increased by 20% for cellulases and 25% for xylanases.

Example 8. The yeast S. cerevisiae diastaticus (PMBC-u-1218 were grown in continuous mode in the industrial unit V-900 m3(Vpa - 450 m3) fermentolizate of SINOSURE.

The amount of the biostimulator was 0.002% of the volume of flow per hour.

The main effect was to reduce the time growing from 8.2 hours to 6.6 hours or 24,24%. The number of ASV increased by 4,88%. Quality target product feed product was evaluated according to the content of crude protein +of 4.44%, and protein on Barnstein - +6,04%. The number of produced forage product grew 80,7 to 113,7 tons per day, i.e. by 40.9%. This supplies the coefficient on Samosir decreased from 1,55 t/t up to 1.37 t/so

It should be noted that the physiological state of yeast cells characterized by an active budding (50-60%) and homogeneous protoplasm. Due to the deep disposal of the substrate (carbohydrates) in the last section was the large number of vacuolation cells.

Example 9. Spent the cultivation of Leptospira serotypes Pomona, Icterohaemorrhagiae, Tarassovi standard serum medium (control) and modified whey medium containing 5% of biostimulant (experience) at a temperature of 28°C for 10 days.

Experienced a series of vaccine prepared from cultures of leptospires grown in modified serum medium containing 450-500 million M.K. cm3by mixing them.

A control series of a vaccine prepared from cultures of leptospires grown in serum medium containing 85-95 million M.K. cm3by concentrating to content 500 million M.K. cm3and subsequent mixing.

Culture Leptospira iactiveaware in the presence of 0.1% formaldehyde for 5 days at 37°C. In the period of experimental work was produced by 3 series of experimental and control vaccines against leptospirosis volume 2 DM3each.

Series of vaccines against leptospirosis controlled for sterility, safety and activity in accordance with accepted standard methods.

Financial p the tat.

Pooled sample of vaccines on August 29, 2009 was administered at a dose of 1.0 cm3intramuscularly five rabbits gray mass of 2.8-3.0 kg

After 25 days in immunized rabbits from the ear vein produced a blood volume of 5.0 cm3. Serum obtained from the blood of rabbits was investigated in the reaction microagglutination with cultures of strains of leptospires included in vaccines.

Recorded results in the reaction microagglutination. In the serum of rabbits immunized with control concentrated culture of leptospires, established the presence of specific antibodies to Leptospira Pomona and Tarassovi - 1:200 and Icterohemorrhagiae 1:50.

In the serum of rabbits immunized experienced the culture of leptospires grown in the presence of biostimulant identified antibodies to Leptospira Pomona and Tarassovi 1:400 and Icterohemorrhagiae 1:100.

Conclusions: the inclusion of the biostimulator in the composition of the nutrient medium for the cultivation of Leptospira significantly increases the accumulation of leptospires and biosynthesis of specific antigens.

Example 10. The fungus Fusarium sambucinum strain MKF-200-3 were grown on a medium containing as a main carbon source sucrose (3-4%, molasses containing sucrose). In molasses contribute additional ammonium sulfate (NH4)2SO4in an amount of 5 g/l (0.5%) and potassium phosphate (KH2PO4in Koli is este 3.5 g/l (0.35 per cent).

As for essential micronutrients (Zn, Mg, Mn, Fe and others), they are contained in the molasses and additionally not recorded.

The cultivation of the device V-30 litres (useful volume 20 l) was carried out for 48 hours with the following parameters: t=30°C, pH=5,0.

The suspension of the fungus Potterville hydrochloric acid to pH=4.2, and was heated for 1.5 hours to 115°C, kept for 1.3 hours to room temperature, made the stimulator in the amount of 0,05%, i.e. the useful volume of the device V-30 - 100 ml of the Obtained suspension of the fungus is declared biostimulant.

5. Technical results

An advantage of the claimed proposal compared to the prototype is a significant stimulation of microbial growth by stimulating the biological activity of the growth factors. The use of growth promoters in the process of biomass as in periodic and continuous cultivation leads to a more complete utilization of the substrate, which improves the yield of the target product at a reduced expenditure ratios by source of carbon and energy. The amounts of feed and other target products due to the proposed use of the stimulant was increased to 40% or more reduction of the time growing up to 50%.

The method of obtaining growth of microorganisms, which grow is their fungal strains Fusarium sambucinum MKF-2001-3 or Fusarium sambucinum BKM F-3051D on the environment, contains a source of carbon - 3-4%, nitrogen - 0,2-0,3%, phosphorus - 0,2-0,3% and trace elements - 0,07-0,08% in sterile conditions at a temperature of 26-30°C With stirring and aeration for 40-48 h, the resulting suspension of the fungus or the culture fluid petitrenaud hydrochloric acid to pH=4.0 to 4.5, heated for 1.0 to 1.5 hours up to 115°C, incubated for 2 h, cooled within 1.0 to 1.5 h to room temperature.



 

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6 ex

FIELD: biotechnology, microbiology.

SUBSTANCE: invention relates, in particular, to preparing nutrient media used for culturing the plague microorganism vaccine strain and can be used in medicinal microbiology. The nutrient medium for culturing the plague microorganism vaccine strain comprises additionally as a stimulating additive sodium sulfite and as a nutrient base - soybean fruits enzymatic hydrolyzate in the following ratio of components, g/l: microbiological agar, 11.0-13.0; soybean fruits enzymatic hydrolyzate, 250.0-350.0; sodium chloride, 4.5-5.5; sodium hydrogen phosphate, 3.5-4.5; sodium sulfite, 0.0003-0.0005; distilled water, the balance. Invention provides enhancing the growth property of nutrient medium.

EFFECT: valuable properties of medium.

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