The growth regulator and a method of increasing the biochemical activity of microorganisms and plants

 

(57) Abstract:

The invention relates to organic chemistry and biotechnology, namely the growth regulator of plants and microorganisms. The growth regulator contains a solution of biologically active substances - 2-oxo-(3H)-5,6,7,8-tetrahydropyrido (Hydroptere) of the formula I in the form of a hydrate or salts at a concentration of 10-6- 10-5g/L. using this growth regulator to improve the biochemical activity of microorganisms specified solution is injected in a fermentation medium, to increase the biomass of microorganisms and accumulation produced by these substances. When using this growth regulator to improve the biochemical activity of the plants with a solution of specified substances treated seed and green part of plants that can increase the length of the stem, roots, number of leaves, reduce the time of germination, growth and development of plants. 3 C. p. F.-ly, 9 PL.

The invention relates to organic chemistry and biotechnology, namely, growth regulators microorganisms and plants on the basis of heterocyclic compounds with tsitokininovykh properties, increasing the biochemical activity of microorganisms and plants.

The disadvantage of this method is that chlorthalidone is used strictly only for spring wheat.

It is known to improve quality and increase yield Sitnik when applying N-6-benzyladenine at a dose of 2.5 to 25 g/ha in the composition of solid or liquid preparations, see application Japan # 56 - 139666 in class. A 01 N 43/90, publ. 1983

Known application of N-6-benzyladenine limited to use only for rush.

Aware of the impact of purine compounds gipoksantina, adenine, kinetin, guanosine at stimulating effect on microorganisms during cultivation dairy bacteria, see Mikhlin E. D., Radina B. N. Activation of lactic acid bacteria // Applied biochemistry and Microbiology. - 1983. - T. XVII, vol. 3. - S. 353-354.

These purine compounds contribute to the accumulation of lactic acid, but the accumulation of long-term, which reduces the efficiency of utilization.

A known method of regulating the growth of alfalfa, including treatment, while the budding aqueous solution containing a growth regulator and groups of the pyrimidine series, see the patent of Russian Federation N 1810036 in class. A 01 N 43/00, publ. 1993.

P> Known growth regulator (-naphthalenyloxy acid - NAA), stimulating the growth of blue-green algae, see Shigaev M. H. Stimulation of microorganisms and viruses, Kazakh SSR: Nauka, 1986. - 43 S.

The regulator NAA slightly increases the number of cells of blue-green algae.

Known N-oxide 6-BAP (N-oxide 6-benzylaminopurine), providing phytohormonal effects on plants on the ageing, the substrate and the protein content in the leaves, see the Physiology of active substances//,P. Brain, W. S. Petrenko, I. C. Boldyrev, V. M. Cherkasov. - Kiev: 1984. - N 16. - S. 57-59.

Known 6-BAP has only hormonal effects on plants, leading only to an earlier distillation of the flowers and the formation of the fruit.

Known growth regulator containing an aqueous solution of biologically active substances, see U.S. patent N 4326877 in class. A 01 N 43/50, publ. 1982, as a biologically active substance take 6-aminopurin at a concentration of 25-300 million hours and use an aqueous solution to stimulate the growth of roots of herbs by spraying grass.

This growth regulator is taken as a prototype of the present invention.

However, he has a number of nedostatki is the less biochemical effects - only to stimulate the growth of roots.

The basis of the present invention it is the task of expanding Arsenal of economic growth regulators of plants and microorganisms, improving the efficiency of growth and development of plants and the increasing intensification of metabolic processes.

According to the invention this problem is solved due to the fact that the growth regulator of plants and microorganisms, containing a solution of biologically active substances, as a biologically active substance contains 2-oxo-(3H)-5,6,7,8-tetrahydropyrido (Hydroptere) formula

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in the form of a hydrate or salts at a concentration of 10-610-5g/l

According to the invention this problem is solved due to the fact that the way to improve the biochemical activity of microorganisms by affecting the processes of growth and metabolism by a solution of biologically active substances, such as biologically active substances take 2 - oxo-(3H)-5,6,7,8-tetrahydropyrido (Hydroptere) formula

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in the form of a hydrate or salts and injected into the fermentation medium to achieve a concentration of 10-6- 10-5g/l

According to the invention this problem is solved due to the fact that the way to improve biochemical the CSOs substances take 2-oxo-(3H)-5,6,7,8-tetrahydropyrido (Hydroptere) formula

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in the form of a hydrate or salts and prepare a solution concentration of 10-6- 10-5g/l

The applicants have not identified the sources of information about growth regulators and methods of their realization, identical with the present invention, which allows to make a conclusion about their compliance with the criterion of "novelty".

Due to the implementation of the distinguishing features of the invention (in conjunction with the features indicated in the restrictive part of the formula) achieved an important new properties of matter and methods for their use. The proposed growth regulator of plants and microorganisms is a new regulator, it is economical, as the cost of production hydroptila are small and the cost of the product is determined by the price of a cheap source of raw materials (uracil, bromine, Ethylenediamine).

The proposed controller improves the efficiency of the growth and development of plants at the stated concentration, as when using the solution hydroptila activated druzhnost seed germination, increases the weight of the stem, the weight of roots and the number of true leaves. Hydroptere used for soaking seeds, as well as for further processing in the vegetative development of plants.

Predlo use solution hydroptila increases the biomass of yeast, increased alcohol yield, increases the accumulation of lactic acid.

Applicants are not aware of any publication that would contain information about the impact of the distinctive features of the invention on the achieved results. In this regard, according to the applicants, it is possible to draw a conclusion on the conformity of the proposed solutions to the criterion of "inventive step".

The invention is illustrated by examples.

Hydroptere environmentally safe for humans and the biosphere. Acute toxicity hydroptila investigated in experiments on white mice and white rats. After subcutaneous administration to mice hydroptila at a dose of 2 g/kg no symptoms of intoxication were observed. In white rats after introducing them through a tube into the stomach mist or warm acidified aqueous solution hydroptila at a dose of 0.5 g/kg no signs of intoxication were not detected, even during a three-week introduction with a total dose of 1400 mg study of the influence hydroptila in the dynamics of the functional state of various physiological systems (cardiovascular, nervous system and organs (spleen, heart, lungs, kidneys, brain, liver) in animals with registration body weight, ECG, analisia statistically significant difference between rats and mice, receiving hydroptere, and animals of the control group. Thus, hydroptere can be attributed to the V class is practically non-toxic substances.

The invention includes the original technology for hydroptila, which describes the chemical scheme (see below).

Example 1 (synthesis).

I stage - 5-bromouracil:

- uracil 504 g (4.5 mol) and 1800 ml of water are loaded into the three-neck flask with a volume of 4 l, equipped with a reflux condenser, a thermometer, an efficient stirrer and addition funnel;

the mixture is heated to 40oC in a water bath and under vigorous stirring was added dropwise 232 ml (4.5 mol) of liquid bromine for 45 min, with a slight heating effect of rise of temperature to 55-60oC and almost complete dissolution of the original uracil;

the mixture is stirred at a temperature of 80-85oC for 1.5 hours with full bleaching of pulp and falls copious precipitate of the product;

- the reaction mass is then cooled first to room temperature, then incubated in ice water for 2 hours and cold filtered off white crystalline precipitate;

- sludge press from the mother liquor and washing the - sludge is squeezed and dried in a drying Cabinet at a temperature of 70-75oC to constant weight;

- get 816,7 g (95%) 5-bromouracil in the form of colorless crystals with so pl. 303-305oC decomp. ; chromatographically homogeneous - Rf= 0.45 in the system methanol-chloroform - 1: 4, Silufol UV 254. Calculated for C5H3BrN2O2(M. M. 191.05), %: Br - 41.83, N - at 14.66. Found, %: Br - 41.67, N - 14.59;

The PMR spectrum, DMSO-d6, , M. D.: 7.75 (C6-H, d, 1H); 11.15 (NH, d, 1H), 11.50 (NH, s, 1H); UV range: 0.1 N. HCl -max= 275 nm (lg 3.88); 0.1 N. NaOHmax= 290 nm (lg 3.86).

II stage - 2-oxo-(3H)-5,6,7,8-tetrahydropyrido (hydroptere)

- 1410 ml of 70% aqueous solution of Ethylenediamine (d=0.975) loaded into a three-neck flask with a volume of 4 l, equipped with a reflux condenser, a thermometer, an efficient stirrer;

the mixture is heated to 70-75oC in a water bath and add in small portions 860 g (4.5 mol) of 5-bromouracil for 1 hour. The temperature of the reaction mixture should not exceed 85oC;

- observed the dissolution of the original 5-bromouracil, and then crystallization of the reaction product;

the mixture was stirred at 80-85oC for 1.5 hours until the disappearance of the original chromatogram of uracil;

reaction is at 70-75oC and stirred for 1 hour;

the mixture is cooled with ice water for 3 hours and the precipitate is filtered off;

- sediment wring out well from the solution, washed with 450 ml of 70% methanol mother liquor separated for regeneration of methanol and Ethylenediamine, wring out from the solvent and dried to constant weight;

- receive 651 g (85%) of 2-oxo-(3H)-5,6,7,8-tetrahydropyridine monohydrate (hydroptila) in the form of light yellow crystals with so pl. 200 - 205oC decomp. ; chromatographically homogeneous Rf= 0.8 in the system 5% NH4Cl, Silufol UV 254. Calculated for C6H10N4O2(M. M. 170.18), %: C - at 42.31, H 5.87, N - 32.94. Found, %: C - 42.26, H 5.81, N - 32.89.

The product is standardized on physico-chemical parameters:

The PMR spectrum, DMSO-d6, , M. D.: 6.45 (C4-N, s, 1H); 2.80 (CH2CH2, s, 4H); UV range: watermax= 233 nm lg = 4.01),max= 300 nm (lg of 3.77), 0.1 N. HCl -max= 299 nm (lg 3.60); 0.1 N. NaOHmax= 292 nm (lg 3.55); IR spectrum (KBr):C=O1645 cm-1, rings1600 cm-1.

Hydroptere soluble in dilute alkalis, mineral acids with the formation of salts, insoluble in chloroform, benzene, ether. Solubility in boiling alcohol 1 g/50 ml, in boiling water to 1 g/30 ml (good and with Ethylenediamine, that is, the process of obtaining hydroptila of uracil simplified and carried out in a single reactor. Production costs hydroptila are small and the cost of the product is determined only by the price of a cheap source of raw materials (uracil, bromine, Ethylenediamine).

The claimed solution contains biologically active substance 2-oxo-(3H)-5,6,7,8 tetrahydropyridine (Hydroptere) formula

< / BR>
in the form of a hydrate or salts at a concentration of 10-6-10-5g/l

Method of intensification of metabolic processes of microorganisms is as follows and is illustrated by examples.

Prepare a water solution hydroptila: hydroptere in the amount of 1 mg dissolved in 1 g of water.

Prepare a fermentation environment: planting material introduced into the sterile environment in the ratio of 10% by volume of the environment.

An aqueous solution hydroptila in the amount of 1 ml injected (10 ml; 100 ml; 1000 ml 10000 ml) of fermentation medium, respectively, and receive concentration hydroptila 10-4; 10-5; 10-6; 10-7g/l

Example 2.

Seed: the yeast Saccharomyces cerevisiae pc. ATS-7754 with a titer of living cells 107cells/ml.

Sterile mineral medium, g/l:

Glucotrol extract - 1,0

Yeast is cultivated aerobically on a rotary shaker at 220 rpm for 4 days and determine the biomass accumulation in the culture medium.

The results of the experiment are presented in table. 1.

From table. 1 shows that the most efficient and economical solution concentration hydroptila 10-6- 10-5g/L. This limit concentrations contributes to the increase in the number of yeast by 53% on the 4th day. Outside a specified range of concentrations using hydroptila becomes less effective and economical.

Note:

1. Seed obtained from the training collection of the Department of technology of microbiological synthesis of Saint-Petersburg state technological Institute (technical University).

2. The control option is not treated with hydroperiod.

Example 3.

Seed: the yeast Saccharomyces cerevisiae pc. ATS-7754 with a titer of living cells 106cells/ml.

Sterile mineral medium hook, g/l:

Glucose - 20,0

NH4H2PO4- 5,0

K2SO4- 0,5

MgSO47H2O - 0,5

Yeast extract - 1,0

Conditions of cultivation medium: yeast, cultive.

The results of the experiment are presented in table. 2.

From table. 2 shows that the most efficient and economical solution concentration hydroptila - 10-6- 10-5g/L. This limit concentrations increases the fermentation activity of the yeast and increases the yield of alcohol on 22-40% on the 3rd day. Outside a specified range of concentrations using hydroptila becomes less effective and economical.

Note:

1. Seed obtained from the training collection of the Department of technology of microbiological synthesis of Saint-Petersburg state technological Institute (technical University).

2. The control option is not treated with hydroperiod.

Example 4.

Seed: algae Clorella vulgaris pieces 157 with a titer of living cells 106cells/ml.

Sterile mineral medium Hata, g/l:

Glucose - 10,00

(NH4)2HPO4- 1,50

K2HPO4- 1,50

MgSO47H2O - 1,00

CaCl2- 0,50

NaCl - 0,50

FeSO47H2O - 0,01

Yeast extract - 1,0

The cultivation conditions environment: algae cultivated under aerobic conditions in daylight 2000 Lux for 4 days and OS="ptx2">

From table. 3 shows that the most effective and economical solution concentration hydroptila - 10-6- 10-5g/L. This limit concentration increases the number of algal cells on 23-31% on the 2nd day. Outside a specified range of concentrations using hydroptila becomes less effective and economical.

Note:

1. The seed material is taken from the collection BNS the Russian Academy of Sciences.

2. The control option is not treated with hydroperiod.

Example 5.

Seed: Streptococcus bovis units ADS-49133 with a titer of living cells 109cells/ml.

Sterile starch-containing medium, g/l:

The flour - 10,00

Drug glucoamylase activity 2000 M. E. - 0,05

The cultivation conditions of the environment: bacteria grown under anaerobic conditions at a temperature of 40oC for 73 hours and determine the accumulation of lactic acid in the culture medium.

The results of the experiment are shown in table 4.

Table 4 shows that the most efficient and economical solution concentration hydroptila - 10-6-10-5g/L. This limit concentrations increases the accumulation of lactic acid in 20-28% 73 through omicini.

Note:

1. Seed obtained from the training collection of the Department of technology of microbiological synthesis of Saint-Petersburg state technological Institute (technical University).

2. The control option is not treated with hydroperiod.

Example 6.

Seed: Streptococcus bovis units ADS-49133 with a titer of living cells 109cells/ml.

Sterile insulinopenia environment, g/l: Jerusalem artichoke flour 100

The cultivation conditions of the environment: bacteria grown under anaerobic conditions at a temperature of 40oC for 73 hours and determine the accumulation of lactic acid in the culture medium.

The results of the experiment are shown in table 5.

From table 5 it is seen that the most efficient and economical solution concentration hydroptila - 10-6- 10-5g/L. This limit concentrations increases the accumulation of lactic acid on 26-31% after 73 hours. Outside a specified range of concentrations using hydroptila becomes less effective and economical.

Note:

1. Seed obtained from the training collection of the Department of technology of microbiological synthesis of Saint-Peterby option is not treated with hydroperiod.

The way to improve the biochemical activity of plants is as follows.

Prepare a water solution hydroptila: hydroptere in the amount of 1 mg dissolved in 1 l of water, 1 ml of solution is transferred into a 10 ml; 100 ml; 1000 ml 10000 ml of water, respectively, and receive aqueous solutions hydropenia with concentrations of 10-4; 10-5; 10-6; 10-7g/l

Example 7.

Plants:

- annual family cereal RYEGRASS;

- pea fodder.

An aqueous solution hydroptila at concentrations: 10-4; 10-5; 10-6; 10-7g/l

Conditions seed treatment:

- the seeds are soaked in an aqueous solution hydroptila the above concentrations, respectively, for 1 hour;

- incubated for 1 hour;

- are planted at a depth of 0.5 cm;

- determine the number of germinated seeds after 3-4 days;

- measure the length of the sprout in 7 days.

The results of the experiment are shown in table 6.

From table 6 it is seen that the most efficient and economical solution concentration hydroptila - 10-6- 10-5g/L. This limit concentration increases the number of germinated seeds at Regresa on 20-23% and peas on 56-59%, and Vanye hydroptila becomes less effective and economical.

Note: the control option is not treated with hydroperiod.

Example 8.

Plants: peas, cucumbers, bargetzi.

An aqueous solution hydroptila at a concentration of 10-6g/l

Conditions seed treatment:

- the seeds are soaked in an aqueous solution hydroptila for 1 hour;

- incubated for 1 hour;

- are planted at a depth of 0.5 cm;

- determine germination in 4 to 7 days.

The results of the experiment are shown in table 7.

Table 7 shows that treatment of seeds with a solution hydroptila significantly increase energy and druzhnost seed germination. Germination of pea seeds on the fifth day reaches 98%. Germination of seeds of cucumbers and bargatze on the fourth day reaches 96% and 98%. While the maximum germination in the control was only observed on the seventh day. Use hydroptila improves germination and reduces the time of germination for three-four days.

Note: the control option is not treated with hydroperiod.

Example 9.

Plants: tomatoes, cabbage, radish, onion, dill;

- peas;

- Lupin.

An aqueous solution hydroptila at a concentration of 110-6g/l

seeds incubated for 1 hour;

seeds are planted at a depth of 0.5 cm

The option of processing of seeds hydroperiod with subsequent spraying green parts of plants:

- the seeds are soaked;

seeds incubated for 1 hour;

seeds are planted at a depth of 0.5 cm;

- green part of plants sprayed 1 time a week with a solution hydroptila;

- green part of plants are sprayed with a solution hydroptila for 5 weeks.

The results of the experiment are presented in table.8.

From table.8 shows that after treatment of seeds with a solution hydroptila in all studied plants compared to the control, an increase in the length of the stem 6 to 133% increase in root length from 12 to 53%, increase in the number of leaves from 33 to 78%.

Even more effective is the combination of seed soaking with subsequent spraying green parts of plants solution hydroptila once a week for 5 weeks. In this processing mode, the increase in the length of the stem compared with the control in all the investigated plants ranges from 10 to 181%, increase in root length from 17 to 110%, the increase in the number of leaves - from 78 to 175%.

Note: the control option is not treated with hydroperiod.

Example 10.

Plant: Plectrantus austrelis.

Growth regulators:

- listenin;

- indelicacy acid (IAA);

- benzylaminopurine (6-BAP) - prototype;

- hydroptere.

The results of the experiment are presented in table 9.

From table. 9 shows that the most efficient and economical is hydroptere. When using hydroptila in the form of a solution with a concentration of 10-6g/l dry weight of root and stalk Plectrantus austrelis increases compared to the control at 119 and 143%, respectively. The use of 6-BAP (prototype) at the same concentration leads to an increase in root mass at 104%, and stem - 106%. Increasing concentrations of 6-BAP 10/ g/l does not lead to weight gain root and stem.

Use of the solution indolylacetic acid (IAA) and santehnika with a concentration of 10-4g/l less efficient than using hydroptila, since it leads to increase root mass, respectively 115 and 94%, and stem - 128 and 117%.

Note: the control option is not treated with growth regulator.

The proposed growth regulator of plants and microorganisms is an effective and economical. Technology receiving it suitable for implementation in promyshlennyy applicants his criterion of "industrial applicability".

New growth regulator - hydroptere is highly efficient and economical regulator, the method of obtaining hydroptila high-performance, cost-effective and the cost of the target product is significantly lower compared to imported drugs of similar action.

Use hydroptila as a growth regulator of plants and microorganisms allows you to:

- to increase the efficiency of biochemical processes;

- to shorten the growth, plant development and metabolism of microorganisms and plants;

- to increase the biomass of microorganisms;

to increase the alcohol yield in the fermentation process;

- to increase the accumulation of lactic acid in the fermentation process;

- to increase the stem length, root mass of plants;

- reduce the time of germination, growth and development of plants.

1. The growth regulator of plants and microorganisms, sackrai solution of biologically active substances, characterized in that the biologically active substances it contains 2-oxo-(3H)-5,6,7,8-tetrahydropyrido(hydroptilidae) formula

< / BR>
in the form of a hydrate or salts at a concentration of 10-6- 10-5g/l

2. ISM solution of biologically active substances, characterized in that the biologically active substances take 2-oxo-(3H)-5,6,7,8-tetrahydropyrido under item 1 and enter in the fermentative medium to achieve a concentration of 10-6- 10-5g/l

3. The way to improve the biochemical activity of plants by affecting the processes of growth and development with a solution of a biologically active substance, characterized in that the biologically active substances take 2-oxo-(3H)-5,6,7,8-tetrahydropyrido under item 1 and prepare a solution concentration of 10-6- 10-5g/L.

 

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SUBSTANCE: invention describes a method for feeding potato and tomato with 6-benzylaminopurine an aqueous solution taken in the concentration 10-4 M and growing pants up to preparing harvest according to technology accepted for the culture crop. Invention proposes 3-fold treatment of plants for vegetation: at the lateral branching phase, at onset of forming economically value organs and immediately after the growth termination. Method provides the effective enhancing the productivity of the most important vegetable crops - tomato and potato.

EFFECT: improved enhancing method.

6 tbl, 4 ex

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