RU2055827C1 - Consortium of bacterium (lactobacillus salivarius var salivarius, streptococcus thermophilus, streptococcus bovis) for seed germination activation - Google Patents

Abstract

FIELD: agriculture microbiology. SUBSTANCE: consortium of lactic acid bacteria (L. salivarium var. salivarium, S. thermophilus, S. bovis) 3844 is isolated from the soil and designed for activation of seed germination and deposited in All-Union Collection of microorganism at N B-5974. For preparation preparing consortium is sown on defatted milk and cultured at 40 C for 12 hr. Prepared culture with titer 2.0 billion/ml containing 10 g/l lactic acid and activity for cytokinin 150% (by biotest) is used for preparation preparing for seed germination activation. EFFECT: increased effectiveness of consortium.

Description

 The invention relates to agricultural microbiology and biotechnology, in particular to the production of bacterial preparations used in crop production and relates to a consortium of bacteria to obtain the above drugs.

 It is known that during bacterialization of plant seeds with lactic acid bacteria adapted to the rhizosphere, they multiply in it, and also contribute to an increase in the titer of other bacteria that use lactic acid. In addition, multiplying in the rhizosphere, lactic acid bacteria contribute to the plant in the absorption of phosphorus.

 An object of the present invention is to provide a consortium of lactic acid bacteria 3844 (Lactobacillus salivarius var. Salivarius, Streptococcus thermophilus, Streptococcus bovis) a producer of a preparation for activating seed germination.

 The proposed consortium does not contain the most common lactic acid bacteria of the rhizosphere and consists of cultures that actively form lactic acid and cytokinins. For example, the bacterium seed proposed by the consortium increases the energy of seed germination and increases their germination.

 The consortium was isolated from soil samples taken in the territory adjacent to the dairy (Fergana, Uzbekistan) in order to obtain lactic acid bacteria, which are active producers of cytokinins and lactic acid. To isolate the consortium, direct seeding from soil samples, accumulative cultures on liquid media with alcohol, and subsequent seeding on agar media with chalk were used. Then, thermophilicity and high productivity of lactic acid and cytokinins were selected (biotest for cytokinins).

When determining the taxonomic composition of the consortium, microscopy of deep colonies allows us to determine the ratio of a) lactic streptococci, b) lactic acid rods in the studied passage of the consortium. Colonies of lactic streptococci in the form of dense regular discs. Colonies of lactic acid rods are star-shaped, with outgrowths on the periphery and a dense center. For deep colonies with a characteristic structure growth in Petri dishes with 2 x overnight culture consortium (up to 100 colonies per dish) culture medium is poured peridotite layer melted and cooled ^to 45 ° C and cultured for another 48 hours.

The dominant component of the consortium are sticks 65-75% streptococci account for 25-35%
Variation of the components of the consortium may depend on the composition of the nutrient medium. With a sharp increase in the percentage of carbohydrates in the medium and a simultaneous decrease in the content of nitrogenous substances, the number of streptococci in liquid culture increases to 90-95%
Cultivation of the consortium in a constant regulatory environment (10% skim milk) provides 65-75% of rods and 25-35% of streptococci without losing the functional integrity of the consortium.

 One of the reasons for the indivisibility of the consortium is to protect crops from bacteriophages. The selectivity of bacteriophages and the small likelihood of simultaneously entering into the culture of a consortium of different phages that are specific to all the constituent cultures of the consortium gives a guarantee of the development of lactic acid to the limit at which bacteriophages cannot exist. In addition, the more vigorous development of certain cultures ensures the rapid accumulation of lactic acid to the specified limit, thus slowly growing lactic acid bacteria are under reliable protection.

 Sowing monocolonial isolates of the consortium on skim milk and studying their acid-forming activity, the ability to ferment carbohydrates and other physiological and biochemical properties makes it possible to determine the species composition of the consortium.

Streptococcus thermophilus grow ^at 50 ° C, does not grow at 10, 13 and 16 ° ^C to ferment maltose, raffinose. Do not ferment arabinose, xylose, some strains do not ferment mannitol and sorbitol; preferred use of sucrose and lactose over glucose.

Streptococcus bovis weak and slow growth ^at 50 ° C, at 10, 13 and 16 ^{° C} do not grow. Maltose, raffinose, inulin are fermented, not always mannitol and sorbitol; glucose is fermented more intensively than lactose and sucrose.

Lactobacillus salivarius var. salivarius grow ^at 50 ° C, typically not grow at ²⁰ ° C and never grow at 15 ° ^C to ferment rhamnose, galactose, lactose, maltose, mannitol, melibiose, sorbitol, sucrose, raffinose, trehalose. Do not ferment salicin.

 A similar consortium is not described in the literature and is highlighted for the first time.

 The consortium was accepted for deposit at the Central Museum of Industrial Microorganisms VNIIgenetika under the number B-5974.

The physiological feature of the consortium, in general, of practical importance, is the production of biologically active substances. All cultures that make up the consortium, thermophiles, the temperature limits of good growth and high activity are 30-45 ^o C, the optimum is 38-40 ^o C. The fermentation is homoenzymatic.

 According to morphology, the bacteria of the consortium are sticks and cocci. Rods often form chains, especially in the late logarithmic phase of growth. Fixed, non-spore-forming, gram-positive, become gram-negative with age and with increased acidity. Cocci cells are spherical or oval, single, in pairs or chains. Fixed, non-spore-forming, gram-positive.

The culture is grown on a nutrient medium of the following composition, Skimmed milk powder 10.0 Water Up to 100.0
The initial pH of the medium is natural.

 The consortium culture is grown for 8-12 hours to a pH of 3.8-4.0.

 A liquid culture with a titer of living cells from 2.0 billion / ml to $ 5.0 billion / ml, lactic acid content from 10 to 12 g / l and bioassay activity for cytokinins (on radish seedlings) from 130 to 150% is used for preparation a drug used in crop production to activate seed germination.

 The culture of the consortium is preserved in lyophilized form in ampoules.

 Before drying, a sucrose-gelatin mixture (4% gelatin, 40% sucrose, water) is added to the liquid culture as a protective medium in an amount of 75% by volume of the liquid culture and poured into 2 ml ampoules.

Shelf life of dry culture up to 2 years at a temperature of (-4) - (+ 4) ^o C.

 PRI me R 1. During industrial cultivation to obtain a seed culture 1 ampoule of dry material of the consortium was seeded in 100 ml of skim 10% milk.

 Flasks with 400 ml of culture medium were inoculated with 10 ml of cultivated inoculation of passage 1 in order to obtain an inoculum of passage 2.

Culturing a seed culture of lactic acid bacteria consortium 1 and 2 passages were ^at 40 ° C for 12 hours. The pH of grown inoculum 1 and 2 passages 3,8.

Seed 2 passage used to prepare the inoculum (10% of the volume of the nutrient medium). Culturing in inoculator was carried out at 40 ^{° C} for 12 hours. The pH was 3.8 at the end of fermentation.

Cultivation in the fermenter was carried out at a temperature of 40 ^about C for 10 hours in 10% skim milk. The pH at the end of fermentation is 4.0.

 The resulting liquid culture with a titer of 2.0 billion / ml, a lactic acid content of 10 g / l and a bioassay activity (on radish seedlings) of 150.0% was used to prepare the preparation.

 PRI me R 2. For the preparation of 2.0 l of the drug, the components were mixed in the following ratio: dry Fucus algae 12.0 g; urea 12.0 g; the culture of the consortium 3844 obtained in example 1 600,0 ml; sodium benzoate 5.0 g; calcium propionate 5.0 g; ascorbic acid 5.4 g; complexonates (mixture) 2.0 g; water up to 2000.0 ml.

To prepare the preparation, a mixture of the following complexonates was used in equal weight ratios: K ₂ ZnOEDP, NH ₄ -FeOEDP, Mo-OEDP, K ₂ CoOEDP, K ₂ MgOEDP, K ₂ MnOEDP.

 Dry algae was pre-soaked in a small amount of water.

 After urea was added, the acidity of the mixture was adjusted to pH 5.0.

 The pH of the finished product is 4.4.

Cytokinin bioassay activity (on radish seedlings) 150.0%
The resulting preparation was treated with potatoes, Nevsky variety, at the rate of 1 liter per hectare seed rate.

 Harvest increased by 36.0% compared with the control (control untreated potatoes). The output of standard products is 98.0% (81.0% in the control).

Claims (1)

 A consortium of bacteria (Lactobacillus salivarius var. Salivarius, streptococcus thermophilus, streptocossus bovis) VKPM B-5974 to activate seed germination.