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Title:
A MICROBIOLOGICAL PREPARATION FOR THE MINERALIZATION OF CELLULOSE -CONTAINING ORGANIC MATTER AND THE APPLICATION OF THE MICROBIOLOGICAL PREPARATION IN GROWING PLANTS AND/OR IN ANIMAL NUTRITION
Document Type and Number:
WIPO Patent Application WO/2017/138824
Kind Code:
A1
Abstract:
A microbial preparation for the mineralization of organic material, in particular cellulose- containing, comprising selected bacterial strains, living or capable of reproduction in the soil, macro- and/or microelements, and also biologically active compounds with antioxidant, antibacterial, and antifungal properties, stabilizers preventing the microbiological preparation from undesired destabilization and optionally water, characterized in that it comprises strains of Bacillus subtilis All (PCM B/00105) and/or Bacillus licheniformis 4/18 (B PCM/00106), deposited in the Polish Collection of Microorganisms of the Polish Academy of Sciences in Wroclaw, in the amount of 101-1012 cells per gram of preparation, preferably 108-109 cells per gram of preparation, immobilized on a carrier in the form of aluminosilicate mineral, preferably from the group of montmorillonite and/or attapulgite bentonites, with an average particle size of 0.001 to 0.07 mm and constituting from 0.01 to 5% by mass of the preparation. The invention also relates to the application of the preparation for the mineralization of organic matter, in particular cellulose-containing, in plant cultivation, for foliar or and/or root application, after the dilution of the preparation with water in the range of 0.02 to 5 % by mass, and/or in animal feed in liquid or dry form, for application with feed or water in the amount of 0.00001 g to 0.1 g preparation per kg of body weight per day.

Inventors:
CIECIERSKI, Wiesław (Dąbrowice 4, 96-124 Maków, PL)
KARDASZ, Hubert (ul. Jaworowa 42/3, 02-798 Warszawa, PL)
SZYCHOWSKA, Katarzyna (ul. Ks. M. Mądrzyka 50, 32-067 Tenczynek, PL)
WILK, Radosław (ul. Inowrocławska 21c/5, 53-635 Wrocław, PL)
Application Number:
PL2017/000006
Publication Date:
August 17, 2017
Filing Date:
February 08, 2017
Export Citation:
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Assignee:
INTERMAG Sp. z o.o. (Al. 1000-lecia 15G, 32-300 Olkusz, PL)
International Classes:
C05F11/08; A01N63/00; C12N1/20; C12R1/10; C12R1/125
Foreign References:
CN105085040A2015-11-25
CN105061117A2015-11-18
CN104140305A2014-11-12
CN102972447A2013-03-20
CN102503665A2012-06-20
CN104403971A2015-03-11
CN104560770A2015-04-29
Attorney, Agent or Firm:
ROSÓŁ, Patrycja (ul. Szlak 65/510, 31-153 Kraków, PL)
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Claims:
PATENT CLAIMS

1. A microbial preparation for the mineralization of organic material, in particular cellulose- containing, selected bacterial strains, living or capable of reproduction in the soil, macro- and/or microelements, and also biologically active compounds with antioxidant, antibacterial, and antifungal properties, stabilizers preventing the microbiological preparation from undesired destabiiization and optionally water, characterized in that it contains strains of Bacillus subtilis All (PCM B/00105) and/or Bacillus licheniformis 4/18 (B PCM/00106), in the amount of 10 -1012 cells per gram of preparation, preferably 08-109 cells per gram of preparation, immobilized on a carrier in the form of aluminosilicate mineral, preferably from the group of montmorillonite and/or attapulgite bentonites with an average particle size of 0.001 to 0.07 mm and constituting from 0.01 to 5% by mass of the preparation.

2. A preparation according to Claim 1 characterized in that the strains of Bacillus subtilis 4/7 (PCM B/00105) and Bacillus licheniformis 4/18 (PCM B/00106) are found in the preparation in a ratio of 1 : 1.

3. A preparation according to Claim 1 characterized in that it contains up to 40% by mass of at least one macro-or microelement, preferably in a water-soluble form, selected from the group consisting of the following elements: nitrogen, phosphorus, potassium, magnesium, calcium, silicon, titanium, boron, copper, iron, manganese, molybdenum, zinc, and cobalt.

4. A preparation according to Claim 1 or 3, characterized in that it contains up to 40% by mass of nitrogen, up to 25% by mass of phosphorus, up to 20% by mass of potassium and 20% by mass of silicon, up to 25% by mass of magnesium, up to 10% by mass of calcium, up to 10% by mass of boron to 12% by mass of iron, up to 10% by mass of manganese, up to 10% by mass of molybdenum, up to 10% by mass of zinc, up to 10% by mass of cobalt, up to 5% by mass of copper, and up to 3% by mass of titanium.

5. A preparation according to Claim 1 characterized in that it contains substances with the ability to chelate microelements, in particular: EDTA in the amount of 0.01 to 5% by mass, HEDTA in the amount of 0.01 to 5% by mass, DTPA in the amount of 0.01 to 5% mass, HEEDTA in the amount of 0.01 to 5% by mass, amino acids in the amount of 0.01 to 5% by mass, fulvic acids in the amount of 0.01 to 5 % by mass, sugars in the amount of 0.01 to 5% mass, polysaccharides in the amount of 0.01 to 5% by mass, polyphenols in the amount of 0.01 to 5% by mass, vitamins in the amount of 0.01 to 5% by mass.

6. A preparation according to Claim 1 characterized in that it contains biologically active antioxidant, antibacterial, and antifungal compounds selected from the group consisting of polyphenols, polyunsaturated fatty acids, phlorotannins, sulphated polysaccharides, carotenoids, and peptides, contained in the preparation in the amount of 0.01 to 5% by mass.

7. A preparation according to Claim 1 , characterized in that it contains plant hormones, including phytosterols-cytokinins, auxins, and gibberellins in the amount of 0.01 to 5% by mass.

8. A preparation according to Claim 1 characterized in that it contains a post-cu!ture fluid and metabolites produced by bacteria during culture in the amount of 0.1 to 95 % by mass.

9. A preparation according to Claim 1 characterized in that its pH is in the range of 2 to 12, preferably from between 6 and 8.

10. A preparation according to Claim 9 characterized in that its pH is adjusted with an acid or a base, preferably citric acid, hydrochloric acid, sulphuric acid, hydroxides and/or carbonates of alkali metals, especially sodium or potassium.

11. The application of the microbiologica! preparation containing selected strains of Bacillus subtilis All (PCM B/00105) and/or Bacillus licheniformis 4/18 (PCM B/00106) bacteria living or capable of reproducing in soil in a proportion of 10 -1012 cells per gram of preparation, preferably 108-109 cells per gram of preparation, for the mineralization of organic matter, in particular cellulose-containing, in plant cultivation, for foliar or and/or root application, after the dilution of the preparation with water in the range of 0.02 to 5 % by mass, and/or in animal feed in liquid or dry form, for application with feed or water in the amount of 0.00001 g to 0.1 g preparation per kg of body weight per day.

12. Application according to Claim 11 , characterized in that the preparation contains liquid from the culture of Bacillus subtilis 4/7 (PCM B/00105) and/or Bacillus licheniformis 4/ 8 (B PCM/00106) bacteria, preferably enriched with mineral and/or organic components.

13. Application according to Claim 1 1 , characterized in that the preparation contains strains of bacteria with known probiotic properties, preferably of the Bacillus and/or the Lactobacillus and/or Streptococcus and/or Lactococcus and/or Pediococcus and/or Enterococcus and/or Saccharomyces genera in the amount of 101-1012 cells per gram of preparation.

Description:
A MICROBIOLOGICAL PREPARATION FOR THE MINERALIZATION OF CELLULOSE - CONTAINING ORGANIC MATTER AND THE APPLICATION OF THE MICROBIOLOGICAL PREPARATION IN GROWING PLANTS AND/OR IN ANIMAL NUTRITION

The present invention relates to a microbiological preparation comprising living micro-organisms or micro-organisms capable of reproducing in soil, intended for the mineralization of organic material, in particular cellulose- containing, and the application of the microbiological preparation comprising living micro-organisms or micro-organisms capable of reproducing in soil in growing plants and/or in animal nutrition.

The present invention relates to a solution in the field of microbiological preparations containing new strains of bacteria designed to intensify agricultural and livestock production.

Microbiological preparations are used in the cultivation of plants to protect them from diseases and pests. Microbiological preparations are also used to enhance the life processes of plants related to their growth and development, and to improve the physical and chemical properties of soils or, e.g., the decomposition and mineralization of organic matter such as post-harvest residues and natural fertilizers. The empirical evidence of farming practices demonstrates that the application of microbiological preparations leads to, among others, increasing yields, reducing the exposure to disease, better utilization by plants of nutrients contained in the soil, and better and faster rooting. Certain microbiological preparations also reduce abiotic stress, i.e., the stress caused by adverse external factors such as e.g. drought, frost and cold, stress caused by the application of herbicides, and environmental pollution with toxic substances or heavy metals.

The influence of microbiological preparations containing microorganisms on soil and plants is not due to the fact that they directly participate in the regulation of

SUBSTITUTE SHEET life processes, but to the effect of the active compounds produced by these bacteria, e.g., amino acids, enzymes, specific proteins, hormones, and other organic, inorganic and mineral compounds. Depending on the type and strain of the microorganism used in the microbial preparation, such products have different properties and are responsible for different functionalities.

In agriculture, a number of microbiological preparations are known and used, e.g. preparations containing symbiotic bacteria of Fabaceae plants obtained on the basis of Rhizobium bacteria, or vaccines mycorrhizing tree seedlings produced on the basis of Trichoderma spp. fungi, in biological plant protection, products containing microorganisms such as Alternaria, Trichoderma, Azotobacter, Bacillus, Pseudomona, Streptomyces, and others are used.

A paper by Truba ., K. Jankowski, and J. Sosnowski entitled„Reakcja roslin na stosowanie preparatow biologicznych", Ochrona Srodowiska i Zasobow Naturalnych 53, 2012, provides an overview of biopreparations known and used in the Polish market. For example, a preparation for soil fertilization, marketed under the trade name UGmax, accelerates the decomposition of organic matter and increases the content of organic matter (humus) in the soil. The preparation contains microorganisms such as lactic acid bacteria, photosynthetic bacteria, Azotobacter, Pseudomonas, yeasts, and actinomycetes and macro- and microelements in the following amounts amount per 1 kg of preparation: 1.2 g N, 0.5 g P, 3.5 g K, 0.1 g g, 0.0003 g Mn, and 0.2 g Na. Microorganisms transform and compost natural and organic fertilizers to produce humus, thereby improving the soil structure. The UGmax preparation has been tested with positive results on potato crops.

Other preparations described in the literature are based on the technology of Effective Microorganisms (EM) and comprise approximately 80 microorganisms, which have positive effects on plants and soil, including: lactic acid bacteria, photosynthetic bacteria, and Azotobacter yeasts and fungi. Increasing the amount of positive microorganisms in the soil increases the absorption of nutrients from organic matter and, consequently, improves soil fertility and health.

Also a preparation supporting the development of plants on poor degraded soils, marketed as Phylazonit M and containing microorganisms such as Azotobacter, Bacillus and microelements of Cd, Co, Cr, Cu, Hg, Ni, Pb, Se, is known.

Another known preparation, marketed under the trade name WG Dipel, is based on the Bacillus thuringiensis var. kurstaki, bacteria which are selectively toxic to caterpillars of many harmful species of butterflies (Lepidoptera).Jhe Dipel WG preparation provides an alternative to chemicals in agricultural plant, vegetable, fruit and ornamental plant cultures, it is not harmful to beneficial predatory and parasitic insects, and therefore is conducive to maintaining biological balance. It is used in a dose of 0.5 to 3.0 kg/ha. The recommended amount is introduced into a spray tank containing e.g. 500-1 ,000 l/ha for spraying fruit.

Bacteria also help processes of waste composting. For example, in the publication by K. Cybulski et al., Inz. Ap. Chem., 2012, 51 , 4, 108-111 , a method of is described for composting pig bristles mixed with wood chips or straw, lignite dust and granulated chicken droppings with the participation of a microbial vaccine containing bacteria of the Bacillus genus in the form of fresh biomass, filamentous Trichoderma sp. fungi and Streptomyces sp. actinomycetes in the form of a wash-out from a culture plate. The application of such a vaccine made it possible to increase the efficiency of bristle decomposition compared to non- vaccinated control compost

Microbiological preparations used for plant cultivation are also the subject of numerous patents.

Patent description PL217740 B1 discloses preparations for the treatment of soil and plant seeds which contain live micro-organisms or micro-organisms capable of reproducing in soil, the manners of producing preparations for the treatment of soil and plant seeds, microorganisms, a method for producing micro-organism, and the method for the treatment of soil and plant seeds with those preparations. The invention relates to preparations for the treatment of soil and plant seeds which contain live micro-organisms or micro-organisms capable of reproducing in soil of various types in the environment of plants, wherein the preparations contain the culture of Bacillus megaterium var. M326 (NCAI /P/B

001291 ) in the amount of 5 x 10 6 -10 11 , preferably 10 7 -10 10 cells per g, and one or more of the micro-organisms among the following: Azospirillum brasilense ssp.SW51 (NCAIM/P/B 001293), Azotobacter vinelandii ssp. M657 (NCAIM/P/B

001292) , Pseudomonas fluorescens var. SW11 (NCAIM/P/B 001296), Bacillus polymyxa var. SW17 (NCAIM/P/B 0012 95), Micrococcus roseus ssp. A21 (NCAIM/P/B 001294), Bradyrhizobium japonicum i ar.PH25 (NCAIM/P/B 001302) and Streptomyces albus var. 0003 LP (NCAIM/P/B 001301 ), as microorganisms capable of reproduction also at low temperatures, preferably below 20°C, as well as in soils with low pH, preferably below 5, deposited at the National Collection of Agricultural and Industrial Micro-organisms, Budapest, Hungary, and wet or dry carriers permitted in agricultural crops and non-toxic for microorganisms. Preferred are preparations in which the role of the carrier is performed by water and/or soya flour and/or starch and/or cellulose and/or glucose. Preparations containing microbial cells in the amount of 10 10 -10 4, preferably 10 1 -10 12 per hectare, are applied to the surface of or into the soil during a season of the year during which temperature does not drop below 0°C or used to treat seeds. The culture of microorganisms can be introduced directly into the treated soil or plants along with the medium used for the culture, or in the form of preparations that retain the biotic potential of the microorganism, including preparations with carriers, which ensure that the bacteria adhere to the seeds by adhesive forces. The patent application PL376766 A1 discloses a biopreparation, especially for the mineralization of waste which contains live bacteria that accelerate the mineralization of organic waste, such as Pseudomonas nitroreducens, Pseudomonas proteolyica, Pseudomonas psychrophila, Pseudomonas azotoformans, Pseudomonas denitriflcans, Pseudomonas putida, Pseudomonas rhizosphaerae, Pseudomonas lutea, Pseudomonas Agarici and the cultures of Bacillus subtilis and Bacillus denitriflcans bacteria. Live bacteria are stored on a sterile field, for example an agar one. Such a composition of the preparation allows significantly reducing the time of mineralization of organic waste and, as a result of the process, obtaining from them a substrate suitable for use in agriculture.

Patent application PL292243 A1 , discloses a biological preservative for ensiling green fodder, intended for properly directing and accelerating the fermentation processes taking place during ensiling animal feed, consisting of a bacterial preparation with a starter and a regulator of cell growth. The preparation contains a composition of lactic acid bacteria and propionic acid bacteria in a ratio of between 1 :0.5 and 1 :1. Glucose is used as a starter, in the amount of 0.4-0.6%. The role of the stabilizer is performed by a mixture of anhydrous monoammonium phosphate, used in the amount of 0.4-0.6% by mass, and anhydrous disodium phosphate, used in the amount of 1.4-1.8 % by mass, wherein the dry preservative contains a total of approximately 10 11 cells per 1 gram.

Patent description R0126081 B1 discloses a biopreparation intended for the control of fungi of the Fusarium genus causing plant diseases, which comprises 1-20% of a suspension of the following micro-organisms: Saccharomyces cerevisiae L30 (deposit number NCAIM Y001350) and Bacillus subtilis B49b (deposit number NCAIM (P) B 001360), preferably between 4-8% of each strain, 2-15% of nutrients 5-15% surfactants, 10-50% solvent, 0.01-1 % stabilizer, 1-10% anticoagulant and distilled water up to 100%. Patent US6232270 B1 discloses a composition for use in agriculture, comprising the culture of Bacillus subtilis, Bacillus cereus, or a new strain of Bacillus ATCC 55675 bacteria. The composition is used in the form of an aqueous solution with bacteria concentration of 300,000 to 1 ,500,000 cfu/ml, preferably between 1 ,000,000 and 1 ,200,000 cfu/ml, also in an admixture with other active compounds used in agriculture, such as fungicides, insecticides or herbicides.

Patent RU2147181 C1 discloses a biopreparation for increasing crop yields and protecting plants against diseases which contains a hydroiysate of Pseudomonas aureofaciens VKM V-1973D bacteria in the amount of 18-20 parts by mass, a hydrolyzate of Bacillus megaterium bacteria in the amount of 39-40 parts by mass, the extract of pine needles in the amount of 5-6 parts by mass, chlorophyll and carotene paste in the amount of 1-2 parts by mass, and a solution of macro- and microelements in the amount of 32-37 parts by mass.

Also known are strains of bacteria with probiotic properties intended for animals, described, e.g., in the paper by L. Mizak, R., Gryko et al. entitled „Probiotyki w zywieniu zwierzaf, Zycie Weterynaryjne 87(9), 2012. These include lactic-acid-producing bacteria of the Lactobacillus genus, gram-positive cocci of the Streptococcus genus, bacteria of the Enterococcus, Pediococcus, Lactococcus, Leuconostoc, Propionibacterium freundenreichii genera, Gram- positive Streptococcus bacilli and yeast of the Saccharomyces, Kluyveromyces, Aspergillus genera, and many more. Depending on the animal's age and species, probiotic preparations are administered in the form of capsules, paste, powder, liquid, gel or pellets, directly or indirectly, with food.

In an era of intensifying agricultural production, farmers are looking for more efficient application of fertilizers introduced into the soil. The content of organic matter in agricultural soils lowers by approximately 2% per year. Lower content of organic matter in soils is caused by the fact that straw is left on the fields, or non-agricultural use, e.g. by burning biomass. It is therefore necessary to periodically introduce additional soil fertilizers in various forms.

As we know from farming practices, crop residues are a valuable source of organic matter.

Studies carried out for a number of years indicate that 5 tons of straw contains on average up to 35 kg N, 15 kg P2O5, 110 kg K 2 O t 10 kg MgO, 20 kg of CaO, 8 kg S, 25 g B 200 g Zn 150 g of Mn and 15 g of Cu. However, these valuable fertilizer ingredients are available for plants in the absorbable form only when crop microorganisms present in the soil, especially cellulolytic bacteria, decompose posthervest crop residues. Therefore, finding a method for quicker decomposition of crop residues would allow increasing yields by making valuable nutrients and humus compounds necessary for the proper growth and development of plants more easily availability for plants, while reducing additional fertilization.

Surprisingly, it turned out that the decomposition of postharvest crop residues may be accelerated by using selected strains of the soil bacteria Bacillus subtilis 4/7 (PCM B/00105) and/or Bacillus licheniformis 4/18 (PCM B/00106) living or capable of reproducing in soil, deposited in the Polish Collection of Microorganisms Polish Academy of Sciences in Wroclaw. During the research it was also found that the microbiological preparation can produce unexpected results when it is used in animal nutrition.

According to the invention, the microbial preparation for the mineralization of organic material containing, in particular, cellulose, also contain selected bacterial strains, living or capable of reproducing in soil, macro- and/or microelements, biologically active compounds with antioxidant, antibacterial, and antifungal properties, as well as stabilizers preventing the microbiological preparation from undesired destabilization, and possibly water.

The proposed solution is characterized in that the preparation contain, in a proportion of 10 1 -10 12 cells per gram of preparation, strains of Bacillus subtilis 4/7 (PCM B/00105) and/or Bacillus licheniformis 4/18 (B PCM/00106), deposited in the Polish Collection of Microorganisms Polish Academy of Sciences in Wroclaw, preferably in a proportion of 10 8 -10 9 cells per gram of preparation, immobilized on a carrier in the form of aluminosilicate mineral, preferably from the group of montmorillonite and/or attapulgite bentonites with an average particle size of between 0.001 and 0.07 mm, which constitute between 0.01 and 5 % by mass of the preparation.

Preferably, the strains of Bacillus subtilis 4/7 (PCM B/00105) and Bacillus licheniformis 4/18 (PCM B/00106) are found in the preparation in a ratio of 1 : .

The product also contains up to 40% by mass of at least one macro- or microelement, preferably in water-soluble form, selected from the group consisting of the following nutrients: nitrogen, phosphorus, potassium, magnesium, calcium, silicon, titanium, boron, copper, iron, manganese, molybdenum, zinc, and cobalt.

Preferably the preparation contain up to 40% by mass of nitrogen, up to 25% by mass of phosphorus, up to 20% by mass of potassium and 20% by mass of silicon, up to 25% by mass of magnesium, up to 10% by mass of calcium, up to 10% by mass of boron, up to 12% by mass of iron up to 10% by mass of manganese, up to 10% by mass of molybdenum, up to 10% by mass of zinc, up to 10% by mass of cobalt, up to 5% by mass of copper, and up to 3% by mass of titanium.

Preferably, the preparation contain substances with the ability to chelate microelements, in particular: EDTA in the amount of 0.01 to 5% by mass, HEDTA in the amount of 0.01 to 5% by mass, DTPA in the amount of 0.01 to 5% by mass, HEEDTA in the amount of 0.01 to 5% by mass, amino acids in the amount of 0.01 to 5% by mass, fulvic acids in the amount of 0.01 to 5 % by mass, sugars in the amount of 0.01 to 5% by mass, polysaccharides in the amount of 0.01 to 5% by mass, polyphenols in the amount of 0.01 to 5% by mass, and vitamins in the amount of 0.01 to 5% by mass. Preferably, the preparation contains biologically active compounds with antioxidant, antibacterial, and antifungal properties selected from the group consisting of polyphenols, polyunsaturated fatty acids, phlorotannins, sulphated polysaccharides, carotenoids, and peptides, contained in the preparation in the amount of 0.01 to 5% by mass.

Preferably, the preparation optionally contains plant hormones, including phytosterols - cytokinins, auxins, and gibberellins in the amount of 0.01 to 5% by mass.

Preferably, the preparation optionally contains post-culture medium and metabolites produced by bacteria during fermentation process in the amount of 0.1 to 95 % by mass.

Preferably, the pH of the preparation is contained in the range between 2 to and 12, ideally between 6 and 8, and is controlled by means of an acid or a base, preferably citric acid, hydrochloric acid, sulphuric acid, hydroxides and/or carbonates of alkali metals, especially sodium or potassium.

The essence of the proposed solution consists in the application of the microbiological preparation containing live or capable of reproducing in soil selected strains of bacteria Bacillus subtilis All {PCM B/00105) and/or Bacillus licheniformis 4/18 (B PCM/00106), in the amount of 10 1 -10 12 cells per gram of preparation, preferably 10 8 -10 9 cells per gram of preparation for the mineralization of organic matter which contains in particular cellulose, in plant cultivation, and foliar and/or root application, after having diluted the preparation with water in the range of 0.02 to 5% by mass, and/or for animal nutrition, in liquid or dry form, to be applied with feed or water in the amount of 0.00001 g to 0.1 g of preparation per kg of body weight per day.

Preferred is application wherein the liquid preparation comprises liquid from a culture of Bacillus subtilis 4/7 (PCM B/00105) and/or Bacillus licheniformis 4/18 (PCM/B00106), preferably enriched with mineral and/or organic components. Preferably, the preparation contain strains with known probiotic properties, preferably from the Bacillus and/or the Lactobacillus and/or Streptococcus and/or Lactococcus and/or Pediococcus and/or Enterococcus and/or Saccharomyces genera, in the amount of 10 1 -10 12 cells per gram of preparation.

A microbial preparation prepared on the basis of strains of Bacillus subtilis 417 (PCM B/00105) and/or Bacillus licheniformis 4/18 (PCM B/00106) shows the ability to accelerate the decomposition of cellulose contained in the organic matter, thanks to which it, among others: prevents the development of pathogens growing on post-harvest leftovers; has a positive effect on soil properties, in particular its structure, the availability of micro and macronutrients, and water capacity; and acts as a stimulant to the growth and development of agricultural and horticultural plants. Bacterial strains used to formulate the microbiological preparation are capable of producing, among others, cellulotytic enzymes, which accelerate the decomposition of crop residues, and at the same time provide the soil with nutrients easily available for the plants.

In addition, it was found that the application of a microbiological preparation according to the present invention in animal nutrition enhances the degree of utylization food, restores the balance of intestinal microflora, strengthens the immune system, and reduces the energy shortages of dairy cows, calves, pigs, laying hens, and chicken, turkey, duck and goose broilers. The application of a microbiological preparation according to the invention increases the biodiversity of beneficial intestinal flora of animals, which increases their weight and stabilizes the gut microflora of monogastric farm animals by eliminating the harmful microorganisms colonizing their intestines, and consequently enhances the health of the animals.

The product maintains a strong probiotic effect when applied in a liquid form for watering animals and as a feed additive sputtered on the feed material, or in a powder form as a preparation supplementing feed mixtures. The strains of Bacillus subtilis 4/7 (PCM B/00105) and/or Bacillus licheniformis 4/18 (B PCM/00106) used in the creation of the microbiological preparation are capable of producing, among others, celluloiytic enzymes, which accelerate the decomposition of the feed, and at the same time provide the animals with easily digestible nutrients.

The preparation is suitable for storage in a liquid or solid form, and at the same time has limited tendency for instability.

Introducing into the preparation at least one macro and/or microelement such as nitrogen, phosphorus, potassium, magnesium, calcium, sulphur, boron, copper, iron, manganese, molybdenum, zinc, cobalt, titanium, or silicon, in a water-soluble form, stimulates bacteria to synthesize celluloiytic enzymes more quickly and more efficiently, and reduces the biopreparation's tendency to become unstable.

Nutrients such as nitrogen, phosphorus, potassium, magnesium, sulphur, carbon, boron, copper, iron, manganese, molybdenum, zinc, sugars and polysaccharides not nly stabilize the solution, but also lead to improving the yield of agricultural and horticultural crops.

The preparation maintains a strong stimulating effect on the growth and development of plants when applied on leaves and/or seeds and/or outside roots.

The invention is further defined in the following examples without limiting the scope thereof,

Example 1

The microbiological preparation in the form of a solution contains the following bacterial strains in the amount of 10 8 -10 9 cells per gram of preparation:

- 5 % by mass of the inoculum of a strain of Bacillus subtilis 4/7 (PCM B/00105),

- 5 % by mass of the inoculum of a strain of Bacillus licheniformis 4/18 (B PCM/00106), - 0.06% bentonite with an average particle size of 0.001 to 0.07 mm,

macro and microelements:

Nitrogen (N) - 3.087% by mass,

Phosphorus (P 2 0 5 ) - 0.067% by mass,

Potassium (K 2 0) - 0.043% by mass,

Magnesium (MgO) - 0.009 % by mass,

Sulphur (S0 3 ) - 0.064 % by mass,

Boron (B) - 0.001 % by mass.

Iron (Fe) - 0.009 % by mass,

Manganese (Mn) - 0.009% by mass,

Zinc (Zn) - 0.009 % by mass,

and

- 0.418% by mass of citric acid,

bacteria nutrients:

- 0.093% by mass of carboxymethyi cellulose CMC,

- 0.093% by mass of Peptone K

and solvent:

- 75.94 % by mass of water.

The detailed procedure for the preparation of the inoculum was carried out according to Polish patent application entitled "Sposob przygotowania inokulum bakterii celulolitycznych do wytwarzania biopreparatow poprawiaja_cych wlasciwosci gleby", nr PL41442 A1 of October 19, 2015.

Strains of Bacillus subtilis 4/7 (PCM B/00105) and Bacillus licheniformis 4/18 (PCM B/00106) bacteria were isolated from the environment by collecting them from the substrate for mushroom production, mixing with sterile physiological saline solution, conducting a series of dilutions, and then multiplication on a substrate containing Peptone K, CMC, agar, gelatin, and water as supplement to 100% by mass. The multiplication of the bacteria was carried out over a period of 48 hours at a temperature not lower than 32 °C until individual colonies of Bacillus subtilis 4/7 (PCM B/00105) and/or Bacillus Hcheniformis 4/18 (PCM B/00106) bacteria strains were obtained on the substrate. The bacteria were then deposited on an agar slant with solidified broth medium, and the inoculum was stored at low temperatures in a cryobank.

To prepare a microbiological preparation in liquid form, in a sterile reactor equipped with a stirrer and the possibility of heating, 50 ml of the inoculum of a strain of Bacillus subti!is B/00105, 50 ml of the inoculum Bacillus licheniformis B/00106, and 306 g of urea, 100 g of water were introduced and stirred at 35-40 °C at a speed of 50 rev/min for 1 minute. Then, after vigorous stirring of the amounts indicated above, the mixture was completed with water up to 1 liter and vigorously stirred.

The activity of Bacillus subtilis Ail (PCM B/00105) and/or Bacillus licheniformis 4/ 8 (B PCM/00106) strains, in the decomposition of cellulose.

Samples of Bacillus subtilis All (PCM B/00105) and/or Bacillus licheniformis 4/18 (PCM B/00106) strains were cultured in a solution comprising: 1 % casein peptone,

1 % CMC,

0.2% K 2 HP0 4>

1.5% agar,

0.03% MgS0 4 -7H 2 0,

0.25% (NH 4 ) 2 S0 4 ,

0.2% gelatin.

Cellulolysis was marked according to the method using 3,5-dinitrosalicy!ic acid (DNS). In order to determine the amount of reduced sugar, a supernatant obtained after the centrifugation of the samples at certain hours of culture was used as an enzyme source (spin parameters: 4,500 rev/min x 10 min/14 °C).The supernatant in the amount of 1 m! was then added to 0.5 ml of the appropriate substrate:

- 1 % CMC suspended in 0.05 M phosphate buffer,

- Wathman paper (1 cm x 6 cm, weight 50 mg) was suspended in 0.05 M of citrate buffer.

The whole was subjected to incubation in a water bath at around 30 °C or 50 °C. The incubation time was 60 minutes. After this time, the reaction was terminated by using DNS (1.5 mi), and the samples were transferred to a water bath at a temperature of 100 °C. After 5 minutes, the samples were cooled and diluted with distilled water (12 ml).

The amount of the released sugars was measured using a spectrophotometer.

Absorbance was measured at a wavelength of 530 nm. Distilled water and DNS were used for blank determination.

The amount of glucose [micromol] released in 1 mi of enzyme during 1 minute was adopted as the unit of activity [U]:

lpmol glucose

1 U = mm

1

Tests demonstrating a significant acceleration of cellulose decomposition by the bacteria! strains of Bacillus subtilis 417 (PCM B/00105) and Bacillus licheniformis 4/18 (PCM B/00106) were carried out in comparison to the other strains of the ceilulolytic activity.

The evaluation of the activity of the isolates of Bacillus subtilis All (PCM B/00105) and Bacillus licheniformis 4/18 (B PCM/00106), in the decomposition of cellulose (source of cellulose-CMC, temp. 30 and 50 °C) are shown in Tables 1 and 2. Table 1

Table 2

The assessments of enzymatic activity by the by DNS method of the isolates from sources # 2 and # 4 (source of cellulose-paper, temp. 30 and 50 °C) are shown in Tables 3 and 4.

Table 3

Table 4

Example 2

The microbiological preparation prepared as in Example 1 , in the form of a solution, comprises the following bacterial strains in the amount of 10 8 -10 9 cells per gram of preparation:

- 15 % by mass of the inoculum of a strain of Bacillus subtilis All (PCM B/00105),

- 5 % by mass of the inoculum of a strain of Bacillus licheniformis 4/18 (B PCM/00106),

- 0.06% bentonite with an average particle size of 0.001 to 0.07 mm,

macro and microelements:

Nitrogen (N) - 23.087% by mass,

Phosphorus (P 2 0 5 ) - 1.067 % by mass,

Potassium (K 2 0) - 0.143% by mass,

Magnesium (MgO) - 0.009 % by mass,

Sulphur (S0 3 ) - 0.064 % by mass,

Manganese (Mn) - 0.009% by mass,

Zinc (Zn) - 0.009 % by mass,

and

- 0.418% by mass of citric acid,

bacteria nutrients:

- 0.093% by mass of carboxymethyl cellulose CMC,

- 0.093% by mass of Peptone K

and solvent:

- 64.83 % by mass of water. Example 3

The microbiological preparation prepared as in Example 1 , in the form of a solution, contains the following bacterial strains in the amount of 10 8 -10 9 ceils per gram of preparation: - 35 % by mass of the inoculum of a strain of Bacillus subtilis All (PCM B/00105),

- 35 % by mass of the inoculum of a strain of Bacillus licheniformis 4/18 (B PCM/00106),

- 0.06% bentonite with an average particle size of 0.001 to 0.07 mm,

and solvent:

- 29.94 % by mass of water. Example 4

The microbiological preparation prepared as in Example 1 , in the form of a solution, contains the following bacterial strains in the amount of 10 9 cells per gram of preparation:

- 75% by mass of the inoculum of a strain of Bacillus subtilis All (PCM B/00105),

- 0.06% bentonite with an average particle size of 0.001 to 0.07 mm

and solvent:

- 24.94 % by mass of water. Example 5

The microbiological preparation prepared as in Example 1 , in the form of a solution, contains strains of Bacillus subtilis All (PCM B/00105) and Bacillus licheniformis 4/18 (B PCM/00106), in a ratio of 1 : 1 in the amount of 10 8 -10 9 cells per gram of preparation used in the cultivation of rape on crop residues in amount of preparation of 1 L/ha and 2L/ha, after having been diluted in 300L of water. The increase in root weight after 7 weeks from the application in comparison to the controls and bacterial preparations by other manufacturers available are shown in the tables below, where Table 5 shows the dry weight of the above-ground part of rape at the dates of sampling, and Table 6, the dry weight of the roots of rape at the dates of sampling. Table 5

Example 6

Autoclaved soil was placed in flowerpots. Then, cut wheat straw was placed in each pot and sprayed with the microbiological preparation, prepared according to the method described in Example 1 , containing the strains of Bacillus subtilis 4/7 (PCM B/00105) and Bacillus licheniformis 4/18 (B PCM/00106), in the proportion of 1 :1 in the amount of 10 8 -10 9 cells per gram of the preparation in an amount corresponding to a dose of 2 l/ha. The straw was then mixed with the soil and left to stand for 8 weeks in a plastic tunnel, in a bright and sunny location. After 8 weeks, the soil and straw in each pot were re-mixed, and then winter wheat of the Bamberka variety was sowed, in the amount of 100 grains of wheat per pot. 4 weeks after sowing, the plants from each pot were collected and of the whole plants and the above-ground parts were weighed.

The root weight increased by 30% in comparison to the control after 4 weeks from sowing.

The weight of the above-ground part increased by 12% in comparison to the control after 4 weeks from sowing.

Example 7

The microbiological preparation prepared as in Example 1 , in the form of a solution, contains strains of Bacillus subtilis 4/7 (PCM B/00105) and Bacillus licheniformis 4/18 (B PCM/00106), in a ratio of 1 : 1 in the amount of 10 8 -10 9 cells per gram of preparation were applied on crop residues in the amount of 2 L/ha preparation, after being diluted in 300 L of water. Approximately 2 months after the application, soil samples were collected and the content of selected elements was analysed.

Copper content increased by 5.91 % in comparison to the control approximately 2 months after the application of the preparation.

Iron content increased by 6.4% in comparison to the control approximately 2 months after the application of the preparation.

Manganese content increased by 8.78% in comparison to the control approximately 2 months after the application of the preparation.

Zinc content increased by 11.39% in comparison to the control approximately 2 months after the application of the preparation.

Potassium content increased by 6.979% in comparison to the control approximately 2 months after the application of the preparation. Example 8

The liquid microbial preparation, prepared as in Example 1 , contains the following bacterial strains in the amount of 10 8 -10 9 cells per gram of preparation:

- 5 % by mass of the inoculum of a strain of Bacillus subtilis 4/7 (PCM B/00105),

- 5 % by mass of the inoculum of a strain of Bacillus licheniformis 4/18 (B PCM/00106),

- 90% of ma!todextrin.

The preparation was fed to a milking cow with a weight of 350 kg with feed, at a dose of 0.001 g of preparation per kilogram of body weight per day, over 90 days. After this period, a weight gain of 5.5% more was observed than in cows that were not fed the microbiological preparation and no gastro-intestinal troubles were recorded throughout the duration of the experiment.

Example 9

The microbiological preparation, prepared as in Example 1 was subjected to freeze-drying. The preparation in the form of a lyophilisate (free flowing powder) contains the following bacterial strains in the amount of 10 8 -10 9 cells per gram of preparation:

- 55 % by mass of the inoculum of a strain of Bacillus subtilis 4/7 (PCM B/00105),

- 35 % by mass of the inoculum of a strain of Bacillus licheniformis 4/18 (B PCM/00106),

- 10 % by mass of the inoculum of a strain of Enterococcus faecium NCIMB 11181.

The preparation was added to pre-weighed portions of standard feed containing no other antibiotics or probiotics, in the amount of 100 grams per 1 ton of feed. It was then fed to a group of 50 broilers, the birds' weight gain was measured, and simultaneously, tests were conducted on a control group of the same size which was fed the feed without the preparation. After 42 days of tests, the mean weight gain of broilers fed a diet containing the microbial preparation was measured at 1 .81 kg, while in the control group, it was 1.77 kg.

Example 10

The microbiological preparation, prepared as in Example 1 was subjected to freeze-drying. The preparation in the form of a lyophilisate (free flowing powder) contains the following bacterial strains in the amount of 0 8 -10 9 cells per gram of preparation:

- 35 % by mass of the inoculum of a strain of Bacillus subtilis 417 (PCM B/00105),

- 35 % by mass of the inoculum of a strain of Bacillus licheniformis 4/18 (B PCM/00106),

- 0.06% Lactobacillus fermentum,

and additionally

- 29.94 % by mass of dextrose (solvent).

The preparation was added during the process of preparing the feed for granulation in the standard manner, in the amount of 100 grams per 1 ton of the granulate. The feed with the addition of the microbiological preparation and without it was used in an experiment using 50 pigs with a weight of 25 kg each, which were divided into two groups of the same size. One group was fed feed with the addition of the microbiological preparation, and the other-without it. Tests was carried out until the pigs reached the weight of 40 kg. It turned out that the pigs which were fed the feed containing the microbiological preparation gained weight faster - on average, by 0.6%, and they were healthier and stronger than those which received feed without the addition of microbiological preparation.