Dried biological compositions and methods thereof

Abstract

To provide dried and stable biological compositions, with high colony- forming units, and methods of making and using the same.SOLUTION: A dried biological composition comprises (1) a substrate and (2) micro-organisms loaded onto the surface of the substrate, wherein the composition has a total moisture content of about 0.01 wt.% to about 15 wt.%.SELECTED DRAWING: None

Description

[Technical Field]

[0001] FIELD OF THE DISCLOSURE The present disclosure relates generally to dry, stable biological compositions having large colony forming units, and methods of making and using the same. [Background technology]

[0002] Background of the Invention Microbial insecticides, herbicides, fungicides, and growth promoters formulated with beneficial viruses, bacteria, yeasts, and fungi to target specific insect or plant species are gaining increasing interest in the agricultural sector due to their minimal impact on non-target species and the environment. However, maintaining the viability of these products is typically a challenge during storage and formulation. Currently, microbial pesticide products may be manufactured as liquid or dry formulations. Liquid formulations typically contain suspensions of these microorganisms in water, oil, or emulsion to maintain viability and efficacy. However, these liquid formulations must be stored and transported at low temperatures, which is often cumbersome and cost-inefficient. On the other hand, dry formulations typically involve formulating microorganisms into wettable powders, granules, prilled, coated, or crystallized forms for easier storage and transportation. However, when formulated into dry forms for easier handling, these microorganisms face cell death and stability issues due to the drying heat of the formulation process.

[0003] U.S. Patent No. 8,409,822 (Trevino et al.) discloses and claims a composition for delivering microorganisms in a dry state, comprising precipitated silica granules having a porous structure and microorganisms loaded throughout the pores of the precipitated silica granules, which can function to allow the growth of microorganisms within the pores of the precipitated silica granules. Similarly, U.S. Patent No. 9,296,989 (Trevino et al.) discloses and claims a composition for delivering live cells in a dry state, comprising an inert carrier substrate having pores, live cells loaded within the pores of the inert carrier substrate, and a surface layer disposed on the outer surface of the inert carrier substrate on which the live cells are loaded, the surface layer being permeable to molecules that support cell growth of the live cells, such that the composition can function to allow increased growth of live cells within the inert carrier substrate compared to an alternative composition lacking the surface layer. Although the composition of Trevino et al. is disclosed as being "dry," it is not actually dried, as it is disclosed that a liquid containing live microorganisms is substantially loaded within the pores of the precipitated silica granules. Trevino et al.'s silica acts as an absorbent and is loaded with 25-75% viable microorganisms. At this level of loading, the loaded silica is free-flowing, defined as dry to the touch. These compositions have relatively limited utility because the microbial concentration and water content in the silica are not optimized, and the microorganisms can still respire, leading to a rapid loss of activity.

[0004] Various protective agents, such as sulfoxides, alcohols, monosaccharides, polysaccharides, amino acids, peptides, glycoproteins, and other additives, have been used to protect microorganisms from dehydration damage. U.S. Patent No. 5,360,607 (Eyal et al.) discloses and claims an improved, stable, dry-prilled, biological insecticide composition, comprising an inert carrier capable of supporting fungal growth and promoting conidiosporulation, and a live insect biomass produced by submerged fermentation of an isolate of the fungus Paecilomyces fumosoroseus. However, this method reduces the moisture content (e.g., water activity (A)) that microorganisms depend on for survival and respiration. w Alginate is used to encapsulate intact prills, which are susceptible to changes in saturation levels. [Prior art documents] [Patent documents]

[0005] [Patent Document 1] U.S. Patent No. 8,409,822 [Patent Document 2] U.S. Patent No. 9,296,989 [Patent Document 3] U.S. Patent No. 5,360,607 Summary of the Invention [Problem to be solved by the invention]

[0006] There remains an unmet need in the art for highly concentrated preparations of microorganisms in a dry, stable form. [Means for solving the problem]

[0007] Summary of the Invention The present inventors have surprisingly discovered that by incorporating microorganisms, such as mold spores and bacteria, and drying them on the surface of various substrates at specific temperatures, dried biological compositions can be obtained that have improved viability and greater concentrations or colony forming units ("CFU") than those of the prior art. To achieve the desired CFU levels in these dried biological compositions (concentrated dried biological compositions), the present invention defines multiple interrelated parameters to create an optimal environment for the attachment of microorganisms without sacrificing viability. First, the substrate is made of a group of porous particles, e.g., 10-400 m. 2 The microorganisms are then selected from sedimented particles having a BET surface area of ​​about 0.01% to about 15% by weight on such a substrate. Since water activity depends on both the total amount of water and the relative availability of water, as controlled by the specific substrate, achieving the total water concentration target in combination with the specific substrate parameters will result in a defined water activity (A w ) is formed. The ability to customize the ideal surface water activity allows biological materials to be stabilized in a desired dormant state, represented by a small change in colony forming units ("CFUs") over time.

[0008] Thus, in a first aspect, the present invention provides a dry biological composition (Composition I) comprising, in certain embodiments consisting essentially of, or in other particular embodiments consisting of, (i) a substrate and (ii) a microorganism supported on said substrate, said composition having a total moisture content of about 0.01% to about 15% by weight. Surprisingly, it has been discovered that microorganisms can be made to survive on certain substrates at high concentrations and with excellent viability in a dormant state caused by the resulting surface water activity level. Preferably, in a first aspect, the present invention provides Composition I as follows: 1.1 Composition I having a total water content of about 0.01% to about 8% by weight; 1.2 Composition I or the composition of 1.1 having a total moisture content selected from about 3% to about 8% by weight, preferably about 5% to about 8% by weight, and more preferably 3%, about 5%, and 7% by weight; 1.3 A water activity value (A) of about 0.01 to about 0.6, preferably about 0.2 to about 0.6, and more preferably about 0.3 to about 0.5 w ) composition I or the composition of 1.1 or 1.2; 1.4 about 10 7 More than 10 CFU / g, preferably about 10 8 CFU / g or more, more preferably about 10 9 CFU / g or more, more preferably about 10 10 CFU / g or more, more preferably about 10 11 CFU / g or more, more preferably about 10 12 Composition I or any of compositions 1.1 to 1.3 having CFU / g or more; 1.5 Composition I or any of compositions 1.1-1.4, wherein the substrate is selected from the group consisting of silica (e.g., precipitated silica, and in certain embodiments, hydrophilic silica, e.g., SIPERNAT® 22 silica), diatomaceous earth, silica gel, silicates (e.g., aluminosilicates or clays such as ZEOLEX® 301), and water-insoluble natural fiber-based materials such as cellulose; 1.6 Composition I or any of compositions 1.1-1.5, wherein the substrate is silica; 1.7 Composition I or any of compositions 1.1-1.6, wherein the substrate is precipitated silica; 1.8 Composition I or any of compositions 1.1-1.7, wherein the substrate is a hydrophilic silica, such as SIPERNAT® 22 silica; 1.9 Composition I or any of compositions 1.1-1.5, wherein the substrate is a water-insoluble natural fiber-based material such as cellulose; 1.10 Composition I or any of compositions 1.1-1.5, wherein the substrate is diatomaceous earth; 1.11 Composition I or any of compositions 1.1 to 1.5, wherein the substrate is silica gel; 1.12 Composition I or any of 1.1-1.5, wherein the substrate is a silicate (e.g., an aluminosilicate such as ZEOLEX® 301, or a clay); 1.13 Composition I or any of compositions 1.1-1.13, wherein the particle size (d50) of the substrate is from about 5 to 200 microns, preferably from about 8 to 160 microns, more preferably from about 9 to 150 microns, even more preferably from about 50 to 150 microns, even more preferably from about 50 to 130 microns, and even more preferably selected from the group consisting of about 50 microns, about 85 microns, and about 120 microns; 1.14 If the BET surface area of ​​the substrate is approximately 2 to 400 m 2 / g, preferably about 5 to 400 m 2 / g, more preferably about 10 to 400 m 2 / g, more preferably about 30 to 400 2 m / g, more preferably about 30 to 300 m 2 / g, more preferably about 40 to 200m 2 / g, more preferably about 180m 2 / g of composition I or any of compositions 1.1 to 1.14; 1.15 If the BET surface area of ​​the substrate is approximately 2 m 2 / g, preferably about 5m 2 / g of composition I or any of compositions 1.1 to 1.14; 1.16 If the BET surface area of ​​the substrate is approximately 180 m 2 / g of composition I or any of compositions 1.1 to 1.14; 1.17 Composition I or any of compositions 1.1-1.16, wherein the pore volume of the substrate is about 0.01-1.20 cc / g, preferably about 0.05-1.20 cc / g, more preferably about 0.10-1.0 cc / g, and even more preferably about 0.20-0.95 cc / g; 1.18 Approximately 50~200m 2 / g, preferably about 180m 2 / g BET surface area and a total moisture content of about 5% to about 8% by weight; 1.19 Approximately 50~200m 2 / g, preferably about 180m 2 / g BET surface area, and a particle size of about 5 to 200 microns, more preferably about 120 microns, and having a total moisture content of about 5% to about 8% by weight; 1.20 Composition I or any of compositions 1.1 to 1.19, wherein the final microbial concentration is about 4 to about 40% by weight, preferably about 4 to about 20% by weight, of the total composition; 1.21 The microorganism is selected from the group consisting of Bacillus subtilis QST713, Pasteuria usgae, Beauveria bassiana, Coniothyrium minitans, Chondrostereum purpureum, Paecilomyces lilacinus, Aschersonia aleyrodis, Beauveria brongniartii, Hirsutella thompsonii, Isaria fumosorosea, Isaria sp. sp., Lecanicillium longisporum, Lecanicillium muscarium, Lecanicillium sp., Metarhizium anisopliae, Metarhizium anisopliae var. acridum, Nomuraea rileyi, Sporothrix insectorum; Cydia pomonella GV; Phytophthora palmivora, Lagenidium giganteum, Bacillus thuringiensis, Pseudomonas fluorescens fluorescens, Bradyrhizobium, Mycorrhiza, Clonostachys rosea, Bacillus spp., and Lactobacillus spp.or any combination thereof, preferably selected from the group consisting of Bacillus thuringiensis, Pseudomonas fluorescens, Bradyrhizobium, Mycorrhiza, Clonostachys rosea, and any combination thereof; Composition I or any of compositions 1.1-1.20; 1.22 Composition I or any of compositions 1.1-1.21, wherein the microorganism is Clonostachys rosea, or in another embodiment Pseudomonas fluorescens; 1.23 Composition I or any of compositions 1.1-1.22, further comprising one or more excipients, in certain embodiments one or more pesticidally acceptable excipients; 1.24 Composition 1.22 in tablet form, in the form of a flowable concentrate, e.g. for seed treatment, or in the form of an oil suspension; 1.25 Composition I or any of compositions 1.1-1.24, which does not require an external protectant such as an alginate encapsulant; 1.26 Composition I or any of compositions 1.1-1.25, further comprising a polymer selected from the group consisting of polyvinyl alcohol, xanthan gum, gum arabic, other polysaccharides such as maltodextrin and guar gum (e.g., hydroxypropyl guar gum), and polyethylene glycol; 1.27 Composition I or any of compositions 1.1-1.26, further comprising a second substrate as an outer layer; 1.28 Composition 1.27, wherein the second substrate is selected from precipitated silica, such as SIPERNAT® 50S silica, or fumed silica, such as AEROSIL® 200, AEROSIL® R972, or AEROSIL® R812S silica; 1.29 The number of colony-forming units per gram of composition (CFU / g) is approximately 10 after 120 days of storage at room temperature. 7 Composition I or any of compositions 1.1-1.28, which remains above CFU / g; 1.30 The number of colony forming units per gram of composition (CFU / g) is approximately 10 after 40 days of storage at 40°C. 7 CFU / g remains above composition I or any of compositions 1.1-1.29; 1.31 The number of colony-forming units per gram of composition (CFU / g) is approximately 10 after 40 days of storage at a relative humidity of 65% or less. 7 Composition I or any of compositions 1.1-1.30, which remains above CFU / g; 1.32 Composition I or any of compositions 1.1-1.31, wherein the tap density of the composition is greater than 150% of the tap density of the pure substrate material; 1.33 Composition I or any of compositions 1.1-1.32, wherein the microorganisms are larger than the pore size of the substrate or the microorganisms are supported on the surface of the substrate; 1.34 Composition I or any of compositions 1.1-1.24 or 1.27-1.33, further comprising (i) a polymer selected from the group consisting of polyvinyl alcohol, xanthan gum, gum arabic, or other polysaccharides such as maltodextrin or guar gum (e.g., hydroxypropyl guar gum), polyethylene glycol, and polyglycerol, or (ii) a non-reducing disaccharide such as trehalose or sucrose, or (iii) skim milk or dimethyl sulfoxide; 1.35 Composition I or any of compositions 1.1-1.24 or 1.27-1.33, further comprising a non-reducing disaccharide such as trehalose or sucrose; 1.36 Composition I or any of compositions 1.1-1.24 or 1.27-1.33, further comprising a polymer such as polyglycerol, particularly a hyperbranched polyglycerol polymer; 1.37 Composition I or any of compositions 1.1-1.36, wherein the second substrate is finely divided hydrophobic or hydrophilic particles, and such particles have been surface treated, e.g., with silane or silicone oil, to modify their wettability or to modify the tendency of the sample to absorb water; 1.38 Composition I or any of compositions 1.1-1.37, wherein the second substrate is silica or clay, and the silica or clay has been surface treated, e.g., with a silane or silicone oil, to modify the wettability or water absorption tendency of the sample; 1.39 If the second substrate has a large BET surface area, e.g., 50-750 m 2 / g, in certain embodiments, 50 to 380 m 2 / g BET surface area of ​​composition I or any of compositions 1.1 to 1.38; 1.40 Composition I or any of compositions 1.1-1.39, wherein the second substrate is hydrophobic silica; 1.41 Composition I or any of compositions 1.1 through 1.40, wherein the second substrate is precipitated silica; 1.42 The second substrate has a large BET surface area, e.g., 50–750 m 2 / g, in certain embodiments, 50 to 380 m 2 Composition I or any of compositions 1.1-1.41, wherein the composition is a precipitated silica having a BET surface area of ​​1.1 / g; 1.43 Composition I or any of compositions 1.1-1.42, wherein the second substrate is SIPERNAT® 50 or ZEOFREE® silica, and in certain embodiments SIPERNAT® 50 silica; 1.44 Composition I or any of compositions 1.1 through 1.40, wherein the second substrate is fumed silica; 1.45 Composition I or any of compositions 1.1-1.44, wherein the second substrate is fumed silica; 1.46 Composition I or any of compositions 1.1-1.44, wherein the second substrate is hydrophobic fumed silica; 1.47 The second substrate is 180-220 m 2 Composition I or any of compositions 1.1-1.44, which is a hydrophobic fumed silica such as AROSIL® R202 silica having a BET surface area of ​​1.0 / g and a carbon content of 3.5-5%; 1.48 The first substrate is 400 to 600 m 2 / g, preferably 500m 2 Composition I or any of compositions 1.1-1.6, 1.13, 1.17, or 1.20-1.47, wherein the composition is silica having a BET surface area of ​​1.1 / g; 1.49 Composition 1.49, wherein the silica has a pore volume by Barrett-Joyner-Halenda model greater than 1 cc / g, preferably greater than 1.4 cc / g, or a pore volume by mercury pore volume greater than 2 cc / g, preferably greater than 2.2 cc / g; 1.50 Composition 1.49, wherein the silica is SIPERNAT® 50 silica;

[0009] In a second aspect, the present invention provides a method for producing a dry biological composition having a total moisture content of about 0.01% to about 15% by weight, comprising, in certain embodiments consisting essentially of, and in other specific embodiments consisting of, a substrate and a microorganism supported on the substrate, the method comprising: (1) combining a mixture, solution, or suspension containing the microorganism with the substrate; and (2) drying the substrate-microorganism mixture to reach a total moisture content of about 0.01% to about 15% by weight (Method I). Preferably, the present invention provides Method I as follows: 2.1 Method I, in which microorganisms are harvested from the surface of seeds by mechanically crushing or grinding the surface of the seeds (step (a)), to obtain a fine fraction containing the microorganisms, preferably fungal spores, and a plurality of seed portions. Preferably, the yield of microorganisms in the fine fraction is 10% or more per gram of seed that is initially crushed or ground. 9cfu. Further preferably, the method comprises sieving the obtained fine fraction (step (b)) to obtain a powder with a defined particle size distribution for subsequent processing steps. Preferably, the powder is used to prepare a microbial mixture, solution or suspension (step (c)); 2.2 Method 2.1, in which step (a) comprises grinding with a grinding stone to separate the seeds from the fine fraction; 2.3 Method 2.1, wherein step (a) comprises crushing under pressure with a rotating shaft in a sealed tube of a slotted screen, followed by separation of the seeds from the fine fraction by sieving and filtering; 2.4 Method I or any of methods 2.1 to 2.3, wherein the step (b) of sieving the fine fraction comprises sieving through a sieve mesh size of 20 to 800 μm, preferably 100 μm to 300 μm; 2.5 Method I, in which microorganisms are harvested from the surface of seeds by rinsing the seed surface with water and separating the seeds from a liquid microbial solution or suspension. Preferably, the seeds are stirred in water for 1 to 20 minutes. More preferably, solid-liquid separation is performed in a pressure Nutze filter, more preferably using a mesh size of 1 to 3 mm in the pressure Nutze filter. More preferably, the dewatering time in the pressure Nutze filter is 20 to 200 seconds. More preferably, the filtration pressure in the Nutze filter is 1 to 3 bar. More preferably, the microbial solution or suspension is concentrated by separating the microorganisms from the liquid in a centrifugal field. More preferably, the concentration step includes separation in a disc stack separator. More preferably, the concentration step again involves diluting the concentrate with water and subsequent second concentration in a centrifugal field to separate the soluble portion from the microorganisms; 2.6 Method I or any of methods 2.1-2.5, wherein step (2) dries the substrate-microorganism mixture to a total moisture content selected from about 0.01% to about 15% by weight, preferably about 0.01% to about 8% by weight, more preferably about 3% to about 8% by weight, even more preferably about 5% to about 8% by weight, and even more preferably 3%, 5%, and 7% by weight; 2.7 Method I or any of methods 2.1 through 2.6, wherein the drying step (2) comprises fluidized bed drying of the substrate-microorganism mixture; 2.8 Method I or any of methods 2.1 through 2.6, wherein the drying step (2) comprises spray drying the substrate-microorganism mixture; 2.9 Method I or any of methods 2.1 through 2.6, wherein the drying step (2) comprises contact drying the substrate-microorganism mixture; 2.10 Method I or any of methods 2.1-2.6, wherein the drying step (2) comprises freeze-drying the substrate-microorganism mixture; 2.11 Method I or any of methods 2.1 to 2.10, wherein the temperature of the dry air is about 130°C or less, preferably about 90°C or less, more preferably about 80°C or less, even more preferably about 50°C or less, even more preferably about 30°C to 50°C, even more preferably about 40°C to 50°C, even more preferably about 40°C to 45°C, even more preferably about 43°C; 2.12 Method I or any of methods 2.1-2.11, wherein the powder bed is maintained at about 35°C or less, preferably about 30°C or less, more preferably about 25°C-35°C; 2.13 Method I or any of methods 2.1 through 2.7, in which the spray volume is approximately 2 mL per 1 g of substrate; 2.14 The resulting composition has a water activity (A) of about 0.01 to about 0.6, preferably about 0.2 to about 0.6, and more preferably about 0.3 to about 0.5. w ) Method I or any of methods 2.1 to 2.13; 2.15 If the resulting composition is, for example, about 10 7 CFU / g, preferably greater than 10 8 Colony forming units per gram (CFU / g) or more, preferably about 10 9 CFU / g or more, more preferably about 10 10 CFU / g or more, more preferably about 10 11 CFU / g or more, more preferably about 10 12Method I or any of methods 2.1 through 2.14, having a microbial colony forming units per gram of composition (CFU / g) equal to or greater than CFU / g; 2.16 Method I or any of methods 2.1-2.15, wherein the substrate is selected from the group consisting of silica (e.g., precipitated silica, and in certain embodiments, hydrophilic silica, e.g., SIPERNAT® 22 silica), diatomaceous earth, silica gel, silicates (e.g., aluminosilicates or clays such as ZEOLEX® 301), and water-insoluble natural fiber-based materials such as cellulose; 2.17 Method I or any of methods 2.1 through 2.16, wherein the substrate is silica; 2.18 Method I or any of methods 2.1 through 2.17, wherein the substrate is precipitated silica; 2.19 Method I or any of methods 2.1-2.18, wherein the substrate is a hydrophilic silica, such as SIPERNAT® 22 silica; 2.20 Method I or any of methods 2.1 through 2.16, wherein the substrate is a water-insoluble natural fiber-based material such as cellulose; 2.21 Method I or any of methods 2.1 through 2.16, wherein the substrate is diatomaceous earth; 2.22 Method I or any of methods 2.1 through 2.16, wherein the substrate is silica gel; 2.23 Method I or any of methods 2.1-2.16, wherein the substrate is a silicate (e.g., an aluminosilicate such as ZEOLEX® 301, or a clay); 2.24 Method I or any of methods 2.1-2.23, wherein the particle size (d50) of the substrate is about 5-200 microns, preferably about 8-160 microns, more preferably about 9-150 microns, even more preferably about 50-150 microns, even more preferably about 50-130 microns, and even more preferably selected from the group consisting of about 50 microns, about 85 microns, and about 120 microns; 2.25 The BET surface area of ​​the substrate is approximately 2 to 400 m 2 / g, preferably about 5 to 400 m 2 / g, more preferably about 10 to 400 m 2 / g, more preferably about 30 to 400m 2 / g, more preferably about 30 to 300m 2 / g, more preferably about 40 to 200m 2 / g, more preferably about 180m 2 / g, Method I or any of Methods 2.1 to 2.24; 2.26 If the BET surface area of ​​the substrate is approximately 2 m 2 / g, preferably about 5m 2 / g, Method I or any of Methods 2.1 to 2.25; 2.27 If the BET surface area of ​​the substrate is approximately 180 m 2 / g, Method I or any of Methods 2.1 to 2.25; 2.28 Method I or any of methods 2.1 through 2.27, wherein the pore volume of the substrate is about 0.01 to 1.20 cc / g, preferably about 0.05 to 1.20 cc / g, more preferably about 0.10 to 1.0 cc / g, and even more preferably about 0.20 to 0.95 cc / g; 2.29 The composition is about 50 to 200 m 2 / g, preferably about 180m 2 / g BET surface area and a total moisture content of about 5% to about 8% by weight; 2.30 The composition is about 50 to 200 m 2 / g, preferably about 180m 2 / g BET surface area, and a particle size of about 5 to 200 microns, more preferably about 120 microns, and having a total moisture content of about 5% to about 8% by weight; 2.31 Method I or any of methods 2.1 to 2.30, wherein step (1) comprises loading from about 4 to about 40% by weight, preferably from about 4 to about 20% by weight, of the total composition; 2.32 Method I or any of methods 2.1-2.31, wherein step (1) further comprises adding a polymer selected from the group consisting of polyvinyl alcohol, xanthan gum, gum arabic, and other polysaccharides such as maltodextrin and guar gum (e.g., hydroxypropyl guar gum), and polyethylene glycol; 2.33 Method I or any of methods 2.1-2.32, wherein step (1) further comprises a second substrate as an outer layer; 2.34 Method 2.33, wherein the second substrate is selected from precipitated silica, such as SIPERNAT® 50S silica, or fumed silica, such as AEROSIL® 200, AEROSIL® R972, or AEROSIL® R812S silica. 2.35 The microorganisms are Bacillus subtilis QST713, Pasteuria usgae, Beauveria bassiana, Coniothyrium minitans, Chondrostereum purpureum, Paecilomyces lilacinus, Aschersonia aleyrodis, Beauveria brongniartii, Hirsutella thompsonii, Isaria fumosorosea, Isaria sp. sp., Lecanicillium longisporum, Lecanicillium muscarium, Lecanicillium sp., Metarhizium anisopliae, Metarhizium anisopliae var. acridum, Nomuraea rileyi, Sporothrix insectorum; Cydia pomonella GV; Phytophthora palmivora, Lagenidium giganteum, Bacillus thuringiensis, Pseudomonas fluorescens fluorescens, Bradyrhizobium, Mycorrhiza, Clonostachys rosea, Bacillus spp., and Lactobacillus spp.), or any combination thereof, selected from the group consisting of method I or any of methods 2.1 to 2.34;. 2.36 Method I or any of methods 2.1 through 2.34, wherein the microorganism is selected from the group consisting of Bacillus thuringiensis, Pseudomonas fluorescens, Bradyrhizobium, Mycorrhiza, and Clonostachys rosea; 2.37 Method I or any of methods 2.1 through 2.34, wherein the microorganism is Clonostachys rosea; 2.38 Method I or any of methods 2.1-2.34, wherein the resulting composition does not require an external protectant, such as an alginate encapsulant; 2.39 The number of colony-forming units per gram of composition (CFU / g) is approximately 10 after 120 days of storage at room temperature. 7 CFU / g remains above Method I or any of the methods 2.1-2.38; 2.40 The number of colony-forming units per gram of composition (CFU / g) is approximately 10 after 40 days of storage at 40°C. 7 CFU / g remains above Method I or any of the methods 2.1-2.39; 2.41 The number of colony-forming units per gram of composition (CFU / g) is approximately 10 after 40 days of storage at a relative humidity of 65% or less. 7 CFU / g remains above Method I or any of Methods 2.1-2.40; 2.42 Method I or any of methods 2.1 through 2.41, wherein the tap density of the composition is greater than 150% of the tap density of the pure substrate material; 2.43 Method I or any of methods 2.1-2.31 or 2.33-2.37 or 2.39-2.42, wherein the composition further comprises (i) a polymer selected from the group consisting of polyvinyl alcohol, xanthan gum, gum arabic, or other polysaccharides such as maltodextrin or guar gum (e.g., hydroxypropyl guar gum), polyethylene glycol, and polyglycerol, or (ii) a non-reducing disaccharide such as trehalose or sucrose, or (iii) skim milk or dimethyl sulfoxide; 2.44 Method I or any of methods 2.1-2.31 or 2.33-2.37 or 2.39-2.42, wherein the composition further comprises a non-reducing disaccharide, such as trehalose or sucrose; 2.45 Method I or any of methods 2.1-2.31 or 2.33-2.37 or 2.39-2.42, wherein the composition further comprises a polymer such as a polyglycerol, particularly a hyperbranched polyglycerol polymer; 2.46 Method I or any of methods 2.1-2.33 or 2.35-2.45, wherein the second substrate is finely divided hydrophobic or hydrophilic particles, and such particles have been surface treated, e.g., with silane or silicone oil, to modify their wettability or to modify the tendency of the sample to absorb water; 2.47 Method I or any of methods 2.1-2.33 or 2.35-2.45, wherein the second substrate is silica or clay, and such silica or clay has been surface treated, e.g., with a silane or silicone oil, to modify its wettability or to modify the water absorption tendency of the sample; 2.48 The second substrate has a high BET surface area, e.g., 50 to 750 m 2 / g, in certain embodiments, 50 to 380 m 2 / g BET surface area of ​​Method I or any of Methods 2.1-2.33 or 2.35-2.45; 2.49 Method I or any of methods 2.1-2.33 or 2.35-2.48, wherein the second substrate is hydrophobic silica; 2.50 Method I or any of methods 2.1-2.33 or 2.35-2.49, wherein the second substrate is precipitated silica; 2.51 The second substrate has a large BET surface area, e.g., 50–750 m 2 / g, in certain embodiments, 50 to 380 m 2 / g BET surface area of ​​precipitated silica, Method I or any of Methods 2.1-2.33 or 2.35-2.50; 2.52 Method I or any of methods 2.1-2.33 or 2.35-2.50, wherein the second substrate is SIPERNAT® 50 or ZEOFREE® silica, and in one particular embodiment SIPERNAT® 50 silica; 2.53 The second substrate is 180-220 m 2 Method I or any of methods 2.1-2.33 or 2.35-2.45, wherein the silica is a hydrophobic fumed silica such as AROSIL® R202 silica having a BET surface area of ​​1 / g and a carbon content of 3.5-5%; 2.54 Method I or any of methods 2.1-2.33 or 2.35-2.45, wherein the second substrate is AROSIL® R202 silica; 2.55 The BET surface area of ​​the first substrate is 400 to 600 m 2 / g, preferably 500m 2 / g, Method I or any of the methods 2.1 to 2.18, 2.24, 2.28, or 2.31 to 2.54; 2.56 Method 2.55, wherein the substrate has a pore volume by Barrett-Joyner-Halenda model of greater than 1 cc / g, preferably greater than 1.4 cc / g, or a pore volume by mercury pore volumetric model of greater than 2 cc / g, preferably greater than 2.2 cc / g; 2.57 Method I or any of methods 2.33-2.56, wherein a second substrate is added to the microbial suspension prior to said drying step (2); 2.58 Method I or any of methods 2.33-2.56, wherein a second substrate is added to the microbial suspension during the drying step (2); 2.59 Method I or any of methods 2.33-2.56, wherein a second substrate is added to the microbial suspension after the drying step (2); 2.60 Method I or any of methods 2.33-2.59, wherein a polymer or polysaccharide or non-reducing disaccharide is added to the microbial suspension prior to said drying step (2); 2.61 Method I or any of methods 2.33-2.59, wherein a polymer or polysaccharide or non-reducing disaccharide is added to the microbial suspension during the drying step (2); 2.62 Method I or any of methods 2.33-2.59, wherein a polymer or polysaccharide or non-reducing disaccharide is added to the microbial suspension after the drying step (2); 2.63 Method I or any of the methods described above, wherein microorganisms are harvested from the surface of the seeds by mechanically crushing or grinding the surface of the substrate (step (a)), to obtain a fine fraction containing the microorganisms, preferably fungal spores, and a plurality of seed portions. Preferably, the yield of microorganisms in the fine fraction is greater than 10 per gram of seed that is initially crushed or ground. 9 cfu. Further preferably, the method comprises sieving the obtained fine fraction (step (b)) to obtain a powder with a defined particle size distribution for subsequent processing steps. Further preferably, the powder is used to prepare a microbial mixture, solution or suspension (step (c)); 2.64 Method 2.63, in which step (a) includes grinding the seeds with a grinding stone to separate them from the fine fraction; 2.65 Method 2.63, in which step (a) involves crushing under pressure with a rotating shaft in a sealed tube of a slotted screen, followed by separation of the seeds from the fine fraction by sieving and filtering; 2.66 Method 2.63, in which the sieving step (b) of the fine fraction comprises sieving through a sieve mesh size of 20 to 800 μm, preferably 100 μm to 300 μm; 2.67 Methods 2.1 to 2.63, in which microorganisms are harvested from the surface of seeds by rinsing the surface with water and separating the seeds from a liquid microbial solution or suspension. Preferably, the seeds are stirred in water for 1 to 20 minutes. More preferably, solid-liquid separation is performed in a pressure Nutze filter. Preferably, a mesh size of 1 to 3 mm is used in the pressure Nutze filter. More preferably, the dewatering time in the pressure Nutze filter is 20 to 200 seconds. More preferably, the filtration pressure in the Nutze filter is 1 to 3 bar. More preferably, the microbial solution or suspension is concentrated by separating the microorganisms from the liquid in a centrifugal field. More preferably, the concentration step includes separation in a disc stack separator. More preferably, the concentration step again includes dilution of the concentrate with water and subsequent second concentration in a centrifugal field to separate the soluble portion from the microorganisms; 2.68 Method I or any of the foregoing methods, wherein the drying step (2) comprises fluidized-bed drying the substrate-microorganism mixture; 2.69 Method I or any of the foregoing methods, wherein the drying step (2) comprises spray-drying the substrate-microorganism mixture; 2.70 Method I or any of the foregoing methods, wherein the drying step (2) comprises contact drying the substrate-microorganism mixture; 2.71 Method I or any of the foregoing methods, wherein the drying step (2) comprises freeze-drying the substrate-microorganism mixture; 2.72 Method I or any of the foregoing methods, wherein the temperature of the drying air is about 130°C or less, and in certain embodiments about 90°C or less, preferably about 80°C or less, more preferably about 50°C or less, even more preferably about 30°C to 50°C, even more preferably about 40°C to 50°C, even more preferably about 40°C to 45°C, and even more preferably about 43°C; 2.73 Method I or any of the foregoing methods, wherein the powder bed is maintained at about 35°C or less, preferably about 25°C to 35°C; 2.74 The resulting composition has a water activity (A) of about 0.01 to about 0.6, preferably about 0.2 to about 0.6, and more preferably about 0.3 to about 0.5. wMethod I or any of the methods described above, 2.75 If the resulting composition is, for example, about 10 7 CFU / g, preferably greater than 10 8 Colony forming units per gram (CFU / g) or more, preferably about 10 9 CFU / g or more, more preferably about 10 10 CFU / g or more, more preferably about 10 11 CFU / g or more, more preferably about 10 12 Method I or any of the foregoing methods, having a microbial colony forming units per gram of composition (CFU / g) of equal to or greater than CFU / g; 2.76 Method I or any of the aforementioned methods, wherein the temperature of the dry air is about 130°C or less, specifically about 90°C or less, preferably about 80°C or less, more preferably about 50°C or less, even more preferably about 30°C to 50°C, even more preferably about 40°C to 50°C, even more preferably about 40°C to 45°C, and even more preferably about 43°C; 2.77 Method I or any of the foregoing methods, wherein the powder bed is maintained at about 35°C or below, preferably about 25°C to 35°C; 2.78 The resulting composition has a water activity (A) of about 0.01 to about 0.6, preferably about 0.2 to about 0.6, and more preferably about 0.3 to about 0.5. w Method I or any of the methods described above, 2.79 If the resulting composition is, for example, about 10 7 CFU / g, preferably greater than 10 8 Colony forming units per gram (CFU / g) or more, preferably about 10 9 CFU / g or more, more preferably about 10 10 CFU / g or more, more preferably about 10 11 CFU / g or more, more preferably about 10 12 Method I or any of the foregoing methods, having a microbial colony forming units per gram of composition (CFU / g) of equal to or greater than CFU / g;

[0010] In a third aspect, the present invention provides a dry biological composition (Composition II') prepared by any of Methods I or 2.1-2.79 of the present invention. In another embodiment of the third aspect, the present invention provides a dry biological composition (Composition II-A) prepared by any of Methods I or 2.1-2.42 of the present invention. In yet another embodiment of the third aspect, the present invention provides a dry biological composition (Composition II-B) prepared by any of Methods I or 2.43-2.79 of the present invention.

[0011] The compositions of the present invention are useful for application to seeds to protect them from pests or to provide biostimulatory functions, such as releasing phosphorus or supplying nitrogen, to microorganisms. Thus, in a fourth aspect, the present invention provides Composition I or any of 1.1-1.50, or Composition II' or any of 2.1-2.79, further comprising, or in certain embodiments consisting essentially of, or in other embodiments consisting of seeds to be treated (Composition III'). In yet another embodiment of the fourth aspect, the present invention provides Composition I or any of 1.1-1.33, or Composition II-A, further comprising, or in certain embodiments consisting essentially of, or in other embodiments consisting of seeds to be treated (Composition III-A). In yet another embodiment of the fourth aspect, the present invention provides Composition I or any of 1.34-1.50, or Composition II-B, further comprising, or in certain embodiments consisting essentially of, or in other embodiments consisting of seeds to be treated (Composition III-B). These compositions may optionally contain a colorant.

[0012] In a fifth aspect, the present invention provides a method for controlling insects, fungi, or nematodes on a treated area, comprising optionally rehydrating a concentrated, dry biological composition of the present invention (i.e., Composition I or any of 1.1-1.50, or Composition II' or any of 2.1-2.79, or any of Composition III'), and applying an effective amount of the concentrated, dry biological composition of the present invention (optionally rehydrated) to the area affected by the treatment. In a further embodiment of the fifth aspect, the present invention provides a method for controlling insects, fungi, or nematodes on a treated area, comprising optionally rehydrating a concentrated, dry biological composition of the present invention (i.e., Composition I or any of 1.1-1.33, or Composition II-A, or Composition III-A), and applying an effective amount of the concentrated, dry biological composition of the present invention (optionally rehydrated) to the area affected by the treatment. In yet another embodiment of the fifth aspect, the present invention provides a method for controlling insects, fungi, or nematodes on a treated area, comprising optionally rehydrating a concentrated, dry biological composition of the present invention (i.e., Composition I or any of 1.34-1.50, Composition II-B or any of 2.43-2.79, or Composition III-B), and applying an effective amount of the (optionally rehydrated) concentrated, dry biological composition of the present invention to the affected area. In one embodiment, the treated area is a part of a plant, such as, but not limited to, plant cuttings, plant roots, bulbs, tubers, stems, fruits, flowers, and / or leaves, including, for example, corn, wheat, sorghum, soybeans, citrus and non-citrus fruits, nut trees, and the like. In another embodiment, the treated area is soil or seeds, or a mixture thereof. [Brief explanation of the drawings]

[0013] [Figure 1] FIG. 1 is a plot of log CFU over time for Examples 12-23 stored at 40° C. Samples are labeled with additives. [Figure 2]Figure 1 shows the decimal decay times in weeks for Examples 12-23 stored at 40° C. Error bars are the standard error of the regression. [Figure 3] FIG. 1 shows the decrease in log(CFU) over time for Examples 12 to 23 stored at high humidity. [Figure 4] Decimal decay times for each sample stored at high humidity are shown. Error bars represent the standard error of the regression. DETAILED DESCRIPTION OF THE INVENTION

[0014] Detailed Description The present invention provides a system for delivering microorganisms (e.g., microbial pesticides such as mold spores and other bacteria) in a dry, stable form and at higher CFUs than those of the prior art. It has been found that, for example, using the methods disclosed herein, microorganisms can be dried on certain substrates to a target total moisture concentration of about 0.01% to about 15% by weight to form a suitable surface for the biological material to become dormant. Exemplary substrates useful in the present invention include, but are not limited to, silica, in certain embodiments, precipitated silica, and in yet other specific embodiments, hydrophilic silica, in certain embodiments, SIPERNAT® 22 silica. Other exemplary substrates include diatomaceous earth, silica gel, silicates (e.g., aluminosilicates such as ZEOLEX® 301, or clays), and water-insoluble natural fiber-based materials such as cellulose. In yet other embodiments, the substrate is 400-600 m 2 / g, preferably 500m 2 In a further embodiment, the silica has a pore volume by Barrett-Joyner-Halenda model of greater than 1 cc / g, preferably greater than 1.4 cc / g, or a pore volume by mercury pore volume of greater than 2 cc / g, preferably greater than 2.2 cc / g, and is preferably SIPERNAT® 50 silica.

[0015] Typical particle sizes of the substrate of the compositions of the present invention may have a d50 selected from the group consisting of about 5-200 microns, preferably about 8-160 microns, preferably about 9-150 microns, more preferably about 50-150 microns, more preferably about 50-130 microns, and even more preferably about 50 microns, about 85 microns, and about 120 microns. The particle size of the silica can be measured by any method known to those skilled in the art, such as, for example, dry particle size analysis using laser light scattering or scanning electron microscope (SEM) analysis.

[0016] Typical BET surface areas of the substrates of the compositions of the present invention are about 2 to 400 m 2 / g, preferably about 5 to 400 m 2 / g, preferably about 10 to 400 m 2 / g, more preferably about 30 to 400m 2 / g, more preferably about 30 to 300m 2 / g, more preferably about 40 to 200m 2 / g, more preferably about 180m 2 / g. The BET surface area of ​​the silica substrate is approximately 10-400 m 2 / g, preferably about 30 to 400m 2 / g, preferably about 30 to 300m 2 / g, more preferably about 40 to 200m 2 / g, more preferably about 180m 2 / g. The natural fiber substrate is approximately 2 m 2 / g, preferably about 5m 2 / g. In another embodiment, the substrate of the compositions and methods of the present invention may have a lower BET surface area, such as 350 m / g. 2 / g, preferably about 500m 2 / g. Preferably, it is medium (150 to 350 m 2 / g) to high (350m 2 Silicas having a BET surface area of ​​400-600 m / g or greater are useful in the compositions and methods of the present invention. Such silicas are believed to have better control of water activity and better preservation of CFU. Thus, in yet another embodiment, the substrate is 400-600 m2 / g, preferably 500m 2 The silica has a BET surface area of ​​1000-1500 m / g. When the compositions or methods of the present invention include a substrate with a high BET surface area, such as SIPERNAT® 50S silica, the compositions and methods preferably further include (i) a polymer selected from the group consisting of polyvinyl alcohol, xanthan gum, gum arabic, or other polysaccharides such as maltodextrin or guar gum (e.g., hydroxypropyl guar gum), polyethylene glycol, and polyglycerol, or (ii) a non-reducing disaccharide such as trehalose or sucrose, or (iii) skim milk or dimethyl sulfoxide. A substrate with a low BET surface area (e.g., 50-150 m) is preferred. 2 / g) of silica is also useful in the compositions of the present invention.

[0017] Typical pore volumes of the substrates of the compositions of the present invention are about 0.01 to 1.20 cc / g, preferably about 0.05 to 1.20 cc / g, more preferably about 0.10 to 1.0 cc / g, and even more preferably about 0.20 to 0.95 cc / g. Substrates such as silica having pore volumes of about 0.05 to 1.20 cc / g, preferably about 0.10 to 1.0 cc / g, and even more preferably about 0.20 to 0.95 cc / g, are useful in the present invention. Substrates such as cellulose having smaller pore volumes, such as 0.01 to 1.2 cc / g, are useful in the present invention. Such pore volume values ​​are measured according to the Barrett-Joyner-Halenda model. In another embodiment, the substrate of the present invention is a silica having a pore volume by Barrett-Joyner-Halenda model of greater than 1 cc / g, preferably greater than 1.4 cc / g, or a pore volume by mercury pore volume of greater than 2 cc / g, preferably greater than 2.22 cc / g.

[0018] The substrates disclosed herein provide a suitable surface for attachment of microorganisms and then efficient drying in a dryer to achieve the target total moisture content disclosed herein, avoiding prolonged exposure to heat, which reduces microbial survival and viable microorganisms after storage. In particular, microorganisms are dried on the surface of the substrate, for example, using the methods disclosed herein, to a target total moisture content of about 0.01% to 15% by weight, in certain embodiments about 0.01% to about 8% by weight, preferably 5% and about 8% by weight, more preferably 5% and about 8% by weight, and even more preferably selected from about 3%, about 5%, and about 7% by weight. Moisture content levels can be measured by methods known in the art, such as by assessing the amount of moisture present in a particular product and measuring the weight percent moisture lost per gram of product over a period of time at about 100°C to a constant weight (i.e., loss on drying).

[0019] The selection of the substrate of the present invention, along with the target moisture content levels provided herein, is based on an optimum defined water activity (A) of about 0.01 to about 0.6, preferably about 0.2 to about 0.6, more preferably about 0.2 to about 0.5, and even more preferably about 0.3 to about 0.5. w ) level, in which the microorganisms are believed to be dormant but still viable, thereby providing a dry, stable system for the delivery of such microorganisms without sacrificing viability. Water activity is defined as the ratio of the partial vapor pressure of water in the product to the standard partial vapor pressure of pure water. Water activity (A w ) assesses the equilibrium amount of water available for hydration (i.e., water availability) of a particular material. Certain substances with the same water content may have different water activity levels. w ) levels can be measured by methods known in the art, such as by use of resistance-electrolyte hygrometers (REH), capacitance hygrometers, and dew point hygrometers.

[0020] The present invention allows for the concentration of microorganisms on a substrate at a high colony forming unit concentration, and in a particular embodiment, the microorganisms are concentrated on a substrate (particularly silica) in the form of crusts. Thus, the composition of the present invention can be used to concentrate microorganisms at a high colony forming unit concentration on a substrate at a high colony forming unit concentration of about 10 7 CFU / g, preferably about 10 8 CFU / g or more, more preferably about 10 9 CFU / g or more, more preferably about 10 10 CFU / g or more, more preferably about 10 11 CFU / g or more, more preferably about 10 12 The compositions of the present invention are particularly stable, and in certain embodiments, the number of colony forming units per gram of composition (CFU / g) is greater than or equal to about 10 CFU / g after 120 days of storage at room temperature. 7 CFU / g, and in another embodiment, after 40 days of storage at 40° C. 7 and in yet another embodiment, the CFU / g remains above about 10 after 40 days of storage at 65% or less relative humidity. 7 CFU / g remains greater than 5 log, preferably less than 3 log, more preferably less than 2 log, and most preferably less than 1 log loss of CFU over 10 weeks at ambient temperature, e.g., 25° C. In another particular embodiment, the compositions of the present invention have a CFU loss of less than 5 log, preferably less than 3 log, more preferably less than 2 log, and most preferably less than 1 log loss over 10 weeks at ambient temperature (e.g., 25° C.) and high humidity, e.g., 70% relative humidity.

[0021] Microorganisms useful in the present invention include natural or recombinant microorganisms that can act as predators of other undesirable microorganisms, intervene in their life cycle, beneficially affect the area being treated, or produce biologically active substances that act beneficially as pesticides. Exemplary microorganisms useful in the present invention include those that can be used in agriculture, including, but not limited to, Bacillus thuringiensis, Pseudomonas fluorescens, Bradyrhizobium, Mycorrhiza, Clonostachys rosea, and the like, or any combination thereof.Additional microorganisms useful in the present invention include, but are not limited to, bacteria such as Bacillus subtilis QST713 and Pasteuria usgae; Beauveria bassiana, Coniothyrium minitans, Chondrostereum purpureum, Paecilomyces lilacinus, Aschersonia aleyrodis, Beauveria brongniartii, Hirsutella thompsonii, Isaria fumosorosea, Isaria sp. fungi such as Lecanicillium sp., Lecanicillium longisporum, Lecanicillium muscarium, Lecanicillium sp., Metarhizium anisopliae, Metarhizium anisopliae var. acridum, Nomuraea rileyi, and Sporothrix insectorum; viruses such as Cydia pomonella GV; and Phytophthora palmivora, Lagenidium giganteum, Bacillus spp. spp., and oomycetes such as Lactobacillus spp.; or any combination thereof.Further examples of fungi and subspecies useful in the present invention can be found in Faria, et al., Biological Control 43 (2007) 237-256, the contents of which are incorporated herein by reference in their entirety. This list is not intended to be exhaustive and may include other microorganisms useful not only in the agricultural sector, but also in other sectors such as the food, medical or pharmaceutical, detergent, and energy sectors.

[0022] Although the substrate-microorganism mixtures of the present invention do not require external protectants such as alginate encapsulation, they can be optionally treated with polymers or other materials, such as fumed silica (e.g., AEROSIL®) or a combination of polymer and fumed silica, to provide additional moisture protection and insulation from high-temperature storage. Thus, in one embodiment, the composition further contains (i) a polymer selected from the group consisting of polyvinyl alcohol, xanthan gum, gum arabic, or other polysaccharides such as maltodextrin and guar gum (e.g., hydroxypropyl guar gum), polyethylene glycol, and polyglycerol; or (ii) a non-reducing disaccharide such as trehalose or sucrose; or (iii) skim milk or dimethyl sulfoxide. In another embodiment, the composition of the present invention further contains a polymer selected from the group consisting of polyvinyl alcohol, xanthan gum, gum arabic, or other polysaccharides such as maltodextrin and guar gum (e.g., hydroxypropyl guar gum), and polyethylene glycol. In yet another embodiment, the polymer is polyglycerol, e.g., a hyperbranched polyglycerol polymer. In yet another embodiment, the composition further comprises a non-reducing disaccharide, such as trehalose or sucrose. In yet another embodiment, the composition further comprises a polymer in combination with a second substrate, as further described below. The amount of polymer can be about 0.1-3% by weight of the microbial suspension, about 0.1-1% by weight in certain embodiments, and about 1-1.5% by weight in certain embodiments. The polymer or polysaccharide or non-reducing disaccharide herein can be added before, during, or after the drying step (2).

[0023] The substrate-microorganism mixture of the present invention may be treated with a second substrate, such as an inorganic material, for additional moisture protection during storage. In one embodiment, the second substrate is selected from precipitated silicas, such as SIPERNAT® 50S silica, for example, at less than 3% as an outer layer. In another embodiment, the composition of the present invention further comprises adding a second substrate, such as a fumed silica, such as AEROSIL® 200, AEROSIL® R972, or AEROSIL® R812S silica, for example, at less than 2% as an outer layer. In yet another embodiment, the second substrate is 180-220 m 2 The second substrate is a hydrophobic fumed silica such as AROSIL® R202 silica having a BET surface area of ​​1 / g and a carbon content of 3.5-5%. The amount of the second substrate can be about 0.1-3% by weight of the total composition, about 0.1-1% by weight in certain embodiments, and about 0.1% by weight in certain embodiments. In certain embodiments, the compositions of the present invention comprise a mixture of microorganisms and 350 ml of water. 2 / g or more BET surface area, e.g., 400-600 m 2 / g, preferably 500m 2 and a substrate, such as silica, having a BET surface area of ​​180-220 m / g, such substrate coated with one or more of (i) a polymer selected from the group consisting of polyvinyl alcohol, xanthan gum, gum arabic, or other polysaccharides, such as maltodextrin or guar gum (e.g., hydroxypropyl guar gum), polyethylene glycol, and polyglycerol, or (ii) a non-reducing disaccharide, such as trehalose or sucrose, or (iii) skim milk or dimethyl sulfoxide. In another embodiment, the compositions of the present invention comprise one or more microorganisms and a substrate, such microorganism-substrate coated with a second substrate (such as AROSIL® R202 silica, 180-220 m / g). 2 / g BET surface area and a carbon content of 3.5-5%, as further described below), and optionally one or more of (i) a polymer selected from the group consisting of polyvinyl alcohol, xanthan gum, gum arabic, or other polysaccharides such as maltodextrin or guar gum (e.g., hydroxypropyl guar gum), polyethylene glycol, and polyglycerol, or (ii) a non-reducing disaccharide such as trehalose or sucrose, or (iii) skim milk or dimethyl sulfoxide. In yet another embodiment, the composition of the invention comprises a microorganism, a first silica substrate, a second silica substrate, and one or more polymers selected from the group consisting of (i) polyvinyl alcohol, xanthan gum, gum arabic, or other polysaccharides such as maltodextrin or guar gum (e.g., hydroxypropyl guar gum), polyethylene glycol, and polyglycerol, or (ii) non-reducing disaccharides such as trehalose or sucrose, or (iii) skim milk or dimethyl sulfoxide. In yet another embodiment, the composition of the present invention comprises a microorganism, a hydrophilic silica substrate (e.g., a hydrophilic precipitated silica such as SIPERNAT® 22 silica), a second silica substrate (e.g., a hydrophilic or hydrophobic fumed silica such as AEROSIL® R202 or 200 silica), and optionally one or more of (i) a polymer selected from the group consisting of polyvinyl alcohol, xanthan gum, gum arabic, or other polysaccharides such as maltodextrin or guar gum (e.g., hydroxypropyl guar gum), polyethylene glycol, and polyglycerol, or (ii) a non-reducing disaccharide such as trehalose or sucrose, or (iii) skim milk or dimethyl sulfoxide. In certain embodiments, the second substrate is added after the drying step (2).

[0024] The compositions of the present invention can be applied directly to the treatment area, such as plants, seeds, or pests, or they can be formulated into biological formulations, for example, for application to such treatment areas. Traditionally, aqueous formulations containing microorganisms or other biologically active materials have been difficult to stabilize during the shelf life of typical suspension concentrate formulations. The compositions described herein aim to lower the formulation barriers overall. The present invention teaches an approach for creating biological compositions with both higher activity levels (CFU) and stability (low CFU change over time). A more stable composition lowers the formulation hurdles required to produce a usable product suitable for use in agricultural applications. Due to their advantages, the compositions of the present invention can be formulated into both liquid and dry pesticide formulation types. Examples of these formulations include, but are not limited to, WP (wettable powder), WG (water dispersible granule), SC (suspension concentrate), OD (oil dispersion), and FS (seed treatment). Thus, in another aspect, the present invention provides a biological formulation containing a composition of the present invention, e.g., any of Compositions I, 1.1-1.33, or 1.34-1.50, and one or more excipients. Because the microorganisms of the present invention may be useful in agricultural applications, pesticide-acceptable excipients or adjuvants, such as wetting agents, are contemplated. Additionally, combinations with other pesticide active ingredients may be used.

[0025] Aqueous formulations of SC form may contain one or more dispersants, polymers, spreading agents, surfactants, colorants, and / or antifreeze compounds. The selection of specific formulation aids is well within the knowledge of one skilled in the art.

[0026] Dry formulations include dusts (DP), seed coating powders (DS), granules (GR), microgranules (MG), water dispersible granules (WG), wettable powders (WP), which may contain one or more binders, dispersants, and wetting agents. The selection of specific formulation aids is well within the knowledge of one skilled in the art.

[0027] The compositions of the present invention are particularly useful in tablet form for formulation types ST (water-soluble tablets) and TB (tablets). Accordingly, in certain embodiments, the present invention provides biological tablets containing a composition of the present invention, e.g., Compositions I or any of 1.1-1.33 or 1.34-1.50, and one or more excipients. Excipients useful in tablet formulations of the present invention can include one or more lubricants, binders, disintegrants, and fillers. Useful lubricants include, but are not limited to, talc, magnesium stearate, calcium stearate, stearic acid, boric acid, polyethylene glycol, and sodium stearyl fumarate. Useful binders include, but are not limited to, microcrystalline cellulose, cellulose acetate, carrageenan, dextrin, glucose, ethylcellulose, and polyvinylpyrrolidone. Useful fillers include, but are not limited to, corn starch, potato starch, sodium starch, glycolate, amylose, primogel, crospovidone, and croscarmellose sodium. Useful disintegrants include, but are not limited to, calcium silicate. An exemplary tablet contains 10g of disintegrant per gram. 9 It can be manufactured using 2-30% microbial powder containing CFU of microorganisms. 2 gram tablets are compressed at 20 KN. Tablets can be rapidly disintegrated using 7.5% FM1000.

[0028] The oil dispersion formulations of the present invention contain a composition of the present invention, e.g., Compositions I or any of 1.1-1.33 or 1.34-1.50, dispersed in a non-aqueous or non-water-soluble liquid such as mineral oil, paraffinic oil, or vegetable oil, which may include one or more dispersants, emulsifiers, polymers, spreading agents, surfactants. The selection of specific formulation aids is well within the knowledge of one skilled in the art.

[0029] Agricultural oils useful in the formulations of the present invention include paraffinic oils such as octane, nonane, decane, undecane, dodecane, tridecane, tetradecane, pentadecane, hexadecane, and mixtures thereof, or such oils mixed with higher boiling homologues such as hepta-, octa-, nona-decane, eicosane, heneicosane, docosane, tricosane, tetracosane, pentacosane, and their branched chain isomers; vegetable oils such as olive oil, kapok oil, castor oil, papaya oil, camellia oil, palm oil, sesame oil, corn oil, rice bran oil, peanut oil, cottonseed oil, soybean oil, rapeseed oil, linseed oil, tung oil, sunflower oil, safflower oil, or transesterification products thereof such as rapeseed oil methyl ester and rapeseed oil ethyl ester; whale oil, cod liver oil, or mint oil. animal oils such as tallow oil; butanol, n-octanol, i-octanol, dodecanol, cyclopentanol, cyclohexanol, cyclooctanol, ethylene glycol, propylene glycol or benzyl alcohol, caproic acid, capric acid, caprylic acid, pelargonic acid, succinic acid, glutaric acid, benzoic acid, toluic acid, salicylic acid and phthalic acid, benzyl acetate, caproic acid ethyl ester, pelargonic acid ethyl ester, benzoic acid methyl or ethyl ester, salicylic acid methyl, propyl, or butyl ester, phthalic acid diesters with saturated aliphatic acids, phthalic acid dimethyl ester, dibutyl ester, diisooctyl ester, or any combination thereof.

[0030] The present invention also contemplates the use of compositions of the present invention for seed treatment or seed coating. Thus, in one embodiment, the compositions of the present invention provide, for example, a flowable concentrate form (FS form) for seed treatment, which can be prepared by blending Composition I or any of Compositions 1.1 to 1.33, or any of Compositions 1.34 to 1.50 with one or more dispersants, film-forming polymers, spreading agents, surfactants, and colorants, and then adding the blend to seeds. Ingredients that help the formulation adhere to the seeds, enhance coating, and reduce dust may also be included. The selection of specific formulation aids is well within the knowledge of those skilled in the art.

[0031] In another aspect, the present invention also provides a method for preparing a dried biological composition, comprising, in certain embodiments consisting essentially of, and in other embodiments consisting of, (1) combining a microbial mixture, solution, or suspension with a substrate; and (2) drying the substrate-microbial mixture to reach a total moisture content of about 0.01 to about 15% by weight, preferably about 3% to about 8% by weight, more preferably about 5% to about 8% by weight, and even more preferably selected from 3%, 5%, and 7% by weight. In yet another embodiment, step (2) of the method of the present invention provides a method for preparing a dried biological composition, comprising: ...i) drying the substrate-microbial mixture to a total moisture content of about 0.01 to about 0.6, preferably about 0.2 to about 0.6, and even more preferably about 0.3 to about 0.5. w The composition is dried until

[0032] The microorganisms used in the compositions of the present invention can be obtained by various means. In one embodiment, the microorganisms can be harvested from the surface of the seeds by washing the seeds with water, for example, in a 1:1 ratio of water:seed. In another embodiment, the microorganisms are harvested from the surface of the seeds by mechanically crushing or grinding the surface of the seeds (step (a)), resulting in a fine fraction containing the microorganisms, in certain embodiments, fungal spores, and a plurality of seed portions. In certain embodiments, the yield of the microorganisms in the fine fraction is 10% or more per gram of seed initially crushed or ground. 9cfu. In certain embodiments, the method includes sieving the resulting fine fraction to obtain a powder with a defined particle size distribution for subsequent processing steps (step (b)). In certain embodiments, the powder is used to prepare a microbial mixture, solution, or suspension (step (c)). In another specific embodiment, step (a) includes grinding with a grinding stone to separate the seeds from the fine fraction. In another specific embodiment, step (a) includes grinding under pressure using a rotating shaft in a sealed tube with a slotted screen, followed by separating the seeds from the fine fraction using a sieve and a filter. The sieving step (b) of the fine fraction includes sieving through a sieve mesh size of 20 to 800 μm, in certain embodiments, 100 μm to 300 μm.

[0033] In another embodiment, microorganisms can be harvested from the surface of seeds by rinsing the surface of the seeds with water and separating the seeds from a liquid microbial solution or suspension. In a specific embodiment, the seeds are stirred in water for 1 to 20 minutes. In another specific embodiment, solid-liquid separation is performed in a pressure Nutze filter, and in another specific embodiment, a pressure Nutze filter with a mesh size of 1 to 3 mm is used. In another specific embodiment, the dewatering time in the pressure Nutze filter is 20 to 200 seconds. In a specific embodiment, the filtration pressure in the Nutze filter is 1 to 3 bar. In another specific embodiment, the microbial solution or suspension is concentrated by separating the microorganisms from the liquid in a centrifugal field. In a specific embodiment, the concentration step includes separation in a disc stack separator. In a specific embodiment, the concentration step again involves diluting the concentrate with water and subsequent second concentration in a centrifugal field to separate the soluble portion from the microorganisms.

[0034] Substrates useful in step (1) of the method of the present invention can be selected from the group consisting of water-insoluble natural fibrous materials such as silica (e.g., precipitated silica, and in certain embodiments, hydrophilic silica, e.g., SIPERNAT® 22 silica), diatomaceous earth, silica gel, silicates (e.g., aluminosilicates such as ZEOLEX® 301, or clays), and cellulose. In one embodiment, the substrate in step (1) of the method of the present invention is silica, and in a further embodiment, it is precipitated silica, e.g., having a particle size (d50) of about 5 to 200 microns, preferably 8 to 160 microns, more preferably about 9 to 150 microns, even more preferably about 50 to 150 microns, even more preferably about 50 to 130 microns, even more preferably about 50 microns, about 85 microns, and about 120 microns. In another further embodiment, the substrate in step (1) of the method of the present invention is hydrophilic precipitated silica. In a further embodiment, the silica in step (1) of the method of the present invention is (i) about 2 to 600 microns 2 / g, and in a further embodiment 500m 2 / g, and in another further embodiment, 2 to 400 m 2 / g, preferably about 5 to 400 m 2 / g, more preferably about 10 to 400 m 2 / g, more preferably about 30 to 400m 2 / g, more preferably about 30 to 300m 2 / g, more preferably about 40 to 200m 2 / g, more preferably about 180m 2 / g BET surface area; and / or (ii) a pore volume by Barrett-Joyner-Halenda model of about 0.01 to 1.20 cc / g, preferably about 0.05 to 1.20 cc / g, more preferably about 0.10 to 1.0 cc / g, even more preferably about 0.20 to 0.95 cc / g; and / or (iii) a pore volume by Barrett-Joyner-Halenda model of greater than 1 cc / g, preferably greater than 1.4 cc / g, or a pore volume by mercury pore volume of greater than 2 cc / g, preferably greater than 2.2 cc / g. In a further embodiment, the substrate of step (1) is selected from SIPERNAT® 22 or SIPERNAT® 50S silica.

[0035] The methods of the invention described herein may further comprise adding after step (1), but in one embodiment before step (2), in another embodiment during step (2), or in yet another embodiment after step (2): (i) a polymer selected from the group consisting of polyvinyl alcohol, xanthan gum, gum arabic, or other polysaccharides such as maltodextrin or guar gum (e.g., hydroxypropyl guar gum), polyethylene glycol, and polyglycerol, or (ii) a non-reducing disaccharide such as trehalose or sucrose, or (iii) skim milk or dimethyl sulfoxide, in certain embodiments polyvinyl alcohol or polyglycerol (e.g., hyperbranched polyglycerol); and / or a second substrate as an outer layer. In certain embodiments, the second substrate is selected from precipitated silica, such as SIPERNAT® 50S silica, or fumed silica, such as AEROSIL® 200, AEROSIL® R972, AEROSIL® R812S, or AEROSIL® 202, preferably AEROSIL® 200 or R202, more preferably AEROSIL® R202 silica. The polymer disclosed herein can be added without a second substrate. In another embodiment, the polymer disclosed herein can be added together with the second substrate, and can be added before or after the second substrate. The polymer and / or second substrate can be added before, during, or after the drying step (2).

[0036] Drying step (2) of the method of the present invention can be performed by fluidized bed drying, spray drying, contact drying, or freeze drying. Fluidized bed drying can be achieved by setting the inlet air temperature to about 90°C or less, preferably about 80°C or less, preferably about 50°C or less, more preferably about 30°C to 50°C, even more preferably about 40°C to 50°C, even more preferably about 40°C to 45°C, and even more preferably about 43°C. In a specific embodiment, drying step (2) of the method of the present invention can be achieved by preheating the spray dryer at an inlet air temperature of about 50°C or less, preferably about 30°C to 50°C, even more preferably about 40°C to 50°C, even more preferably about 40°C to 45°C, using a very low fan speed, and spraying the microbial mixture onto the substrate in the chamber. Preferably, the pump rate on a laboratory scale is 1 mL / min, more preferably 2 mL / min of substrate. Optionally, drying step (2) also includes drying under reduced pressure (e.g., 0.1 bar).

[0037] Spray drying can be achieved by setting the inlet air temperature to about 130° C. or less, preferably 110° C. or less, more preferably 100° C. or less, more preferably 90° C. or less, more preferably 80° C. or less, more preferably 50° C. or less, and even more preferably about 30° C. to 50° C. Spray drying can be carried out using a gas stream.

[0038] Preferably, drying step (2) of the method of the present invention comprises maintaining the powder bed temperature at about 35°C or below, preferably about 30°C or below, more preferably between about 25°C and 35°C.

[0039] It is believed that drying time is proportional to the surface area of ​​the silica, and control of water activity is inversely proportional to the surface area of ​​the silica. Therefore, in one embodiment, a medium (150-350 m 2 / g) to large (400-600m 2 / g etc. 350m 2 / g or more, e.g., 500m 2 / g) BET surface area, better control of water activity and better preservation of cfu is obtained. In another embodiment, large (400-600 m) silica containing humectants or polymers or polysaccharides is used. 2 / g etc. 350m 2 / g or more, e.g., 500m 2 The use of silica with a BET surface area of ​​1000 s / g also results in better control of water activity and better preservation of cfu over time. Preferably, the high BET surface area silica is dried at shorter times and higher temperatures, for example using spray drying at 100°C for short periods of time (e.g., 2-80 second residence times).

[0040] The microbial mixture, solution, or suspension of step (1) of the method of the present invention can be fermented in a stirred batch fermenter by adding sugars and other nutrients to a batch reactor that is aerated or maintained under anaerobic conditions to allow the microorganisms to grow and reach optimal conditions for harvest depending on the nature of the microorganisms. In another embodiment, the microorganisms can be grown on solid media, such as cellulosic materials, seeds, and other solid materials, suspended in a stirred reactor. In yet another embodiment, the microorganisms can be grown on solid media in a dry but humidified environment and washed off the seeds at the optimal time.

[0041] For the purposes of this application, AEROSIL® 200 silica refers to 200 ml 2 / g。 AEROSIL® R202, R972, R812 refer to hydrophobic fumed silica.

[0042] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art. In case of conflict, the present specification, including definitions, will prevail. Preferred methods and materials are described below, but methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. The materials, methods, and examples disclosed herein are illustrative only and are not intended to be limiting.

[0043] As used herein, the terms "comprise(s)," "include(s)," "having," "has," "can," "contain(s)," and variations thereof are intended to be open-ended transitional phrases, terms, or words that do not exclude the possibility of additional features or structures. The singular forms "a," "an," and "the" include plural references unless the context clearly dictates otherwise. The present disclosure contemplates other embodiments that "comprise," "consist," and "consist essentially of" the embodiments or elements presented herein, whether explicitly stated or not.

[0044] The connector term "or" includes any combination of one or more of the listed elements associated by the connector term. For example, the phrase "a device comprising A or B" can refer to a device comprising A without B, a device comprising B without A, or a device in which both A and B are present. "At least one of A, B, ... and N" or "at least one of A, B, ... N or combinations thereof" are defined in the broadest sense to mean one or more elements selected from the group including A, B, ... and N. That is, any combination of one or more of the elements A, B, ... or N includes any one element alone or in combination with one or more other elements, which may also include additional, unlisted elements in combination.

[0045] The modifier "about" used in connection with a quantity is inclusive of the stated value and has the meaning dictated by the context (e.g., it includes at least the degree of error associated with measurement of the particular quantity). The modifier "about" should also be considered to disclose a range defined by the absolute values ​​of the two endpoints. For example, the phrase "about 2 to about 4" also discloses a range of "2 to 4." The term "about" can refer to plus or minus 10% of the recited number. For example, "about 10%" can indicate a range of 9% to 11%, and "about 1" can mean 0.9 to 1.1. Other meanings of "about" can be apparent from the context, such as rounding; thus, for example, "about 1" can mean 0.5 to 1.4. [Example]

[0046] Example The foregoing description may be better understood with reference to the following examples, which are presented for illustrative purposes and are not intended to limit the scope of the invention.

[0047] Examples 1 to 10. Fluid-bed dried compositions of the present inventionApproximately 50 grams of suspension extracted from a 1:1 water wash of seeds containing Chlonostachys rosea is sprayed onto approximately 25 grams of substrate in a fluidized bed dryer. The pump speed, atomizing air pressure, and fan speed are adjusted accordingly to dry the substrate-spores at an inlet temperature of approximately 45°C and a powder bed temperature of less than 28°C (e.g., 1 mL / min pump speed and 0.1 bar atomizing air pressure). The sample is heated until the target moisture reading is achieved. The sample is analyzed using the following method:

[0048] Dry particle size test The particle size of the substrate is measured using a HORIBA Laser Scattering Dry Particle Size Distribution Analyzer LA-950, based on the angle of the scattered laser light.

[0049] Total moisture content measurement Moisture determination of the dried substrate-microbial powder is carried out with a Satorius moisture balance. A mass of 0.1 g of sample powder is weighed and placed in an aluminum plate. The sample is heated to a temperature of 105°C for a period of 2 minutes, typically three times, until a constant weight is reached.

[0050] water activity The water activity (A w Water activity is measured by placing the sample in a water activity measuring device, which consists of a mirror above the test sample in a sealed sample chamber. Once the relative humidity reaches equilibrium, the mirror is cooled until condensation forms on the mirror due to the dew point. This temperature can then be calculated as the water activity level.

[0051] Scanning electron microscope (SEM) images A Hitachi™ 3000 electron microscope was used to capture images of the substrate-microorganisms of the present invention and demonstrate the morphology and composition of the product particles. The images show that the spore cells are attached to the silica particles.

[0052] Mercury Pore Volume and Pore Size TestMercury intrusion pore volume (Hg) is measured by mercury porosimetry using a Micromeritics AutoPore IV 9520 instrument. Pore size can be calculated using the Washburn equation, which uses a contact angle theta (θ) equal to 130° and a surface tension gamma equal to 485 dynes / cm. Mercury is forced into the particle voids as a function of pressure, and the amount of mercury intruded per gram of sample is calculated at each pressure setting. The pore volumes presented herein represent the cumulative volume of mercury intruded at pressures ranging from 171 to 18,000 psia. Mercury intruded at these pressures corresponds to pore diameters ranging from 100 to 10 nm. The volume increment (cm3 / g) at each pressure setting is plotted against the pore diameter corresponding to the pressure setting increment. The peak of the intruded volume versus pore radius or diameter curve corresponds to the mode of the pore size distribution and identifies the most common pore size in the sample. Specifically, the sample size is adjusted to achieve 25-75% stem volume in a powder penetrometer with a 5 mL bulb and approximately 1.1 mL stem volume. The sample is evacuated to a pressure of 50 μm Hg and held for 5 minutes. Mercury fills the pore at 1.5-60,000 psia with a 10-second equilibration time at each of approximately 103 data collection points.

[0053] BET surface area and pore volume substrates The BET surface area of ​​particles (e.g., silica or silicate particles) is determined using a Micromeritics TriStar 3020 instrument by the BET nitrogen adsorption method of Brunaur et al., J. Am. Chem. Soc., 60, 309 (1938), which is well known in the field of particulate materials, such as silica and silicate-based materials. Nitrogen adsorption / desorption isotherms were obtained at 77 K. Prior to measurement, 50–100 mg of powder samples were degassed at 105°C for 2 hours. The Barrett-Joyner-Halenda (BJH) model was used to calculate the pore volume and BET surface area. The total pore volume was calculated from the total amount of nitrogen adsorbed at a partial pressure (P / P) of 0.99.

[0054] CFU testMicrobial concentrations are determined by plate count using a serial dilution technique. Microbial substrate powder is stirred in sterile water with Triron surfactant present to concentrate the microorganisms. The resulting suspension of microorganisms is serially diluted multiple times, 10-fold each time. After each dilution, a sample of the dilution is plated on a sterile agar plate and incubated. After several days, any microorganisms present can be seen as dots on the agar. When the dilution is sufficient to reduce the number on the plate to a countable amount, the number of colonies is counted and multiplied by the dilution factor to determine the population of the original population.

[0055] Using methods described or similar to those described above, the physical properties of various substrates are measured and are summarized in Table 1 below.

[0056] [Table 1]

[0057] The total moisture content and water activity (A) of the test samples after fluidized bed drying were determined using the analytical methods described or similar to those mentioned above. w ) levels are measured, which are summarized in Table 2. The BET surface area, drying time, final moisture content, and water activity (A w ), and initial CFU are reported in Table 3. Water activity (A) for CFU / g after 5 months at 25°C w ) versus moisture content is summarized in Table 4.

[0058] [Table 2]

[0059] [Table 3]

[0060] [Table 4]

[0061] As can be seen from the table above, substrates with large BET surface areas and larger pore volumes, such as SIPERNAT® 50, surprisingly do not dry very quickly in a fluidized bed dryer, resulting in excessively long exposure times for microorganisms in the dryer. Substrates with lower BET surface areas and pore volumes dry faster in a fluidized bed dryer, potentially reducing stress on the microorganisms and allowing for higher CFU / g after drying, potentially resulting in lower cost per CFU / g due to lower drying costs. Table 4 also shows that the higher the total moisture content, the greater the decline in CFU / g during storage. Silica must maintain low moisture during storage to maintain high CFU / g. Thus, the present invention demonstrates that selecting a substrate with optimal BET surface area and pore volume can achieve fast drying times with low total moisture content, thereby reducing stress on the microorganisms and resulting in higher initial CFU / g and potentially higher CFU / g values ​​after 5 months.

[0062] Example 11. Spray-dried composition of the present invention Bacterial biomass of Pseudomonas fluorescens is harvested from overnight cultures in shake flasks by centrifugation at 8000 g for 10 min. The cell pellet is resuspended in sodium chloride solution (0.9% w / w) and added to a suspension of SIPERNAT® 50 silica substrate and gum arabic. The resulting suspension contains approximately 8% silica, 7% gum arabic, 3% dry biomass, and 81% water. The suspension is then spray-dried in a Büchi B-290 laboratory spray dryer at a gas inlet temperature of 78°C. Atomization is performed using a two-fluid nozzle at an atomization pressure of approximately 1.35 bar. The drying air flow rate is 38 m / s. 3 / h. The spray volume is approximately 5 mL / min. The set parameters result in an outlet temperature of 53°C and a residual moisture content of the product of 6.3%. The cfu count of the resulting powder is 3.4 x 10 7 cfu / g.

[0063] Aqueous collection of fungal spores: 15 g of the initial seeds with fungal spores on their surface are washed with water. The mass of the water is 3 to 10 times the mass of the seeds. The resulting suspension is mixed using a stirrer (disk stirrer) and filtered through a 3 mm mesh after a mixing time of 20 minutes. The suspension is filtered using a 380 ml laboratory pressure nutze. The operating conditions are room temperature and 1 bar (abs). The dehydration time is 120 seconds. The filtrate is analyzed by spore count analysis. To reduce the water content before use in fluidized bed spraying, the filtrate is further concentrated by separation in a laboratory centrifuge at 2100 g for 5 minutes.

[0064] Dried collection of fungal spores 100 g of seeds with fungal spores on their surface are ground in a grinder equipped with a rotating grinding stone for a residence time of 20 seconds. The fine fraction is generated by grinding the seed surface. This is collected separately from the seed residue, weighed, and the number of cfu in the sample is determined. The cfu reached in the fine fraction is 5 x 10 per gram of seed. 9 The resulting fine fraction is sieved through a 300 μm mesh sieve. The resulting powder is mixed with water to obtain a suspension for subsequent spraying and drying in a fluidized bed.

[0065] Examples 12-23 The following examples are conducted to determine the effect of additives on improving the heat and humidity stability of microorganisms.

[0066] Washing Procedure: Seeds are washed by mixing them with an equal mass of water until the water turns light brown. The spore suspension is strained from the seeds until half the original amount of water is recovered, and more water is added, if necessary, to recover the final volume, which includes the amount added from the stock solution. Additives are mixed directly into the suspension (AEROSIL® 200 silica and HPG) or in concentrated stock solutions (PVA) from 2% grams to the final volume (mL).

[0067] Fluidized Bed Drying. The recovered spore suspension is sprayed at approximately 4 g / min onto SIPERNAT® 22 silica, with a weight equal to the suspension being sprayed. The fan speed is 8 Hz, and the inlet air temperature is set at 45°C for samples without additives in the suspension and 55°C for samples with additives. The starting powder temperature is 28°C. The powder is considered dry if, after a few minutes, there is a rapid increase in temperature from the starting temperature (28°C), indicating drying.

[0068] CFU Count. CFU, or colony-forming units, are the number of viable spores per gram of product. Spore powder is mixed with Triton solution and plated by serial dilution onto potato dextrose agar containing 0.1% streptomycin and incubated at room temperature for 5 days. CFU is determined by counting plates containing 30 to 300 spores multiplied by the dilution factor.

[0069] Post-addition of AEROSIL® R202 silica. AEROSIL® R202 silica is added to the final powder at 1% g / g for selected samples. This is mixed in a Turbula low-energy mixer for 5 minutes to ensure a uniform coating of the spore powder.

[0070] Heat stability: Spore powder with sufficiently low water activity is stored in an oven at 40°C and CFU counts are performed at various time points to measure the decrease in viable cell density on the powder.

[0071] Humidity Stability. Spore powder is stored in a humidity chamber (Associated Environmental Systems) at 70% relative humidity and 25°C in Tubulin semi-porous bags that are permeable to water vapor but impermeable to spores. CFU are measured at various time points to measure progression.

[0072] Water activity. Water activity is defined as the vapor pressure of water within a closed sample. It is measured by the dew point of a cooled mirror in a sealed chamber as the temperature of the mirror decreases. Water activity is measured with an AquaLab Model 3.

[0073] Decimal Decay Time. Decimal decay time is defined as the time to reduce the viable population of a microorganism by 90%. It is calculated using the inverse slope of the survival curve, which is a plot of log CFU over time.

[0074] Results. Samples used in the stability experiments are first screened to have both high CFU and low water activity. Samples that do not meet these requirements are discarded and remade. The water activity of each sample used can be seen in Table 6 below. Samples that meet these two requirements are split and one half is mixed with AEROSIL® R202 silica. The resulting powder is then stored in a 40°C oven or a humidity chamber at 25°C / 70% relative humidity.

[0075] The sample preparation is summarized in Table 5 below: [Table 5]

[0076] [Table 6]

[0077] Samples tested for thermal stability were stored in an oven and observed for 10 weeks. CFUs were measured at multiple time points, as shown in Figure 1. Decimal decay times (D values) were calculated and can be seen in Figure 2. As can be seen, values ​​did not diverge significantly during the first 6 weeks. However, at the 10-week point, samples mixed with AEROSIL® R202 silica appeared more stable than untreated samples. Samples not post-treated with AEROSIL® R202 silica had low CFU values ​​at this point, too low for very accurate counting. This indicates that the addition of AEROSIL® R202 silica helps improve the stability of the spore powder and extend its shelf life. The combination of PVA and AEROSIL® R202 silica was the most stable over the long term. However, the initial CFU values ​​for PVA were low. While some of this may be due to processing variability, it also means that PVA is difficult to dissolve and is added to the spore suspension as a concentrated stock solution, resulting in a more dilute spore suspension.

[0078] Most samples show comparable trends, but the control performs the worst in the heat stability test. The sample with no additive also starts with the lowest CFU. The sample with only AEROSIL® R202 silica also starts with a low CFU, but it shows much better stability. These trends in CFU can be seen in Figure 3.

[0079] As shown in Figure 4, the sample containing HPG performs best. Samples using only AEROSIL® R202 silica have similar decimal decay times. Samples in which the spore solution was mixed with additives (AEROSIL® 200 silica, HPG, PVA) show no further improvement when subsequently mixed with AEROSIL® R202 silica. While AEROSIL® R202 silica improves humidity stability compared to the control, it does not show any additional improvement in humidity stability when used in addition to other additives. Samples containing additives appear to be better able to keep spores insulated from moisture under humid conditions.

[0080] The later addition of AEROSIL® R202 silica shows a clear improvement in thermal stability compared to the unadded sample. Similarly, while AEROSIL® R202 silica also has improved humidity stability, it does not show any additional improvement in humidity stability when used in conjunction with other additives. Therefore, adding AEROSIL® R202 silica is the most effective way to improve long-term heat and humidity stability.

[0081] Examples 24-25 are prepared as described below: [Table 7]

[0082] [Table 8]

[0083] Pseudomonas fluorescens biomass was fermented in minimal medium and harvested using a disc centrifuge to obtain a concentrated cell suspension. A saline solution was prepared and mixed with trehalose, gum arabic, and SIPERNAT® 50 silica. The harvested cell suspension was then mixed with the trehalose / gum arabic / SIPERNAT® silica suspension. After mixing, the composition of the suspension in Example 24 was 8% silica, 7% gum arabic, 3% dry biomass, 77% sodium chloride solution, and 5% trehalose. The composition of the suspension in Example 25 was 4% silica, 4% gum arabic, 8% dry biomass, 75% sodium chloride solution, and 9% trehalose. The suspensions in Examples 24 and 25 were separately spray-dried using a two-fluid nozzle in a Niro Minor spray dryer at a spray pressure of 2.3 bar. For Example 24, the gas inlet temperature was 100°C and the mass flow rate of the suspension was 0.9 kg / h. For Example 25, the gas inlet temperature was 110°C and the mass flow rate of the suspension was 1.6 kg / h. This resulted in an outlet temperature of 50°C for both Examples 24 and 25. The gas flow of the drying gas was 45 m 3 / h. The moisture content of the products of Examples 24 and 25 is 7% by weight, with a water activity of 0.3. The final product of Example 24 had a cfu of 2 x 10 10 CFU / g, and Example 25 was 3.6 x 10 10 CFU / g.

Claims

1. A dry biological composition having improved storage stability, comprising: (1) a substrate; and (2) a microorganism supported on a surface of the substrate, the composition has a total water content of 3% to 8% by weight; the pore volume of the substrate is 0.10 to 1.0 cc / g based on the Barrett-Joyner-Halenda model; The BET surface area of ​​the substrate is 40 to 200 m ２ / g, The composition comprises: ７ CFU / g and the composition has a water activity value (A w ) of 0.2 to 0.6; The composition.

2. The composition of claim 1 , wherein the substrate is selected from the group consisting of silica, diatomaceous earth, silica gel, silicates, clay, and water-insoluble natural fiber-based materials.

3. The microorganism is selected from the group consisting of Bacillus subtilis QST713, Pasteuria usgae, Beauveria bassiana, Coniothyrium minitans, Chondrostereum purpureum, Paecilomyces lilacinus, Aschersonia aleyrodis, Beauveria brongniartii, Hirsutella thompsonii, Isaria fumosorosea, Isaria sp. (Isaria sp.), Lecanicillium longisporum, Lecanicillium muscarium, Lecanicillium sp., Metarhizium anisopliae, Metarhizium anisopliae var. Metarhizium anisopliae var. acridum, Nomuraea rileyi, Sporothrix insectorum; Cydia pomonella GV; Phytophthora palmivora, Lagenidium giganteum, Bacillus thuringiensis, Pseudomonas fluorescens, Bradyrhizobium, Mycorrhiza, Clonostachys rosea, Bacillus spp., and Lactobacillus spp.

3. The composition of claim 1 or 2, wherein the composition is selected from the group consisting of:

4. 3. The composition of claim 1 or 2, further comprising: (i) a polymer selected from the group consisting of polyvinyl alcohol, xanthan gum, gum arabic, maltodextrin, guar gum, polyethylene glycol, and polyglycerol; or (ii) a non-reducing disaccharide; or (iii) skim milk or dimethyl sulfoxide.

5. The present invention further includes a second substrate as an outer layer, and the second substrate has a thickness of (i) 50 to 750 mm. ２ 3. The composition of claim 1, wherein the silica is selected from (i) precipitated silica having a high BET surface area of ​​1 / g; or (ii) fumed silica.

6. 3. The composition of claim 1, wherein the tap density of the composition is greater than 150% of the tap density of the pure substrate material.

7. 3. The dry biological composition of claim 1 or 2, further comprising seeds to be treated.

8. 10. A method for controlling insects, fungi, or nematodes on a treated area, comprising optionally reconstituting the dried biological composition of claim 1 or 2, and applying an effective amount of the optionally reconstituted composition to the area to be affected by the treatment.

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