AQUEOUS AGROCHEMICAL FORMULATIONS COMPRISING BACTERIAL SPORES

Abstract

Process for making liquid dispersion formulations comprising a) At least one type of bacterium spores, b) An aqueous continuous phase comprising at least one glycol, wherein said glycol is comprised in the formulation in a weight amount that exceeds the weight amount of water, c) optionally at least one surface active compound S, and d) Optionally further auxiliaries, B. Providing a solid powder of Bacterium spores, B1. Dispersing said spores in a continuous phase comprising at least one glycol, wherein the weight ratio of said at least one glycol to water is below or equal to 1:1, B2. Adding further glycol to the dispersion obtained in step B 1. to obtain a ratio of said at least one glycol to water from 4:1 to 1.2:1, C. Adding at least one surfactant S, D. Optionally adding at least one thickener, E. Optionally adding further auxiliaries, Wherein steps C), D) and E) can be carried out at any time during the process.

Claims

1. A process for making liquid dispersion formulations comprising a) at least one type of bacterium spores, b) an aqueous continuous phase comprising at least one glycol, wherein said glycol is comprised in the formulation in a weight amount that exceeds the weight amount of water, c) optionally at least one surface active compound S, and d) optionally further auxiliaries, comprising: A. providing a solid powder of Bacterium spores, B1. dispersing said spores in a continuous phase comprising at least one glycol, wherein a weight ratio of said at least one glycol to water is below or equal to 1:1, B2. adding further glycol to the dispersion obtained in step B1. to obtain a ratio of said at least one glycol to water from 4:1 to 1.2:1, C. optionally adding at least one surfactant S, D. optionally adding at least one thickener, E. optionally adding further auxiliaries, wherein steps C), D) and E) can be carried out at any time during the process, and where the term “glycol” shall include glycerol.

2. A liquid dispersion formulation comprising a) at least one type of bacterium spores, b) an aqueous continuous phase comprising at least one glycol, c) optionally at least one surface active compound S, d) optionally further auxiliaries, wherein said bacterium spores are dispersed in the continuous phase and wherein the formulation comprises glycol in a weight amount that exceeds the weight amount of water comprised in said formulation, and where the term “glycol” shall include glycerol.

3. The formulation according to claim 2, wherein said formulation comprises at least 40 wt % of said at least one glycol based on the formulation and wherein the formulation comprises at least 15% but maximally 40% of water.

4. The formulation according to claim 2, wherein the formulation comprises at least one nonionic surfactant S.

5. The formulation according to claim 2, wherein said surfactant S is an ethoxylate of a sugar alcohol.

6. The formulation according to claim 2, wherein said bacterium spores are present in the formulations in the form of solid particles having an average particle size of 1 to 25 μm.

7. The formulation according to claim 2, wherein said glycol is selected from 1,2-propylene glycol, ethylene glycol, diethylene glycol, 1,2-butylene glycol, polyethylene glycol having an average molecular mass Mn of 150 to 600 g/mol, and glycerol.

8. The formulation according to claim 2, wherein said at least one bacterium is selected from Bacillus subtilis, Bacillus velezensis, Bacillus amyloliquefaciens, Bacillus firmus, Bacillus pumilus, Bacillus simplex, Paenibacillus polymyxa, Bacillus megaterium, Bacillus aryabhattai, Bacillus thuringiensis, Bacillus megaterium, Bacillus aryabhattai, Bacillus altitudinis, Bacillus mycoides, Bacillus toyonensis, Bacillus safensis, Bacillus methylotrophicus, Bacillus mojavensis, Bacillus psychrosaccharolyticus, Bacillus galliciensis, Bacillus lentus, Bacillus siamensis, Bacillus tequilensis, Bacillus firmus, Bacillus aerophilus, Bacillus altitudinis, Bacillus stratosphericus, Bacillus velezensis, Brevibacillus brevis, Brevibacillus formosus, Brevibacillus laterosporus, Brevibacillus nitrificans, Brevibacillus agri, Brevibacillus borstelensis, Lysinibacillus xylanilyticus, Lysinibacillus parviboronicapiens, Lysinibacillus sphaericus, Lysinibacillus fusiformis, Lysinibacillus boronitolerans, Paenibacillus alvei, Paenibacillus Validus, Paenibacillus amylolyticus, Paenibacillus lautus, Paenibacillus peoriae, Paenibacillus tundrae, Paenibacillus daejeonensis, Paenibacillus alginolyticus, Paenibacillus pini, Paenibacillus odorifer, Paenibacillus endophyticus, Paenibacillus xylanexedens, Paenibacillus illinoisensis, Paenibacillus thiaminolyticus, Paenibacillus barcinonensis, Sporosarcina globispora, Sporosarcina aquimarina, Sporosarcina psychrophila, Sporosarcina pasteurii, and Sporosarcina saromensis.

9. The formulation according to claim 2, wherein said at least one bacterium is selected from Bacillus subtilis, Bacillus velezensis, Bacillus amyloliquefaciens, Bacillus firmus, Bacillus pumilus, Bacillus simplex, and Paenibacillus polymyxa.

10. The formulation according to claim 2, further comprising Xanthan Gum.

11. The formulation according to claim 2, further comprising Magnesium Aluminum Silicate, bentonite, attapulgite, or silica as a thickener.

12. A process for making a formulation according to claim 2 comprising: A) providing a solid powder of Bacterium spores, B) dispersing said spores in a continuous phase comprising at least one glycol, C) adding at least one surfactant S, D) optionally adding at least one thickener, E) optionally adding further auxiliaries, wherein steps C), D) and E) can be carried out at any time during the process.

13. A method of combating fungi, insects or nematodes using a formulation according to claim 2.

14. A method of treating seeds using a formulation according to claim 2.

15. Aqueous formulation comprising at least one active ingredient and xanthan gum, wherein said formulation comprises less than 50 wt % of water.

Description

EXAMPLES

[0198] Materials Used:

[0199] Bacillus amyloliquefaciens MBI 600 NRRL B-50595 spore powder with a viable spore content of approximately 1 E+12 cfu/g.

[0200] Bacillus subtilis BU 1814 ATCC PTA-11857 spore powder

[0201] Ethylene glycol

[0202] 1,2-Propylene glycol

[0203] 1,3-Propyene glycol

[0204] Diethylene glycol

[0205] Triethylene glycol

[0206] PEG200 (polyethylene glycol with an average molar mass of 200 g/mol (from OH number)).

[0207] Glycerol (Propane-1,2,3-triol)

[0208] Surfactant A (Polyoxyethylene (20) sorbitan monooleate)Silicone antifoam emulsion

[0209] Xanthan gum powder

[0210] Magnesium Aluminum Silicate A: Magnesium Aluminum Silicate NF Type IA

[0211] Magnesium Aluminum Silicate B: Magnesium Aluminum Silicate NF Type IA, non-irradiated

[0212] Biocide solution (containing Methylisothiazolinone and Benzisothiazolinone, 250 ppm each)

[0213] The analysis of colony forming units follows standard microbiological procedures (e.g. FDA Bacteriological Analytical Manual, Chapter 3, Aerobic Plate Count). First, a dilution series of the sample is produced and the dilutions are plated onto agar and incubation at a characteristic growth temperature. Upon incubation, colonies form and are counted for all plates with 20 to 200 colonies. Each colony is defined to represent one initial viable spore or cell. The count is multiplied by the dilution factor to obtain the number of cfu per gram or milliliter of the original sample.

Examples 1a to 1h: Preparation of Aqueous Formulations of Bacterium Spores and Microbiological Stability

[0214] Water and glycol were mixed and half of the antifoam was added to the mixture. The Bacillus amyloliquefaciens spores were added under stirring and then homogenized with a rotor-stator equipment. The formulation samples were finalized by adding the second half of antifoam and in case of recipe 1d and 1h the isothiazolinone biocide.

[0215] All formulation samples were evaluated for cfu/g initially. The samples were then stored in an incubator for 12 days at 30° C. and reevaluated for cfu/g and visually. After additional 17 days storage at room temperature (21+−2° C.), all formulation samples were evaluated again. All formulation samples except those containing 500 g/kg of 1,2-propylene glycol or glycerol had developed a strange characteristic scent. In the case of 1,2-propylene glycol the scent was similar to that of alcohol or ketone solvents. In the case of glycerol, the scent was that of decomposed organic matter, and moreover, a reddish color developed in the suspension. From examples 1a to 1h (table 1, amounts given in grams [g]), only the formulations containing 500 g/kg glycol (i.e. propylene glycol or glycerol) (recipes 1c and 1g) can be considered microbiologically stable.

TABLE-US-00001 TABLE 1 Ingredients (g) Recipe 1a Recipe 1b Recipe 1c Recipe 1d Recipe 1e 1,2-Propylene 50.00 150.00 500.00 50.00 glycol Glycerol 50.00 Biocide (MIT/BIT) 2.00 B. amyloliquefaciens MBI600 110.00 110.00 110.00 110.00 110.00 Silicone Antifoam 5.00 5.00 5.00 5.00 5.00 Water, Deionized ad 1000 g ad 1000 g ad 1000 g ad 1000 g ad 1000 g cfu/g initial 1.1E+11 1.7E+11 6.3E+10 1.3E+11 1.3E+11 12 days @30° C. cfu/g 1.1E+11 7.1E+10 5.6E+10 7.8E+10 1.1E+11 visual appearance homogeneous homogeneous homogeneous homogeneous reddish color scent characteristic gas development. scent bad smell 17 days @21° C. cfu/g 8.0E+10 6.7E+10 6.0E+10 9.5E+10 6.3E+10 visual homogeneous homogeneous homogeneous homogeneous reddish color scent characteristic characteristic characteristic gas development, scent scent scent bad smell Ingredients (g) Recipe 1f Recipe 1g Recipe 1h 1,2-Propylene glycol Glycerol 150.00 500.00 50.00 Biocide (MIT/BIT) 2.00 B amyloliquefaciens MBI600 110.00 110.00 110.00 Silicone Antifoam 5.00 5.00 5.00 Water, Deionized ad 1000 g ad 1000 g ad 1000 g cfu/g initial 1.2E+11 8.4E+10 1.1E+11 12 days @30° C. cfu/g 9.4E+10 1.0E+11 1.4E+10 visual appearance homogeneous homogeneous reddish color scent gas development. bad smell 17 days @21° C. cfu/g 8.8E+10 7.5E+10 1.3E+10 visual reddish color homogeneous reddish color scent gas development, bad gas development, bad smell smell

Examples 2a to 2f: Preparation of Bacillus Spore Dispersions with High Propylene Glycol Content

[0216] The samples 2a, 2b, 2c and 2d were prepared as follows: A direct dispersion of Bacillus spore powder in the respective complete mixture of 1,2-propylene glycol and water was carried out. First, water and glycol were mixed, and the spore powder was added and dispersed by stirring and further homogenized by a rotor-stator equipment with a target particle size d50 value of about 2 μm. Without a sufficient amount of water, a sufficient deagglomeration was not possible (2a and 2b), while with increasing water amount (2c and 2d) the deagglomeration with shear becomes much more efficient and at a ratio of water to 1,2-propylene glycol of 1:1 the desired particle size can be reached.

[0217] For samples 2e and 2f, a premix of 50 g Bacillus spore powder dispersed and homogenized in a mixture of 199.5 g water and 199.5 g 1,2-propylene glycol was prepared in a first step. The particle size distribution measured for this 11% Bacillus spore premix had a d50 of 2.1 μm and a d90 of 54.2 μm. The final formulation 2e then was prepared by addition of 241.9 g 1,2-propylene glycol to 197.5 g of Bacillus spore premix. In the case of formulation 2f, 0.5 g Xanthan Gum dispersed in 241.9 g of 1,2-propylene glycol were added to 197.5 g of Bacillus spore premix. The sample 2f was stirred for 1 h for the Xanthan Gum to hydrate. The initial viscosity increased from 26 to 80 mPas by addition of the Xanthan Gum, showing that even with the low amount of water in this formulation the Xanthan gum could still be hydrated.

[0218] After 11 days storage at room temperature, samples 2a and 2b showed phase separation, whereas samples 2c and 2d, with a higher water content and deagglomeration, as well as samples 2e and 2f with a similar water to 1,2-propylene glycol content as 2b, but prepared from a deagglomerated premix were still homogenous. In case of the samples 2c, 2e and 2f, the particle size distribution showed some agglomeration with the d50 value increasing.

[0219] After further 52 days room temperature storage, samples 2e and 2f were reevaluated visually. While sample 2e was completely sedimented, sample 2f was still perfectly homogeneous, showing the physical stabilization effect of the Xanthan gum added.

[0220] Compositions and properties of examples 2a to 2f are given in table 2 (amounts given in % w/w).

TABLE-US-00002 TABLE 2 Recipe % (wfw) 2a 2b 2c Bacillus 5 5 5 amyloliquefaciens MBI 600 1,2-Propylene 95 75 60 glycol Water 20 35 Xanthan Gum Premix Not possible No No initial Visual evaluation fast sedimentation homogeneous homogeneous D90 (μm) 68.5 65.5 42.7 D50 (μm) 33.0 28.8 5.11 viscosity 97 57 119 (mPas at 100s − 1) 11 d RT Visual evaluation flocculated, serum phase separation, homogeneous 54% sediment D90 (μm) 70.6 67.6 46.5 D50 (μm) 33.6 30.0 13.6 viscosity 99 58 117 (mPas at 100s − 1) 63 d RT Visual evaluation — — — Recipe % (w/w) 2d 2e 2f Bacillus 5 5 5 amyloliquefaciens MBI 600 1,2-Propylene 47.5 75 75 glycol Water 47.5 20 19.9 Xanthan Gum 0.1 Premix No Yes Yes initial Visual evaluation homogeneous homogeneous homogeneous D90 (μm) 39.3 54 (Premix) 54 (Premix) D50 (μm) 1.68 2.1 (Premix) 2.1 (Premix) viscosity 135 26 80 (mPas at 100s − 1) 11 d RT Visual evaluation homogeneous homogeneous homogeneous D90 (μm) 40.9 56.4 60.3 D50 (μm) 1.8 19.6 25.8 viscosity 138.7 26 88 (mPas at 100s − 1) 63 d RT Visual evaluation — completely homogeneous sedimented, redispersible

Examples 3a to 3d: Preparations with and without Spore Premix in Comparison

[0221] To show the effect of premix preparation, 1% Bacillus spore dispersions were prepared from two different batches of Bacillus amyloliquefaciens MB1600 powder with and without premix. The samples 3a and 3c were prepared as follows: A direct dispersion of Bacillus spore powder in the respective complete mixture of 1,2-propylene glycol and water was carried out. First, water and glycol were mixed, and the spore powder was added and dispersed by stirring and further homogenized by a rotor-stator equipment with a target particle size d50 value of about 2 μm. A sufficient deagglomeration was not possible and the samples showed quick sedimentation even directly after preparation.

[0222] For samples 3b and 3d, a premix of 15 g Bacillus spore powder dispersed and homogenized in a mixture of 285 g water and 75 g 1,2-propylene glycol was prepared in a first step. The final formulations 3b and 3d then were prepared by addition of the residual 1125 g 1,2-propylene glycol to 375 g of Bacillus spore premix.

[0223] For samples 3b and 3d, prepared from a premix, lower particle size values (d50 and d90) could be reached as compared to samples 3a and 3c, where the dispersion was carried out directly in the final 1,2-propylene-glycol to water mixture. Samples 3b and 3d also were homogeneous after preparation in contrast to samples 3a and 3c that displayed quick sedimentation. Compositions and properties of examples 3a to 3d are given in table 3 (amounts given in % w/w).

TABLE-US-00003 TABLE 3 Recipe % (w/w) 3a 3b 3c 3d Bacillus amyloliquefaciens 1 1 1 1 MBI 600 1,2-Propylene glycol 80 80 80 80 Water 19 19 19 19 Premix No Yes No Yes Spore batch A A B B initial Visual evaluation Inhomogeneous, homogeneous Inhomogeneous, homogeneous fast sedimentation opaque liquid fast sedimentation opaque liquid D90 (μm) (with sonication) 83.6 (40.4) 37.2 (35.2) 78.6 (62.1) 46.1 (50.7) D50 (μm) (with sonication) 43.9 (4.2) 3.4 (2.8) 38.7 (25.7) 1.4 (1.4) viscosity (mPas at 100s − 1) 26 47 26 51 cfu/g 8.1E+9 1.4E+10 1.7E+10 1.8E+10

Examples 4a to 4f: Preparation of Microbiologically and Physically Stable Spore Dispersions

[0224] The samples 4a to 4f were prepared as follows: First, a 5% premix of spore powder was dispersed and homogenized with a rotor-stator equipment in a mixture of 77.2 parts of water and 17.9 parts of 1,2-propylene glycol. Then, the remaining amount of 1,2-propylene glycol was added and the sample stirred until homogeneous. In case of samples 4b and 4e, part of the 1,2-propylene glycol was replaced by the surfactant A. In case of 4c and 4f the Xanthan gum thickener in the remaining 1,2-propylene glycol was added to the premix and stirred for one hour. The samples were stored under increased temperature stress for 14 days at 54° C. In case of samples 4a and 4d without Xanthan gum, strong flocculation and accordingly phase separation occurred. Samples 4c and 4f on the contrary stayed homogeneous. The particle size distribution after storage changed slightly, with some soft flocculation (visible or redispersible under sonication). This shows that formulations with a low particle size can be prepared by a premix concept with sufficient amount of water for hydration and kept homogeneous by the addition of Xanthan gum. Surfactant A is compatible with the system but provides no significant advantage over the formulation without any surface active compound. A separate subsample of each formulation 4a to 4f was exposed to 35° C. for 1 week, and the viable spore count in cfu/g evaluated. It did not decrease or increase significantly, and no odor or coloring was developed, showing that the formulations are also microbiologically stable.

[0225] Compositions and properties of examples 4a to 4f are given in table 4 (amounts given in % w/w).

TABLE-US-00004 TABLE 4 Recipe (% w/w) 4a 4b 4c 4d Bacillus amyloliquefaciens 1 1 1 1 MBI 600 1,2-Propylene glycol 59 54 59 80 Water 40 40 39.9 19 Surfactant A 5 Xanthan Gum 0.1 initial Visual evaluation homogeneous homogeneous homogeneous homogeneous opaque liquid opaque liquid opaque liquid opaque liquid D90 (μm) (with sonication) 45.7 (45.4) 50.1 (43.1) 49.8 (46.4) 47.6 (48.0) D50 (μm) (with sonication) 5.1 (1.6) 6.7 (1.6) 6.6 (1.6) 2.2 (1.9) viscosity (mPas at 100s − 1) 12 22 56 35 cfuig 8.1E+9 1.4E+10 1.3E+10 1.3E+10 14 d 54° C. Visual evaluation flocculation flocculation homogeneous flocculation and phase and phase opaque liquid. and phase separation, separation, with slight separation, sediment, sediment, syneresis sediment, red ispersible red ispersible red ispersible D90 (μm) (with sonication) 46.7 (47.6) 46.7 (47.6) 48.3 (45.7) 44.9 (49.9) D50 (μm) (with sonication) 10.3 (2.1) 11.3 (1.9) 9.7 (2.1) 6.9 (2.4) viscosity (mPas at 100s − 1) 20 24 60 42 1 week 35° C. cfu/g 9.6E+9 7.4E+9 1.2E+10 1.4E+10 Recipe 4e 4f Bacillus amyloliquefaciens 1 1 MBI 600 1,2-Propylene glycol 75 80 Water 19 18.9 Surfactant A 5 Xanthan Gum 0.1 initial Visual evaluation homogeneous homogeneous opaque liquid opaque liquid D90 (μm) (with sonication) 56.2 (44.8) 55.6 (39.4) D50 (μm) (with sonication) 4.8 (1.6) 2.3 (1.7) viscosity (mPas at 100s − 1) 45 101 cfu/g 1.4E+10 1.5E+10 14 d 54° C. Visual evaluation flocculation homogeneous and phase opaque liquid separation, with few sediment, translucent redispersible spots, easily redispersible D90 (μm) (with sonication) 46.7 (47.6) 51.2 (45.0) D50 (μm) (with sonication) 13.7 (2.1) 8.6 (1.9) viscosity (mPas at 100s − 1) 49 121 1 week 35° C. cfu/g 1.6E+10 1.3E+10

Examples 5a to 5d: Preparation of Microbiologically and Physically Stable Spore Dispersions with Alternative Glycol and Thickening Agents

[0226] The samples 5a to 5c were prepared as follows: First, a 10% premix of spore powder of Bacillus amyloliquefaciens MBI 600 was dispersed in a mixture of 80 parts of water and 10 parts of polyethylene glycol with a mean molecular weight of 200 g/mol (from OH number) (PEG 200). A thickener premix was added to the spore powder premix and the complete mixture was homogenized with a rotor-stator equipment until a final particle size of D50 of around 2 μm was reached. In the case of fumed silica, the thickener premix consisted of 9.1% thickening agent, 60.6% PEG 200 and 30.3% water. In the case of Magnesium Aluminum Silicate the thickener premix consisted of 9.1% thickening agent in 90.9% water.

[0227] Finally, the remaining amount of polyethylene glycol and water were added and the sample stirred until homogeneous.

[0228] The sample 5d was prepared as follows:

[0229] First, a 10% premix of spore powder was dispersed in a mixture of 80 parts of water and 10 parts of polyethylene glycol with a mean molecular weight of 200 (PEG 200). A thickener premix was added to the spore powder premix and the complete mixture was homogenized with a rotor-stator equipment until a final particle size of D50 of around 2 μm was reached.

[0230] Finally, the remaining amount of polyethylene glycol, water and antifoam were added and the sample stirred until homogeneous.

[0231] Compositions and properties of examples 5a to 5d are given in table 5 (amounts given in grams [g]).

[0232] The samples were stored for 2 weeks at 40° C. They stayed homogeneous showing only slight syneresis and could be completely rehomogenized with only three inversions. Also, no odor or coloring was developed, showing that the formulations are microbiologically stable.

TABLE-US-00005 TABLE 5 Recipe (g) 5a 5b 5c 5d Bacillus amyloliquefaciens 30 30 30 30 MBI 600 PEG 200 600 600 600 604 Water 418 418 418 389.5 Fumed Silica 10 10 Magnesium Aluminum 10 Silicate A Magnesium Aluminum 10 Silicate B Surfactant A 19.5 Antifoam vegetable 5.0 oil basis initial Visual evaluation homogeneous homogeneous homogeneous homogeneous opaque liquid opaque liquid opaque liquid opaque liquid D90 (μm) (with sonication) 46.9 (48.1) 44.6 (42.9) 45.3 (43.0) 44.0 (48.8) D50 (μm) (with sonication) 1.7 (1.5) 2.9 (2.0) 2.9 (1.6) 1.7 (1.5) viscosity (mPas at 100s − 1) 157 151 187 137 2 weeks at 40° C. Visual evaluation slight synere- slight synere- slight synere- slight synere- sis, easily sis, easily sis, easily sis, easily red ispersible red ispersible red ispersible red ispersible D90 (μm) (with sonication) 48.1 (48.9) 44.4 (41.9) 45.2 (45.5) 43.8 (46.5) D50 (μm) (with sonication) 1.9 (1.6) 4.0 (2.2) 2.1 (1.8) 2.2 (1.9) viscosity (mPas at 100s − 1) 120 112 161 134

Examples 6a to 6d: Preparation of Microbiologically and Physically Stable Bacillus Amyloliquefaciens Spore Dispersions with Alternative Glycol and Thickening Agents, 12 Weeks 40° C. Shelf Life

[0233] For examples 6a to 6d, Bacillus amyloliquefaciens MBI 600 spore powder was dispersed in pure water to give a 10% spore premix. To 120 grams of this spore premix complete the samples, 40 grams of thickener premix containing 8 parts of Xanthan Gum in 392 parts of water, were added. To finalize the samples, 193.6 g of the respective glycol and 46.4 g of water were added.

[0234] Compositions and properties of examples 6a to 6d are given in table 6 (amounts given in grams [g]).

[0235] The samples were stored for 12 weeks at 40° C. They stayed homogeneous showing only slight syneresis and could be completely rehomogenized with only three inversions. Also, no odor or coloring was developed, showing that the formulations are microbiologically stable. Also, a determination of cfu every 4 weeks during storage showed that the spores stay viable in this mixture at elevated temperatures.

TABLE-US-00006 TABLE 6 6a 6b 6c 6d Bacillus amyloliquefaciens 3 3 3 3 MBI 600 Ethylene glycol 48.4 1.3-Propylene glycol 48.4 Triethylene glycol 48.4 PEG 200 48.4 Xanthan Gum 0.2 0.2 0.2 0.2 Water 48.4 48.4 48.4 48.4 initial Homogeneous yes yes yes yes Serum/redispersible no/— no/— no/— no/— Sediment/redispersible no/— no/— no/— no/— Cfu/g 3.5 E+10 3.1 E+10 3.5 E+10 3.7 E+10 4 weeks at 40° C. Homogeneous no no no no Serum/redispersible yes/yes yes/yes yes/yes yes/yes Sediment/redispersible no/— no/— no/— no/— Cfu/g 2.9 E+10 3.2 E+10 3.1 E+10 3.1 E+10 8 weeks at 40° C. Homogeneous no no no no Serum/redispersible yes/yes yes/yes yes/yes yes/yes Sediment/redispersible no/— no/— no/— no/— Cfu/g 2.4E+10 2.5 E+10 3.1 E+10 3.1 E+10 12 weeks at 40° C. Homogeneous no no no no Serum/redispersible yes/yes yes/yes yes/yes yes/yes Sediment/redispersible no/— no/— no/— no/— Cfu/g 1.5E+10 1.7 E+10 2.1 E+10 2.0 E+10

Examples 7a to 7d: Preparation of Microbiologically and Physically Stable Bacillus subtilis BU 1814 Spore Dispersions with Alternative Glycol and Thickening Agents, 12 Weeks 40° C. Shelf Life

[0236] For examples 6a to 6d, Bacillus subtilis BU1814 spore powder was dispersed in pure water to give a 10% spore premix. To 120 grams of this spore premix complete the samples, 40 grams of thickener premix containing 8 parts of Xanthan Gum in 392 parts of water, were added. To finalize the samples, 193.6 g of the respective glycol and 46.4 g of water were added.

[0237] Compositions and properties of examples 7a to 7d are given in table 7 (amounts given in grams [g]).

[0238] The samples were stored for 12 weeks at 40° C. They stayed homogeneous showing only slight syneresis and could be completely rehomogenized with only three inversions. Also, no odor or coloring was developed, showing that the formulations are microbiologically stable. Also, a determination of cfu every 4 weeks during storage showed that the spores stay viable in this mixture at elevated temperatures.

TABLE-US-00007 TABLE 7 Recipe (g) 7a 7b 7c 7d Bacillus amyloliquefaciens 3 3 3 3 MBI 600 1.2-Propylene glycol 48.4 1.2-Butylene glycol 48.4 Triethylene glycol 48.4 PEG 200 48.4 Xanthan Gum 0.2 0.2 0.2 0.2 Water 48.4 48.4 48.4 48.4 initial Homogeneous yes yes yes yes Serum/redispersible no/− no/− no/− no/− Sediment/redispersible no/− no/− no/− no/− Cfu/g 1.4E+10 1.8E−10 1.4 E+10 1.4 E+10 4 weeks at 40° C. Homogeneous no no no no Serum/redispersible yes/yes yes/yes yes/yes yes/yes Sediment/redispersible no/− no/− no/− no/− Cfu/g 2.2E+10 2.2E+10 1.6E+10 1.7E+10 8 weeks at 40° C. Homogeneous no no no No Serum/redispersible yes/yes yes/yes yes/yes yes/yes Sediment/redispersible no/− no/− no/− no/− Cfu/g 1.1E+10 1.1 E+10 1.2E+10 1.0 E+10 12 weeks at 40° C. Homogeneous no no no no Serum/redispersible yes/yes yes/yes yes/yes yes/yes Sediment/redispersible no/− no/− no/− no/− Cfu/g 1.1E+10 1.9E+10 1.3E+10 9.8E+9