Biocompatible siloxanes for formulation of microorganisms

Abstract

Compositions containing at least one siloxane and at least one active microbiological ingredient are useful for the treatment of plants, of seeds or of soils, as biostimulant or as probiotic food supplement or animal feed additive, or as probiotic medicament. In addition, the siloxane improves the storage stability of an active microbiological ingredient.

Claims

1. A composition, comprising: at least one active microbiological ingredient, and at least one siloxane of Formula (I)
M.sup.1.sub.aM.sup.2.sub.bD.sup.1.sub.cD.sup.2.sub.d  Formula (I) wherein: M.sup.1=R.sup.1.sub.3SiO.sub.1/2; M.sup.2=R.sup.1.sub.2R.sup.2.sub.1SiO.sub.1/2; D.sup.1=R.sup.1.sub.2SiO.sub.2/2; D.sup.2=R.sup.1R.sup.2SiO.sub.2/2; a=0 to 2; b=0 to 2; c=1.5 to 100; d=0.5 to 25; with the proviso that: a+b=0 to 2; and a+b+c+d≥4; R.sup.1 is in each case independently a monovalent hydrocarbyl radical; R.sup.2 is in each case independently a monovalent polyether radical.

2. The composition according to claim 1, wherein, as a further proviso: c+d=2 to 125; and/or 1≤c/d≤6.

3. The composition according to claim 1, wherein the at least one active microbiological ingredient is selected from the group consisting of microorganisms, organs of microorganisms and mixtures thereof.

4. The composition according to claim 1, wherein the at least one active microbiological ingredient has an antagonistic and/or hyperparasitic effect directed against a particular pathogen.

5. The composition according to claim 1, wherein the at least one active microbiological ingredient increases resistance and/or stress tolerance and/or nutrient availability in plants.

6. The composition according to claim 1, wherein the at least one active microbiological ingredient is selected from the group consisting of fungi, fungal organs and mixtures thereof.

7. The composition according to claim 1, wherein the at least one active microbiological ingredient is selected from the group consisting of Trichoderma spp. and its fungal organs and mixtures thereof.

8. The composition according to claim 1, wherein the at least one active microbiological agent comprises a fungal organ selected from the group consisting of spores, conidia, blastospores, chlamydospores, sclerotia, hyphal segments and mixtures thereof.

9. The composition according to claim 1, wherein the at least one active microbiological ingredient is or comprises a bacterium or a mixture of various bacteria.

10. The composition according to claim 1, wherein the at least one active microbiological ingredient comprises spores.

11. The composition according to claim 1, wherein the at least one active microbiological ingredient comprises vegetative cells.

12. The composition according to claim 1, wherein the at least one active microbiological ingredient comprises spores of Trichoderma harziamum and/or of Bacillus amyloliquefaciens and/or vegetative cells of Pseudomonas fluorescens.

13. The composition according to claim 1, wherein the at least one active microbiological ingredient is or comprises a virus or a mixture of various viruses.

14. A process for producing the composition according to claim 1, the process comprising: mixing the at least one active microbiological ingredient and the at least one siloxane of Formula (I).

15. The process according to claim 14, wherein i) the at least one active microorganism is cultivated; ii) optionally the cultivated microorganism is processed by suitable separation, drying, grinding and/or dispersion methods; and iii) the cultivated and optionally processed microorganism is suspended and/or dispersed in a siloxane of Formula (I).

16. The process according to claim 15, wherein the at least one microorganism and/or at least one organ of the microorganism, is processed ii) and after processing, is isolated by sieving, filtration, windsifting and/or centrifugation methods.

17. The process according to claim 14, further comprising reacting a composition comprising at least one SiH-functional siloxane of Formula (IV)
M.sup.1.sub.aM.sup.3.sub.bD.sup.1.sub.cD.sup.3.sub.d  Formula (IV) wherein: M.sup.1=R.sup.1.sub.3SiO.sub.1/2; M.sup.3=R.sup.1.sub.2HSiO.sub.1/2; D.sup.1=R.sup.1.sub.2SiO.sub.2/2; D.sup.3=R.sup.1HSiO.sub.2/2; wherein: the indices a, b, c and d are as defined for Formula (I); R.sup.1 is as defined for Formula (I); in the manner of a hydrosilylation with at least one unsaturated, polyether, so as to obtain a composition comprising the at least one siloxane of Formula (I).

18. A method, comprising: i) treating plants; or ii) treating seeds; or iii) treating soils; or iv) producing as biostimulant; or v) producing as probiotic food supplement and/or probiotic animal feed additive with the composition according to claim 1.

19. A probiotic medicament, comprising: a composition according to claim 1.

20. A process for improving the storage stability of at least one active microbiological ingredient, the process comprising: producing the composition of claim 1 by combining the at least one active microbiological ingredient with the at least one siloxane of Formula (I).

Description

EXAMPLES

General Methods:

Determination of the Composition of the SiH-Functional Siloxanes:

[0203] The siloxanes can be characterized with the aid of .sup.1H NMR and .sup.29Si NMR spectroscopy. These methods, especially taking account of the multiplicity of the couplings, are familiar to the person skilled in the art.

[0204] The proportion by mass of the SiH-functional siloxanes can be determined with the aid of a gas chromatography method (GC method) in which the substances are separated according to their boiling point and detected by means of a thermal conductivity detector. This is done by analysing an aliquot of the sample to be examined without further dilution by means of GC. This is conducted in a gas chromatograph equipped with a split/splitless injector, a capillary column and a thermal conductivity detector, under the following conditions:

TABLE-US-00001 Injector: 290° C., split 40 ml Injection volume: 1 μl Column: 5 m * 0.32 mm HP5 1 μm Carrier gas: helium, const, flow, 2 ml/min Temperature program: 1 minute at 80° C., then 80° C.-300° C. at 30° C./min, then conditioning at 300° C. for 10 minutes. Detector: TCD at 320° C. Make-up gas 6 ml/min Reference gas 18 ml/min

[0205] The SiH-functional siloxanes are separated according to their boiling point. The proportion by mass of the individual substances is determined as the percentage of the peak areas determined for the respective substance by comparison with the total area of all substances detected (area % method).

Determination of the Number of Colony-Forming Units (CFU)

[0206] To determine the number of colony-forming units (CFU), 1.0 g of the compositions according to the invention is diluted with sterile physiological saline (0.9% by weight of NaCl in water) in a decimal dilution series down to the level of 10.sup.−8. Typically, the 10.sup.−8, 10.sup.−7 and 10.sup.−8 dilution levels (1.0 ml of each) are plated onto ready-made nutrient medium (Compact Dry YM for yeasts and mould fungi or Compact Dry Total Count from Nissui Pharmaceutical Co., Ltd.). Fungal spores are incubated at 25° C. for three days, bacteria at 30° C. for one day. Plates on which 10-100 CFU are visible are evaluated.

SiH-Functional Siloxanes:

Example a-0

[0207] 29.7 g of poly(methylhydrosiloxane) (CAS: 63148-57-2, Gelest Inc., Code HMS-992 M.sub.eq.=63.8 g/mol SIH, i.e. 63.8 g based on the number of SiH groups) were mixed with 32.7 g of hexamethyldisiloxane and 37.6 g of octamethylcyclotetrasiloxane, and 0.1 g of trifluoromethanesulfonic acid (purity: 99%) was added. The mixture was stirred at room temperature for 24 h. Subsequently, 2 g of NaHCO.sub.3 were added and the mixture was stirred for 4 h. The mixture was filtered. A clear liquid was obtained. The siloxane was characterized with the aid of .sup.29Si NMR spectroscopy. A siloxane of the general empirical formula Me.sub.3SiO[SiMe.sub.2O].sub.2.4[SiMeHO].sub.2.2SiMe.sub.3 was obtained.

Example b-0

[0208] 800 g of octamethylcyclotetrasiloxane were mixed with 180 g of polymethylhydrosiloxane (CAS 63148-57-2. M.sub.eq.=63.8 g/mol SiH, i.e. 63.8 g based on the number of SiH groups) and 35 g of hexamethyldisiloxane under a protective gas blanket. Subsequently, 2 g of trifluoromethanesulfonic acid (purity: 99%) were added. The mixture was stirred at room temperature for 24 h. Subsequently, 20 g of NaHCO.sub.3 were added and the mixture was stirred for 4 h. Thereafter, the product was filtered and the filtrate obtained was freed of volatile constituents on a rotary evaporator at 130° C. at a pressure of 1 mbar. The product obtained was characterized with the aid of .sup.29Si NMR spectroscopy and the GC method. The empirical formula is ([SiMe.sub.2O].sub.0.61[SiMeHO].sub.0.19).sub.x (x=4 to 6). The distillate contains 92% by weight of SiH-functional cyclic siloxanes, i.e. siloxanes of formula (I) with a+b=0. The siloxanes are in the form of a mixture of 4-, 5- and 6-membered rings. The siloxanes are thus of formula (I) with c+d=4, 5 or 6.

Example c-0

[0209] 17.8 g of poly(methylhydrosiloxane) (CAS: 63148-57-2, Gelest Inc., Code HMS-992 M.sub.eq.=63.8 g/mol SiH, i.e. 63.8 g based on the number of SiH groups) were mixed with 3.5 g of hexamethyldisiloxane and 78.7 g of octamethylcyclotetrasiloxane, and 0.1 g of trifluoromethanesulfonic acid (purity: 99%) was added. The mixture was stirred at room temperature for 24 h. Subsequently, 2 g of NaHCO.sub.3 were added and the mixture was stirred for 4 h. The mixture was filtered. A clear liquid was obtained. The siloxane was characterized with the aid of .sup.29Si NMR spectroscopy. A siloxane of the empirical formula Me.sub.3SiO[SiMe.sub.2O].sub.38[SiMeHO].sub.10SiMe.sub.3 was obtained.

Polyethersiloxanes

General Remarks:

[0210] The polyethersiloxanes were prepared in the examples which follow by hydrosilylation. This was done by reacting SiH-functional siloxanes with an unsaturated polyether. The hydrosilylation reaction was conducted in the presence of a complete platinum(0)-1,3-divinyl-1,1,3,3-tetramethydisiloxane solution in xylene (purchased from Sigma-Aldrich, Pt content: 2% by weight) as Karstedt catalyst. The hydrosilylation reaction was brought to full conversion in relation to the hydrogen content of the SiH-functional siloxanes. In the context of the present invention, full conversion is understood to mean that more than 99% of the SiH functions were converted. Detection is effected in the manner familiar to the person skilled in the art by gas-volumetric means after alkaline breakdown.

Example a-1

[0211] 152.3 g of a polyether of the empirical formula CH.sub.2═CHCH.sub.2O[C.sub.2H.sub.5O].sub.6[CH.sub.2CH(CH.sub.3)O].sub.3H were mixed with 47.7 g of an SiH-functional linear siloxane from Example a-0 in a 500 ml three-neck flask with precision glass stirrer and reflux condenser under a nitrogen blanket. The mixture was then heated to 90° C. Subsequently, 0.04 g of a solution of the Karstedt catalyst in xylene (Pt content 2% by weight) was added to the mixture. An exothermic reaction set in. The mixture was then stirred at 90° C. for 2 h. A clear liquid was obtained. It was no longer possible to detect any SiH functions by gas-volumetric means. The conversion of SiH functions was 100%. A siloxane of the empirical formula Me.sub.3SiO[SiMe.sub.2O].sub.2.4[SiMeR.sup.2O].sub.2.2SiMe.sub.3 with R.sup.2═—CH.sub.2CH.sub.2CH.sub.2O[C.sub.2H.sub.5O].sub.6[CH.sub.2CH(CH.sub.3)O].sub.3H was obtained.

Example b-1

[0212] 109.9 g of a polyether of the empirical formula CH.sub.2═CHCH.sub.2O[C.sub.2H.sub.5O].sub.6[CH.sub.2CH(CH.sub.3)O].sub.3H were mixed with 60 g of the cyclic SiH-functional siloxane of the empirical formula ([SiMe.sub.2O].sub.0.81[SiMeHO].sub.0.19).sub.x (x=4 to 6) from Example b-0 in a 250 ml three-neck flask with precision glass stirrer and reflux condenser under a nitrogen blanket. The mixture was then heated to 90° C. Subsequently, 0.03 g of a solution of the Karstedt catalyst in xylene (Pt content 2% by weight) was added to the mixture. An exothermic reaction set in. The mixture was then stirred at 90° C. for 2 h. A clear liquid was obtained. The conversion of SiH functions was 100%. A siloxane of the empirical formula ([SiMe.sub.2O].sub.0.81[SiMeR.sup.2O].sub.0.19).sub.x (x=4 to 6) with R.sup.2═—CH.sub.2CH.sub.2CH.sub.2O[C.sub.2H.sub.5O][CH.sub.2CH(CH.sub.3)O].sub.3H was obtained.

Example a-1

[0213] 262 g of a polyether of the empirical formula CH.sub.2CHCH.sub.2O[C.sub.2H.sub.5O].sub.13.9[CH.sub.2CH(CH.sub.3)O].sub.5.3H were mixed with 70 g of a siloxane of the empirical formula Me.sub.3Si[SiMe.sub.2O].sub.38[SiMeHO].sub.10SiMe.sub.3 from Example c-0 in a 500 ml three-neck flask with precision glass stirrer and reflux condenser under a nitrogen blanket. The mixture was heated to 90° C. Subsequently, 0.16 g of a solution of the Karstedt catalyst in xylene (Pt content 2% by weight) was added to the mixture. An exothermic reaction set in. The mixture was then stirred at 90° C. for 2 h. A clear liquid was obtained. The conversion of SiH functions was 100%. A siloxane of the empirical formula Me.sub.3SiO[SiMe.sub.2O].sub.38[SiMeR.sup.2O].sub.10SiMe.sub.3 with R.sup.2═—CH.sub.2CH.sub.2CH.sub.2O[C.sub.2H.sub.5O].sub.13.9[CH.sub.2CH(CH.sub.3)O].sub.5.3H was obtained.

Production of the Compositions with T. harzianum Spores

Examples a-2 to c-2

[0214] Spores of the Trichoderma harzianum fungus were sourced from Rhizo-Mic UG and contained, according to elemental analysis, apart from the spores, about 75% by weight of SiO.sub.2. The powder contained 3×10.sup.9 germinable spores/g of product. The compositions of polyethersiloxanes and spores of T. harzianum were produced as follows: 5.00 g of spores were weighed into a 50 ml centrifuge tube (e.g. sterile 50 ml tubes from Greiner Bio-One GmbH), and blanketed with 20.00 g of the polyethersiloxane a-1, b-1 or c-1. The mixture was mixed on a vortex shaker (lab dancer from ika) for 30 seconds. After homogenization with a spatula, the composition, after a wait time of 15 minutes, was mixed again on a vortex shaker for 30 seconds. The composition produced contained 6×10.sup.8 germinable spores/g.

Determination of Storage Stability

[0215] The compositions a-2 to c-2 produced, which comprise spores of Trichoderma harzianum in polyethersiloxanes, were incubated at 40° C. for four weeks, and the number of colony-forming units was determined immediately after production (starting value) and after 7, 14, 21 and 28 days. The corresponding procedure was followed with the comparative examples. The number of colony-forming units (CFU) is a measure of the number of spores that were able, before or after storage, to germinate and form colonies. Table 1 shows the percentage of colony-forming units (in CFU/g) based on the starting value, as a measure of the viability rate or for the storage stability of the composition. The results shown are arithmetic averages from a triple determination.

TABLE-US-00002 TABLE 1 Storage stability of compositions with spores of Trichoderma harzianum Proportion of germinable spores after storage at 40° C.:.sup.1) After 7 After 14 After 21 After 28 Composition days days days days Example a-2 Example a-1 91% 77% 66% 20% (80% by wt.) + T. harzianum spores (20% by wt.) Example b-2 Example b-1 94% 89% 62% 41% (80% by wt.) + T. harzianum spores (20% by wt.) Example c-2 Example c-1 72% 58% 50% 18% (80% by wt.) + T. harzianum spores (20% by wt.) Comparative T. harzianum 73% 30%  7%  2% Example 1 spores Comparative Commercially 19%  7%  2%  2% Example 2 available WP formulation of T. harzianum .sup.1)expressed as the percentage of colony-forming units (in CFU/g) based on the starting value

[0216] The results show a distinct improvement in the compositions according to the invention over the pure spores (Comparative Example 1) and a commercial P formulation (Comparative Example 2).

Preparation of the Composition with Pseudomonas fluorescens

Example b-3

[0217] Pseudomonas fluorescens was cultivated in anaerobic submerged fermentation on double-concentrated LB medium at 25° C. and pH 7 up to an optical density of 13. Thereafter, the bacteria biomass was harvested from the fermentation broth by centrifugation at 8000 g for 10 minutes. The cell pellet was then resuspended in a sodium chloride solution (0.9% by weight) and added to a suspension of Sipernat®50 and gum arabic. The resulting suspension contained about 8% by weight of silica, 7% by weight of gum arabic, 3% by weight of dry biomass and 81% by weight of water. The suspension was then spray-dried in a laboratory spray-dryer (Büchi B-290) at a gas inlet temperature of 72° C. The spray application was effected with a two-phase nozzle at an atomization pressure of about 1.35 bar. The flow rate of the drying air was 38 m.sup.3/h. The spray rate was about 5 ml/min. The parameters set led to an exit temperature of 52° C. and a residual moisture content of the product of 4-7% by weight of water. To produce the composition from polyethersiloxane b-1 and spray-dried biomass of Pseudomonas fluorescens, 1 g of dried vegetative Pseudomonas fluorescens cells was mixed into 9 g of the polyethersiloxane from Example b-1. The Example b-3 composition thus prepared was incubated at 40° C. for four weeks and the number of colony-forming units was determined immediately after the preparation (start value) and after 7, 14, 21 and 28 days. The procedure was the same with the comparative example. Table 2 shows the percentage of colony-forming units (in CFU/g) based on the start value, as a measure of the survival rate or of the storage stability of the composition. The results shown are arithmetic averages from a double determination.

TABLE-US-00003 TABLE 2 Storage stability of compositions comprising Pseudomonas fluorescens Proportion of vegetative cells after storage at 40° C:.sup.1) After 7 After 14 After 21 After 28 Composition days days days days Example b-3 Example b-1 52% 39% 34% 23% (90% by weight) + spray-dried biomass of Pseudomonas fluorescens (10% by weight) Comparative spray-dried biomass 39% 18% 21% 11% example 3 of Pseudomonas fluorescens .sup.1)represented as percentage of colony-forming units (in CFU/g) based on the start. value

[0218] Even though Gram-negative bacterial cells such as Pseudomonas fluorescens are not known to be stable to heat or drying, the results show a clear improvement in the survival rate of Pseudomonas fluorescens as a constituent of the composition according to the invention compared to the spray-dried biomass on its own.

Germination Test in the Presence of Adjuvants

[0219] With the aid of a germination test in the presence of 1.0% by weight of adjuvant, the effect of the siloxanes of formula (I) on the germinability of various commercial microorganisms was examined under application-relevant conditions. For this purpose, the commercial formulations were diluted with sterile aqueous adjuvant solution (1.0% by weight) in a decimal dilution series in a ratio of 1:100 000 to 1:1 000 000 000 and plated out on a suitable ready-made nutrient medium (Compact Dry from Nissui Pharmaceutical Co., Ltd.). Fungal spores were incubated at 25° C. for three days, bacterial spores at 30° C. for one day. Plates on which 10-100 CFU are visible were evaluated. The results summarized in Table 3 show the germination rate as a percentage of colony-forming units (in CFU/g) based on the value without adjuvants.

TABLE-US-00004 TABLE 3 Germination test for various commercial microorganisms Germination rate in the presence of 1.0% by wt. of siloxane of formula (I) .sup.1) FZB24 ® WG Trichoderma (Bacillus Adjuvant harzianurn amyloliquefaciens) Example a-1 118% 124% Example b-1  18%  93% Example c-1  97% 100% Break Thru ® S240 .sup.2)  8%  9% .sup.1) expressed as the percentage of colony-forming units (in CFU/g) based on the value without adjuvants .sup.2) commercially available polyether-modified trisiloxane, from Evonik

[0220] The inventive adjuvants a-1, b-1 and c-1 show distinctly improved germination rates over the comparative example Break Thru@ S240. They show improved biocompatibility.

[0221] It is also found that the siloxanes of formula (I) retain moisture for much longer than polyether-modified trisiloxanes and hence offer better conditions for microorganisms.