Use of polyglycerol esters as carriers for microbiological active ingredients

Abstract

Compositions containing at least one polyglycerol ester and preferably, at least one emulsifier, can be used as a carrier for an active microbiological ingredient. Compositions containing both the carrier and the active microbiological ingredient are useful; and methods of increasing the storage stability of the active microbiological ingredient can be used with the composition. These compositions can be also used for the treatment of plants, for the treatment of seed, for the treatment of soils, as a biostimulant, as a probiotic food supplement, or as a probiotic animal feed additive.

Claims

1: A composition, comprising at least one active microbiological ingredient, and a carrier, comprising at least one polyglycerol ester, and preferably at least one for the at least one active microbiological ingredient.

2. The composition according to claim 1, wherein the at least one polyglycerol ester has an HLB value of not more than 8.

3. The composition according to claim 1, whereinacyloxy radicals of the at least one polyglycerol ester have 4 to 40 carbon atoms.

4. The composition according to claim 1, wherein the at least one polyglycerol ester is a compound of the general formula (I)
M.sub.aD.sub.bT.sub.c  Formula (I); with: M=[C.sub.3H.sub.5(OR).sub.2O.sub.1/2]; D=[C.sub.3H.sub.5(OR).sub.1O.sub.2/2]; T=[C.sub.3H.sub.5O.sub.3/2]; a=1 to 10 b=0 to 10, c=0 to 3, preferably 0 to 1, especially 0; with the proviso that: a+b+c=2 to 20; and wherein the R radicals are each independently selected from the group consisting of acyl radicals R′—C(═O)— and H, with the proviso that at least one R radical is not H; and wherein the R′ radicals are each independently selected from the group consisting of monovalent aliphatic, saturated or unsaturated hydrocarbon radicals having 3 to 39, carbon atoms.

5. The composition according to claim 1, wherein the carrier comprises at least one emulsifier and wherein the at least one emulsifier has an HLB value of not less than 9.

6. The composition according to claim 1, wherein the carrier comprises at least one emulsifier. and wherein the at least one emulsifier is selected from the group consisting of a sorbitan fatty acid ester and an ethoxylated sorbitan fatty acid ester.

6. The composition according to claim 6 wherein the acyloxy radicals of the sorbitan fatty acid ester or the ethoxylated sorbitan fatty acid ester have 4 to 40, carbon atoms, and/or wherein the sorbitan fatty acid ester or the ethoxylated sorhitan fatty acid ester has 0 to 40, oxyethylene groups.

8. The composition according to claim 1, wherein the at least one polyglycerol ester is triglycerol trioleate- and/or wherein the carrier comprises at least one emulsifier, and the at least one emulsifier is polyethylene glycol-20 sorbitan trioleate.

9. The composition according to claim 1, wherein a proportion by mass of the at least one polyglycerol ester based on the total mass of the carrier is 60% to 100%; and a proportionby mass of emulsifier based on the total mass of the carrier is 0% to 40%.

10. 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.

11. The composition according to claim 1, wherein the at least one active microbiological ingredient is selected from the group consisting of Triehoderina harzianum, Bacillus angloliquefaciens, Beauveria bass ,Metarnizium rrieyl Aletarhizium anisopliae, Clonostachys rosea, Aureobasidiuin puilulans, Coillothyrium minitans, and their organs.

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

13. The composition according to claim 1, wherein the at least one active microbiological ingredient comprises spores of Trichoaerina harzianum.

14. The composition according to claim 1, wherein the a.sub.w value of the carrier is less than 0.4.

15. (canceled)

16. A method of increasing the storage stability of at least one active microbiological ingredient, the method comprising: storing at least one active microbiological ingredient by using the composition according to claim 1.

17. (canceled)

18: The composition according to claim 1, wherein the composition is a composition for treatment of plants, seed, or soil; a biostimulant; a probiotic food supplement, or a probiotic animal feed additive.

19: The composition according to claim 1, wherein the composition is a probiotic medicament.

20: The composition according to claim 1, wherein the carrier comprises at least one emulsifier.

21: The composition according to claim 9, wherein the carrier comprises at least one emulsifier, and wherein the proportion by mass of the at least one polyglycerol ester based on the total mass of the carrier is 75% to 85%, and the proportion by mass of the at least one emulsifier based on the total mass of the carrier is 15% to 25%.

11: The composition according to claim 11, wherein the organs are selected form the group consisting of spores, conidia, blastospores, chlamydospores, sclerotia, and hypha segments.

Description

EXAMPLES

Carrier Composition (Inventive)

[0174] The inventive carrier composition used was BREAK-THRU® SP 133 from Evonik, a mixture of 80% by weight of triglycerol trioleate and 20% by weight of polyethylene glycol-20 sorbitan trioleate.

Determining the Activity of Water of Compositions

[0175] To determine the activity of water of a sample, the air humidity is measured directly above a sample after attainment of equilibrium relative humidity (partial water vapour pressure differential). Equilibrium relative humidity (ERF) is measured in % relative humidity and is related to the a.sub.w value by the following relationship: a.sub.w=ERH/100. The activity of water in the compositions was determined using the LabMaster-aW neo from Novasina.

Production of the Compositions with Trichoderma harzianum

[0176] 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 1.97×10.sup.9 germinable spores/g of product. The inventive active ingredient composition composed of BREAK-THRU® SP 133 and spores of Trichoderma harzianurn was produced as follows: 3.60 g of the powder containing spores were weighed into a 50 ml centrifuge tube (e.g. sterile 50 ml tubes from Greiner Bio-One GmbH), and blanketed with 26.40 g of BREAK-THRU® SP 133. 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 1.95×10.sup.8 germinable spores/g. The compositions of the comparative examples were produced analogously. For the comparative examples, glycerol (ultrapure, min. 98%, anhydrous; Bernd Kraft GmbH), PEG 400 (Kollisolve® PEG E 400; Sigma Aldrich, average molar mass 320-420 g/mol), Pluronic® PE 6400 (BASF) and sunflower oil (food quality) were used as liquid carrier. The commercial WP formulation of Trichoderma harzianum was Trianum® P (Koppert).

Determination of Storage Stability

[0177] The compositions produced with spores of Trichoderma harzianum were incubated at 40° C. for four weeks, and the number of colony-forming units (CFU) was determined immediately after production (starting value) and after 7, 14, 21 and 28 days. 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. To determine the number of colony-forming units (CFU), by the plating method, 1.0 g of the sample material was 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, The three dilution levels of 10.sup.6, 10.sup.−7 and 10.sup.−8 (1.0 ml of each) were plated onto ready-made nutrient medium (Compact Dry YM for yeasts and mould fungi or Compact Dry Total Count from Nissui Pharmaceutical Co., Ltd.). The fungal spores were incubated at 25° C. for three days. Those plates on which 10-100 CFU are visible were evaluated. Table 1 shows the percentage of colony-forming units (in CFU/g) based on the starting value, as a measure of the survival rate or for the storage stability of the composition. The results shown are arithmetic averages from a triple determination.

TABLE-US-00001 TABLE 1 Storage stability experiments Proportion of germinable spores after storage at 40° C.: .sup.3) After After After After 7 14 21 28 Composition a.sub.w days days days days BREAK-THRU ® SP133 0.22 21% 19% 20% 12% (88% by wt.) + T. harzianum spores (12% by wt.) .sup.1) T. harzianum spores.sup.2) 0.33 73% 30%  7%  2% Glycerol (80% by wt,) + T. hazianum 0.10  2% 0.2%  n.d..sup.4) n.d..sup.4) spores (20% by wt.).sup.2) PEG 400 (80% by wt.) + T. harzianum 0.05 n.d..sup.4) 21%  9%  2% spores (20% by wt.).sup.2) Pluronic ® PE 6400 (80% by wt) + 0.20 26% 18%  8%  1% T. harzianum spores (20% by wt).sup.2) Sunflower oil (80% by wt.) + 0.40 50% 15% 10% 0.4%  T. harzianum spores (20% by wt.).sup.2) Commercially available WP 0.27 19%  7%  2%  2% formulation of T. harzianum.sup.2) .sup.1) inventive example .sup.2) comparative example .sup.3) expressed as the percentage of colony-forming units (in CFU/g) based on the starting value .sup.4) not determined

[0178] The inventive example BREAK-THRU® SP 133 shows a distinct improvement in the survival rate after 28 days at 40° C. compared to the comparative examples including a commercially available Trichoderma Harzianum WP formulation.

Determination of Microbe Count in the Presence of Adjuvants

[0179] A determination of microbe count in the presence of 1% by weight of adjuvants was used to determine the effect thereof on the germinability or viability of different commercial microorganisms under application-relevant conditions. Comparative examples used were the Nu-film®-P (Intrachem Bio Deutschland GmbH) and Wetcit (Oro Agri) adjuvants that have likewise been approved for biological cultivation. The commercial formulations were diluted with sterile physiological NaCl solution (0.9% by weight) with addition of 1.0% by weight of adjuvant 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 Nissul Pharmaceutical Co., Ltd,), Fungal spores and yeast cells 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, Microbe count was determined as the arithmetic average from a triple determination. The microbe count was used to determine the percentage change in the number of colony-forming units by using the microbe count in the presence of BREAK-THRU® SP 133 as reference and setting it to 100%.

TABLE-US-00002 TABLE 2 Relative change in the number of colony-forming units of various commercial microorganisms with addition of adjuvants with BREAK-THRU ® SP 133 as reference BREAK- THRU ® SP Active ingredient 133.sup.1) Nu-film ®.sup.2) WETCIT ® .sup.2) Aureobasidium 100% 44% <0.01% pullulans (Botectol) Bacillus 100% 97% <0.01% amyloliquefaciens (FZB24) Coniothyrium minitans 100% 53% <0.01% (Contans) Trichoderma 100% 74% <0.02% harziarium (Trianum P) .sup.1) inventive example .sup.2) comparative example

[0180] The results show that BREAK-THRU® SP 133 is very mild compared to other adjuvants and the growth of various microorganisms is not impaired.

Determination of Retention

[0181] In order to verify whether the inventive carrier composition BREAK-THRU® SP 133 is able to improve the retention of spray liquors on the crop plant, a study was conducted at the Plant Protection Chemistry institute in New Zealand (PPCNZ) The model plant used was spinach (Spinacia oleracea var. Perpetual). Based on contact angle measurements on the adaxial leaves of the plant, spinach is rated as “moderately difficult to wet”. For comparison, a polyether-modified trisiloxane (BREAK-THRU® 240, Evonik) was employed as a conventional adjuvant known from the prior art. The spinach plants were bought as 4-week-old seedlings from a garden centre and sown in individual pots. These were then cultivated further in a growing chamber under controlled environmental conditions (20° C. by day, 15° C. by night, 75% rh, with a photoperiod lasting for 12 h). At the time of the experiment, the plants were then 6 weeks old and 7 cm high.

[0182] Retention was determined using a moving head track sprayer that sprayed the spray liquors made up onto the trial plants. The dye tartrazine was added to the spray liquors used in an amount of 8 g/l. Each variant included 25 repetitions/plant and was treated at 100 l/ha by means of a tlat-jet air induction nozzle (Al 95015EVS) at pressure 250 kPa and a flow rate of 0.56 l/min. The nozzles were secured 50 cm above the height of the plants (7 cm). In order to verify the application rate, artificial collectors (plastic dishes repeated four times per variant) were secured horizontally at the level of the average plant height.

[0183] After deployment of the spray liquor, three leaves were collected by sampling from each repetition and washed immediately with deionized water. The artificial collectors were also washed with deionized water immediately after application.

[0184] The tartrazine dye added to the spray liquor was then quantified by spectrophotometry at 427 nm.

[0185] The leaf area was determined by means of a leaf area measuring device (Licor LI 3100A). Then the amount of spray remaining was calculated (table 3). The results were calculated by means of variance analysis and LSD test (P=0.05) using the Statistix computer program. If necessary, the data were transformed prior to the analysis.

TABLE-US-00003 TABLE 3 Retention trials Retention Concentration in Retention Additive % w/v μl/cm.sup.2 in % BREAK-THRU ® SP 133.sup.1) 0.1 0.68 a 68.2 a BREAK-THRU ® S 240.sup.2) 0.1 0.56 b 55.6 b .sup.1) inventive example .sup.2) comparative example

[0186] The results given in Table 3 show that the retention of the spray liquor with the inventive carrier BREAK-THRU® SP 133 is significantly improved compared to the comparative example BREAK-THRU® S 240 on spinach leaves.