Coated product form

Abstract

A product form comprises an inanimate substrate comprising i) an active agent; and ii) carrier particles including at least an outer surface comprising an organic matter constituent, wherein the said active agent is combined within and/or on the surface of the carrier particles, the carrier particles being a) in dry, particulate form and carrying at least an electrostatic surface charge, and b) being at least substantially uniformly distributed over the outer surface of the substrate.

Claims

1. A product form, comprising an inanimate substrate comprising i) an active agent; and ii) carrier particles comprising at least an outer surface comprising an organic matter constituent, wherein the said active agent is combined within and/or on the surface of the carrier particles, the carrier particles being a) in dry, particulate form and carrying at least an electrostatic surface charge, and b) being at least substantially uniformly distributed over the outer surface of the inanimate substrate, wherein the substrate is a fertiliser prill and wherein the active agent comprises a chemical pesticide or a biofertiliser, or a mixture thereof.

2. A product according to claim 1, wherein the biofertiliser comprises live bacteria, yeasts, fungi for aflatoxin management, blue green algae, live bacterial spores, yeast spores, fungal spores of use in the promotion of root and/or shoot growth from germinating seeds and/or enhancing root and shoot growth from germinated seeds, or mixtures of two or more thereof.

3. A product according to claim 1, wherein the carrier particles are particles comprising natural waxes, synthetic waxes, mineral waxes having a melting point of ≥50° C., polymers, biopolymers, soluble or insoluble starches, proteinaceous compounds, chitin, waxes, paraffin wax, beeswax, carnauba wax, lanolin, shellac wax, bayberry wax, sugar cane wax, ozocerite, ceresin wax, montan wax, candelilla wax, castor wax, microcrystalline wax, ouricury wax, rice bran wax, polyethylene wax, polypropylene wax, or mixtures of two or more thereof.

4. A product according to claim 1 wherein the active agent comprises live bacterial spores, yeast spores or fungal spores.

5. A product according to claim 1, wherein the particles have a median diameter of from 5 μm to 300 μm.

6. A method of producing a product form comprising i) mixing carrier particles comprising at least an outer surface comprising an organic matter constituent, said carrier particles being in dry, particulate form and carrying at least an electrostatic surface charge, with at least one active agent comprising a chemical pesticide or a biofertiliser, or a mixture thereof, in a ratio of 1:99 to 99:1 wt. %; and ii) adding the particles of i) to a substrate comprising a fertilizer prill to produce the product form wherein the active agent is combined within and/or on the surface of the carrier particles, and wherein the carrier particles are at least substantially uniformly distributed over the outer surface of the substrate.

7. A method according to claim 6, wherein the ratio of step i) is at least about 30:90 wt %.

8. A method according to claim 6, wherein the biofertiliser comprises live bacteria, yeasts, fungi for aflatoxin management, blue green algae, live bacterial spores, yeast spores, fungal spores of use in the promotion of root and/or shoot growth from germinating seeds and/or enhancing root and shoot growth from germinated seeds, or mixtures of two or more thereof.

9. A method according to claim 6, wherein step ii) comprises the substrate.

10. The method according to claim 6, wherein the carrier particles have a median diameter from 5 μm to 300 μm.

11. A product form comprising fertiliser prills, live biological agent and solid wax carrier particles wherein the carrier particles are adhered to the prills, the agent is adhered to the carrier particles, and the carrier particles are in dry form, carry an electrostatic surface charge and are at least substantially uniformly distributed over the outer surface of the fertiliser prills.

12. A product according to claim 11, wherein the carrier particles have a volume mean diameter in the range of 5-100 μm.

13. A product according to claim 11, wherein the solid wax carrier particles have a melting point of 50° C.

14. A product according to claim 11, wherein the size of the prills is in the range of 1-10 mm.

15. A product according to claim 11, wherein the solid wax particles further comprise a chemical agent dispersed within the solid wax particles.

16. A product according to claim 11, wherein the carrier particles have a volume mean diameter in the range of 5-100 μm.

Description

EXAMPLES

(1) The invention is illustrated in the following examples, with reference to the accompanying drawings, in which:

(2) FIG. 1 shows the loading of Bacillus onto pellets was two-fold higher in the Entostat® treatment group, compared to the group where spores only were applied;

(3) FIG. 2 shows that Bacillus spores were more uniformly distributed on pellets in the Entostat® treatment group, compared to the group where spores only were applied;

(4) FIG. 3 is a photograph of NPK fertiliser pellets prior to addition of Entostat®;

(5) FIG. 4 is another photograph of the NPK fertiliser pellets of FIG. 3;

(6) FIG. 5 is a photograph of the NPK fertiliser pellets after addition of Entostat® (carnauba variety);

(7) FIG. 6 is another photograph of the NPK fertiliser pellets after addition of Entostat® (carnauba variety);

(8) FIG. 7 is a photograph of the NPK fertiliser pellets after addition of Entostat® (polyethylene variety);

(9) FIG. 8 is a photograph of Polyhalite Sirius fertiliser prior to addition of Entostat®;

(10) FIG. 9 is a photograph of the Polyhalite Sirius fertiliser after addition of Entostat® (carnauba variety);

(11) FIG. 10 is a photograph of urea fertiliser prills prior to addition of Entostat®; and

(12) FIG. 11 is a photograph of the urea fertiliser prills after addition of Entostat® (carnauba variety);

(13) FIG. 12 is another photograph of the urea fertiliser prills after addition of Entostat® (carnauba variety);

(14) FIG. 13 is a photograph of the urea fertiliser prills after addition of Entostat® (polyethylene variety);

(15) FIG. 14 is a photograph of animal feed pellets prior to addition of Entostat®;

(16) FIG. 15 is a photograph of animal feed pellets after addition of Entostat® (carnauba variety);

(17) FIG. 16 is a photograph of animal feed pellets after addition of Entostat® (polyethylene* variety);

(18) FIG. 17 is a photograph of animal feed pellets after addition of Entostat® (polyethylene variety); and

(19) FIG. 18 is a photograph of animal feed pellets after addition of Entostat® (rice bran variety).

OBJECTIVES

(20) To demonstrate that inputs (biological or chemical) applied to a substrate (e.g. fertiliser prills/animal feed pellets) using Entostat® carrier (electrostatically charged) in a dry powder formulation are more uniformly distributed on the substrate (e.g. fertiliser prills/animal feed pellets) compared to application of inputs alone (e.g. spores only), or inputs in a typical dry powder formulation (e.g. spores in a Talc formulation or a chemical compound in a powder formulation).

Introduction and Study Outline

(21) Direct inoculation of a substrate (e.g. fertiliser prills/animal feed pellets) allows inputs such as nutrient use efficiency bacteria or probiotics to be applied directly to the substrate (e.g. fertiliser prills/animal feed) closer to the point of use. While the ability of the inputs to stick to the substrate is critical, even distribution of inputs is also vital. Uneven distribution of inputs may result in variation in their efficacy, as some plants or animals fail to receive the minimum effective dose while other plants or animals receive significantly more inputs than are actually required.

(22) Entostat® can be made from a range of micronized waxes, as discussed above, and has been developed as a carrier for bacteria, fungi, viruses and chemistries. Previous results have demonstrated that Entostat® facilitates better placement of biological and chemical input as the tribocharged Entostat® waxes adhere to the seeds and arthropods. The purpose of this study is to demonstrate that when applied to an inanimate substrate (e.g. fertiliser/animal feed) that the wax, and therefore the input, is also more uniformly distributed on the substrate compared to unformulated technical grade inputs. The tribocharged Entostat® wax used in this study is carnauba wax, unless otherwise stated.

(23) The Heubach Dustmeter is designed to simulate the mechanical stress associated with handling and distributing. This equipment was used in this study to establish the adhesion of test material to the substrate following mechanical stress.

Example 1

Application of a Probiotic/Direct Fed Microbe to Domesticated Animal Feed Comprising Cellulose and Lignin Content

(24) Test & Reference Item Details

(25) TABLE-US-00001 Test items 25% Bacillus subtilis spores (strain PY79) combined with 75% Entostat ® (1:3 w/w). Applied at a rate of 0.5000 g formulation per kg of animal feed pellet (substrate). Bacillus subtilis (strain PY79) was provided by Sporegen, UK. Entostat ® (carnauba) wax particles had a median diameter (X50) of 30 μm. Formulations were prepared at Exosect facilities by mixing the ingredients in a random orbital powder blender (e.g. turbula T10), with a blending time 1-8 hours. Reference 100% Bacillus subtilis (strain PY79) without a carrier (i.e. items pure spores)
Substrate—Animal Feed Pellets

(26) TABLE-US-00002 TABLE 1 Animal feed details Animal Feed Variety Source Batch Number Pellet Pasture cubes Dodson & Horrell, (12) 26C 105224 Northamptonshire, UK
Experimental Design

(27) Bacillus subtilis spores, formulated with Entostat®, were tested against unformulated Bacillus subtilis spores. There were 12 experiments, within each experiment there was 2-3 (bottles) per treatment (spores v Entostat®). For the purpose of analysis, experiment is the replicate (n=12). Treatments were mechanically stressed in the Heubach Dustmeter in a fully randomised order.

(28) The rate of spore material was kept at a constant 0.125 g per kg of animal feed in all formulations, which was equivalent to approximately 1.625×10.sup.8 spores per g of animal feed.

(29) TABLE-US-00003 TABLE 2 Experimental treatments Ratio spores Spore rate Carrier rate Formulation to carrier (g per 500 g (g per 500 g rate (g per Formulation (w/w) pellets) pellets) 500 g pellets) Entostat ® 1:3 0.0625 0.1875 0.2500 Spores only 1:0 0.0625 0.00 0.0625

(30) Treatments were weighed into sterile 1 L Duran bottles along with 500 g of animal pellets. Prior to use, the animal pellets had been equilibrated for a minimum of 48 hours at 20° C. After application of the treatment to the pellets the contents of the Duran bottles were homogenised for 30 seconds by gently agitating the bottles using the Stuart Rotator with MIX2040 attachment. Two pellets (approx. weight 0.3000-0.6000 g) were removed from each 500 g batch and placed into individual empty dilution bottles for enumeration to give a measure of adhesion before mechanical stress (‘Before Heubach’). One 100 g sample was removed from each 500 g batch to undergo mechanical stress (Heubach).

(31) The 100 g sample was mechanically stressed in the Heubach Dustmeter (Heubach GmbH, Heubachstrasse7, 38685 Langelsheim). Samples were poured into the drum of the Heubach. Heubach settings were: rotation speed 30 rpm, rotation time 120 seconds and airflow rate 20 l/min. A vacuum pump created an air flow through the rotating drum, the connected glass cylinder and the attached filter unit. Abraded dust particles were transported out of the drum via the air flow, through the glass cylinder, and subsequently through the filter unit. Coarse non-floating particles were separated and collected in the glass cylinder while the floating dust particles were deposited onto a filter. After the cycle period (120 seconds), treated pellets were removed from the rotating drum. Two pellets (approx. weight 0.3000-0.6000 g) were taken from each sample (bottle) and placed into individual empty dilution bottles for Colony Forming Unit (CFU) enumeration to give a measure of adhesion after mechanical stress (‘After Heubach’). Work involving the Heubach Dustmeter was conducted between 20° C. and 25° C. and 30% and 70% relative humidity.

(32) Sampling/Measurement Regime

(33) CFU Enumeration

(34) For each treatment, the four pellets collected for assessment (2 before and 2 after Heubach) were individually weighed and each of the individual pellets were transferred in a sterile 15 mL centrifuge tube containing 10 mL of sterile 5% Tween 80. In order to detach the spores from the pellet, the pellet was left to soak in this solution for 20 minutes. The disintegrated pellet was then suspended by vortexing for 30 seconds followed by sonicating for 3 minutes. From this solution 1 mL of the spore suspension was pipetted into a sterile vial containing 9 mL of dH.sub.2O (D1 dilution). To create a serial dilution 1 mL of D1 was pipetted into a sterile vial containing 9 mL of dH.sub.2O (D2 dilution). 1 mL of the D2 dilution was then pipetted to a sterile vial containing 9 mL of 0.05% Tween solution (D3 dilution).

(35) The number of CFUs per pellet was determined using the spread plate method. Nutrient agar plates (9 cm petri dishes containing 25 mL agar per plate) containing Cycloheximide (40 mg/L agar) were inoculate with 0.1 μL of each dilution using a sterile pipette (spread plate method). Cycloheximide was added to the agar prior to plate pouring (agar temp <50° C.) to reduce fungal contamination. The rate of cycloheximide (40 mg/L agar) was as per supplier recommendation (Thermo Scientific). Agar plates used to culture the bacteria were poured at least 4 hours prior to inoculation and incubated at room temperature (approx. 20° C.) to ensure that bacteria were not exposed to high temperatures. For each pellet 3 agar plates were inoculated for each dilution (D1-D3). Post inoculation, plates were incubate at 30° C. for 24 hours, until discrete colonies were distinguishable, after which time colonies were counted to determine the loading (CFU per g of pellet). Only plates with between 20 and 400 CFU's per plate were used in the assessment (minimum 3 plates used to generate an average value per treated pellet). CFU counts were conducted on treated pellets collected before and after the Heubach Test. Within each experiment, counts for each set of two pellets were averaged to generate a single value per bottle for pre and post Heubach.

(36) The standard deviation (SD) is a measure that is used to quantify the amount of variation in a set of data values. A low standard deviation indicates that the data points tend to be close to the mean or average value of the set, while a high standard deviation indicates that the data points are spread out over a wider range of values. To compensate for possible differences between sample means (i.e. loading rates) relative variation can be expressed as the coefficient of variation. The coefficient of variation (CV), also known as relative standard deviation (RSD), is a standardized measure of dispersion which shows the extent of variability in relation to the mean of the population. Coefficient of variation is calculated according to the following equation:

(37) $\mathrm{Cv}=\frac{\sigma }{\mu }$

(38) Where CV=coefficient of variation, σ=standard deviation and μ=sample mean

(39) The lower the coefficient of variation value, the more uniform the distribution on the pellet. Where coefficient of variation is expressed as a percentage, a value of 0% indicates complete uniformity/zero variation.

(40) Results

(41) The loading of Bacillus onto the pellet was twice as high in the Entostat® treatment, compared to where the spores only were applied (1.7×10.sup.8 CFU per g compared with 7.6×10.sup.7 CFU per g). This ratio was not influenced by mechanical stress.

(42) TABLE-US-00004 TABLE 3 The coefficient of variation (CV) for the number of Bacillus spores per g of animal feed Before Heubach (% After Heubach (% Formulation variation) variation) Spores only 32.6 42.5 Entostat ® & spores 25.4 23.2

(43) Even with a mixing time of just 30 seconds (‘Before Heubach’) spores were more uniformly distributed on the fertiliser prill where Entostat® was used as a carrier: variation in the Entostat® treatment was 22% lower than in the spores only treatment (coefficient of variation in spores only was 32.6, compared to 25.4% in the Entostat® treatment). Once pellets were subjected to mechanical stress (Heubach), which included an additional 120 seconds mixing time, the mixing time/adding mechanical stress actually decreased the uniformity in the spores only treatment (coefficient of variation rose from 32.6 to 42.5%, respectively). Conversely, increasing the mixing time/adding mechanical stress did not have an effect on the distribution of the bacteria on the animal feed pellets where the bacteria were formulated with Entostat® (coefficient of variation 25.4 and 23.2%). If we compare the distribution after mechanical stress (‘After Heubach’), the bacteria were almost twice as uniform when delivered with Entostat® as when delivered as pure spores (coefficient of variation 42.5 and 23.2%, respectively). This result demonstrates that Entostat® formulations deliver a more uniform distribution.

(44) The coefficient of variation data was non-normal in distribution, so was transformed (square root transformation) prior to analysis using a two-way ANOVA, with Heubach and Formulation as main effects. Formulation significantly affected how uniform the bacteria were distributed onto the animal feed pellets (F=4.104, df=1, P=0.0489), though mechanical stress (Heubach) did not (F=0.062, df=1, P=0.8045). There was no interaction between the main effects (F=0.897, df=1, P=0.3487).

Example 2

Application of Phosphate-Solubilizing Bacteria to Fertiliser Prills

(45) Phosphorus is one of the major nutrients required for plant growth, however, much of the phosphorus in the soil is unavailable to plants. Chemical fertilisers are commonly used to fill this deficiency, though restrictions around the use of chemical fertilisers are becoming common (e.g. E.U. Drinking water directive and Water Framework directive). Bolan and Duraisamy (2003) outlined how phosphate solubilizing bacteria (PSB) play a significant part in phosphorus nutrition by improving phosphorus accessibility to the plants through the release of organic and inorganic soil phosphorus pools by mineralization and solubilisation. Specifically, improved accessibility of phosphorus can be achieved by the microorganism increasing the solubility of inorganic phosphorus compounds; mineralizing organic compounds with release of inorganic phosphate; converting inorganic phosphate into cell components, and; oxidation or reduction of organic phosphorus compounds. Microorganisms which are known to be capable of solubilizing phosphate include certain bacteria (e.g. Alcaligenes, Burkholderia, Enterobactor, Pseudomonas and Bacillus) and fungi (e.g. Aspergillus, Fusarium, Penicillium and Rhizopus) (Khan et al., 2014).

(46) In field experiments conducted on maize investigating the integrated use of chemical, organic and biofertilisers, Jilan et al (2007) concluded that integration of half dose of chemical (NP) fertiliser with biofertilisers (including Bacillus sp.) can give similar crop yield as with full rate of the chemical fertiliser. In a study on wheat and maize, Kaur and Reddy (2015) reported that soil fertility, in the context of available P, enzyme activities and phosphate-solubilizing bacteria population, was significantly improved when phosphate-solubilizing bacteria were applied in conjunction with rock phosphate fertiliser, compared to plots which received a chemical P fertiliser (diammonium phosphate, DAP). The authors concluded that the combined use of the bacteria and rock phosphate was also more economical. Co-delivery of biofertilisers and chemical fertilisers may therefore offer opportunities to increase crop yields with reduce fertiliser inputs, especially given the increasingly stringent environmental compliance standards growers must now comply with in terms of where and when fertilisers can be applied.

(47) Test & Reference Item Details

(48) TABLE-US-00005 Test items 25% Bacillus subtilis spores (strain PY79) are combined with- 75% Entostat ® (1:3 w/w) and applied at a rate of 0.5000 g formulation per kg of fertiliser prill (substrate). Bacillus subtilis (strain PY79) is sourced from Sporegen, UK. Entostat ® (carnauba) wax particles have a median diameter (X50) of 30 μm. Formulations are prepared at Exosect facilities by mixing the ingredients in a random orbital powder blender (e.g. turbula T10), with a blending time 1-8 hours. Reference 100% Bacillus subtilis (strain PY79) without a carrier (i.e. items pure spores)
Substrate—Fertiliser Prills

(49) Fertiliser prills were acclimatised at room temperature for at least 48 hours prior to testing. Details of the prills used are in Table 4.

(50) TABLE-US-00006 TABLE 4 Fertiliser Prill details Fertiliser Type Variety Source Urea Prill Krista Yara, UK
Experimental Design

(51) Bacillus subtilis spores, formulated with Entostat®, are tested against unformulated spores (Table 5). The Entostat® formulation was applied at 0.0625 g per 500 g batch of prills, to give the same application weight as the unformulated spores. There were two experiments, and within each experiment there were 12 replicates per treatment (500 g batches). The two experiments used different batches of spores and formulations.

(52) The rate of formulation was kept at a constant 0.125 g per kg of fertiliser prill. This was equal to 1.63×10.sup.8 CFU/g fertiliser prills for the ‘spores only’ treatment and 0.41×10.sup.8 CFU/g fertiliser prills for the Entostat® 1:3 formulation.

(53) TABLE-US-00007 TABLE 5 Experimental treatments Ratio spores to Application rate Formulation carrier (w/w) (g per 500 g fertiliser) Entostat ® 1:3 0.0625 Spores only 1:0 0.0625

(54) Fertiliser prills were sieved using a 425 micron sieve before each experiment to reduce the amount of dust produced by the prills during the mechanical process. Treatments were weighed into 10 mL weighboats and transferred to sterile 1 L Duran bottles containing 500 g of sieved fertiliser prills. The treatments were homogenized for 30 seconds by gently agitating using the Stuart Rotator with MIX2040 attachment. One 100 g sample was removed from each 500 g batch.

(55) The 100 g sample is mechanically stressed in the Heubach Dustmeter (Heubach GmbH, Heubachstrasse7, 38685 Langelsheim). Samples are poured into the drum of the Heubach. Heubach settings are: rotation speed 30 rpm, rotation time 120 seconds and airflow rate 20 L/min. A vacuum pump creates an air flow through the rotating drum, the connected glass cylinder and the attached filter unit. Abraded dust particles are transported out of the drum via the air flow, through the glass cylinder, and subsequently through the filter unit. Coarse non-floating particles are separated and collected in the glass cylinder while the floating dust particles are deposited onto a filter. Work involving the Heubach Dustmeter was conducted at room temperature. After the mechanical stress cycle, treated fertiliser prills were removed from the rotating drum. Fifteen fertiliser prills (1 replicate) were removed from each 100 g batch and placed into individual empty dilution bottles for enumeration.

(56) CFU enumeration was conducted on treated fertiliser prills collected after the Heubach Test. For each treatment, fifteen fertiliser prills per replicate were collected for assessment and transferred in a sterile 15 mL centrifuge tube containing 10 mL of sterile 5% Tween 80. In order to detach the spores from the prill, the prills are left to soak in this solution for 20 minutes, after which time the centrifuge tube is vortexed for 30 seconds. From this solution 1 mL of the spore suspension is pipetted into a sterile vial containing 9 mL of dH.sub.2O (D1 dilution). To create a serial dilution 1 mL of D1 is pipetted into a sterile vial containing 9 mL of dH.sub.2O (D2 dilution). 1 mL of the D2 dilution is then pipetted to a sterile vial containing 9 mL of 0.05% Tween solution (D3 dilution).

(57) The number of CFUs per fertiliser prill are determined using the spread plate method. Nutrient agar plates (9 cm petri dishes containing 25 ml agar per plate) containing Cycloheximide (40 mg/L agar) are inoculated with 0.1 μL of each dilution using a sterile pipette (spread plate method). Cycloheximide is added to the agar prior to plate pouring (agar temp <50° C.) to reduce fungal contamination. The rate of cycloheximide (40 mg/l agar) is as per supplier recommendation (Thermo Scientific). Agar plates used to culture the bacteria were poured at least 4 hours prior to inoculation and incubated at room temperature (approx. 20° C.) to ensure that bacteria are not exposed to high temperatures. For each fertiliser prill 3 agar plates are inoculated for each dilution (D1-D3). Post inoculation, plates are incubated at 30° C. for 24 hours, until discrete colonies are distinguishable, after which time colonies are counted to determine the loading (CFU per g of pellet). Only plates with between 20 and 400 CFU's per plate are used in the assessment (2-3 plates used to generate an average value per treated prill). The CFU values in the Entostat® formulation were multiplied by 4 to correct for a different application rate to the unformulated spores.

(58) The standard deviation (SD) is a measure that is used to quantify the amount of variation in a set of data values. A low standard deviation indicates that the data points tend to be close to the mean or average value of the set, while a high standard deviation indicates that the data points are spread out over a wider range of values. To compensate for possible differences between sample means (i.e. loading rates) relative variation can be expressed as the coefficient of variation. The coefficient of variation (CV), also known as relative standard deviation (RSD), is a standardized measure of dispersion which shows the extent of variability in relation to the mean of the population. Coefficient of variation is calculated according to the following equation:

(59) $\mathrm{Cv}=\frac{\sigma }{\mu }$

(60) Where CV=coefficient of variation, σ=standard deviation and μ=sample mean

(61) The lower the coefficient of variation value, the more uniform the distribution on the pellet. Where coefficient of variation is expressed as a percentage, a value of 0% indicates complete uniformity/zero variation.

(62) Results

(63) Coefficient of Variation

(64) The Entostat® formulation had a lower coefficient of variation than unformulated spores in experiment 1 and experiment 2 (Table 6).

(65) TABLE-US-00008 TABLE 6 Coefficient of variation between the replicates in experiment 1 and 2 after mechanical stress created by the Heubach Dust Meter Coefficient of variation (%) Experiment Spores (0.0625 g) Entostat ® (0.0625 g) 1 111.451 53.718 2 71.793 35.633

(66) If we compare the distribution after mechanical stress (Table 6), the bacteria were twice as uniform when delivered with Entostat® as when delivered as pure spores, a trend that was repeated across both experiments (Experiment 1 coefficient of variation 54%% and 111%; Experiment 2 36% versus 72%). This result demonstrates that Entostat® formulations deliver a more uniform distribution.

Example 3

Application of Wax Particles to a Variety of Substrates

(67) While Heubach is a useful test for quantitatively demonstrating the uniformity of adherence of carrier particles to an inanimate substrate such as a fertiliser prill or animal feed pellet, it is an aggressive technique that results in the destruction of many prills, pellets and granules. The test is designed to simulate the effects of mechanical handling and distribution of the substrates, such as application to a field, but is generally more aggressive, leading to greater damage to the prills, pellets or granules than is typically observed. Another way of demonstrating the uniformly of the adherence of the carrier is by photographing coated prills, pellets and granules under a microscope.

(68) Experimental Design

(69) Entostat® was added to several types of fertiliser and animal feed as substrates set out below in Table 7. The Entostat® varieties and VMD are set out in Table 8, and the application rates are set out in Table 9. The Entostat® was added at a rate as set out below in Table 9. The Entostat® was weighted into sterile 1 L Duran bottle along with the substrate, and the contents of the bottles were shaken gently for approximately 30 seconds to distribute. A quantity of the substrate was then placed in a small dish and photographed under a microscope.

(70) TABLE-US-00009 TABLE 7 Fertilisers compositions Substrate Fertiliser pellets comprising 10% nitrogen, 2% phosphorous, 5% potassium, 2% MgO and 2% Fe, and obtained from by Progreen Weed Control. Mineral granules comprising 14% K2O, 19% Sulphur, 6% MgO, 17% CaO, sold under the name Polyhalite Sirus and obtained from by Sirius Minerals Polyhalite Sirus-http://siriusminerals.com/polyhalite/poly4-explained/ Urea prills comprising 46% nitrogen, and obtained from by Yara UK Limited. Prilled Urea Yara-http://www.yara.co.uk/crop-nutrition/fertiliser/nitrate/ 0149-yarabela-prilled-n/ Animal feed pellets comprising wheatfeed, nutritionally improved straw, barley, cane molasses, wheat, maize, limestone flour, peas, oatfeed, vegetable oil, salt, extracted sunflower, mint (0.8%), vitamin/trace mineral premix, garlic granules (0.5%), dried carrots (0.5%), calcined magnesite, l-lysine. The product is sold under the name “Pasture mix” for feeding to horses and ponies, and was obtained from Dodson & Horrell.

(71) TABLE-US-00010 TABLE 8 Entostat ® varieties and size Entostat ® Variety Size (VMD) Carnauba 24.89 μm Polyethylene  7.40 μm Polyethylene*  8.01 μm Rice bran 10.08 μm

(72) The wax was loaded onto the pellets, granules and prills as set out in the table below, using the Fig. number for identification.

(73) TABLE-US-00011 TABLE 9 loading of wax onto substrate Fig. No. Substrate Wax Loading Rate 3 NPK fertiliser — pellets 4 NPK fertiliser — pellets 5 NPK fertiliser carnauba 500 g substrate + 0.1875 g pellets wax 6 NPK fertiliser carnauba 500 g substrate + 0.1875 g pellets wax 7 NPK fertiliser polyethylene 10 g substrate + 0.1 g wax pellets 8 Polyhalite Sirius — granules 9 Polyhalite Sirius carnauba 500 g substrate + 0.1875 g granules wax 10 Urea prills — 11 Urea prills carnauba 500 g substrate + 0.1875 g wax 12 Urea prills carnauba 500 g substrate + 0.1875 g wax 13 Urea prills polyethylene 10 g substrate + 0.1 g wax 14 Animal feed pellets — 15 Animal feed pellets carnauba 15 g substrate + 0.1 g wax 16 Animal feed pellets polyethylene* 500 g substrate + 0.1875 g wax 17 Animal feed pellets polyethylene 15 g substrate + 0.1 g wax 18 Animal feed pellets rice bran 500 g substrate + 0.1875 g wax
Results

(74) The photographs are shown in FIGS. 3-18. As can be seen the simply act of gently shaking the pellets, granules and prills with the Entostat® results in a uniform distribution of the Entostat® over the surface of the prills and pellets. Importantly the distribution is substantially uniform over the surface of each pellets, granule or prill and over the mass of pellets, granules and prills.

(75) This experiment demonstrates that Entostat®, namely electrostatic wax particles made from a range of different types of wax, adheres to a variety of different substrates, namely fertilizer pellets, granules and prills and animal feed pellets, providing a substantially uniform coating across the substrate. Thus Entostat® is able to be used to append a range of chemicals, micronutrients and biological agents to the substrates that enables them to be tailored to their particular use, namely soil type, soil condition, intended use of soil, particular animal or condition of animal. This is not possible using mass manufacture as generally it would not be possible to incorporate live biological agents into these substrates as the live agents would not survive the manufacturing process, and the different range of chemicals, including pesticides for example, and micronutrients that can be added is too broad for commercial manufacturing.

(76) Additional Protocol

(77) TABLE-US-00012 Test 30% Beauvaria bassiana spores combined with 70% Entostat ® - items paraffin wax variety (3:7 w/w). Applied to milled maize kernels at a rate of 0.500 g formulation per kg. Entostat ® has a median diameter (X50) of 15 μm. Formulations were prepared at Enosect facilities by mixing the ingredients in a random orbital powder blended, with a blending time of 1 hour.
Results

(78) After mixing the kernels were viewed under a microscope and a uniform distribution of the particles over the milled kernels was observed.

(79) The invention hence provides product forms comprising substrates uniformly coated with particles carrying active agents, methods of obtaining such product forms, and uses thereof.

REFERENCES

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