COMPOSITIONS CONTAINING A HOLLOW GLUCAN PARTICLE OR A CELL WALL PARTICLE ENCAPSULATING A TERPENE COMPONENT, METHODS OF MAKING AND USING THEM
20200236927 · 2020-07-30
Inventors
Cpc classification
A01N49/00
HUMAN NECESSITIES
B01J13/203
PERFORMING OPERATIONS; TRANSPORTING
A61P31/00
HUMAN NECESSITIES
A01N31/08
HUMAN NECESSITIES
A01N31/16
HUMAN NECESSITIES
A61K9/5036
HUMAN NECESSITIES
A01N31/16
HUMAN NECESSITIES
A01N2300/00
HUMAN NECESSITIES
A01N35/02
HUMAN NECESSITIES
A61P43/00
HUMAN NECESSITIES
A01N35/06
HUMAN NECESSITIES
A01N2300/00
HUMAN NECESSITIES
A01N31/08
HUMAN NECESSITIES
A01N35/06
HUMAN NECESSITIES
A61K9/5068
HUMAN NECESSITIES
A01N35/02
HUMAN NECESSITIES
A01N49/00
HUMAN NECESSITIES
International classification
A01N49/00
HUMAN NECESSITIES
B01J13/20
PERFORMING OPERATIONS; TRANSPORTING
A61K9/50
HUMAN NECESSITIES
A01N31/08
HUMAN NECESSITIES
A01N35/02
HUMAN NECESSITIES
A01N31/16
HUMAN NECESSITIES
Abstract
The present invention relates to compositions comprising a hollow glucan particle or cell wall particle encapsulating a terpene component, methods of their manufacture and their use. The compositions are suitable for preventing and treating infections in plants and animals, including humans.
Claims
1. A composition comprising a hollow glucan particle or cell wall particle encapsulating a terpene component.
2. A composition according to claim 1 wherein the hollow glucan particle or cell wall particle is a fungal cell wall.
3. A composition according to claim 2 wherein the hollow glucan particle or cell wall particle is a yeast cell wall.
4. A composition according to claim 3 wherein the yeast cell wall is derived from a Baker's yeast cell.
5. A composition according to any preceding claim wherein the hollow glucan particle or cell wall particle is an insoluble waste product from a yeast extract manufacturing process.
6. A composition according to any one of claims any preceding claim wherein the hollow glucan particle or cell wall particle has been alkali extracted.
7. A composition according to any preceding claim wherein the hollow glucan particle or cell wall particle has been acid extracted.
8. A composition according to any preceding claim wherein the hollow glucan particle or cell wall particle has been organic solvent extracted.
9. A composition according to any preceding claim wherein the hollow glucan particle or cell wall particle has a lipid content of 1% or greater.
10. A composition according to claim 9 wherein the lipid content of the hollow glucan particle or cell wall particle is 5% w/w or greater.
11. A composition according to claim 10 wherein the lipid content is 10% w/w or greater.
12. A composition according to any preceding claim wherein the terpene component comprises one or more of the terpenes selected from the group consisting of citral, pinene, nerol, b-ionone, geraniol, carvacrol, eugenol, carvone (for example L-carvone), terpeniol, anethole, camphor, menthol, thymol, limonene, nerolidol, farnesol, phytol, carotene (vitamin A.sub.1), squalene, thymol, tocotrienol, perillyl alcohol, borneol, myrcene, simene, carene, terpenene, linalool or a mixture thereof.
13. A composition according to any preceding claim wherein the terpene component comprises a terpene having have the general structure C.sub.10H.sub.16.
14. A composition according to any preceding claim wherein the terpene component comprises one or more terpenes selected from the group consisting of geraniol, thymol, citral, carvone (for example L-carvone), eugenol, b-ionone or a mixture thereof.
15. A composition according to any preceding claim wherein the terpene component comprises a mixture of geraniol, thymol and eugenol.
16. A composition according to claim 14 wherein the terpene component comprises 100% thymol.
17. A composition according to claim 14 wherein the terpene component comprises 50% geraniol and 50% thymol w/w.
18. A composition according to claim 14 wherein the terpene component comprises 50% eugenol and 50% thymol w/w.
19. A composition according to claim 14 wherein the terpene component comprises 33% geraniol, 33% eugenol and 33% thymol w/w.
20. A composition according to claim 14 wherein the terpene component comprises 33% eugenol, 33% thymol and 33% citral w/w.
21. A composition according to claim 14 wherein the terpene component comprises 25% geraniol, 25% eugenol, 25% thymol and 25% citral w/w.
22. A composition according to claim 14 wherein the terpene component comprises 20% geraniol, 20% eugenol, 20% citral, 20% thymol and 20% L-carvone w/w.
23. A composition according to any preceding claim wherein the terpene component is associated with a surfactant.
24. A composition according to any preceding claim wherein the surfactant is selected from the group consisting of sodium lauryl sulphate, polysorbate 20, polysorbate 80, polysorbate 40, polysorbate 60, polyglyceryl ester, polyglyceryl monooleate, decaglyceryl monocaprylate, propylene glycol dicaprilate, triglycerol monostearate, polyoxyethylenesorbitan monooleate, Tween, Span 20, Span 40, Span 60, Span 80, Brig 30 or a mixture of two or more thereof.
25. A composition according to any preceding claim comprising 1 to 99% by volume terpenes, 0 to 99% by volume surfactant and 1 to 99% hollow glucan particles or cell wall particles.
26. A composition according to claim 25 comprising from about 10 to about 67% w/w terpenes, from about 0.1 to about 10% w/w surfactant and from about 40 to about 90% w/w hollow glucan particles or cell wall particles.
27. A composition according to any preceding claim suitable for killing bacteria or fungi.
28. A composition according to any preceding claim suitable for killing mold.
29. A composition according to any preceding claim, suitable for killing mycoplasma.
30. A composition according to any preceding claim wherein the terpenes used are food grade.
31. A composition according to any preceding claim comprising an additional food grade active compound.
32. A composition according to claim 31 wherein the additional food grade active compound is an antimicrobial agent or enzyme.
33. A composition according to any preceding claim comprising an antimicrobial agent, an anti-fungal agent, an insecticidal agent, an anti-inflammatory agent or an anaesthetic.
34. A composition according to any preceding claim further comprising an antioxidant.
35. A composition according to claim 34 wherein the antioxidant is rosemary oil, vitamin C or vitamin E.
36. A composition according to any preceding claim in the form of a dry powder.
37. A composition according to any one of claims 1 to 35 in a pellet, tablet or other solid form.
38. A composition according to any preceding claim comprising a dispersal agent which promotes dispersal of the composition when placed into a liquid.
39. A composition according to any preceding claim in combination with an agriculturally, food or pharmaceutically acceptable carrier or excipient in a liquid, solid or gel-like form.
40. A composition according to any one of claims 1 to 35 suspended or dissolved in a liquid.
41. A composition according to claim 40 wherein the liquid is water.
42. A composition according to either claim 40 or 41 comprising from about 500 to about 10,000 ppm hollow glucan particles or cell wall particles, where the particles contain from about 1 to about 67% terpene component.
43. A composition according to claim 42 comprising from about 1000 to about 2000 ppm hollow glucan particles or cell wall particles, where the particles contain from about 10 to about 50% terpene component w/w.
44. A composition according to any one of claims 40 to 43 comprising between about 1 ppm and about 25 ppt of the terpene component.
45. A composition according to claim 44 comprising between about 100 to 1000 ppm of the terpene component.
46. A composition according to any one of claims 1 to 39 which is dispersed in water, saline, aqueous dextrose, glycerol or ethanol to form a solution or suspension.
47. A composition according to claim any preceding claim which includes a wetting agent, an emulsifying agent or a pH buffering agent.
48. A composition according to any preceding claim dispersed in a liquid human or animal food or drink material.
49. A composition according to any preceding claim in a form suitable for oral administration.
50. A composition according to any one of claims 1 to 46 in a form suitable for parental administration.
51. A composition according to any one of claims 1 to 46 in a form suitable for topical administration.
52. A method of preparing a hollow glucan particle or cell wall particle encapsulating a terpene component, said method comprising the steps of; a) providing a terpene component; b) providing a hollow glucan particle or cell wall particle; c) incubating the terpene component with the glucan particle or cell wall particle under suitable conditions for terpene encapsulation; and d) recovering the glucan particle or cell wall particle encapsulating the terpene component.
53. A method according to claim 52 further comprising the step of drying the glucan particle or cell wall particle encapsulating the terpene component.
54. A method according to claim 53 wherein drying is achieved by freeze drying, fluidised bed drying, drum drying or spray drying.
55. A method according to any one of claims 52 to 54 wherein in step a) the terpene component is provided as a suspension in an aqueous solvent.
56. A method according to claim 55 wherein the terpene component is provided in association with a surfactant.
57. A method according to claim 56 wherein the surfactant is polyoxyethylenesorbitan monooleate at a concentration of about 0.1 to 10% by volume of the total reaction mixture.
58. A method according to any one of claims 52 to 54 wherein in step a) the terpene component is provided as a true solution in the aqueous solvent.
59. A method according to any one of claims 52 to 58 wherein in step b) the hollow glucan particle or cell wall particle is provided as a suspension in water or other suitable liquid.
60. A method according to claim 59 wherein the suspension comprises approximately 1 to 1000 mg glucan particle or cell wall particles per ml.
61. A method according to claim 59 wherein the particles are dispersed in a volume of from the hydrodynamic volume (HV) to 1.5 HV of liquid.
62. A method according to any one of claims 52 to 58 wherein in step b) the hollow glucan particle or cell wall particle is provided as a dry powder.
63. A method according to any one of claims 52 to 62 wherein in step c) the reaction is carried out at atmospheric pressure at a temperature of about 20 to 37 C.
64. A method of killing a microorganism, said method comprising the step of; contacting said microorganism with a composition comprising a hollow glucan particle or cell wall particle encapsulating a terpene component.
65. A method of treating or preventing infection of a plant, said method comprising the step of; administering, in a therapeutically effective dose, a composition comprising a hollow glucan particle or cell wall particle encapsulating a terpene component to the plant or to soil in proximity to the plant.
66. A method according to claim 65 wherein the infection of the plant is caused by a nematode.
67. A method according to claim 65 wherein the infection of a plant is caused by a fungus.
68. A method according to claim 67 wherein the fungus is downy mildew, powdery mildew or botrytis bunch rot.
69. A method according to any one of claims 65 to 68 wherein the plant is a grape vine.
70. A method according to any one of claims 65 to 69 wherein the composition is administered 21 days or less prior to harvest of a crop from the plant.
71. A method according to claim 70 wherein the composition is administered 14 days or less prior to harvest.
72. A method according to claim 71 wherein the composition is administered 7 days or less prior to harvest.
73. A method according to claim 72 wherein the composition is administered 3 days or less prior to harvest.
74. A method according any one of claims 65 to 73 wherein the composition is administered by spraying.
75. A method according to claim 74 wherein the composition is sprayed at a rate of 500 L/Ha or greater.
76. A method according to claim 75 wherein the composition is sprayed at a rate of 900 L/Ha or greater.
77. A method according to claim 76 wherein the composition is sprayed at a rate of 1200 L/Ha or greater.
78. A method according to any one of claims 65 to 73 wherein the composition is administered via irrigation.
79. The present invention further provides a method of preventing or treating an infection in a patient, said method comprising the step of; administering to said patient in a therapeutically effective dose, a composition comprising a hollow glucan particle or cell wall particle encapsulating a terpene component.
80. A method according to claim 79 wherein the infection of the patient is caused by Staphylococcus aureus, Aspergillius fumigatus, Mycoplasma iowae, Penicillium sp. or Mycoplasma pneumoniae.
81. A method according to claim 80 wherein the composition is administered orally, vaginally, rectally, by inhalation, topically or by parenteral routes.
82. A composition comprising a hollow glucan particle encapsulating a terpene component for use in the prevention or treatment of an infection in a patient or a plant.
83. Use of a hollow glucan particle encapsulating a terpene component in the manufacture of a medicament for the treatment of an infection in patient.
84. The use of claim 83 wherein the infection is caused by Aspergillius fumigatus, Scierotinta homeocarpa, Rhizoctonia solani, Colletotrichum graminicola or Penicillium sp.
Description
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[0144] The following examples are provided to further enable those of ordinary skill in the art to make or perform the present invention. They are purely exemplary and are not intended to limit the scope of the invention. Unless indicated otherwise, parts are parts by volume or parts by weight, as indicated, temperature is in degrees Celsius ( C.) or is at ambient temperature, and pressure is at or near atmospheric. There are numerous variations and combinations of the compositions and conditions for making or using them, e.g., component concentrations, desired solvents, solvent mixtures, temperatures, pressures, and other ranges and conditions that can be used to optimise the results obtained from the described compositions and methods. Only reasonable and routine experimentation will be required to optimise these.
Example 1Demonstration of Terpene Loading into Baker's Yeast Particles and Purified Yeast Glucan Particles
[0145] The following protocol was performed to demonstrate that terpenes would load into yeast cell walls and other hollow glucan particles.
[0146] Emulsions of citral and L-carvone were prepared by mixing 150 l of the terpene with 100 l of 10% Tween 80 in water and 250 l of water.
[0147] Baker's yeast particles (YP) or Levacan yeast glucan particles (YGP), available from Savory Systems International, Inc., Branchburg, N.J., were mixed with water to form a 250 mg/ml suspension.
[0148] 500 l of the YP or YGP suspension and 250 l of the terpene emulsion were mixed together and incubated overnight under constant agitation. 500 l YP or YGP suspension and 500 l of water were used as a control. The particles were then washed with water until free from external emulsion. The particle preparations were then frozen and lyophilised until dry.
[0149] The particles were then rehydrated and examined under light microscope. The results are shown in
[0150]
Example 2Determination of Maximal Citral and L-Carvone Loading Levels in Baker's Yeast Cell Wall Particles (YP)
[0151] The following protocol was performed to determine the maximal amounts of terpenes that would load into YP. [0152] L-carvone and citral emulsions were prepared by sonicating 4.5 g of the terpene with 0.3 ml water. [0153] 10% Tween-80 solution was prepared by sonicating 4.5 g Tween-80 in 40.5 mls water. [0154] YP suspension was prepared by mixing YP with water to form 20 mg/ml suspension. [0155] Encapsulation reactions were set up as described in Table 1.
[0156] Citral or L-carvone-water emulsion was mixed with YP and Tween 80 surfactant overnight at room temperature. Samples were centrifuged at 14,000g for 10 minutes and the appearance of free terpene floating on the aqueous layer was scored. The results are shown in the right hand column labelled free terpene of Table 1.
[0157] The expression free terpene refers to the visible presence of terpene in the centrifuged reaction mixture. The absence of free terpene indicates complete absorption of the terpene by the particles. The highest volume of terpene absorbed by the particles, as evidenced by the absence of free terpene, was recorded as the maximal volume of absorbed terpene emulsion.
TABLE-US-00001 TABLE 1 20 mg/ml 10% YP Terpene Vol Tween-80 Free Tube l Emulsion l l Terpene 1 500 500 2 500 L-carvone 0.5 500 3 500 L-carvone 1.65 500 4 500 L-carvone 5 495 5 500 L-carvone 16.5 483.5 6 500 L-carvone 50 450 + 7 500 L-carvone 165 335 + 8 500 L-carvone 500 + 9 500 Citral 0.5 500 10 500 Citral 1.65 500 11 500 Citral 5 495 12 500 Citral 16.5 483.5 +/ 13 500 Citral 50 450 + 14 500 Citral 165 335 + 15 500 Citral 500 +
[0158] As can be seen from the results, YP is capable of absorbing and encapsulating at least 16.5 l of L-carvone terpene emulsion or at least 5 l of citral emulsion per 10 mg of YP.
Example 3Demonstration of Improved Terpene Loading with Surfactant and Determination of Optimal Tween-80:Terpene Ratio
[0159] The following protocol was performed to demonstrate that the presence of surfactant improves terpene loading and to determine the minimum level of Tween-80 surfactant required for the YP terpene loading reaction. [0160] L-carvone and citral emulsions were prepared by sonicating 4.5 g of the terpene with 0.3 ml water. [0161] 10% Tween-80 solution was prepared by sonicating 4.5 g Tween-80 in 40.5 ml water. [0162] Baker's YP suspension was prepared by mixing YP with water to form 250 mg/ml suspension.
[0163] Loading reactions were set up as shown in Table 2 below.
[0164] Citral or L-carvone-water emulsion was mixed with YP with 0-10% v/v Tween 80 surfactant overnight at room temperature. Samples were centrifuged at 14,000g for 10 minutes and the appearance of free terpene floating on the aqueous layer was scored. The results are shown in the right hand column labelled free terpene of Table 2.
[0165] The expression free terpene refers to the visible presence of terpene in the centrifuged reaction mixture. The absence of free terpene indicates complete absorption and encapsulation of the terpene by the YP. The highest volume of terpene absorbed by the YP, as evidenced by the absence of free terpene, was recorded as the maximal volume of absorbed terpene emulsion.
TABLE-US-00002 TABLE 2 250 mg/ml 10% YP Terpene Vol Tween-80 Water Free Tube ml Emulsion l l l Terpene 1 500 500 2 500 L-carvone 150 0 350 Sl 3 500 L-carvone 150 5 345 Sl 4 500 L-carvone 150 10 340 Sl 5 500 L-carvone 150 33 317 Sl 6 500 L-carvone 150 100 250 7 500 L-carvone 150 200 150 8 500 L-carvone 150 350 9 500 L-carvone 400 0 100 ++ 10 500 L-carvone 400 5 95 ++ 11 500 L-carvone 400 10 90 ++ 12 500 L-carvone 400 33 77 ++ 13 500 L-carvone 400 100 + 14 500 L-carvone 400 20 l 100% 30 + 15 500 Citral 113 0 387 + 16 500 Citral 113 5 382 + 17 500 Citral 113 10 377 + 18 500 Citral 113 33 354 Sl 19 500 Citral 113 100 287 Sl 20 500 Citral 113 200 187 21 500 Citral 113 350 37 22 500 Citral 250 0 250 ++ 23 500 Citral 250 5 245 ++ 24 500 Citral 250 10 240 ++ 25 500 Citral 250 33 217 + 26 500 Citral 250 100 150 + 27 500 Citral 250 20 l 100% 230 + Sl = slight
[0166] As can be seen from the results a Tween-80 concentration of 1% (i.e. 100 l of 10% Tween-80 in 1000 l of reaction mixture) is sufficient to allow complete uptake of the terpene in the above reaction. A 2% Tween-80 causes no improvement in results, whereas with a 0.33% concentration free terpene was observed. This indicates that: [0167] a) Terpenes are absorbed into YP particles in the absence of a surfactant, but the presence of surfactant significantly increases terpene absorption. [0168] b) A Tween-80 concentration of around 1% is optimum for YP loading as it ensures proper loading whilst maximising the terpene payload of the YP particles.
Example 4Determination of Maximal Terpene Loading and Encapsulation at High Baker's Yeast Cell Wall Particles (YP) Levels
[0169] The following protocol was performed to determine the maximal amounts of terpenes that would load into YP at high YP levels. [0170] L-carvone and citral emulsions were prepared by sonicating 4.5 g of the terpene with 3 ml 1% Tween. [0171] 5% Tween-80 solution was prepared by sonicating 0.5 g Tween-80 in 9.5 ml water. [0172] YP suspension was prepared by mixing YP with water to form 250 mg/ml suspension. [0173] Encapsulation reactions were set up as shown in Table 3.
[0174] Citral or L-carvone-water emulsion was mixed with YP and Tween 80 surfactant overnight at room temperature. Samples were centrifuged at 14,000g for 10 minutes and the appearance of free terpene floating on the aqueous layer was scored. The results are shown in the right hand column labelled free terpene of Table 3.
[0175] The expression free terpene refers to the visible presence of terpene in the centrifuged reaction mixture. The absence of free terpene indicates complete absorption of the terpene by the YP. The highest volume of terpene absorbed by the YP, as evidenced by the absence of free terpene, was recorded as the maximal volume of absorbed terpene emulsion.
TABLE-US-00003 TABLE 3 250 1% mg/ml YP Terpene Vol Tween-80 Free Tube l Emulsion l l Terpene 1 500 500 2 500 L-carvone 15 485 3 500 L-carvone 37.5 462.5 4 500 L-carvone 75 425 5 500 L-carvone 112.5 387.5 6 500 L-carvone 150 350 Sl+ 7 500 L-carvone 225 275 + 8 500 L-carvone 450 50 + 9 500 Citral 15 485 10 500 Citral 37.5 462.5 11 500 Citral 75 425 12 500 Citral 112.5 387.5 Sl+ 13 500 Citral 150 350 + 14 500 Citral 225 275 + 15 500 Citral 450 50 +
[0176] As can be seen from the results in Table 3, YP is capable of absorbing and encapsulating terpenes at high YP concentration. YP absorbed and encapsulated at least 112.5 l of L-carvone terpene emulsion or at least 75 l of citral emulsion per 125 mg of YP. This demonstrates that the terpene encapsulation reaction is independent of YP concentration within the ranges tested.
Example 5Screen Commercially Available Particles for Terpene Absorption
[0177] The following protocol was performed to analyse the loading properties of different types of particles. The particles studied were Baker's Yeast Cell Wall Particles (Sigma Chemical Corp., St. Louis, Mo.), Nutrex Walls (Sensient Technologies, Milwaukee, Wis.), SAF-Mannan (SAF Agri, Minneapolis, Minn.), Nutricept Walls (Nutricepts Inc., Burnsville, Minn.), Levacan (Savory Systems International, Inc., Branchburg, N.J.) and WGP (Alpha-beta Technology, Inc. Worcester, Mass.).
[0178] L-carvone and citral emulsions were prepared by sonicating 7 g terpene+3 ml 3.3% Tween-80.
[0179] Table 4 below compares the purity with the number of yeast particles per mg and the packed solids weight/volume ratio.
TABLE-US-00004 TABLE 4 Purity Yeast % Beta 1,3- No. Mg Particle glucan particles/mg particles/ml Bakers 11.2 4 10.sup.7 250 Nutrex 24.5 1.7 10.sup.8 58.8 SAF Mannan 33.4 2.4 10.sup.8 41.7 Nutricepts 55.7 5.2 10.sup.8 37 Levacan 74.6 1 10.sup.8 19.2 WGP 82.1 3.5 10.sup.8 10
[0180] From Table 4 it can be concluded that the number of particles per mg is inversely proportional to purity. Thus the number of particles per mg of WGP is almost 10-fold higher than Baker's YP.
[0181] The YP suspensions were prepared as follows: [0182] Baker's yeast cell wall particle suspension (YP) was prepared by mixing 250 mg YP/ml 1% Tween 80. [0183] Nutrex suspension was prepared by mixing 163 mg Nutrex YGP/ml 1% Tween 80. [0184] SAF Mannan suspension was prepared by mixing 234 mg Biospringer YGP/ml 1% Tween 80. [0185] Nutricepts suspension was prepared by mixing 99 mg Nutricepts YGP/ml 1% Tween 80. [0186] Levacan suspension was prepared by mixing 217 mg Lev YGP/ml 1% Tween 80. [0187] WGP suspension was prepared by mixing 121 mg WGP YGP/ml 1% Tween 80.
[0188] The packed volume of the above particles is identical which means that equal numbers of particles were assayed.
[0189] Loading reactions were set up as shown in Table 5 and left to incubate overnight. Samples were centrifuged at 14,000g for 10 minutes and the appearance of free terpene floating on the aqueous layer and the color of the encapsulated terpenes in the pellet was scored. The results are shown in the two right hand columns of Table 5. The highest volume of terpene absorbed by particles as evidenced by the absence of free terpene was recorded as the volume of absorbed terpene emulsion.
TABLE-US-00005 TABLE 5 conc Terpene Vol 1% Tween Free Tube Particle mg/ml l Emulsion l 80 l Terpene Colour 1 Baker's 250 500 L-carvone 125 375 W 2 Nutrex 163 500 L-carvone 125 375 W 3 SAF Mannan 234 500 L-carvone 125 375 W 4 Nutricepts 99 500 L-carvone 125 375 + W 5 Levacan 217 500 L-carvone 125 375 + W 6 WGP 121 500 L-carvone 125 375 + W 7 Baker's 250 500 Citral 100 375 Y 8 Nutrex 163 500 Citral 100 375 Y 9 SAF Mannan 234 500 Citral 100 375 W 10 Nutricepts 99 500 Citral 100 375 + Y 11 Levacan 217 500 Citral 100 375 + int 12 WGP 121 500 Citral 100 375 + int 13 L-carvone 125 875 + 14 Citral 100 900 + Y W = white; Y = yellow; sl = slight; int = intermediate
[0190] From the results the following conclusions were reached: [0191] Purified particles with a low lipid content were less effective at absorbing terpenes. [0192] Less pure particles were more effective at absorbing terpenes. [0193] Yellow degradation product of citral was not formed when encapsulated in SAF-Mannan. [0194] Based on qualitative loading at the single terpene level tested, SAF Mannan appears to be best, Nutrex second and Baker's third.
Example 6Kinetics of Terpene Loading into Various Types of Particles and Different Incubation Temperatures
[0195] The following protocol was adopted to compare the loading kinetics of various types of yeast particles.
[0196] L-carvone and citral emulsions were prepared by sonicating 7 g terpene with 3 ml 3.3% Tween-80.
[0197] 1% Tween-80 solution was prepared by sonicating 1 ml 10% Tween-80 in 10 ml water. [0198] Baker's YP was prepared by mixing 5 g of bakers YP in 20 ml 1% Tween-80. [0199] Nutrex YGP suspension was prepared by mixing 2 g Nutrex YGP in 20 ml 1% Tween-80. [0200] SAF Mannan suspension was prepared by mixing 2 g SAF Mannan in 20 ml 1% Tween-80.
[0201] Loading reactions were set up as shown in Table 6.
[0202] The reactions were incubated for 1, 3, 6, 9 and 24 hours at room temperature or 37 C. After incubation samples were centrifuged at 14,000g for 10 minutes and the appearance of free terpene floating on the aqueous layer was scored. The results are shown in the two right hand columns of Table 6. The highest volume of terpene absorbed by the particles as evidenced by the absence of free terpene was recorded as the volume of absorbed terpene emulsion. Colour of the encapsulated pellet was scored at 24 hours.
TABLE-US-00006 TABLE 6 T conc Terpene Vol 1% Free Terpene (hr) Tube C. Particle mg/ml l Emulsion l Tween-80 1 3 6 9 24 Color 1 Rt Bakers 250 3500 L-carvone 788 2712 + W 2 37 Bakers 250 3500 L-carvone 788 2712 + W 3 Rt Nutrex 100 3500 L-carvone 1050 2450 + W 4 37 Nutrex 100 3500 L-carvone 1050 2450 + W 5 Rt SAF 100 3500 L-carvone 1050 2450 <+ W 6 37 SAF 100 3500 L-carvone 1050 2450 <+ W 7 Rt Bakers 250 3500 Citral 525 2975 + Y 8 37 Bakers 250 3500 Citral 525 2975 + VY 9 Rt Nutrex 100 3500 Citral 788 2712 + Y 10 37 Nutrex 100 3500 Citral 788 2712 + VY 11 Rt SAF 100 3500 Citral 788 2712 + W 12 37 SAF 100 3500 Citral 788 2712 + W White, W; Yellow, Y; Very Yellow, VY; Room Temperature, Rt
[0203] From the results shown in Table 6 and other observations the following conclusions can be made: [0204] Terpene loading reaction takes between 1 and 3 hours. [0205] Terpene loading occurs faster at 37 C. than at room temperature. [0206] SAF Mannan appears to be preferable particles for two reasons: [0207] Faster and more complete uptake of both terpenes. [0208] Citral remains stable when loaded as evidenced by the absence of yellow colour, characteristic of citral degradation, after 24 hours at 37 C.
Example 7Screen a Range of Single Terpenes and Terpene Combinations for Particle Loading
[0209] The following protocol was adopted to compare the loading efficiency of Baker's YP versus SAF Mannan.
[0210] Terpene emulsions were prepared as follows: [0211] L-carvone4.5 g L-carvone in 1.5 ml 3.3% Tween-80. [0212] Citral4.5 g citral in 1.5 ml 3.3% Tween-80. [0213] Thymol/L-carvone mixture (T/L)2.25 g thymol and 2.25 g L-carvone in 1.5 ml 3.3% Tween-80. [0214] Eugenol4.5 g eugenol in 1.5 ml 3.3% Tween-80. [0215] Geraniol4.5 g geraniol in 1.5 ml 3.3% Tween-80. [0216] Citral/L-carvone/Eugenol mixture (C/L/E)1.5 g citral, 1.5 g L-carvone, 1.5 g eugenol in in 1.5 ml 3.3% Tween-80.
[0217] Emulsions composed of terpene:water:surfactant ratio of 0.75:0.3:0.05 were used for these experiments.
[0218] Increasing volumes of terpene emulsion were mixed with 250 mg/ml Baker's YP or 250 mg/ml SAF Mannan overnight at room temperature as shown in Tables 7 and 8. Samples were centrifuged at 14,000g for 10 minutes and the appearance of free terpene floating on the aqueous layer was scored. The highest volume of terpene emulsion absorbed by Baker's YP or SAF Mannan as evidenced by the absence of free terpene was recorded as the volume of absorbed terpene emulsion. Colour of encapsulated terpenes in the pellet was recorded. The results in Tables 7 and 8 show that all single and terpene combinations were efficiently loaded into both Baker's YP or SAF Mannan particles.
TABLE-US-00007 TABLE 7 Evaluation of Baker's YP Loading of Different Terpenes and Terpene Mixtures. Baker Terpene Vol 1% Tween- Free Tube (l) Emulsion (l) 80 (l) Terpene Colour 1 500 500 W 2 500 L-carvone 15 485 W 3 500 L-carvone 37.5 462.5 W 4 500 L-carvone 7 425 +/ W 5 500 L-carvone 112.5 387.5 +/ W 6 500 L-carvone 150 350 + W 7 500 L-carvone 225 275 + W 8 500 L-carvone 450 50 ++ W 9 500 Citral 15 485 Y 10 500 Citral 37.5 462.5 Y 11 500 Citral 75 425 Y 12 500 Citral 112.5 387.5 +/ Y 13 500 Citral 150 350 + Y 14 500 Citral 225 275 + Y 15 500 Citral 450 50 + Y 16 500 T/L 15 485 W 17 500 T/L 37.5 462.5 W 18 500 T/L 75 425 W 19 500 T/L 112.5 387.5 +/ W 20 500 T/L 150 350 + W 21 500 T/L 225 275 + W 22 500 T/L 450 50 + W 23 500 Eugenol 15 485 W 24 500 Eugenol 37.5 462.5 W 25 500 Eugenol 75 425 W 26 500 Eugenol 112.5 387.5 +/ W 27 500 Eugenol 150 350 + W 28 500 Eugenol 225 275 + W 29 500 Eugenol 450 50 + W 30 500 Geraniol 15 485 W 31 500 Geraniol 37.5 462.5 W 32 500 Geraniol 75 425 W 33 500 Geraniol 112.5 387.5 + W 34 500 Geraniol 150 350 + W 35 500 Geraniol 225 275 + W 36 500 Geraniol 450 50 + W 37 500 C/L/E 15 485 Y 38 500 C/L/E 37.5 462.5 Y 39 500 C/L/E 75 425 Y 40 500 C/L/E 112.5 387.5 +/ Y 41 500 C/L/E 150 350 + Y 42 500 C/L/E 225 275 + Y 43 500 C/L/E 450 50 + Y
TABLE-US-00008 TABLE 8 Evaluation of SAF Mannan Loading of Different Terpenes and Terpene Mixtures. SAF Terpene 1% Tween- Free Tube (l) Emulsion Vol 80 (l) Terpene Colour 1 500 500 W 2 500 L-carvone 15 485 W 3 500 L-carvone 37.5 462.5 W 4 500 L-carvone 75 425 W 5 500 L-carvone 112.5 387.5 W 6 500 L-carvone 150 350 +/ W 7 500 L-carvone 225 275 +/ W 8 500 L-carvone 450 50 + W 9 500 Citral 15 485 W 10 500 Citral 37.5 462.5 W 11 500 Citral 75 ul 425 W 12 500 Citral 112.5 387.5 W 13 500 Citral 150 350 +/ W Inverted 14 500 Citral 225 275 + W Inverted 15 500 Citral 450 50 + W Inverted 16 500 T/L 15 485 W 17 500 T/L 37.5 462.5 W 18 500 T/L 75 425 W 19 500 T/L 112.5 387.5 W 20 500 T/L 150 350 +/ W 21 500 T/L 225 275 + W 22 500 T/L 450 50 + W 23 500 Eugenol 15 485 W 24 500 Eugenol 37.5 462.5 W 25 500 Eugenol 75 425 W 26 500 Eugenol 112.5 387.5 +/ W 27 500 Eugenol 150 350 + W 28 500 Eugenol 225 275 + W 29 500 Eugenol 450 50 + W 30 500 Geraniol 15 485 W 31 500 Geraniol 37.5 462.5 W 32 500 Geraniol 75 425 W 33 500 Geraniol 112.5 387.5 W 34 500 Geraniol 150 350 W 35 500 Geraniol 225 275 W Inverted 36 500 Geraniol 450 50 + W Inverted 37 500 C/L/E 15 485 W 38 500 C/L/E 37.5 462.5 W 39 500 C/L/E 75 425 W 40 500 C/L/E 112.5 387.5 W 41 500 C/L/E 150 350 W 42 500 C/L/E 225 275 +/ W 43 500 C/L/E 450 50 + W Inverted = Phase Inverted - solids floating on top - no free oil; W = white; Y = yellow.
[0219] From the results the following observations were made: [0220] All terpenes appeared to load into Baker's YP and SAF Mannan. [0221] SAF Mannan has a higher terpene loading capacity than bakers YP. [0222] The two and three way mixtures of terpenes also appear to efficiently load. [0223] The terpene Eugenol appears to have a higher density than the particles and water as it was found associated with the pellet. [0224] For the SAF Mannan, the higher load levels and lighter particles resulted in loaded particles floating on the surface of the aqueous layer for citral and geraniol. [0225] Citral was protected from oxidation by the SAF Mannan but not by the Baker's YP.
[0226] The approximate maximal loading for each particle type was determined and is shown in Tables 9 and 10 below. Percentage loaded represents a ratio of the amount of terpene loaded to the amount of particle present (weight for weight).
TABLE-US-00009 TABLE 9 Maximal terpene loading in Baker's YP. Terpene Vol. Loaded l % Loaded w/w L-carvone 37.5 33.3 Citral 75 67% Thymol/L-carvone 1:1 75 67% Eugenol 75 67% Geraniol 75 67% Citral/L-carvone/ 75 67% Eugenol (1:1:1)
TABLE-US-00010 TABLE 10 Maximal terpene loading in SAF Mannan. Terpene Vol. loaded l % Loaded w/w L-carvone 112.5 100% Citral 150 133% Thymol/L-carvone 1:1 112.5 100% Eugenol 112.5 100% Geraniol 150 133% Citral/L-carvone/ 150 133% Eugenol (1:1:1)
Example 8Evaluation of Terpene Stability in Aqueous Emulsions and Encapsulated Terpene Formulations
[0227] Terpene stability was assessed by the observation of citral formulations for the formation of a yellow colored oxidation product. As noted in the right hand column in Tables 5-8 citral emulsions and citral encapsulated Bakers YP turned a progressively increasing yellow color over time. However, citral encapsulation in SAF Mannan increased citral stability as evidenced by a reduction or absence of yellow color over time.
Example 9Loading of Terpenes in Minimal Water
[0228] The following protocol was carried out to evaluate the possibility that terpene loading and encapsulation into YP could be carried out at a very high Yeast Particles (YP) solids level to allow for direct extrusion of the loaded formulation into a fluidised bed drier. The minimal amount of water to completely hydrate the SAF Mannan particles was determined to be 3.53 g water per g solids. This defines the hydrodynamic volume (HV) or water absorptive capacity of the particles. At this level of water the hydrated particles have a consistency of a stiff dough which is thixotropic, i.e. shear thinning like mayonnaise. Addition of water up to 40% above the HV results in a thick flowable paste. The standard reaction that has been used in the above examples was carried out at 3HV water.
[0229] A series of terpene (L-carvone) loading reactions were carried out keeping the ratio of particle:terpene:Tween (1:0.44:0.04) constant and varying the amount of water in the system from the HV (3.53 g) to HV+40% water (4.92 g). Controls were the standard loading system which uses 3HV water, particles only and terpene only reactions. Following overnight incubation samples of the mixtures were evaluated microscopically for free terpene and evidence of terpene uptake into the particles and for material flow characteristics by assessing flow in inverted tubes over 15 minutes. In addition, the presence of free oil was assessed by hydrating the reaction mixture with 5HV, vortexing to obtain a complete dispersion of particles and centrifugation to sediment the particle encapsulated terpene. The results are shown in Table 11 and
TABLE-US-00011 TABLE 11 SAF Terpene Weight Water Free Tube g Emulsion (g) (g) Terpene Flow 1 L-carvone 4.64 4.5 + + 2 1 8.0 + 3 1 L-carvone 4.64 4.5 + 4 1 L-carvone 4.64 5 1 L-carvone 4.64 0.17 6 1 L-carvone 4.64 0.35 7 1 L-carvone 4.64 0.52 Sl 8 1 L-carvone 4.64 0.7 Mod 9 1 L-carvone 4.64 0.87 High 10 1 L-carvone 4.64 1.05 High 11 1 L-carvone 4.64 1.39 High
[0236] The results shown in Table 11 and
[0237] These results extend our understanding of the conditions to load terpenes into hollow glucan particles. The flexibility to use a minimal volume of water to hydrate the particles during the loading process will allow loading of the terpenes under conditions where the reaction mixture is a malleable dough-like consistency using standard food-grade swept surface dough mixers. The consistency of the final high solids terpene loaded mixture is suitable for direct extrusion to form noodles and pellets for fluidised bed drying.
[0238] Suitable facilities to scale up production in this manner would require: [0239] Gaulin homogeniser, or equivalent to produce stable terpene emulsion. [0240] Swept surface dough mixing tank. [0241] Extruder. [0242] Fluidised bed drier.
Example 10Evaluation of an Interstitial Hydrocolloid Agent to Aid Dispersion in Dried Hollow Glucan Particles Encapsulating a Terpene Component Dispersion when Re-Hydrated
[0243] The following protocol was adopted to evaluate the effect of an interstitial hydrocolloid to increase dried hollow glucan particle encapsulated terpene formulations to disperse when hydrated. [0244] SAF Mannan particles [0245] 0.1% Tween 80 [0246] L-carvone [0247] Xanthan Gum1% w/v in 0.1% Tween 80
[0248] The effect of increasing xanthan gum levels on dry hollow glucan particle encapsulated L-carvone dispersion in water was assessed by loading L-carvone into SAF Mannan by incubating 1.1 g of an L-carvone emulsion (L-carvone:water:surfactant ratio of 0.75:0.3:0.05) with 1 g SAF Mannan and 4.4 g 0.1% Tween 80 containing 0-1% xanthan gum as shown in Table 12.
TABLE-US-00012 TABLE 12 L-carvone 0.1% 1% SAF Emulsion Tween-80 Xanthan Visual Tube g (g) (g) (g) Observations 1 1 1.1 4.4 0 Large non- uniform clumps 2 1 1.1 4.33 0.07 Uniform suspension 3 1 1.1 4.26 0.14 Uniform suspension 4 1 1.1 4.12 0.28 Uniform suspension 5 1 1.1 3.85 0.55 Uniform suspension 6 1 1.1 3.3 1.1 Finer Uniform suspension 7 1 1.1 2.2 2.2 Finer Uniform suspension 8 1 1.1 0 4.4 Finer Uniform suspension
[0249] The results in Table 12 and
[0250] It may also be worthwhile to include a pellet coating to increase the stability of the loaded terpenes, and to provide a sustained release of terpene.
Example 11Evaluation of Minimum Inhibitory Concentration (MIC) of Terpene Emulsions, Fresh Baker's YP and SAF Mannan Encapsulated Terpenes and Freeze-Dried Baker's YP and SAF Mannan Encapsulated Terpenes Against S. aureus
[0251] The results of a protocol performed to compare the MIC of fresh versus freeze dried hollow glucan particle encapsulated terpene formulations are shown below in Table 13. A simple terpene emulsion was also tested and the results are shown for comparison.
TABLE-US-00013 TABLE 13 MIC g/ml terpene Bakers SAF Mannan Freeze Freeze Terpene Emulsion Fresh Dried Fresh Dried L-carvone 3.75 0.1 >0.04 0.01 >0.02 Citral 0.94 0.01 0.05 0.01 >0.03 L-carvone/ 0.23 0.01 0.03 0.01 0.05 Thymol Eugenol 0.12 0.03 0.05 0.01 0.05 Geraniol 0.47 0.03 0.06 0.02 >0.03 L-carvone/ 0.23 0.03 0.06 0.02 0.05 Citral/Eugenol
[0252] The conclusions taken from the above results were: [0253] Terpene loading into hollow glucan particles appears to enhance terpene MIC. Generally the fresh terpene emulsions are-4-375 fold less potent than the encapsulated formulations [0254] Terpenes loaded in SAF Mannan perform slightly better than Baker's YP. [0255] Freshly loaded terpene compositions perform slightly better than freeze dried compositions (there may be some volatilisation of terpenes from dry compositions during freeze drying). [0256] Terpenes in aqueous emulsions are stable for at least 3 weeks.
Example 12Efficacy of Encapsulated Terpenes at Pilot Plant Scale Against S. aureus
[0257] Anti-microbial assays were carried out with encapsulated terpenes and mixtures produced at the pilot plant scales against S. aureus. Both the fresh and freeze dried encapsulated terpene samples containing materials demonstrated strong anti-microbial activities. The results are summarised in Table 14 below.
[0258] Terpenes were encapsulated in SAF-Nannan at a 2.5 Kg scale. A mixture of three terpenes (Geraniol, 275 g; Eugenol, 385 g; and thymol, 440 gram was dissolved and homogenized with 100 g Tween-80 and 8 L of water. SAF-Mannan (2.5 Kg) was added to form a homogenous suspension. The suspension was passed through a Gaulin homogenizer to reduce particle size and the homogenate was incubated overnight at room temperature. A sample of the encapsulated terpene was removed and stored at room temperature. The remaining encapsulated terpene was then frozen in trays and freeze dried. The freeze dried encapsulated terpene powder was ground and stored at room temperature.
TABLE-US-00014 TABLE 14 Material MIC (ppm) Staphylococcus aureus assays YGP empty shell control >2500 Pilot Plant - Fresh 100 Pilot Plant - Freeze dried 100
[0259] At the pilot plant scale both the fresh and freeze dried samples were equally potent on a w/w terpene basis.
[0260] Based on the large scale preparation results, the predicted effective dose of the freeze dried formulation against S. aureus is 200 ppm (the formulation contains 50% terpene w/w) or 0.2 g/L water.
Example 13Efficacy of Encapsulated Terpenes Against Mycobacterium
[0261] Terpene emulsions were prepared as follows: [0262] Citral4.5 g citral in 1.5 ml 3.3% Tween-80. [0263] L-carvone/eugenol2.25 g L-carvone and 2.25 g Eugenol in 1.5 ml 3.3% Tween-80. [0264] Eugenol4.5 g eugenol in 1.5 ml 3.3% Tween-80. [0265] Geraniol4.5 g geraniol in 1.5 ml 3.3% Tween-80. [0266] Geraniol/thymol mixture2.25 g geraniol and 2.25 g thymol in 1.5 ml 3.3% Tween-80. [0267] Control emulsion6 ml 1% Tween-80.
[0268] SAF-Mannan (2.5 g) was mixed with 3 ml of each emulsion and 7 ml of 1% Tween 80 and incubated overnight to encapsulate the terpenes and terpene mixtures. The encapsulated terpene formulations were frozen and freeze dried and the powders ground to a fine powder. Suspensions of encapsulated terpenes (25 mg/ml) and unencapsulated terpene emulsions were assayed for antibacterial activity against Mycobacterium. The results are set out in Table 15
TABLE-US-00015 TABLE 15 Material MIC (ppm) Mycobacterial assays YGP Citral FD 250 YGP L-Carvone/Eugenol FD 500 YGP Eugenol FD 500 YGP Geraniol FD 125 YGP Geraniol/Thymol FD 62.5 Control Emulsion >1000 Citral Emulsion 35 L-carvone/Eugenol Emulsion 53 Eugenol Emulsion 105 Gernaniol Emulsion 70 Geraniol/Thymol Emulsion 53 FD = (Freeze Dried)
Example 14Nematocidal Activity of Encapsulated Terpenes
[0269] Preparations of yeast cell walls encapsulating citral were prepared according to the procedures described above. The hollow glucan particles contained 17.5% citral, and the particles were present at in the test preparations at a concentration of 1000 ppm. This means that terpenes were effectively present at a concentration of 175 ppm.
[0270] 1.0 ml of the test preparations was added to 0.1 to 0.15 ml of water containing root-knot nematodes. 1.0 water was added to the nematodes as the control.
[0271] Observations were made as previously described and the kill rate assessed (i.e. percentage dead) after 24 and 48 hrs. The results shown below in Table 16 are an average of 2 sets of results.
TABLE-US-00016 TABLE 16 Nematicidal activity of encapsulated terpene solution (17.5% citral @ 1000 ppm) Kill Rate Time Test Control 24 h 45 17 48 h 56 21
[0272] The results demonstrate that hollow glucan particles encapsulating terpenes are effective at killing root-knot nematodes at a particle concentration of 1000 ppm, which corresponds to a citral concentration of only 175 ppm.
[0273] Thus hollow glucan particles encapsulating terpenes appear to be as effective as terpenes in solution or with surfactant as nematicides. The nematicidal activity is retained despite the terpene being encapsulated within the particle. It can be expected that higher concentrations of terpenes within the hollow glucan particles, or higher concentrations of the particles would result in an even higher kill rate, as is the case for terpenes in solution or with surfactant.
Example 15Fugicidal Properties of Encapsulated and Non-Encapsulated Terpenes
[0274] The following protocols were carried out to assess the fungicidal properties of various terpene combinations, and to compare the efficacy of encapsulated and non-encapsulated compositions.
[0275] Assessment of Anti-Fungal Properties of Different Terpene Formulation
[0276] A microtitre plate assay was used to assess the minimum inhibitory concentration (MIC) of a range of terpene compounds against different pathogenic organisms. The assay used for each organism is described in detail later but important general features are as follows.
[0277] The assay uses two incubation periods to distinguish between static (growth inhibition) and cidal (killing) activities. The first incubation period allows assessment of growth inhibition, but cannot distinguish between merely prevention of growth and killing of the cells. The purpose of the second incubation period is to allow sufficient time and nutrients for any dormant or inhibited cells that survive terpene exposure to proliferate. Any cells that were inhibited by fungistatic effects should respond and grow during the second incubation period, whereas cells that were killed by exposure to terpenes will not grow in the fresh medium.
[0278] Initial screening experiments were carried out using a total of 31 different terpene formulations (Table 17). These experiments were repeated using a subset of strongly active terpene formulations (Table 18).
[0279] A combination of the terpenes geraniol, eugenol and thymol in a ratio of 2:1:2 encapsulated within glucan particles was also tested; this sample is referred to as YP-GET. A non-encapsulated geraniol, eugenol and thymol combination in the same ratio was also tested for comparison with the encapsulated form.
[0280] MIC Assay Using Saccharomyces cerevisiae
[0281] S. cerevisiae (510.sup.5 cells/mL in YPD growth medium) were added to each well of a 96-well microtitre plate in 100 L aliquots. At least one column per plate was designated as a cell-only control and no terpene was added to these wells. Aliquots (100 L) of different terpene formulations were added to the first row of the remaining columns, and serial 2-fold dilutions were performed by transferring 100 L from one row to the next a total of 7 times. Finally, 100 L was discarded from the last row in order to ensure that all wells contained the same volume. Microtitre plates were incubated statically overnight at 30 C.
[0282] Following incubation, plates were scored for inhibition of growth (evidenced by a lack of turbidity). Growth inhibition (75%) was visually confirmed by microscopy.
[0283] Once the MIC had been determined for each formulation, the microtitre plates were centrifuged and the spent medium was removed from non-turbid wells. The cells were resuspended in fresh medium (100 L) and the plates were re-incubated overnight at 30 C. Assessment of growth inhibition was performed as before.
[0284] MIC Assay Using a Mixed Inoculum
[0285] The different terpene formulations were serially diluted in the 96-well microtitre plate as described for S. cerevisiae. Molten YPD agar was then added to the wells, together with 5 L mixed inoculum (prepared from mouldy grape leaves to a concentration of 510.sup.4 cells/mL). The plates were incubated statically for 24 hours at room temperature and spore growth was visually assessed by microscopy.
[0286] Due to the use of solid medium, the second incubation period in fresh media could not be performed.
[0287] MIC Assay Using Colletotrichum graminicola
[0288] The different terpene formulations were serially diluted in the 96-well microtitre plate as described for S. cerevisiae. C. graminicola (300 spores/well) were added to the diluted terpenes and the plates were incubated statically for 48 hours at room temperature. Spore germination and growth were visually assessed by microscopy.
[0289] Once the MIC had been determined for each formulation, the microtitre plates were centrifuged and the spent medium was removed from growth-inhibited wells. The spores were resuspended in fresh medium (100 L) and the plates were re-incubated overnight at room temperature. Assessment of growth inhibition was performed as before.
TABLE-US-00017 TABLE 17 MIC and fungicidal MIC values obtained from initial screening of 31 terpene formulations Saccharomyces Mixed Colletotrichum cerevisiae microbes graminicola Terpene Cidal Cidal Cidal formulation .sup.a MIC MIC MIC MIC MIC MIC 1 Geraniol (G) 500 500 250 NT 63 63 2 Eugenol (E) 500 500 125 NT 125 125 3 Thymol (T) 250 250 63 NT 63 500 4 Citral (C) 250 250 63 NT 125 63 5 L-carvone (L) 250 500 63 NT 125 125 6 GE 1000 2000 125 NT 63 250 7 GT 500 500 250 NT 125 63 8 GC 500 500 125 NT 125 250 9 GL 500 500 125 NT 125 125 10 ET 500 500 125 NT 125 125 11 EC 250 1000 31 NT 125 125 12 EL 500 1000 125 NT 125 125 13 TC 500 500 16 NT 63 63 14 TL 500 1000 63 NT 63 63 15 CL 500 500 8 NT 63 63 16 GET 500 500 23 NT 94 94 17 GEC 250 500 94 NT 94 94 18 GEL 500 1000 188 NT 188 188 19 GTC 500 500 47 NT 188 188 20 GTL 500 1000 94 NT 94 94 21 GCL 250 500 94 NT 47 47 22 ETC 125 250 188 NT 94 94 23 ETL 500 1000 12 NT 94 94 24 ECL 500 1000 12 NT 188 188 25 TCL 500 1000 23 NT 94 375 26 GETC 500 1000 125 NT 250 500 27 ETCL 500 1000 63 NT 125 125 28 GTCL 500 1000 125 NT 250 250 29 GECL 500 1000 16 NT 500 500 30 GETL 1000 1000 125 NT 500 250 31 GECTL 1000 1000 78 NT 625 625 GET (2:1:2 NT NT 98 NT 78 156 ratio, w/w/w) YP-GET 98 391 98 NT 20 20 (G:E:T ratio of 2:1:2, w/w) .sup.b NT, not tested; YP-GET, yeast-encapsulated GET formulation. .sup.a Terpene combinations were mixed in a 1:1 (w/w) ratio unless otherwise indicated. .sup.b MICs calculated by terpene content.
TABLE-US-00018 TABLE 18 Repeat assay to determine MIC and fungicidal MIC values Mixed microbes isolated from Saccharomyces mouldy grape Colletotrichum Terpene cerevisiae leaves .sup.b graminicola formulation .sup.a Cidal Cidal Cidal (by No.) MIC MIC MIC MIC MIC MIC T (3) NT NT 63 NT NT NT L (5) NT NT 250 NT NT NT GE (6) NT NT NT NT 125 500 EC (11) 125 250 NT NT NT NT TC (13) NT NT 250 NT 63 250 TL (14) NT NT 500 NT 250 500 CL (15) NT NT 500 NT 125 500 GET (16) NT NT 375 NT 188 375 GEC (17) 250 500 NT NT NT NT GCL (21) 250 500 NT NT 375 750 ETC (22) 125 250 NT NT 94 188 ETL (23) NT NT 375 NT 188 750 ECL (24) NT NT 750 NT NT NT TCL (25) NT NT 750 NT 94 375 ETCL (27) NT NT 500 NT 63 500 GECL (29) NT NT 1000 NT NT NT YP-GET 98 195 NT NT 39 156 (G:E:T ratio of 2:1:2, w/w) .sup.c NT, not tested; YP-GET, yeast-encapsulated GET formulation. NOTE: Samples were assayed in duplicate. If different values were obtained between duplicate samples, the higher value has been presented. No duplicate samples differed by more than one 2-fold dilution. .sup.a Terpene combinations were mixed in a 1:1 (w/w) ratio unless otherwise indicated. .sup.b 1 10.sup.4 cells/mL stock suspension. .sup.c MICs calculated by terpene content.
[0290] Mixed Inoculum
[0291] Using a mixed inoculum presents a number of problems. The variability in spore content between preparations results in poor interassay reproducibility, and growth of contaminating organisms impedes the scoring of spore germination. Unicellular yeast species are particularly problematic in masking spore growth. Although precise data could not be obtained from this assay, an inhibitory effect of terpenes was observed.
[0292] Identification of spores was easier during scoring of the repeat assay than during the initial screening assay as a larger number of spores were used (approximately 50/well versus approximately 10/well). Therefore, data obtained during the repeat assay may provide a more reliable estimate of MIC.
[0293] Colletotrichum graminicola
[0294] The generally higher MIC values obtained from the repeat assay compared to the initial screening assay may be due to: [0295] use of 1-week-old terpene solutions [0296] use of freshly prepared spores, which had a higher viability than those used in the initial screening assay and may therefore be more difficult to kill.
[0297] Comparison of Terpene Formulations as Free Emulsions with the Same Terpene Formulations when Encapsulated in Hollow Glucan Particles: Saccharomyces cerevisiae MIC Assays
[0298] YPD growth medium (100 L) was added to each well of a 96-well microtitre plate and aliquots of different terpene formulations were added to the first row, giving a total volume of 200 L in this row. One column was designated as a cell-only control and no terpene was added to these wells. Serial 2-fold dilutions were performed by transferring 100 L from one row to the next a total of 7 times. Finally, 100 L was discarded from the last row in order to ensure that all wells contained the same volume. S. cerevisiae (510.sup.5 cells/mL in YPD growth medium) were then added to each well in 100 L aliquots, and the absorbance at 620 nm (A.sub.620) was measured for each well using a microtitre plate reader. Microtitre plates were incubated statically overnight at 30 C.
[0299] Following incubation, the A.sub.620 was measured again and plates were scored for inhibition of growth (75%). Growth inhibition was visually confirmed by microscopy.
[0300] For the free terpene emulsions, once the MIC had been determined for each formulation, the microtitre plates were centrifuged and the spent medium was removed from the growth-inhibited wells. The cells were resuspended in fresh medium (100 L) and the plates were re-incubated overnight at 30 C.
[0301] Assessment of growth inhibition was performed as before.
[0302] MIC and fungicidal MIC results are summarised in Table 19.
Results
[0303]
TABLE-US-00019 TABLE 19 MIC and fungicidal MIC values obtained from screening of 31 terpene formulations against Saccharomyces cerevisiae Terpene formulation .sup.a Yeast-encapsulated (Reference formulations .sup.b, c Free terpene emulsions No) MIC Cidal MIC MIC Cidal MIC G (1) 111 NT 250 250 E (2) 131 NT 125 250 T (3) 115 NT 125 250 C (4) 118 NT 125 250 L (5) 254 NT 250 500 GE (6) 118 NT 250 500 GT (7) 108 NT 125 250 GC (8) 113 NT 125 250 GL (9) 117 NT 250 500 ET (10) 131 NT 125 250 EC (11) 126 NT 125 250 EL (12) 129 NT 125 250 TC (13) 59 NT 63 63 TL (14) 124 NT 63 125 CL (15) 124 NT 125 125 GET (16) 119 NT 63 125 GEC (17) 119 NT 125 250 GEL (18) 121 NT 125 125 GTC (19) 115 NT 125 125 GTL (20) 119 NT 125 125 GCL (21) 234 NT 125 125 ETC (22) 124 NT 125 125 ETL (23) 123 NT 125 125 ECL (24) 63 NT 63 125 TCL (25) 61 NT 125 500 GETC (26) 61 NT 63 250 ETCL (27) 120 NT 63 125 GTCL (28) 124 NT 125 125 GECL (29) 125 NT 125 125 GETL (30) 122 NT 125 250 GECTL (31) 120 NT 125 250 GET (2:1:2 .sup.125 .sup.d NT 125 250 ratio, w/w/w) YP-GET 125 NT .sup.125 .sup.c .sup.250 .sup.c (G:E:T ratio of 2:1:2, w/w) YP-ETC 125 NT .sup.125 .sup.c .sup.250 .sup.c (E:T:C ratio of 1:1:1, w/w) NT, not tested; YP-GET, yeast-encapsulated GET formulation; YP-ETC, yeast-encapsulated ETC formulation. .sup.a Terpene combinations were mixed in a 1:1 (w/w) ratio unless otherwise indicated. .sup.b Yeast-encapsulated formulations unless otherwise indicated. .sup.c MIC calculated by terpene content. .sup.d Non-encapsulated emulsion formulation.
[0304] For both the terpene emulsions and yeast-encapsulated terpenes, MICs were typically 125 ppm, with the most active formulations inhibiting growth at 60 ppm. MIC values obtained for the terpene emulsions were similar to those obtained for their respective yeast-encapsulated formulations. When different values were obtained, they only differed by approximately one 2-fold dilution.
[0305] Many of the free terpene emulsions were fungicidal at the growth inhibitory MIC, with the majority showing fungicidal activity at a 2-fold higher concentration.
[0306] These results demonstrate that terpenes encapsulated in glucan particles are at least as effective at killing fungus as non-encapsulated forms.
[0307] Additionally the encapsulated compositions used may have had reduced potency due to having been stored for 45 days at 4 C. and having a sub-optimal terpene content of 4% w/w.
[0308] The assay to determine fungicidal activity involves a centrifugation step, which attempts to separate the microbial cells from any residual terpene in the growth medium by producing a pellet of cells at the bottom of the well. This pellet is then resuspended in fresh media and incubated for a second time in the absence of terpene. However, the centrifugation step cannot discriminate between microbial cells and yeast particles, therefore when yeast-encapsulated terpenes are used, the cell pellet will also contain terpene-loaded yeast particles. As a result, both the yeast particles and the microbial cells are then resuspended in the fresh medium.
[0309] This methodology issue is not considered to affect the results obtained in the experiments described above for the following reasons. [0310] In previous experiments, terpene emulsions have been used instead of terpene-loaded yeast particles and fungicidal activity has been clearly shown. [0311] Encapsulated terpenes are released by diffusion, and an equilibrium between the concentration of encapsulated terpenes and the concentration of released terpenes in the surrounding medium is quickly reached. Thus, following centrifugation and resuspension in fresh medium, the concentration of released terpene in the growth medium is likely to be well below that required for growth inhibitory activity. [0312] There was no growth when the contents of the fungicidal MIC well were plated onto solid agar growth medium. When plated onto solid growth medium, diffusion of any residual terpene throughout the large volume of the agar plate results in a local terpene concentration that is too low to cause growth inhibition. The lack of growth from the contents of the fungicidal MIC well must therefore be due to initial fungicidal activity. In contrast, when an MIC was obtained that was lower than the fungicidal MIC and the contents of the MIC well were plated onto solid agar growth medium, growth was observed, indicating a fungistatic effect.
Example 16Preparation of Encapsulated Terpene Compositions for Field Trials
[0313] The purpose of the following protocol was to encapsulate a terpene composition into hollow glucan particles for subsequent field trials.
[0314] Materials:
[0315] Thymol (supplied by Alpha-Gamma Corporation)
[0316] Eugenol (supplied by Alpha-Gamma Corporation)
[0317] Geraniol (supplied by Alpha-Gamma Corporation) 1% Tween-80 (supplied by Alpha-Gamma Corporation)
[0318] Yeast Cell Wall Particles
[0319] Xanthan Gum.
[0320] The yeast cell wall particles were obtained from Biorigin (Sao Paolo, Brazil) under the trade name Nutricell MOS 55, and were manufactured by Acucareira Quat S. A, Usina Quat, QuatSao PaoloBrazilZip Code 19780 000. The particles are a spray dried cell wall extract of S. cerevisiae and are a free flowing powder of light beige to tan colour.
[0321] Protocol: The following protocol was suitable for a 1 Kg of particles, but can simply be scaled up for larger production. [0322] 1. Prepare terpene mixturemix 375 grams of Geraniol+525 grams Eugenol+600 grams of Thymol and stir in a glass flask. [0323] 2. Prepare 6.2 L of 1% Tween 80 by mixing 62 grams Tween 80 in 6.2 L water in 2 gallon white bucket. Mix to form solution. [0324] 3. Add 6.2 grams Xanthan Gum to Tween solution and stir to dissolve. [0325] 4. Prepare terpene emulsion by mixing 1.5 Kg terpene mixture+6.2 L 1% Tween 80/0.1% Xanthan gum in white bucket using polytron mixer. [0326] 5. Add 1,000 grams of yeast cell wall particlesmix using paint mixer to form uniform suspension. [0327] 6. Add the terpene emulsion of step 4 to the yeast cell wall particles while mixing to form a thin mayonnaise-like consistency. [0328] 7. Pour terpene mixture into cans and incubate overnight.
[0329] Results: Encapsulated geranoil, eugenol and thymol in hollow glucan particles was obtained as a paste. The paste was easily converted to a dry powder by conventional spray drying techniques. The paste is the liquid composition referred to in the following protocols, and the powder is the spray dried form.
Example 17Field Trials of Encapsulated Terpene Composition on Downy Mildew
[0330] In grapes, downy mildew is caused by the fungus Plasmopara viticola, which infects vineyards worldwide and can cause devastating losses for grape-growers in terms of crop yield and wine quality. The fungus attacks the fruits and all green parts of the vine, causing the leaves to wither and the flowers and berries to rot. The disease manifests as irregular pale yellow or yellow-green spots on the upper surface of leaves, with dense, white-grey, cotton-like fungal growth covering the underside of the leaf lesions. Berries may also be covered with the downy growth and, depending on the time of infection, may turn brown and soft or may not soften at all. Downy mildew is spread through the dispersal of spores by the wind and rain, and requires wet conditions for infection. It is particularly problematic in environments with high humidity. Preventative measures are recommended for management of the disease, with early applications of fungicides followed by repeat applications at appropriate intervals. Resistance has arisen to some treatments, and although the development of resistance can be minimised by rotating the use of different fungicides, it remains a problem.
[0331] The purpose of this trial was to investigate the efficacy of the encapsulated terpene formulation of Example 16 (YGP-GET) supplied as a liquid or powder (spray dried) formulation, for the prevention of downy mildew in grapes.
[0332] Four adjacent blocks, each covering 0.1 ha, were identified on site 20 in the Kir-Yianni vineyard.
[0333] Kir-Yianni is a 35 ha vineyard at an elevation of 300 m. It is bordered by a mixed oak forest on the north and west, and overlooks orchards and vineyards to the south and east.
[0334] All four blocks had been treated with multiple products prior to application of the terpene formulation. On 26 Jun. 2004, two of the four blocks were sprayed with the terpene powder formulation at a dose of either 0.5 g/L or 2 g/L (see schematic illustration in
[0335] Four further adjacent blocks, each covering 0.1 ha, were identified on site 18 in the Kir-Yianni vineyard. All four blocks had been treated with multiple products prior to application of the terpene formulation. On 26 Jun. 2004, two of the four blocks were sprayed with the terpene liquid formulation at a dose of either 1 g/L or 4 g/L (
[0336] For both sites, the terpene product was applied at a rate of 1200 L/ha.
[0337] The following growth stages of the grapes were recorded: [0338] bud break, 26 Mar. 2004 [0339] bloom, 1 Jun. 2004 [0340] veraison, 6 Aug. 2004
[0341] The study applications took place pre-veraison.
[0342] The 2004 growing season was exceptionally late and was wet throughout. Disease pressure from downy mildew was extremely high, botrytis levels were elevated, and powdery mildew pressure was moderate. Both the powder and liquid YGP-GET formulations were stored at room temperature. No special storage conditions were used.
[0343] Details of Comparator Products
[0344] Powder formulation trial: Bordeaux mix, manufactured by Manica Spa, Italy, packed in Greece by Moscholios Chemicals SA; wettable sulphur.
[0345] Liquid formulation trial: Mikal (fosetyl-al 50%, folpet 25%), manufactured by Bayer CropScience, distributed in Greece by Bayer Hellas SA.
[0346] The comparator products were applied as follows: One application before bud-break at a dosage of 15 g/L followed by two more applications per year at a dosage of 6.5 g/L. A spraying rate of 1000 L/ha was used for all three applications.
[0347] Powder formulation trial: Bordeaux mix (2 g/L) and Wettable sulphur (2.2 g/L) were applied on 26 Jun. 2004.
[0348] Liquid formulation trial: Mikal (3.2 g/L) was applied on 28 Jun. 2004.
[0349] Vines were visually examined for symptoms of downy mildew. Onset of the disease was marked by an average of two oily spots per leaf. Treatments that prevented the appearance of further spots were considered to provide effective protection against downy mildew.
[0350] Results
[0351] YGP-GET Powder Formulation (Spray Dried)
[0352] The conventional treatment of Bordeaux mixture provided good protection against downy mildew. Mild symptoms of downy mildew were observed in the control vines. The 0.5 g/L terpene product concentration did not provide protection, and the 2 g/L terpene product concentration provided only slightly better protection than the control. Note: the disease pressure at this site was very low because of the recent pesticide treatment.
[0353] Difficulties were encountered in dissolving the powder formulation as it was very fine, resulting in dispersion in the air. This may have adversely affected the efficacy of the product.
[0354] YGP-GET Liquid Formulation
[0355] When administered at a dose of 4 g/L, the terpene product provided excellent protection against downy mildew on exposed canopy. No protection was provided by the 1 g/L dosage. Serious symptoms of downy mildew were observed in the control block.
[0356] The liquid formulation was easy to use and had a pleasant odour.
[0357] Discussion
[0358] Downy mildew can cause devastating losses for grape-growers because of its effects on crop yield and wine quality. Management of the disease focuses on prevention because, once established, the infection can quickly spread. At the site sprayed with the powder formulation, YGP-GET did not exhibit efficacy at the lower dosage (0.5 g/L), and the dose of 2 g/L was less effective than the conventional treatment. At this site, the recent pesticide applications resulted in low disease pressure, which may have limited the apparent efficacy of the terpene treatment. However, it was considered that a dosage of less than 2 g/L of the terpene product was inadequate.
[0359] At the site sprayed with the liquid formulation, excellent protection of exposed canopy was provided by the higher dose level of 4 g/L. Excessive vegetative growth at this site resulted in more effective treatment of the outer, younger branches compared with the older growth in the inner canopy. Complete foliar coverage by the terpene product is useful, as the treatment is not systemic. It is estimated that an approximately 30% increase over the volume used for conventional systemic treatments would achieve good coverage using the terpene treatment.
[0360] Conclusions:
[0361] Foliar application of YGP-GET liquid formulation was highly effective at controlling downy mildew at a concentration of 4 g/L. The lower concentrations of 0.5 g/L powder and 1 g/L liquid were not effective.
Example 18Field Trials of Encapsulated Terpene Composition on Powdery Mildew
[0362] Powdery mildew of grapes is caused by the fungus Uncinula necator, and causes reductions in vine growth, fruit quality and winter hardiness of vines. In wine grapes, an infection level of only 3% of berries can affect wine quality. The disease is characterised by small white-grey patches of fungal growth that enlarge into a powdery, white coating on the leaves. The fungal growth can also occur on the berries, which may split. In contrast to downy mildew, which requires warm wet conditions, powdery mildew can be a problem in drier growing seasons, as it favours shaded areas with humid but not rainy weather conditions. Preventative measures are recommended for management of powdery mildew, with early applications of fungicides followed by repeat applications at appropriate intervals.
[0363] This study aimed to investigate the efficacy of application of the YGP-GET composition for the prevention of powdery mildew in grapes.
[0364] Three adjacent blocks, each covering 0.1 ha, were identified on site 18 in the Kir-Yianni vineyard. On 19 Jul. 2004, one of the three blocks was sprayed with the YGP-GET liquid formulation at a dose of 2 ml/L and one was left untreated. The remaining block was sprayed with the conventional treatment of Equesion (2.5 g/L), Alliete (0.9 g/L) and Punch (0.075 mL/L) (see
[0365] Three further adjacent blocks, each covering 0.1 ha, were identified on site 20 in the Kir-Yianni vineyard. On 20 Jul. 2004, one of the three blocks was sprayed with the YGP-GET liquid formulation at a dose of 2 mL/L and the two remaining blocks were left untreated (see
[0366] At both sites, the blocks had previously been treated with multiple products, including a prior application of terpene product.
[0367] All terpene treatments were applied at a rate of 1200 L/ha to ensure complete coverage.
[0368] The following growth stages of the grapes were recorded [0369] bud break, 26 Mar. 2004 [0370] bloom, 1 Jun. 2004 [0371] veraison, 6 Aug. 2004
[0372] The study applications took place pre-veraison.
[0373] The 2004 growing season was exceptionally late and was wet throughout. Disease pressure from downy mildew was extremely high, botrytis levels were elevated, and powdery mildew pressure was moderate.
[0374] Details of Comparator Products
[0375] No comparator product was used at site 20. The comparator treatment used at site 18 is detailed below.
[0376] Punch (flusilazole 40%), DuPont.
[0377] On 19 Jul. 2004, Punch was applied at a dose of 0.075 ml/L as a preventative treatment for powdery mildew according to the manufacturer's instructions.
[0378] Details of Additional Products
[0379] No additional products were used at site 20. The additional products used at site 18 are detailed below.
[0380] Equesion system (famoxadone 22.5% plus cymoxanil 30%) Alliete (fosetyl-al 80%)
[0381] On 19 Jul. 2004, Equesion (2.5 g/L) and Alliete (0.9 g/L) were applied as preventative treatments for downy mildew. The dose was determined according to the manufacturer's instructions.
[0382] The comparator and additional products represent conventional treatments in the integrated pest management schedule.
[0383] Vines were visually examined for symptoms of powdery mildew.
[0384] Results:
[0385] Site 18
[0386] Approximately 20% of the peduncles and stems in the control block were black, indicating moderate infection from powdery mildew. In both the conventional treatment block and the terpene-treated block, all stems and bunches were green, indicating that adequate protection had been provided.
[0387] Site 20
[0388] No evidence of powdery mildew infection was observed in any of the blocks.
[0389] Additional Observations
[0390] At the end of the growing season, the blocks at sites 18 and 20 generally showed less stress due to disease than the rest of the vineyard.
[0391] Powdery mildew infections cause considerable losses to growers through reductions in vine growth, fruit quality and winter hardiness of vines. Furthermore, wine quality can be affected by an infection level of as little as 3% of berries. Management of the disease focuses on prevention because, once established, the infection can quickly spread. In this study, the application of terpene product YGP-GET at site 18 effectively prevented powdery mildew infection, and the level of control exhibited by the terpene product was comparable to that provided by the conventional treatment. The results from site 20 are inconclusive, however, due to the lack of powdery mildew infection. This lack of infection is likely to be due to the extensive application of pesticides prior to the study, which resulted in low disease pressure.
[0392] The lower level of stress due to disease at sites 18 and 20 suggests that the earlier terpene treatment applied at these sites may have been beneficial in control of infection in the long term.
[0393] Conclusions:
[0394] YGP-GET effectively prevented powdery mildew infection, with a comparable level of control to that provided by the conventional treatment.
Example 18Further Field Trials of Encapsulated Terpene Composition on Powdery Mildew
[0395] The study aimed to further investigate the efficacy of YGP-GET for the treatment of powdery mildew in Grimson Seedless table grapes.
[0396] A 0.1 ha plot on the Tsigaras vineyard (approximately 80 km south of the Kir-Yianni vineyard) was inadvertently left untreated during an application of Cisteine on 1 Jul. 2004. The vines in this plot subsequently showed severe symptoms of powdery mildew on the leaves, stems and grapes. On 12 Jul. 2004, the untreated plot was sprayed with 3 ml/L liquid YGP-GET formulation at a rate of 1200 l/ha, and the rest of the vineyard was sprayed with the comparator product Rogana. The vines were assessed for symptoms of powdery mildew after 24 hours.
[0397] Vines were trained in a high lyre trellis system.
[0398] Details of Comparator Product
[0399] Rogana (fenbuconazol 5%, binocap 16%), manufactured by BASF (BASF Agro Hellas S. A., Athens, Greece) On 12 Jul. 2004, Rogana was applied to the Tsigaras vineyard as a treatment for powdery mildew. The dose was determined according to the manufacturer's instructions.
[0400] Vines were visually examined for symptoms of powdery mildew.
[0401] Results
[0402] Severe symptoms of powdery mildew were evident prior to application of YGP-GET. Only 24 hours after YGP-GET application, the white bloom of the powdery mildew turned black, indicating effective antifungal activity. As the disease was effectively halted at this time, no further treatments were applied. YGP-GET showed comparable efficacy to the conventional treatment.
[0403] Discussion
[0404] In this study, an established powdery mildew infection was treated quickly and effectively using YGP-GET. Only 24 hours after application, the previously severe powdery mildew infection was halted by application of the terpene product, with comparable efficacy to the conventional treatment. The preliminary data obtained from this study suggest that YGP-GET may be efficacious in treating established fungal infections in addition to showing preventative ability.
Example 19Further Field Trials of Encapsulated Terpene Composition on Powdery Mildew
[0405] Background and Rationale
[0406] In the current trial, the use of YGP-GET was investigated as part of a Tasmanian vineyard's (Frogmore Creek Vineyard, Hathaway Trading Pty Ltd, Box 187, Richmond TAS 7025, Australia) experimental programme to control powdery mildew using organic products. The aim of this study was to investigate the short-term efficacy of the application of YGP-GET in the organic control of powdery mildew in Chardonnay grapevines.
[0407] In this trial grapevines (Chardonnay variety) were either treated with the terpene product YGP-GET or left untreated (control) on 7 Feb. 2005. Although suppressed by previous organic treatments, the pre-trial severity of powdery mildew was at a level considered unacceptable commercially and was equivalent in the 6 active-treatment plots and 6 control plots. The crop stage was approximately E-L 33-34 (pre-veraison).
[0408] YGP-GET (4 mL/L) (liquid formulation) was sprayed onto 6 Chardonnay plots, which had been treated previously with milk. Six Chardonnay plots served as untreated controls, but they had been treated previously with oil/whey. The number of vines per plot was typically 7.
[0409] Details of the composition of the YGP-GET used in this protocol are given in Table 20.
TABLE-US-00020 TABLE 20 Formulation of Batch Used in Present Study Raw material mix details Weight in lbs % by Weight Geraniol 323.52 6.88 Eugenol 161.76 3.44 Thymol 323.52 6.88 Yeast particles 722.13 15.35 Xanthan 3.17 0.07 Polysorbate 3.17 0.07 Water 3166.62 67.32 TOTAL 4703.89 100.00
[0410] The severity of powdery mildew was assessed 3 days before terpene treatment and again 3 days post-treatment. In each plot, 20 grape bunches were selected at random (10 bunches per panel side), and disease severity was estimated as the percentage area of the bunches covered with active mildew colonies. No further assessment was possible because the grower subsequently sprayed the entire trial area with sulphur and a vegetable oil-based spraying adjuvant (Synertrol Horti Oil).
[0411] Number/Area of Plants to be Treated
[0412] Test product: YGP-GET (4 mL/L) to be applied to 6 Chardonnay plots (total of approximately 42 vines), which had been treated previously with milk.
[0413] Control: No treatment was applied to 6 Chardonnay plots (total of approximately 42 vines) to be used as controls, but they had been treated previously with oil/whey.
[0414] Cultivation Methods
[0415] Vitis vinifera (Chardonnay) vines in Block B2: vertical shoot positioning with arched canes.
[0416] Cultivation Arrangement
[0417] Spacing: Distance of 2.5 m between rows and 1.25 m between vines (within row), with 3,200 vines per hectare. Row orientation was north to south.
[0418] Canopy Density
[0419] The point-quadrat method was used to characterise the pre-trial canopy density of the Chardonnay vines (Table 21). Measurements were taken on 13 Jan. 2005 by selecting representative sections of the canopy within the Chardonnay plots that previously had been either treated with sulphur or left untreated. Ten measurements were taken in each of the 6 plots of each prior treatment (i.e. a total of 60 measurements for the sulphur-treated plots and 60 measurements for the untreated control plots). In addition, the length and number of nodes on 3 upright shoots (per plot) were measured.
TABLE-US-00021 TABLE 21 Pre-trial canopy density of the Chardonnay vines Mean Mean Leaf layer Interior Interior number shoot Prior Gaps number leaves clusters of length treatment (%) (LLN) (%) (%) nodes (cm) Untreated 12 1.5 22 26 21 110 Sulphur 5 2.0 27 40 21 104 Optimum 20-40% 1.0-1.5 <10% <40% NA NA values NA, not applicable.
[0420] General Condition
[0421] Previous treatment of these plots with experimental materials suppressed powdery mildew in comparison to the untreated control. However, the level of powdery mildew was considered commercially unacceptable, although equivalent in both the milk- and oil/whey-treated plots.
[0422] Application Method, Dose and Regimen YGP-GET treatment (4 mL/L) was applied on 7 Feb. 2005 with a hand gun connected to a hose reel and pump mounted on the flat tray of a utility vehicle. The spray was propelled with a pump pressure of 1500-1600 kPa (200-230 psi), delivering approximately 63 mL/second. The standard spray volume for conventional treatments (approximately 900 L/ha) was used.
[0423] The severity of powdery mildew, estimated as the area (%) of the grape bunches covered with active mildew colonies, was assessed for 20 bunches selected at random within each plot (10 bunches per panel side). Disease severity was assessed on 4 Feb. 2005, 3 days before application of the YGP-GET treatment, and again on 10 Feb. 2005, 3 days after terpene application.
[0424] Data were transformed using arcsin transformation to obtain mean separations.
[0425] Results
[0426] Prior to treatment, the mean severity of powdery mildew on Chardonnay grape bunches in the 6 plots to be treated with terpene (20.4%) was similar to that in the 6 control plots (23.2%; Table 22). Statistical analysis based on arcsin transformation of these data found that there was no significant difference in disease severity before treatment (Table 23).
[0427] Three days after treatment, however, the mean severity of powdery mildew was 23.8% on the YGP-GETtreated bunches versus 37.8% on the controls (Table 22). Arcsin transformation of these data showed a statistically significant difference in favour of the terpene-treated grape bunches, which had a smaller area covered with active mildew colonies (p=0.058; Table 23).
TABLE-US-00022 TABLE 22 Mean severity of powdery mildew (%) on Chardonnay bunches before and after treatment with YGP-GET Treatment applied Mean severity on 7 Feb. 2005 On 4 Feb. 2005 On 10 Feb. 2005 YGP-GET 20.4 23.8 None 23.2 37.8
TABLE-US-00023 TABLE 23 Statistical separation of treatments following arcsin transformation of data Treatment applied Mean severity (SEM) on 7 Feb. 2005 On 4 Feb. 2005 On 10 Feb. 2005 YGP-GET 0.2063 (0.03857) 0.2411 (0.04303) None 0.2401 (0.08534) 0.3954 (0.07852) t = 0.36 t = 1.72 df = 10 df = 10 p = 0.726 p = 0.058 Two-sided test: One-sided test: difference not untreated > significant treated
[0428] Discussion:
[0429] Infection of grapevines with powdery mildew can cause considerable losses to growers through detrimental effects on vine growth and hardiness, as well as on the quality of the fruit and wine. In organically managed vineyards, growers are searching for alternatives to treatments such as elemental sulphur.
[0430] This study investigated the efficacy of encapsulated terpene formulations (4 mL/L) as a liquid formulation in controlling powdery mildew in an organic vineyard in Tasmania, Australia. While other experimental treatments had been used as little as 3 weeks before terpene application, the level of powdery mildew infection was still considered commercially unacceptable. Three days after treatment of Chardonnay vines with YGP-GET, the severity of powdery mildew on treated grapes was significantly less than that on untreated controls. While the severity of infection in untreated controls worsened during the 6 days between pre- and post-treatment assessments, it remained steady in treated vines. Therefore, YGP-GET appeared to have slowed the rate of disease increase on grape bunches that had well-established colonies of sporulating powdery mildew before treatment. Presumably, colony expansion was inhibited, although existing colonies continued to sporulate to some degree. More long-term assessment of efficacy was not possible because the grower subsequently sprayed the entire trial area with sulphur.
[0431] These encouraging results demonstrate the efficacy of YGP-GET in controlling powdery mildew in grapevines.
Example 20Field Trials of Encapsulated Terpene Composition on Botrytis
[0432] Botrytis bunch rot of grapes is caused by Botrytis cinerea, a common fungus that can cause serious losses in fruit yield. Berries are the predominant site of infection, although the disease can also affect blossom and leaves. Initially, infected berries appear soft and watery, and may become covered with grey fungal growth in conditions of high humidity and moisture. Over time, infected berries shrivel and drop. Botrytis favours humid conditions with poor air circulation, and split or damaged berries are particularly susceptible to the spread of infection. Management strategies for botrytis include promotion of good air circulation, prevention of wounding and application of fungicides at appropriate times during the growing season.
[0433] The aim of this study was to investigate the efficacy of YGP-GET in the treatment of botrytis infection in grapes.
[0434] The emergence of botrytis in the Kir-Yianni vineyard in mid October 2004 (3 weeks after an application of Teldor could not be treated with conventional agrochemicals because the associated re-entry time restrictions would prevent the planned harvest. Two adjacent 0.1 ha plots were therefore identified on site 7 of the vineyard, and, on 12 Oct. 2004, one of these plots was treated with 4 mL/L YGP-GET liquid formulation and the other was left untreated (see
[0435] Site 7 had been treated with multiple products prior to the application of the terpene formulation but still showed botrytis infection.
[0436] Vines were given a single application of 4 ml/L YGP-GET liquid formulation at a rate of 1200 l/ha.
[0437] The following growth stages of the grapes were recorded: [0438] bud break, 26 Mar. 2004 [0439] bloom, 1 Jun. 2004 [0440] veraison, 6 Aug. 2004 [0441] harvest, 15 Oct. 2004
[0442] The study applications took place 3 days before harvest.
[0443] The 2004 growing season was exceptionally late and was wet throughout. Disease pressure from downy mildew was extremely high, powdery mildew pressure was moderate and botrytis levels were elevated. YGP-GET was applied at this time to assess its potential efficacy against a botrytis infection that could not otherwise have been treated because of pesticide time restrictions prior to harvest.
[0444] Visual assessment of the site prior to terpene product application revealed evidence of botrytis infection. After harvest, the berries were displayed on a conveyor belt and infected berries were manually separated from uninfected berries prior to crushing. The proportion of infected berries was calculated as a percentage of the total yield (by weight) for each plot.
[0445] Results
[0446] Visual assessment of the site prior to YGP-GET application revealed evidence of botrytis infection. Following harvest (3 days after YGP-GET application), the proportions of infected berries were 13% and 23% in the treated and untreated plots, respectively. The tested areas were not sufficient to assess statistical significance; however, YGP-GET treatment clearly slowed the progression of the disease.
[0447] Fermentation was not affected by the mixing of uninfected berries from the untreated and terpene-treated plots.
[0448] Discussion
[0449] Conventional treatments for botrytis must be halted 3 weeks before harvest, leaving time for considerable damage to crop yield and quality to occur. The development of a treatment that could be used until harvest, or that could be continued closer to harvest than the existing products, could result in significant improvements in crop yield and wine quality, and would be of considerable benefit to growers. In this study, treatment with the terpene product YGP-GET visibly slowed progression of an established botrytis infection only 3 days prior to harvest, resulting in a lower proportion of infected berries in the terpene-treated plot than in the untreated plot. Furthermore, despite the use of YGP-GET close to harvest, fermentation was unaffected by the combination of treated and untreated grapes.
[0450] These results suggest that YGP-GET is efficacious in reducing the impact of established botrytis infections and can be used near to harvest without detrimental effects on subsequent fermentation.
Example 21Evaluation of Encapsulated Terpenes for the Treatment of Established Downy Mildew and Subsequent Evaluation of Grape Quality
[0451] A trial of YGP-GET was carried out on 25/08/04 applying the composition at a rate of 1000 g per 250 liters.
[0452] A vineyard of Cabernet Sauvignon which was 100% infected and suffering substantial leaf loss due to Downy Mildew was sprayed. Any remaining leaves were infected with spots of Downy Mildew as evidenced by the yellow spot on top of the leaf and the fuzzy growth on the leaf bottom; the classical indication of Downy Mildew. Many of the leaves were almost entirely yellow indicating substantial infection.
[0453] This leaf loss and the infection in general delays the maturity of the grapes and in many cases the grapes never fully ripen for winemaking purposes. Observation of totally unripened (i.e. hard dark green berries 1 cm diameter and oval in shape) bunches occasionally in the vines indicated that the vines were likely infected before veraison, and likely at bloom or before. No early copper (Bordeaux or basic Copper sulfate) application has been used. This vineyard was heavily infected in the previous harvest to the point that no crop was produced from the Cabernet Sauvignon. Leaf loss last year was 100% despite Potassium Bi-carbonate treatment in an attempt to contact kill the Downy Mildew, followed by Stilbourin application for longer term systemic protection.
[0454] On 19/09/04 the grapes treated in this trial were picked and crushed and the following observations were made on the must (Table 24):
TABLE-US-00024 TABLE 24 Control Treated Desirable pH 3.28 3.30 3.3-3.5 TA 0.92 0.85 0.7-0.75 Brix 17.4 18.7 20-22
[0455] These results indicate the grapes from the treated vines are riper than those of the untreated vines. Observation of the grapes themselves indicated that the untreated grapes were, on average, lighter in color, some with a transparent pinkish/purple/green tint, indicative of grapes just past veraison, whereas the treated grapes were dark purple on average and opaque, typical of fully or nearly fully ripened grapes.
[0456] Tasting of these grapes revealed the treated grapes to have a fuller fruitier taste typical of ripe Cabernet Sauvignon, whereas the untreated grapes did not have the full fruity taste. The untreated grapes had a green apple sour taste indicating probable a high malic/tartaric ratio unsuitable for good winemaking.
[0457] These grapes were crushed and destined in preparation for producing a wine from these grapes to demonstrate the difference in these grapes and to demonstrate the suitability of the treated grapes for winemaking. The grape grower was concerned that this treatment would affect the flavor of the wine, although at my suggestion he tasted treated grapes the day after application of YGP-GET and found no lingering taste or aroma.
[0458] The difference in the treated and untreated grapes is further demonstrated in the color of the must. The juice of the untreated grapes was light greenish/uncolored (somewhat like a white wine must) whereas the must from the treated grapes was a pinkish color typical of ripe Cabernet Sauvignon grapes immediately after crushing.
[0459] These results indicate that YGP-GET is efficacious in late summer vineyard treatment by killing and stopping Downy Mildew re-infection, in at least the short term.
[0460] Further research into the long term efficacy of the YGP-GET in controlling downy mildew would be useful, but the results presented show that YGP-GET is a useful treatment.
[0461] Late onset Downy Mildew can completely ruin a crop and there are currently no effective treatments which can be applied shortly before harvest and that retain their ability to provide protection. The great strength of YGP-GET is the ability to provide a quick kill and maintain this efficacy over a longer time than other contact fungicides.
[0462] There are a number of anti-fungals in this market which have an established track record against Downy Mildew, but all need some time after application before the crop can be harvested. Some treatments (like sulfur containing products) cannot be used if the temperature rises above 85 F. Phytotoxicity of copper containing fungicides is also significant depending on the variety of grape. Contact fungicides do not have a long term effect so a second application of a longer active fungicide is often needed, but may be restricted by relevant regulation (e.g. PHI or REI).
[0463] Many conventional treatments for Downy Mildew have a restricted reentry (REI and or PHI) which means the grower cannot apply the treatment in fear that he will apply something like Mancozeb, which has a PHI of 66 days; the grower would then be unable to harvest his grapes at peak maturity.
[0464] Downy Mildew is implicated as the primary cause of the many poor wines being produced east of the Mississippi. YGP-GET could allow affected grapes to ripen properly and be picked at peak maturity in this rapidly growing industry.
[0465] Advantageously YGP-GET should be eligible for approval by the various organic committees (many self-appointed) that this product is suitable for use on grapes grown under organic guidelines. This opens another niche in a rapidly growing market segment in the US and worldwide.
Example 22In Vitro Assessment of the Fungicidal Properties of Encapsulated and Non-Encapsulated Terpenes
[0466] Further tests were conducted to assess the 31 non-encapsulated terpene preparations set out in Example 15 and preparations 16 and 22 encapsulated in glucan particles.
[0467] To conduct these assays, 20,000 spores were placed in strength potato dextrose broth (PDB) and sufficient quantities of selected terpene formulations were added to give concentrations ranging from 10 to 1000 ppm. These test materials were placed in separate sterile capped Eppendorf tubes with Botrytis cinerea (B.c.) spores, incubated for 24 hr, then the spores were recovered by centrifugation, and the terpene solutions were discarded. The spores/biomass were rinsed with sterile water, centrifuged again and then taken back up in 300 l of strength PDB and transferred to 96 well plates. The optical density of the surviving spores growing into mycelia was measured over time. Fungicidal activity is defined as total killing of 20,000 spores after 24 hours terpene exposure, as evidence by the absence of mycelial growth.
[0468] The results suggest that certain formulations were not fungicidal at a statistically significant level under the present test conditions (results not shown). These were:
[0469] 1, 2, 4, 5, 6, 8, 9, 11, 12, 14, 15, 17, 18, 19, 20, 21, 23, 24, 25, 27, 28, 29, 30. Refer to Example 15 (Table 17) for details of the compositions.
[0470] The minimum inhibitory concentration for the most effective compounds is set out in Table 26.
TABLE-US-00025 TABLE 25 Minimum inimum inhibitory inhibitory concentration concentration Material (ppm) Material (ppm) 3 <1000; >750 7 <1000; >750 10 <1000; >500* 13 <1000; >750 16 <1000; >750 22 <750; >500 26 <1000; >750 31 <1000; >750 *In different tests, the lowest concentration that gave no growth was either 500 or 750 ppm.
[0471] Comparative Testing of Compounds in Water and Encapsulated in Hollow Glucan Particles.
[0472] Samples of formulations 16 (geraniol, eugenol and thymol) and 22 (eugenol, thymol and citral) encapsulated in hollow glucan particles were prepared in accordance with techniques previously described. The fungicidal properties were then assessed for encapsulated and non-encapsulated formulations using the protocol previously described for the non-encapsulated formulations.
[0473] The results were quite different with encapsulated terpene formulations as compared with the terpenes suspended in water, as shown in
[0474] The minimum effective concentration is shown below in Table 26.
TABLE-US-00026 TABLE 26 MIC in yeast Material MIC in suspension particles 16 <1000, >750 <100, >250 22 <750, >500 <500, >250
[0475] Thus, the results with materials 16 and 22 are quite different when in aqueous suspension and when tested encapsulated in glucan particles. (Note: as mentioned later, there was some variability in the results with terpenes suspended in water, the experiment noted above is an example of this). The MIC values are composites from several trials. Importantly, the results with encapsulated terpene formulations do not suffer from the problems of variability associated with aqueous terpene suspensions. There have been five separate tests of terpenes suspended in water and three with the YPs.
[0476] Encapsulated terpene formulations are readily miscible with water and provide a slow release terpene formulation into the aqueous medium. This results in a longer exposure time of the spores to the terpenes.
[0477] Problems monitoring the non-encapsulated terpene formulations in suspension in the test media were encountered which may have affected the results in this regard.