U.S. patent application number 16/828530 was filed with the patent office on 2020-07-30 for compositions containing a hollow glucan particle or a cell wall particle encapsulating a terpene component, methods of making an.
The applicant listed for this patent is Eden Research PLC. Invention is credited to Lanny FRANKLIN, Gary OSTROFF.
Application Number | 20200236927 16/828530 |
Document ID | 20200236927 / US20200236927 |
Family ID | 1000004753957 |
Filed Date | 2020-07-30 |
Patent Application | download [pdf] |
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United States Patent
Application |
20200236927 |
Kind Code |
A1 |
FRANKLIN; Lanny ; et
al. |
July 30, 2020 |
COMPOSITIONS CONTAINING A HOLLOW GLUCAN PARTICLE OR A CELL WALL
PARTICLE ENCAPSULATING A TERPENE COMPONENT, METHODS OF MAKING AND
USING THEM
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.
Inventors: |
FRANKLIN; Lanny; (Atlanta,
GA) ; OSTROFF; Gary; (Worcester, MA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Eden Research PLC |
Poulton |
|
GB |
|
|
Family ID: |
1000004753957 |
Appl. No.: |
16/828530 |
Filed: |
March 24, 2020 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11597116 |
Oct 27, 2008 |
10638750 |
|
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PCT/GB2005/002011 |
May 20, 2005 |
|
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16828530 |
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60572892 |
May 20, 2004 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A01N 35/06 20130101;
A01N 31/08 20130101; A01N 25/28 20130101; A01N 31/16 20130101; B01J
13/203 20130101; A01N 49/00 20130101; A61K 9/5068 20130101; A01N
25/26 20130101; A61K 9/5036 20130101; B01J 13/206 20130101; A01N
35/02 20130101; A01N 31/02 20130101 |
International
Class: |
A01N 25/26 20060101
A01N025/26; A01N 31/08 20060101 A01N031/08; A01N 31/16 20060101
A01N031/16; A01N 31/02 20060101 A01N031/02; A61K 9/50 20060101
A61K009/50; A01N 35/02 20060101 A01N035/02; A01N 25/28 20060101
A01N025/28; B01J 13/20 20060101 B01J013/20; A01N 35/06 20060101
A01N035/06; A01N 49/00 20060101 A01N049/00 |
Foreign Application Data
Date |
Code |
Application Number |
Jan 24, 2005 |
GB |
PCT/GB2005/000240 |
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.RTM., Span.RTM. 20, Span.RTM. 40, Span.RTM. 60, Span.RTM. 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.degree. 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
[0001] The present invention relates to compositions comprising
terpenes and hollow glucan particles or cell wall particles and
methods for preparing such compositions. The compositions increase
terpene stability and activity and provide a suitable carrier for
the terpenes. The invention also relates to methods of using such
compositions in the medical, veterinary and agricultural
fields.
[0002] Terpenes are chemical compounds that are widespread in
nature, mainly in plants as constituents of essential oils. Their
building block is the hydrocarbon isoprene (C.sub.5H.sub.8).sub.n.
Examples of terpenes include citral, pinene, nerol, b-ionone,
geraniol, carvacrol, eugenol, carvone, terpeniol, anethole,
camphor, menthol, limonene, nerolidol, farnesol, phytol, carotene
(vitamin A.sub.1), squalene, thymol, tocotrienol, perillyl alcohol,
borneol, myrcene, simene, carene, terpenene, and linalool.
[0003] Terpenes are classified as Generally Recognized as Safe
(GRAS) and have been used for many years in the flavouring and
aroma industries. The LD.sub.50 in rats of citral is approximately
5 g/kg, which is a further indication of the relative safety of
these compounds. Furthermore, terpenes have a relatively short life
span of approximately 28 days once exposed to oxygen (e.g. air).
Terpenes will decompose to CO.sub.2 and water. This decomposition
or break down of terpenes demonstrates the safety and environmental
friendliness of the compositions and methods of the invention.
[0004] Terpenes have been found to inhibit the growth of cancerous
cells, decrease tumour size, decrease cholesterol levels, and have
a biocidal effect on micro-organisms in vitro. Owawunmi, (Letters
in Applied Microbiology, 1993, 9(3): 105-108), showed that growth
media with more than 0.01% citral reduced the concentration of E.
coli, and at 0.08% there was a bactericidal effect. U.S. Pat. No.
5,673,468 describes a terpene formulation, based on pine oil, used
as a disinfectant or antiseptic cleaner. U.S. Pat. No. 5,849,956
teaches that a terpene found in rice has antifungal activity. U.S.
Pat. No. 5,939,050 describes an oral hygiene antimicrobial product
with a combination of 2 or 3 terpenes that showed a synergistic
effect. Several (U.S. Pat. Nos. 5,547,677, 5,549,901, 5,618,840,
5,629,021, 5,662,957, 5,700,679, 5,730,989) teach that certain
types of oil-in-water emulsions have antimicrobial, adjuvant, and
delivery properties. Terpenes have been found to be effective and
nontoxic dietary anti-tumor agents, which act through a variety of
mechanisms of action (Crowell et al. Crit. Rev. Oncog., 1994, 5(1):
1-22; Crowell et al. Adv. Exp. Med. Biol., 1996, 401: 131-136). The
terpenes geraniol, tocotrienol, perillyl alcohol, b-ionone, and
d-limonene, suppress hepatic HMG-CoA reductase activity, a rate
limiting step in cholesterol synthesis, and modestly lower
cholesterol levels in animals (Elson et al, J. Nutr., 1994, 124:
607-614). D-limonene and geraniol reduced mammary tumors (Elegbede
et al. Carcinogenesis, 1984, 5(5): 661-664; Elegbede et al., J.
Natl. Cancer Inst., 1986, 76(2): 323-325; Karlson et al. Anticancer
Drugs, 1996, 7(4): 422-429) and suppressed the growth of
transplanted tumors (Yu et al., J. Agri. Food Chem., 1995, 43:
2144-2147).
[0005] Terpenes have also been found to inhibit the in vitro growth
of bacteria and fungi (Chaumont et al.), Ann. Pharm. Fr., 1992,
50(3): 156-166; Moleyar et al., Int. J. Food Microbiol, 1992,
16(4): 337-342; and Pattnaik et al. Microbios, 1997, 89(358):
39-46) and some internal and external parasites (Hooser et al., J.
Am. Vet. Med. Assoc., 1986, 189(8): 905-908). Geraniol was found to
inhibit growth of Candida albicans and Saccharomyces cerevisiae
strains by enhancing the rate of potassium leakage and disrupting
membrane fluidity (Bard et al., Lipids, 1998, 23(6): 534-538).
B-ionone has antifungal activity which was determined by inhibition
of spore germination, and growth inhibition in agar (Mikhlin et
al., A. Prikl. Biokhim. Mikrobiol, 1983, 19: 795-803; Salt et al.,
Adam. Physiol. Molec. Plant Path, 1986, 28: 287-297). Teprenone
(geranylgeranylacetone) has an antibacterial effect on H. pylori
(Ishii, Int. J. Med. Microbiol. Virol. Parasitol. Infect. Dis.,
1993, 280(1-2): 239-243). Rosanol, a commercial product with 1%
rose oil, has been shown to inhibit the growth of several bacteria
(Pseudomonas, Staphylococus, E. coli, and H. pylori). Geraniol is
the active component (75%) of rose oil. Rose oil and geraniol at a
concentration of 2 mg/L inhibited the growth of H. pylori in vitro.
Some extracts from herbal medicines have been shown to have an
inhibitory effect in H. pylori, the most effective being decursinol
angelate, decursin, magnolol, berberine, cinnamic acid, decursinol,
and gallic acid (Bae et al., Biol. Pharm. Bull., 1998, 21(9)
990-992). Extracts from cashew apple, anacardic acid, and
(E)-2-hexenal have shown bactericidal effect against H. pylori.
[0006] Diterpenes, i.e., trichorabdal A (from R. Trichocarpa), have
shown a very strong antibacterial effect against H. pylori (Kadota
et al., Zentralbl. Bakteriol, 1997, 287(1): 63-67).
[0007] Solutions of 11 different terpenes were effective in
inhibiting the growth of pathogenic bacteria in in vitro tests;
levels ranging between 100 ppm and 1000 ppm were effective. The
terpenes were diluted in water with 1% polysorbate 20 (Kim et al.,
J. Agric. Food Chem., 1995, 43: 2839-2845).
[0008] There may be different modes of action of terpenes against
microorganisms; they could (1) interfere with the phospholipid
bilayer of the cell membrane, (2) impair a variety of enzyme
systems (HMG-reductase), and (3) destroy or inactivate genetic
material. It is believed that due to the modes of action of
terpenes being so basic, e.g., blocking of cholesterol, that
infective agents will not be able to build a resistance to
terpenes.
[0009] There are, however, a number of drawbacks to the use of
terpenes. These include: [0010] Terpenes are liquids which can make
them difficult to handle and unsuitable for certain purposes.
[0011] Terpenes are not very miscible with water, and it generally
requires the use of detergents, surfactants or other emulsifiers to
prepare aqueous emulsions. A stable solution can, however, be
obtained by mixing the terpenes under high shear. [0012] Dry powder
terpene formulations generally only contain a low percentage w/w of
terpenes. [0013] Terpenes are prone to oxidation in aqueous
emulsion systems, which make long term storage a problem.
[0014] There are limitations to the current techniques of spray
coating, extrusion, coacervation, molecular encapsulation, and
spray drying/cooling to provide ingredient delivery systems.
[0015] Baker's yeast cell walls are derived from baker's yeast
cells and are composed of the insoluble biopolymers
.beta.-1,3-glucan, .beta.-1,6-glucan, mannan and chitin. They are
typically 2-4 micron in diameter microspheres with a shell wall
that is only 0.2-0.3 micron thick surrounding an open cavity. This
material has considerable liquid holding capacity, typically
absorbing 5-25 times its weight in liquid. The shell is
sufficiently porous that payloads up to 150,000 Daltons in size can
pass through the outer shell and be absorbed into the hollow cavity
of the spherical particle. Baker's yeast cell walls have several
unique properties, including heat stability (e.g. to 121.degree.
C.), shear stability, pH stability (e.g. pH 2-12), and at high
concentrations they do not build significant viscosity. In addition
to its physical properties this composition contains natural and
healthy dietary fibres that deliver cardiovascular and
immunopotentiation health benefits.
[0016] Yeast cell walls are prepared from yeast cells by the
extraction and purification of the insoluble particulate fraction
from the soluble components of the yeast cell. The fungal cell
walls can be produced from the insoluble byproduct of yeast extract
manufacture. Further, the yeast cells can be treated with an
aqueous hydroxide solution, without disrupting the yeast cell
walls, which digests the protein and intracellular portion of the
cell, leaving the yeast cell wall component devoid of significant
protein contamination, and having substantially the unaltered cell
wall structure of .beta.(1-6) and .beta.(1-3) linked glucans. A
more detailed description of whole glucan particles and the process
of preparing them is described by Jamas et al. in U.S. Pat. No.
4,810,646 and in co-pending patent applications U.S. Ser. No.
166,929, U.S. Ser. No. 297,752 and U.S. Ser. No. 297,982. U.S. Pat.
No. 6,242,594, assigned to Novogen Research Pty Ltd., describes a
method of preparing yeast glucan particles by alkali extraction,
acid extraction and then extraction with an organic solvent and
finally drying. U.S. Pat. No. 5,401,727, assigned to AS
Biotech-Mackzymal, discloses the methods of obtaining yeast glucan
particles and methods of using them to promote resistance in
aquatic animals and as an adjuvant for vaccinations. U.S. Pat. No.
5,607,677, assigned to Alpha-Beta Technology Inc., discloses the
use of hollow whole glucan particles as a delivery package and
adjuvant for the delivery of a variety of pharmaceutical agents.
The teachings of the abovementioned patents and applications are
incorporated herein by reference.
[0017] Other types of yeast and fungi cells have cell walls that do
not contain glucan. The cell walls of such yeast and fungi can be
isolated by similar techniques to those mentioned above to obtain
cell wall particles.
[0018] Additionally, the cells of many plants, algae, bacteria and
other micro-organisms also comprise a cell wall. The structure and
composition of the cell wall varies between micro-organism, but in
general it is a robust and relatively inert structure. It is
possible to obtain cell wall particles derived from such cells
through conventional techniques, such as those mentioned above in
relation to yeast.
[0019] We have now found that terpenes can be taken up and stably
encapsulated within hollow glucan particles or cell wall particles.
Encapsulation of terpenes into such particles can be achieved by
incubation of the particles with the terpene.
[0020] According to the present invention there is provided a
composition comprising a hollow glucan particle or a cell wall
particle encapsulating a terpene component.
[0021] The term "hollow glucan particle" as used herein includes
any hollow particle comprising glucan as a structural component.
Thus, in particular, the term includes yeast cell walls (in
purified or crude forms) or hollow whole glucan particles. The term
"cell wall particle" refers to a particle comprising the wall of a
cell (in a purified or crude form), wherein glucan is not a
structural component. Suitable particles include the cell walls of
plant, algal, fungal or bacterial cells. Cell wall particles
generally retain the shape of the cell from which they are derived,
and thus, like a hollow glucan particle, provide a hollow central
cavity suitable for encapsulating the terpene component.
[0022] For the present invention it is necessary that the hollow
glucan particle or cell wall particle is able to stably encapsulate
the terpene component. In general this means the hollow glucan
particle or cell wall particle must be able to maintain its
structure during incubation with the terpene component (generally
the terpene component is at a relatively high concentration), and
that terpene component must be able to migrate into the particle.
Hollow glucan particles and cell wall particles are generally
formed from relatively inert materials and are porous, and thus it
can be assumed that, in general, hollow glucan particles and cell
wall particles will be able to encapsulate a terpene component.
[0023] Compositions according to the present invention are
effective against various infective agents including bacteria,
viruses, mycoplasmas, fungi and/or nematodes.
[0024] The compositions according to the present invention can
provide the following advantages: [0025] maximise terpene payload;
[0026] minimise unencapsulated payload; [0027] control payload
stability; [0028] control payload release kinetics; [0029] creation
of a solid form of a liquid terpene to increase the mass and
uniformity; [0030] simplify handling and application of terpenes;
and [0031] mask the smell and taste of the terpene.
[0032] Particularly suitable hollow glucan particles or cell wall
particles are fungal cell walls, preferably yeast cell walls. Yeast
cell walls are preparations of yeast cells that retain the
three-dimensional structure of the yeast cell from which they are
derived. Thus they have a hollow structure which allows the terpene
component to be encapsulated within the yeast cell walls. The yeast
walls may suitably be derived from Baker's yeast cells (available
from Sigma Chemical Corp., St. Louis, Mo.). Yeast cell wall
particles with desirable properties can also be obtained from
Biorigin (Sao Paolo, Brazil) under the trade name Nutricell MOS 55.
These particles are a spray dried extract of S. cerevisiae.
[0033] Alternative particles are those known by the trade names
SAF-Mannan (SAF Agri, Minneapolis, Minn.) and Nutrex (Sensient
Technologies, Milwaukee, Wis.). These are hollow glucan particles
that are the insoluble waste stream from the yeast extract
manufacturing process. During the production of yeast extracts the
soluble components of partially autolyzed yeast cells are removed
and the insoluble residue is a suitable material for terpene
loading. These hollow glucan particles comprise approximately
25-35% beta 1,3-glucan w/w. A key attribute of these materials are
that they contain more than 10% lipid w/w and are very effective at
absorbing terpenes. In addition, as a waste stream product they are
a relatively cheap source of hollow glucan particles.
[0034] Alternative hollow glucan particles which have higher purity
are those produced by Nutricepts (Nutricepts Inc., Burnsville,
Minn.) and ASA Biotech. These particles have been alkali extracted,
which removes additional intracellular components as well as
removes the outer mannoprotein layer of the cell wall yielding a
particle of 50-65% glucan w/w.
[0035] Higher purity hollow glucan particles are the WGP particles
from Biopolymer Engineering. These particles are acid extracted
removing additional yeast components yielding a product 75-85%
glucan w/w.
[0036] Very high purity hollow glucan particles are Adjuvax.TM.
from Alpha-beta Technology, Inc. (Worcester, Mass.) and
microparticulate glucan from Novogen (Stamford, Conn.). These
particles are organic solvent extracted which removes residual
lipids and sd: the particles comprise more than 90% glucan w/w.
[0037] In some embodiments a high purlity glucan particle or cell
wall particle may be required, for example where strict control
over possible contaminants is required. In these instances the
higher purity particles would be preferred aver other less pure
products. For other embodiments, the less pure particles would be
preferred for economic reasons; those particles have also been
found to be more effective at absorbing terpenes.
[0038] Preferably the hollow glucan particle or cell wall particle
has a slight lipid content, such as 1 or 2% w/w lipid. A slight
lipid content can increase the ability of the particle to
encapsulate the terpene component. Preferably the lipid content of
the hollow glucan particle or cell wall particle is 5% w/w or
greater, more preferably 10% w/w or greater.
[0039] Optionally the terpene component of the present invention
can be associated with a surfactant. The surfactant can be
non-ionic, cationic, or anionic. Examples of suitable surfactants
include sodium lauryl sulphate, polysorbate 20, polysorbate 80,
polysorbate 40, polysorbate 60 polyglyceryl ester, polyglyceryl
monooleate, decagilyceryl monocaprylate, propylene glycol
dicaprilate, triglycerol monostearate, polyoxyethylenesorbitan,
monooleate, Tween.RTM., Span.RTM. 20, Span.RTM. 40, Span.RTM. 60,
Span.RTM. 80, Brig 30 or mixtures thereof. The surfactant acts to
hold the terpene component in an emulsion and also assists
encapsulation of the terpene component into the hollow glucan
particle or cell wall particle.
[0040] The terpene component of the present invention can comprise
a single terpene or a mixture of terpenes. Mixtures of terpenes can
result in synergistic effects.
[0041] The term "terpene" as used herein refers not only to
terpenes of formula (C.sub.5H.sub.8)--, but also encompasses
terpene derivatives, such as terpene aldehydes or terpene polymers.
Natural and synthetic terpenes are included, for example
monoterpenes, sesquiterpenes, diterpenes, triterpenes, and
tetraterpenes. In addition, reference to a single name of a
compound will encompass the various isomers of that compound. For
example, the term citral includes the cis-isomer citral-a (or
geranial) and the trans-isomer citral-b (or neral).
[0042] It should be noted that terpenes are also known by the names
of the extract or essential oil which contain them, e.g. lemongrass
oil (contains citral).
[0043] The terpenes which are exempted from US regulations and
which are listed in EPA regulation 40 C.F.R. Part 152 (incorporated
herein by reference in its entirety) are suitable for use in this
invention.
[0044] Particularly suitable terpenes for use in the present
invention include those 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 and mixtures
thereof.
[0045] Preferably the terpenes used in the present invention have
the general structure C.sub.10H.sub.16 as this sub-group is
generally more effective against infective agents.
[0046] More preferably the terpene component comprises a terpene
selected from the group consisting of geraniol, thymol, citral,
carvone (for example L-carvone), eugenol and b-ionone.
[0047] The terpene component can suitably comprise thymol, as this
terpene has been shown to be particularly effective in treating or
preventing fungal plant infections.
[0048] Another particularly suitable terpene is citral which has
demonstrated particular efficacy against a number of
micro-organisms.
[0049] A combination of geraniol, thymol and eugenol has
demonstrated particular efficacy in combating plant infections, and
is thus a particularly suitable terpene component.
[0050] Other terpene formulations which have shown high efficacy in
treating plant infections include (percentages are w/w): [0051]
100% thymol; [0052] 50% geraniol and 50% thymol; [0053] 50% eugenol
and 50% thymol; [0054] 33% geraniol, 33% eugenol and 33% thymol;
[0055] 33% eugenol, 33% thymol and 33% citral; [0056] 25% geraniol,
25% eugenol, 25% thymol and 25% citral; [0057] 20% geraniol, 20%
eugenol, 20% citral, 20% thymol and 20% L-carvone.
[0058] Accordingly a terpene component comprising any of the above
formulations is particularly suitable for use in the present
invention.
[0059] In one embodiment the terpene component includes one or more
terpenes which contain oxygen. Citral, for example citral 95, is an
oxygenated C.sub.10H.sub.16 terpene, C.sub.10H.sub.16O CAS No.
5392-40-5 (3,7-dimethyl-2,6-octadien-1-al). A stable suspension of
citral can be formed up to about 2500 ppm. Citral can be made into
a solution at up to about 500 ppm. A stable suspension of hollow
glucan particles incorporating citral of 25 ppt citral can be
made.
[0060] The composition of the invention can comprise 1 to 99% by
volume terpenes, 0 to 99% by volume surfactant and 1 to 99% hollow
glucan particles or cell wall particles. More specifically the
composition can comprise about 10% to about 67% w/w terpenes, about
0.1-10% surfactant and about 40-90% hollow glucan particles or cell
wall particles.
[0061] Suitably a composition of the present invention comprises
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. Preferably the composition comprises 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.
[0062] Specific compositions can include e.g., for bacteria and
fungi, hollow glucan particles or cell wall particles encapsulating
terpenes in water or standard 0.9% saline with up to 67% L-carvone,
up to 67% eugenol, up to 67% citral, up to 67% thymol and
L-carvone, up to 67% geraniol, or up to 67% citral and L-carvone
and eugenol, and 1% Tween.RTM. 80; for mold, hollow glucan
particles or cell wall particles encapsulating terpenes in water or
standard 0.9% saline with up to 67% citral and 1% Tween.RTM. 80; or
for mycoplasma, hollow glucan particles or cell wall particles
encapsulating terpenes in water or standard 0.9% saline with up to
67% citral, up to 67% L-carvone and eugenol, up to 67% eugenol, up
to 67% geraniol, or up to 67% geraniol, thymol, and 1% Tween.RTM.
80.
[0063] Concentrations of hollow glucan particles or cell wall
particles encapsulating terpenes of 1, 5, 10, 20, 30, 40, 50, 60,
70, 80, 90, 100, 110, 125, 130, 140, 150, 160, 175, 190, 200, 225,
250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 525, 550,
575, 600, 625, 650, 675, 700, 725, 750, 775, 800, 825, 850, 875,
900, 925, 950, 975, 1000, 1100, 1250, 1375, 1425, 1500, 1600, 1750,
or 2000 ppm can be used as effective concentrations in the
compositions and methods of the current invention. Even higher
concentrations (up to 25 ppt, i.e. parts per thousand) can be made
and may be useful in the current invention.
[0064] The composition of the present invention can comprise
between about 1 ppm and about 25 ppt (25000 ppm) of the terpene
component, preferably 100 to 2000 ppm of the terpene component, for
example, 250, 500, 1000, 2000 ppm thereof.
[0065] The terpenes, surfactants, and other components of the
invention may be readily purchased or synthesised using techniques
generally known to synthetic chemists.
[0066] It is highly preferred that terpenes used in the present
invention, for safety and regulatory reasons, are at least food
grade terpenes (as defined by the United States FDA or equivalent
national regulatory body outside the USA).
[0067] Optionally the composition can comprise other food-grade
active compounds in addition to the terpene component, for example
other antimicrobial agents, enzymes, or the like.
[0068] Optionally the composition can comprise a further active
agents in addition to the terpene component, for example an
antimicrobial agent, an anti-fungal agent, an insecticidal agent,
an anti-inflammatory agent, an anaesthetic or the like. Suitable
agents include: [0069] Anti-fungal: Cell wall hydrolyases (assuming
they do not degrade the hollow glucan particle or cell wall
particle), cell wall synthesis inhibitors, standard antifungals.
[0070] Anti-bacterial: Antiseptics, cell wall hydrolases, synthesis
inhibitors, antibiotics. [0071] Insecticidal: Natural insecticides,
chitinase.
[0072] The composition can comprise an antioxidant to reduce
oxidation of the terpene. An example of such an anti-oxidant might
be rosemary oil, vitamin C or vitamin E.
[0073] The composition of the present invention can be in the form
of a dry powder. The composition can be provided in combination
with an agriculturally, food or pharmaceutically acceptable carrier
or excipient in a liquid, solid or gel-like form.
[0074] For solid compositions, suitable carriers include
pharmaceutical grades of mannitol, lactose, starch, magnesium
stearate, sodium saccharin, talc, cellulose, glucose, sucrose,
magnesium carbonate, and the like. Suitably the formulation is in
tablet or pellet form. As suitable carrier could also be a human or
animal food material. Additionally, conventional agricultural
carriers could also be used.
[0075] A pellet, tablet or other solid form of the composition can
preferably also contain a dispersal agent which promotes dispersal
of the composition when placed into a liquid, e.g. water. Suitable
dispersal agents include xanthan gum, maltodextrin, alginates, or
the like.
[0076] Liquid compositions can, for example, be prepared by
dispersing the composition in water, saline, aqueous dextrose,
glycerol, ethanol, or the like, to form a solution or suspension.
If desired, these compositions can contain minor amounts of
non-toxic auxiliary substances such as wetting or emulsifying
agents, pH buffering agents (for example, sodium acetate, sorbitan
monolaurate, triethanolamine sodium acetate or triethanolamine
oleate). The methods of preparing such liquid compositions are
known, or will be apparent, to those skilled in this art; for
example see Remington: The Science and Practice of Pharmacy;
Lippincott, Williams & Wilkins; (Dec. 15, 2000)--which is
incorporated herein by reference. Again a liquid composition could
be prepared by dispersing the composition in a liquid human or
animal food or drink material. Additionally a suitable liquid
agricultural excipient could be used.
[0077] For oral administration tablets and granules are generally
preferred. Tablets may contain binders and lubricants. Fine powders
or granules may contain diluting, dispersing and/or surface active
agents and can be presented in water or in a syrup. Capsules or
sachets can conveniently contain the composition in a dry state.
Non-aqueous solutions or suspensions of the composition are also
suitable and may contain suspending agents. Where desirable or
necessary, flavouring, preserving, suspending, thickening, or
emulsifying agents can be included. Of course, it would be suitable
to use a food or drink material as an oral delivery method.
[0078] Parental administration is generally characterised by
injection. For injectables it will be appreciated that, in general,
all materials used in the composition and any excipient used must
be of pharmaceutical grade. Injectables can be prepared in
conventional forms, either as liquid solutions, emulsions or
suspensions, solid forms suitable for dissolution, suspension in
liquid prior to injection, or as emulsions. An alternative approach
for parental administration involves use of a slow release or
sustained release system, such that a constant level of dosage is
maintained. See, for example, U.S. Pat. No. 3,710,795, which is
incorporated by reference herein. Preparations for parenteral can
also contain buffers, diluents and other suitable additives.
Examples of non-aqueous solvents are propylene glycol, polyethylene
glycol, vegetable oils (such as olive oil), and injectable organic
esters (such as ethyl oleate). Aqueous carriers include water,
alcoholic/aqueous solutions, emulsions, or suspensions, including
saline and buffered media. Other parenteral vehicles include sodium
chloride solution, Ringer's dextrose, dextrose and sodium chloride,
lactated Ringer's, or fixed oils. Vehicles for intravenous use
include fluid and nutrient replenishers, electrolyte replenishers
(such as those based on Ringer's dextrose) and the like.
[0079] Preservatives and other additives can also be present such
as, for example, antimicrobials, anti-oxidants, chelating agents,
inert gases, and the like.
[0080] For topical administration liquids, suspension, lotions,
creams, gels, ointments, drops, suppositories, sprays and powders
may be used. Conventional pharmaceutical carriers, aqueous, powder
or oily bases, thickeners, and the like can be used as necessary or
desirable.
[0081] The present invention further provides a method of preparing
a hollow glucan particle or cell wall particle encapsulating a
terpene component, said method comprising the steps of; [0082] a)
providing a terpene component; [0083] b) providing a hollow glucan
particle or cell wall particle; [0084] c) incubating the terpene
component with the glucan particle or cell wall particle under
suitable conditions for terpene encapsulation; and [0085] d)
recovering the hollow glucan particle or cell wall particle
encapsulating the terpene component.
[0086] Optionally the above method can further comprise the step of
drying the particles encapsulating the terpene component. Drying
may be achieved in a number of ways and mention may be made of
freeze drying, fluidised bed drying, drum drying or spray drying,
all of which are well known processes.
[0087] In step a) of the above method, the terpene component is
suitably provided as a suspension in an aqueous solvent, and
optionally in the presence of a surfactant. Suitably the solvent is
water. A suitable surfactant is Tween-80 (polyoxyethylenesorbitan
monooleate), and preferably the surfactant is present at a
concentration of about 0.1 to 10% by volume of the total reaction
mixture, more preferably about 1%. Alternatively the terpene
component may be provided as a true solution in a solvent, e.g.
water. A true solution of terpene in water can be obtained by
mixing the terpene in water at high shear until a true solution is
obtained. Publication No WO 03/020024 provides further details of
forming true solutions of terpenes in water.
[0088] In step b) of the above method, the hollow glucan particle
or cell wall particle is suitably provided as a suspension in water
or other suitable liquid. Suitably the suspension comprises
approximately 1 to 1000 mg particles per ml, preferably 200 to 400
mg/ml. Alternatively the particles may be provided as a dry powder
and added to the terpene-surfactant suspension.
[0089] Alternatively the particles are provided in sufficient
liquid to minimally hydrate the particles, but not in significant
excess. The term "hydrodynamic volume" (HV) is used to describe the
volume of liquid required to minimally hydrate the particles. Thus
suitably the particles: are provided with a volume ranging from the
HV and a volume of 1.5 times the HV (1.5 HV). This makes the
subsequent drying step more efficient. Also, where a low volume of
liquid is used (i.e. around HV to 1.5 HV), it is also possible to
extrude the finished product into pellet or noodle form, which is
convenient for fluidised bed drying.
[0090] It has been found that the terpene component can become
encapsulated by the hollow glucan particle or cell wall particle at
room temperature. The rate of encapsulation is, however, increased
at 37.degree. C. but the temperature should be kept below the
boiling point or denaturing temperature of any component of the
composition. Suitable conditions for step c) of the above method
are therefore atmospheric pressure at a temperature of 20 to
37.degree. C. Optimisation of the conditions for a particular
encapsulation reaction will be a matter of routine
experimentation.
[0091] The present invention further provides a method of killing a
microorganism, said method comprising the step of; [0092] a)
contacting said microorganism with a composition comprising a
hollow glucan particle or cell wall particle encapsulating a
terpene component.
[0093] Suitable compositions are those defined in more detail
above.
[0094] The present invention further provides a method of
preventing or treating an infection in a patient, said method
comprising the step of; [0095] a) administering to said patient in
a therapeutically effective dose, a composition comprising a hollow
glucan particle or cell wall particle encapsulating a terpene
component.
[0096] Suitable compositions are those defined in more detail
above.
[0097] The infection of the patient may be caused by any infectious
agent. Examples of these infectious agents include, but are not
restricted to Staphylococcus aureus, Aspergillius fumigatus,
Mycoplasma iowae, Penicillium sp., and Mycoplasma pneumoniae.
[0098] For internal administration the composition may be
administered orally, vaginally, rectally, by inhalation, or by
parenteral routes, e.g. by intradermal, subcutaneous,
intramuscular, intraperitoneal, intrarectal, intraarterial,
intralymphatic, intravenous, intrathecal and intratracheal routes.
Suitable formulations of the composition for these routes are
discussed above.
[0099] For external treatment, the composition may be applied
topically, for example as a cream or ointment or as a dry powder
for treatment of a wound.
[0100] The amount of terpene administered in the above method
should clearly be sufficient to achieve the desired result, i.e.
prevention and/or treatment of the infection, but should not be at
a level which will induce serious toxic effects in the patient.
[0101] The amount of composition administered will, of course, be
dependent on the manner of administration, on the patient being
treated, i.e. their weight, their age, condition, sex and extent of
the disease in the subject and on the judgement of the prescribing
physician. The dose, schedule of doses, and route of administration
can be varied. One of skill in the art would readily be able to
determine an anti-infective amount for a given application based on
the general knowledge in the art and the procedures in the Examples
given below. It should be noted that the term "patient" as used
herein refers to any individual, either human or animal, to which
the treatment is applied. Thus, the patient can be a domesticated
animal (e.g., cat, dog, etc.), livestock (e.g., cattle, horse, pig,
sheep, goat, etc.), laboratory animal (e.g., mouse, rabbit, rat,
guinea pig, etc.), birds and fish. Suitably the subject is a mammal
and especially a primate, for example a human.
[0102] In a further embodiment the present invention provides a
method of treating or preventing infection of a plant, said method
comprising the step of; [0103] a) 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.
[0104] Suitable compositions are those defined in more detail
above.
[0105] Terpenes have been shown to eliminate a number of plant
pathogens (see WO 03/020024) and, as described in co-pending
application U.S. 60/538,627 also effectively kill nematodes which
are significant plant parasites. Terpenes alone in suspension or
solution, however, are somewhat unstable and degrade rapidly in the
soil environment, thus losing efficacy.
[0106] Incorporation of a terpene component in a hollow glucan
particle or cell wall particle can reduce the rate of terpene
release and degradation, thus increasing the duration of action of
the terpene in the soil.
[0107] Suitably the infection of a plant which is to be treated or
prevented in the above method is infection by nematodes.
[0108] Other plant infections that may be treated or prevented
include fungal plant infections, especially those affecting the
surface of a plant. Such infections include downy mildew, powdery
mildew or botrytis bunch rot; these infections particularly affect
grape vines.
[0109] In one embodiment, the plant infection may be caused by one
or more of the following: Aspergillius fumigatus, Sclerotinta
homeocarpa, Rhizoctonia solani, Colletotrichum graminicola or
Penicillium sp.
[0110] An advantage of a terpene based treatment of plants is that
it can be applied shortly before harvest.
[0111] Many conventional treatments require an extended period
before re-entry to the treated area (generally 3 weeks). This means
that an outbreak of a plant disease shortly before harvest cannot
be treated with conventional treatments as it would then not be
possible to harvest the crop at the desired time. The compositions
of the present invention can suitably be applied at any time up
until harvest, for example 21 days prior to harvest, 14 days prior
to harvest, 7 days prior to harvest, or even 3 days or less before
harvest.
[0112] Encapsulated terpenes have shown particular efficacy in
treating downy mildew, powdery mildew and botrytis bunch rot in
grapes, and thus the present invention provides a method of
treating or preventing these diseases.
[0113] Prevention of plant infections can be achieved by treating
plants which the encapsulated terpenes regularly as a prophylactic
measure.
[0114] Suitably the composition of the present invention is applied
by spraying. This is particularly suitable for treating a plant
disease which affects the surface of a plant. For spraying, a
preparation comprising 2 g/l of the composition in water may be
used. Concentrations of from 2 to 4 g/l are particularly effective,
and concentrations of greater than 4 g/l can be used as required.
Obviously it is important that the concentration of the composition
used is sufficient to kill or inhibit the disease causing agent,
but not so high as to harm the plant being treated.
[0115] When spraying plants a rate of 500 L/Ha or greater is
suitable to cover the plants. Preferably a rate of 900 L/Ha or
greater, more preferably 1200 L/Ha or greater is used to ensure
good coverage. Where grape vines are being treated, a rate of 1200
L/Ha has proven suitably effective.
[0116] The composition of the present invention may alternatively
be applied via irrigation. This is particularly suitable for
treating nematodes or other soil borne pathogens or parasites.
[0117] In a further embodiment the present invention also provides
a composition comprising a hollow glucan particle or cell wall
particle encapsulating a terpene component for use in the
prevention or treatment of an infection in a patient or a plant.
Suitable compositions are those defined in more detail above.
[0118] In a further embodiment the present invention provides the
use of a composition comprising a hollow glucan particle or cell
wall particle encapsulating a terpene component in the manufacture
of a medicament for the treatment of infection caused by a
micro-organism. Suitable compositions are those defined in more
detail above.
[0119] The present invention will now by further described with
reference to the following, non-limiting, examples and figures in
which:
[0120] FIG. 1 represents a light micrograph of empty yeast cell
walls;
[0121] FIG. 2 represents a light micrograph of yeast cell walls
encapsulating L-carvone;
[0122] FIG. 3 represents a light micrograph of yeast cell walls
encapsulating citral;
[0123] FIG. 4 represents a light micrograph of terpene
emulsion;
[0124] FIG. 5 represents a light micrograph of yeast cell walls in
hydrodynamic volume (HV) water;
[0125] FIG. 6 represents a light micrograph of yeast cell walls
encapsulating terpene in 5 times hydrodynamic volume (HV) of
water;
[0126] FIG. 7 represents a light micrograph of yeast cell walls
encapsulating terpene in HV of water;
[0127] FIG. 8 represents a light micrograph of yeast cell walls
encapsulating terpene in HV plus 5% of water;
[0128] FIG. 9 represents a light micrograph of yeast cell walls
encapsulating terpene in HV plus 10% of water;
[0129] FIG. 10 represents a light micrograph of yeast cell walls
encapsulating terpene in HV plus 20% of water;
[0130] FIG. 11 represents a light micrograph of yeast cell walls
encapsulating terpene in HV plus 30% of water;
[0131] FIG. 12 represents a light micrograph of yeast cell walls
encapsulating terpene in HV plus 40% of water.
[0132] FIG. 13 represents a light micrograph showing the dispersal
of dried hollow glucan particles encapsulating a terpene component
and no xanthan gum.
[0133] FIG. 14 represents a light micrograph as in FIG. 13 where
0.07 g of 1% xanthan gum is included.
[0134] FIG. 15 represents a light micrograph as in FIG. 13 where
0.14 g of 1% xanthan gum is included.
[0135] FIG. 16 represents a light micrograph as in FIG. 13 where
0.28 g of 1% xanthan gum is included.
[0136] FIG. 17 represents a light micrograph as in FIG. 13 where
0.55 g of 1% xanthan gum is included.
[0137] FIG. 18 represents a light micrograph as in FIG. 13 where
1.1 g of 1% xanthan gum is included.
[0138] FIG. 19 represents a light micrograph as in FIG. 13 where
2.2 g of 1% xanthan gum is included.
[0139] FIG. 20 represents a light micrograph as in FIG. 13 where
4.4 g of 1% xanthan gum is included.
[0140] FIG. 21 shows a schematic representation of treatment areas
on sites 18 and 20.
[0141] FIG. 22 shows a schematic representation of treatment areas
on sites 18 and 20.
[0142] FIG. 23 shows a schematic representation of the treatment
areas on site 7.
[0143] FIG. 24 shows a graph showing comparison of encapsulated vs.
non-encapsulated terpene formulations.
[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 (.degree. 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 1--Demonstration 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 .mu.l of the terpene with 100 .mu.l of 10% Tween 80 in water
and 250 .mu.l of water.
[0147] Baker's yeast particles (YP) or Levacan.TM. 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 .mu.l of the YP or YGP suspension and 250 .mu.l of the
terpene emulsion were mixed together and incubated overnight under
constant agitation. 500 .mu.l YP or YGP suspension and 500 .mu.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 FIGS. 1 to 4.
[0150] FIG. 1 shows spherical structures with a dark area at their
centre, these are empty hollow glucan particles. FIGS. 2 and 3
shows spherical structures with a swollen appearance with a light
coloured interior, these are particles with terpene encapsulated in
the central cavity--citral in FIG. 2 and L-carvone in FIG. 3. In
FIGS. 2 and 3 small blobs of free terpene can also be seen, e.g. at
the top of FIG. 2, just left of centre. FIG. 4 shows the terpene
emulsion as small blebs of terpene suspended in water.
Example 2--Determination 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 14,000.times.g 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 .mu.l Emulsion .mu.l .mu.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 .mu.l of L-carvone terpene emulsion
or at least 5 .mu.l of citral emulsion per 10 mg of YP.
Example 3--Demonstration 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 14,000.times.g 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 .mu.l .mu.l .mu.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 .mu.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 .mu.l 100% 230 +
Sl = slight
[0166] As can be seen from the results a Tween-80 concentration of
1% (i.e. 100 .mu.l of 10% Tween-80 in 1000 .mu.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 4--Determination 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 14,000.times.g 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 .mu.l Emulsion .mu.l .mu.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 .mu.l of L-carvone terpene
emulsion or at least 75 .mu.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 5--Screen 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.TM. Walls (Sensient Technologies, Milwaukee,
Wis.), SAF-Mannan.TM. (SAF Agri, Minneapolis, Minn.), Nutricept
Walls.TM. (Nutricepts Inc., Burnsville, Minn.), Levacan.TM. (Savory
Systems International, Inc., Branchburg, N.J.) and WGP.TM.
(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 .times. 10.sup.7 250
Nutrex 24.5 1.7 .times. 10.sup.8 58.8 SAF Mannan 33.4 2.4 .times.
10.sup.8 41.7 Nutricepts 55.7 5.2 .times. 10.sup.8 37 Levacan 74.6
1 .times. 10.sup.8 19.2 WGP 82.1 3.5 .times. 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 14,000.times.g
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 .mu.l Emulsion .mu.l 80 .mu.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.TM.. [0194] Based on qualitative loading at the single
terpene level tested, SAF Mannan.TM. appears to be best, Nutrex.TM.
second and Baker's third.
Example 6--Kinetics 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.TM. YGP
suspension was prepared by mixing 2 g Nutrex.TM. YGP in 20 ml 1%
Tween-80. [0200] SAF Mannan.TM. suspension was prepared by mixing 2
g SAF Mannan.TM. 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.degree. C. After incubation samples were
centrifuged at 14,000.times.g 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
.degree. C. Particle mg/ml .mu.l Emulsion .mu.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.degree. C. than at room temperature. [0206] SAF Mannan.TM.
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.degree.
C.
Example 7--Screen 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.TM..
[0210] Terpene emulsions were prepared as follows: [0211]
L-carvone--4.5 g L-carvone in 1.5 ml 3.3% Tween-80. [0212]
Citral--4.5 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] Eugenol--4.5 g eugenol in 1.5 ml
3.3% Tween-80. [0215] Geraniol--4.5 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.TM. overnight at room
temperature as shown in Tables 7 and 8. Samples were centrifuged at
14,000.times.g 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.TM. 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 (.mu.l) Emulsion (.mu.l) 80 (.mu.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 (.mu.l) Emulsion Vol 80 (.mu.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 .mu.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 .mu.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 8--Evaluation 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.TM. increased citral stability as evidenced by a
reduction or absence of yellow color over time.
Example 9--Loading 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.TM. 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 3.times.HV 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 3.times.HV 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 5.times.HV, vortexing to
obtain a complete dispersion of particles and centrifugation to
sediment the particle encapsulated terpene. The results are shown
in Table 11 and FIGS. 7 to 12. FIGS. 7 to 12 show the loading
results of the following tubes: [0230] FIG. 7--Tube 3 [0231] FIG.
8--Tube 5 [0232] FIG. 9--Tube 6 [0233] FIG. 10--Tube 8 [0234] FIG.
11--Tube 10 [0235] FIG. 12--Tube 11
TABLE-US-00011 [0235] 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 FIGS. 7 to 12 demonstrate
that terpene loading and encapsulation into the particles occurred
at all water ratios evaluated. Surprisingly, equivalent loading
occurred even when the loading reaction was taking place in a
reaction with the consistency of a stiff dough using the minimal
amount of water to hydrate the particles. The absence of free
terpene was observed microscopically (FIGS. 7 to 12) and in the low
level of terpene in the supernatants, as evidenced by a marked
reduction in the turbidity of the supernatant compared to the
terpene only control.
[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 10--Evaluation 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.TM. particles [0245] 0.1% Tween 80
[0246] L-carvone [0247] Xanthan Gum--1% 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 FIGS. 13 to 20 demonstrate that
the inclusion of a high molecular weight hydrocolloid during the
drying of the particle encapsulated terpene aids in the hydration
and dispersion of the microparticles into a uniform suspension.
Other examples of such hydrocolloid agents are maltodextrin,
alginates, or the like.
[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 11--Evaluation 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 .mu.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.TM. 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 12--Efficacy 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.TM. 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.TM. (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 .about.50% terpene w/w) or 0.2
g/L water.
Example 13--Efficacy of Encapsulated Terpenes Against
Mycobacterium
[0261] Terpene emulsions were prepared as follows: [0262]
Citral--4.5 g citral in 1.5 ml 3.3% Tween-80. [0263]
L-carvone/eugenol--2.25 g L-carvone and 2.25 g Eugenol in 1.5 ml
3.3% Tween-80. [0264] Eugenol--4.5 g eugenol in 1.5 ml 3.3%
Tween-80. [0265] Geraniol--4.5 g geraniol in 1.5 ml 3.3% Tween-80.
[0266] Geraniol/thymol mixture--2.25 g geraniol and 2.25 g thymol
in 1.5 ml 3.3% Tween-80. [0267] Control emulsion--6 ml 1%
Tween-80.
[0268] SAF-Mannan.TM. (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 14--Nematocidal 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 15--Fugicidal 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 (5.times.10.sup.5 cells/mL in YPD growth
medium) were added to each well of a 96-well microtitre plate in
100 .mu.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 .mu.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 .mu.L from one row to
the next a total of 7 times. Finally, 100 .mu.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.degree. 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 .mu.L) and the plates were re-incubated overnight at
30.degree. 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 .mu.L mixed
inoculum (prepared from mouldy grape leaves to a concentration of
5.times.10.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 .mu.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 .ltoreq.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 .ltoreq.12 NT
94 94 24 ECL 500 1000 .ltoreq.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 .ltoreq.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 .times. 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 .mu.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 .mu.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 .mu.L from one row to
the next a total of 7 times. Finally, 100 .mu.L was discarded from
the last row in order to ensure that all wells contained the same
volume. S. cerevisiae (5.times.10.sup.5 cells/mL in YPD growth
medium) were then added to each well in 100 .mu.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.degree. C.
[0299] Following incubation, the A.sub.620 was measured again and
plates were scored for inhibition of growth (.gtoreq.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 .mu.L) and the plates were re-incubated overnight at
30.degree. C.
[0301] Assessment of growth inhibition was performed as before.
[0302] MIC and fungicidal MIC results are summarised in Table
19.
Results
TABLE-US-00019 [0303] 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 .ltoreq.125 ppm, with the most active
formulations inhibiting growth at .about.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.degree.
C. and having a sub-optimal terpene content of .about.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 16--Preparation 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 Quata S. A, Usina Quata, Quata--Sao
Paolo--Brazil--Zip 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 mixture--mix 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
particles--mix 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 17--Field 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 FIG. 21). A third block was treated with
conventional Bordeaux mix plus wettable sulphur, and the remaining
block was left untreated. The vines in each block were monitored
for signs of downy mildew over the following week.
[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 (FIG. 21) (note: 1 g of the terpene liquid formulation
has a volume of 1 ml). Of the remaining two blocks, one was left
untreated and one was sprayed with Mikal.RTM., a conventional
treatment for downy mildew, on 28 Jun. 2004. The vines in each
block were monitored for signs of downy mildew over the following
week.
[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.RTM. (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 18--Field 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 FIG. 22). The vines
in each block were monitored for signs of powdery mildew over the
following week.
[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 FIG. 22). The vines in each block were
monitored for signs of powdery mildew over the following week.
[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.RTM. (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 18--Further 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 19--Further 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 use