U.S. patent application number 12/934941 was filed with the patent office on 2011-10-27 for pigment composition comprising anthocyanic vacuolar inclusions.
This patent application is currently assigned to THE NEW ZEALAND INSTITUTE FOR PLANT AND FOOD RESEARCH LIMITED. Invention is credited to Kevin Davies, Simon Deroles, Huaibi Zhang.
Application Number | 20110263723 12/934941 |
Document ID | / |
Family ID | 41114158 |
Filed Date | 2011-10-27 |
United States Patent
Application |
20110263723 |
Kind Code |
A1 |
Deroles; Simon ; et
al. |
October 27, 2011 |
Pigment Composition Comprising Anthocyanic Vacuolar Inclusions
Abstract
The invention provides a pigment composition comprising
anthocyanic vacuolar inclusions or "AVIs" from a plant, and an
acceptable carrier. The invention also provides methods for
colouring products with the pigment composition, and products
comprising the pigment composition or AVIs.
Inventors: |
Deroles; Simon; (Levin,
NZ) ; Zhang; Huaibi; (Palmerston North, NZ) ;
Davies; Kevin; (Manawatu, NZ) |
Assignee: |
THE NEW ZEALAND INSTITUTE FOR PLANT
AND FOOD RESEARCH LIMITED
|
Family ID: |
41114158 |
Appl. No.: |
12/934941 |
Filed: |
March 27, 2009 |
PCT Filed: |
March 27, 2009 |
PCT NO: |
PCT/NZ09/00041 |
371 Date: |
December 10, 2010 |
Current U.S.
Class: |
514/777 ;
426/250; 426/540; 514/772; 514/783; 514/784; 8/646 |
Current CPC
Class: |
A61K 8/498 20130101;
A61K 36/00 20130101; A61K 2800/10 20130101; Y10T 442/20 20150401;
A23L 5/43 20160801; D06P 1/34 20130101; A61K 2800/42 20130101; A23K
20/179 20160501; A61Q 90/00 20130101; C09B 61/00 20130101; A61K
8/9789 20170801 |
Class at
Publication: |
514/777 ;
514/783; 514/772; 514/784; 8/646; 426/540; 426/250 |
International
Class: |
C09B 61/00 20060101
C09B061/00; A23L 1/275 20060101 A23L001/275; A61K 8/60 20060101
A61K008/60; D06P 1/34 20060101 D06P001/34; A61K 8/97 20060101
A61K008/97; A61K 8/49 20060101 A61K008/49 |
Foreign Application Data
Date |
Code |
Application Number |
Mar 28, 2008 |
NZ |
567036 |
Claims
1-24. (canceled)
25. A food product, cosmetic product, fabric or topical cream,
comprising or coloured with at least one anthocyanic vacuolar
inclusion or "AVI" from a plant, wherein the AVI is stable in at
least one of the following conditions: a) acidic pH and 37.degree.
C. for at least about 72 hours; b) a pH range from about 2.5 to
about 8.0; and c) high light intensity for at least about 48
hours.
26. The food product, cosmetic product, fabric or topical cream of
claim 25, wherein the anthocyanic vacuolar inclusion (AVI)
comprises: a) at least one anthocyanin, and b) at least one
lipid.
27. The food product, cosmetic product, fabric or topical cream of
claim 26, wherein the anthocyanin is a glycoside of an
anthocyanidin.
28. The food product, cosmetic product, fabric or topical cream of
claim 27, wherein the anthocyanidin is selected from the group
consisting of cyanidin, peonidin, delphinidin, pelargonidin,
petunidin, malvidin, 3-deoxyathocyanindins, and 6- and
8-hydroxyanthocyanidins, and hydroxylated, acylated, and methylated
derivatives thereof.
29. The food product, cosmetic product, fabric or topical cream of
claim 27, wherein the anthocyanidin is selected from the group
consisting of cyanidin, peonidin, delphinidin, and pelargonidin,
and hydroxylated, acylated, and methylated derivatives thereof.
30. The food product, cosmetic product, fabric or topical cream of
claim 26, wherein the anthocyanin is a glucoside or a galactoside
of an anthocyanidin.
31. The food product, cosmetic product, fabric or topical cream of
claim 27, wherein the anthocyanin is a monoglycoside of the
anthocyanidin.
32. The food product, cosmetic product, fabric or topical cream of
claim 27, wherein the anthocyanin is a diglycoside of the
anthocyanidin.
33. The food product, cosmetic product, fabric or topical cream of
claim 27, in which the anthocyanin is an acylated glycoside of the
anthocyanidin.
34. The food product, cosmetic product, fabric or topical cream of
claim 26, in which the anthocyanin has O-glycosylation at one or
more of the 3-, 5-, 7-, 3'- or 5' positions.
35. The food product, cosmetic product, fabric or topical cream of
claim 26, wherein the anthocyanin has O-glycosylation and acylation
at one or more of the 3-, 5-, 7-, 3'- or 5' positions.
36. The food product, cosmetic product, fabric or topical cream of
claim 26, wherein the anthocyanin is a 3- or 5-O-glycoside.
37. The food product, cosmetic product, fabric or topical cream of
claim 26, wherein the anthocyanin is a 3,5-di-O-glycoside.
38. The food product, cosmetic product, fabric or topical cream of
claim 26, wherein the anthocyanin is a 3,5-di-O-glycoside with
acylation.
39. The food product, cosmetic product, fabric or topical cream of
claim 26, wherein the anthocyanin is selected from those listed in
Table 2.
40. The food product, cosmetic product, fabric or topical cream of
claim 26, wherein the anthocyanin is selected from those from
lisianthus listed in Table 2.
41. The food product, cosmetic product, fabric or topical cream of
claim 26, wherein the lipid is part of a biological membrane.
42. The food product, cosmetic product, fabric or topical cream of
claim 26, wherein the lipid is a fatty acid.
43. A food product of claim 25.
44. A cosmetic product of claim 25.
45. A fabric of claim 25.
46. A topical cream of claim 25.
47. A method of colouring a product, the method comprising adding
an AVI as defined in claim 25 to the product.
48. The method of claim 47, wherein the product is selected from a
food product, cosmetic product, topical cream, or a fabric.
Description
TECHNICAL FIELD
[0001] The invention relates to plant-derived pigment compositions,
and applications thereof.
BACKGROUND ART
[0002] Attractive and stable colouration is an important factor in
the marketability of foods and beverages. Synthetic colouring
agents are often used in the food and beverage industry. However,
the safety of synthetic colouring agents has been questioned.
Consumer confidence in synthetic food colouring agents in general,
has decreased. There thus exists a need for naturally occurring
pigment compositions.
[0003] Anthocyanins are naturally occurring in many plants and can
serve as pigments to impart a wide range of colours. Anthocyanin
pigments are biodegradable and water soluble; in addition they are
reported to possess antioxidant properties.
[0004] Despite such beneficial attributes, anthocyanin pigments
have not been widely used as food additives for various reasons.
Anthocyanins are difficult to purify which makes obtaining
commercially useful quantities difficult. Anthocyanins are also
degraded by unfavourable temperature, light and pH conditions.
[0005] The pH environment heavily influences the colour and
stability of the anthocyanins, and it is this sensitivity in
particular, that limits their use as a natural colourant. Under
ideal pH conditions (acidic) anthocyanins are strongly coloured and
relatively stable. However many processed foods such as dairy
products and soft drinks are less acidic in nature. Isolated
anthocyanins under these conditions can change colour and have much
reduced longevity.
[0006] It would be highly desirable to identify a source or form of
anthocyanin pigments with improved performance as an additive for
food and beverage products. Such pigments may also find use as
natural dyes, for example for fabrics, and particularly in products
that come into human contact, such as clothing and cosmetics.
[0007] It is an object of the invention to provide a source or form
of anthocyanins useful in a pigment composition that overcomes at
least one of the problems associated with the anthocyanin
preparations of the prior art and/or at least to provide the public
with a useful choice.
SUMMARY OF THE INVENTION
[0008] In a first aspect the invention provides a pigment
composition comprising anthocyanic vacuolar inclusions or "AVIs"
from a plant, and an acceptable carrier. Preferably the pigment
composition comprises isolated AVIs.
Stability of Pigment Composition
[0009] In one embodiment the pigment composition, or AVI, is stable
at acidic pH and 4.degree. C. for at least about 24 hours,
preferably at least about 72 hours, more preferably at least about
120 hours, more preferably at least about 240 hours, most
preferably at least about 360 hours.
[0010] In a further embodiment the pigment composition, or AVI, is
stable at acidic pH and 25.degree. C. for at least about 24 hours,
preferably at least about 72 hours, more preferably at least about
120 hours, more preferably at least about 240 hours most preferably
at least about 360 hours.
[0011] In a further embodiment the pigment composition, or AVI, is
stable at acidic pH and 37.degree. C. for at least about 24 hours,
preferably at least about 48 hours, more preferably at least about
72 hours, most preferably at least about 120 hours
[0012] Preferably the pigment composition, or AVI, is stable over a
pH range from about 2.5 to about 8.0, preferably from about 4.0 to
about 7.0, most preferably from about 5.0 to about 6.0.
[0013] In a further embodiment the pigment composition, or AVI, is
stable under high light intensity for at least about 48 hours,
preferably at least about 72 hours, more preferably at least about
120 hours, most preferably at least about 168 hours.
[0014] Preferably high light intensity refers to light intensity of
greater than about 10, more preferably greater than about 50, most
preferably greater than about 90 umoles photons per m2 per sec.
Structure of AVIs in Pigment Composition
[0015] In a preferred embodiment the anthocyanic vacuolar
inclusions (AVIs), in the composition, comprise:
[0016] a) at least one anthocyanin, and
[0017] b) at least one lipid.
Anthocyanin
[0018] In one embodiment the anthocyanin is a glycoside of an
anthocyanidin.
Anthocyanidin Types
[0019] In one embodiment the anthocyanidin is selected from one of:
cyanidin, peonidin, delphinidin, pelargonidin, petunidin, malvidin,
3-deoxyathocyanindins, and 6- and 8-hydroxyanthocyanidins, and
hydroxylated, acylated, and methylated derivatives thereof.
[0020] In a preferred embodiment the anthocyanidin is selected from
one of: cyanidin, peonidin, delphinidin, and pelargonidin, and
hydroxylated, acylated, and methylated derivatives thereof.
[0021] In a preferred embodiment the acylation is aromatic
acylation.
Glycoside Types
[0022] In one embodiment the anthocyanin is a glucoside of an
anthocyanidin.
[0023] In a further embodiment the anthocyanin is a galactoside of
an anthocyanidin.
[0024] In a further embodiment, the anthocyanin is a monoglycoside
of the anthocyanidin.
[0025] In a further embodiment, the anthocyanin is a diglycoside of
the anthocyanidin.
[0026] In a further embodiment the anthocyanin is an acylated
glycoside of the anthocyanidin.
[0027] In a further embodiment, the anthocyanin is an acylated
monoglycoside of the anthocyanidin.
[0028] In a further embodiment, the anthocyanin is an acylated
diglycoside of the anthocyanidin.
[0029] In a further embodiment the anthocyanin has O-glycosylation
at one or more of the 3-, 5-, 7-, 3'- or 5' positions.
[0030] In a preferred embodiment the anthocyanin has
O-glycosylation and acylation at one or more of the 3-, 5-, 7-, 3'-
or 5' positions.
[0031] In a more preferred embodiment the anthocyanin is a 3- or
5-O-glycoside.
[0032] In a more preferred embodiment the anthocyanin is a
3,5-di-O-glycoside.
[0033] In a more preferred embodiment the anthocyanin is a
3,5-di-O-glycoside with acylation.
[0034] In one embodiment the pigment composition comprises at least
one anthocyanin selected from those listed in Table 2.
[0035] In a further embodiment the pigment composition comprises at
least one anthocyanin selected from those from lisianthus listed in
Table 2.
[0036] In a preferred embodiment the acylation is aromatic
acylation.
Lipid
[0037] In one embodiment the lipid is a biological membrane or
biomembrane.
[0038] In a further embodiment the lipid is part of a biological
membrane or biomembrane.
[0039] In a preferred embodiment the lipid is a fatty acid.
Structure Independent of Stability
[0040] Compositions comprising AVIs with the described structure
but without the described stability, products comprising the
compositions or AVIs and methods of use of the AVIs and
compositions, are also included within the scope of the
invention.
The AVIs May Comprise Mixtures of Different Types of
Anthocyanin
[0041] A mixture of different anthocyanins, including at least one
type as discussed above (the first anthocyanin), and one or more
additional anthocyanins, may also be in the AVIs, compositions and
products of the invention.
[0042] In one embodiment the additional anthocyanin/s is of a type
discussed above, but different from the first anthocyanin.
Products
[0043] In a further aspect the invention provides a food product
comprising a pigment composition of the invention.
[0044] In a further aspect the invention provides a cosmetic
product comprising a pigment composition of the invention.
[0045] In a further aspect the invention provides fabric coloured
with a pigment composition of the invention.
[0046] In a further aspect the invention provides a topical cream
comprising a pigment composition of the invention.
[0047] In a further aspect the invention provides a food product
comprising anthocyanic vacuolar inclusions or "AVIs" from a plant.
Preferably the food product comprises isolated AVIs.
[0048] In a further aspect the invention provides a cosmetic
product comprising anthocyanic vacuolar inclusions or "AVIs" from a
plant. Preferably the cosmetic product comprises isolated AVIs.
[0049] In a further aspect the invention provides fabric coloured
with anthocyanic vacuolar inclusions or "AVIs" from a plant.
Preferably the fabric is coloured with isolated AVIs.
[0050] In a further aspect the invention provides a topical cream
comprising anthocyanic vacuolar inclusions or "AVIs" from a plant.
Preferably the topical cream comprises isolated AVIs.
[0051] Preferably the AVIs used in the food product, cosmetic
product, topical cream or in colouration of fabric are stable at
acidic pH for at least about 24 hours, preferably at least about 48
hours, more preferably at least about 72 hours, more preferably at
least about 120 hours, most preferably at least about 168
hours.
[0052] Preferably the AVIs used in the food product, cosmetic
product, topical cream or in colouration of fabric are stable at
acidic pH and 4.degree. C. for at least about 24 hours, preferably
at least about 72 hours, more preferably at least about 120 hours,
more preferably at least about 240 hours, most preferably at least
about 360 hours.
[0053] Preferably the AVIs used in the food product, cosmetic
product, topical cream or in colouration of fabric are stable at
acidic pH and 25.degree. C. for at least about 24 hours, preferably
at least about 72 hours, more preferably at least about 120 hours,
more preferably at least about 240 hours, most preferably at least
about 360 hours.
[0054] Preferably the AVIs used in the food product, cosmetic
product or in colouration of fabric are stable at acidic pH and
37.degree. C. for at least about 24 hours, preferably at least
about 48 hours, more preferably at least about 72 hours, most
preferably at least about 120 hours
[0055] Preferably the AVIs used in the food product, cosmetic
product, topical cream or in colouration of fabric are stable over
a pH range from about 2.5 to about 8.0, preferably from about 4.0
to about 7.0, most preferably from about 5.0 to about 6.0.
[0056] In a further embodiment the AVIs used in the food product,
cosmetic product or in colouration of fabric are stable under high
light intensity for at least about 48 hours, preferably at least
about 72 hours, more preferably at least about 120 hours, most
preferably at least about 168 hours.
[0057] Preferably high light intensity refers to light intensity of
greater than about 10, more preferably greater than about 50, most
preferably greater than about 90 umoles photons per m2 per sec.
[0058] The AVIs in the pigment composition, food product, cosmetic
product, topical cream or used in colouration of fabric may be
derived from any plant that contains AVIs. In a preferred
embodiment the AVIs are isolated from a lisianthus species.
Preferably the AVIs are isolated from Eustoma grandiflorum.
Methods
[0059] In a further aspect the invention provides a method of
colouring a product, the method comprising adding a pigment
composition of the invention to the product.
[0060] In one embodiment the product is selected from a food
product, cosmetic product, topical cream, or a fabric.
[0061] In a further aspect the invention provides a method of
colouring a product, the method comprising adding an AVI as herein
defined to the product.
[0062] In one embodiment the product is selected from a food
product, cosmetic product, topical cream, or a fabric.
DETAILED DESCRIPTION
1. General Definitions
[0063] In this specification where reference has been made to
patent specifications, other external documents, or other sources
of information, this is generally for the purpose of providing a
context for discussing the features of the invention. Unless
specifically stated otherwise, reference to such external documents
is not to be construed as an admission that such documents, or such
sources of information, in any jurisdiction, are prior art, or form
part of the common general knowledge in the art.
[0064] The term "comprising" as used in this specification means
"consisting at least in part of". When interpreting each statement
in this specification that includes the term "comprising", features
other than that or those prefaced by the term may also be present.
Related terms such as "comprise" and "comprises" are to be
interpreted in the same manner.
2.0 AVIs
[0065] Anthocyanin pigments most commonly accumulate in the plant
cell vacuole and are responsible for many of the colours in petals
and fruits within the red to blue range. In most plant species the
anthocyanins remain in solution and are evenly distributed within
the vacuole. However in some species the anthocyanins are
aggregated into small bodies within the vacuole, which enhances the
cells ability to accumulate large amounts of anthocyanin. These
anthocyanin-containing bodies have been described under several
different names in a range of plant tissues as shown in Table 1.
Such anthocyanin-containing bodies are referred to here as
"anthocyanic vacuolar inclusions" or "AVIs".
TABLE-US-00001 TABLE 1 Plant Species and Tissue Description
Reference Rose petals AVIs Gonnet, 2003 Polygonium cuspidatum
Intravacuolar Kuboetal., 1995 seedlings spherical bodies Matthiola
incana petals Crystals Hemleben, 1981 Consolida ambigua petals Blue
crystals Asen et al., 1975 Mung bean hypocotyl Red crystals
Nozzolillo and Ishikura, 1988 Red cabbage leaves Anthocyanoplasts
Small and Pecket, 1982 Ipomea batatas tubers Cyanoplasts Nozue et
al., 1995 Grape cell cultures Anthocyanoplasts Cormier and Do,
1993
[0066] Anthocyanic vacuolar inclusions have also been described by
Markham et al., (2000) and Zhang et al., (2006). The same papers
describe methods for the isolation of AVI's from Purple lisianthus
(Eustoma grandiflorum) and the Blue-grey carnation (Dianthus
caryophyllus) and are incorporated herein by reference.
[0067] AVIs are globular or irregular in shape. AVIs display
different colours according to their anthocyanin composition.
3.0 AVIs in the Pigment Composition of the Invention
[0068] The present invention results from the surprising discovery
that the colour intensity of AVI-bound anthocyanins is stable under
environmental conditions normally associated with a loss of the
colour intensity of free, or un-bound, anthocyanins.
[0069] The applicants have elucidated the structure/composition of
AVIs. The applicants have shown that anthocyanins and lipids are
the two main classes of compounds found in the AVIs.
[0070] Preferably the "anthocyanic vacuolar inclusion" or "AVI" as
used herein means a structure found in the vacuole in a plant,
comprising an anthocyanin and a lipid.
[0071] The lipid may be part of a biological membrane or
biomembrane.
[0072] The biological membrane or biomembrane may also include
other components such as proteins and sugar groups.
3.1 Stability of the AVIs in the Pigment Composition of the
Invention
[0073] Preferably, the anthocyanin is more stable when part of the
AVI, than when not part of the AVI, such as when in solution.
[0074] Preferably, the lipid is more stable (less susceptible to
oxidation) when part of the AVI, than when not part of the AVI,
such as when in solution.
[0075] Typically both the anthocyanin and the lipid are more stable
when forming part of an AVI than they are when either the
anthocyanin or lipid are separate, such as in solution.
[0076] The term "stable" in relation to AVIs or AVI containing
pigment compositions refers to a reduced rate of loss of
anthocyanic colour in environments normally associated with a loss
of anthocyanic colour. Preferably stable refers to retention of at
least 50% (more preferably at least 60%, more preferably at least
70%, more preferably at least 80%, more preferably at least 90%) of
the anthocyanin's colour (as defined by optical density at 530 nm)
under the specified conditions.
[0077] The term "stable" in relation to AVI containing pigment
composition refers to the stability of the AVIs in the pigment
composition.
3.2 Structure of the AVIs in the Pigment Composition of the
Invention
[0078] AVIs are typically composed of single or multiple forms of
anthocyanin molecules that are bound to by a lipid (such as a fatty
acids or biomembranes), where such an interaction results in the
increased stability of both the anthocyanins, and preferably also
the lipid.
[0079] The term "anthocyanic vacuolar inclusion" or "AVI"
preferably means a structure comprising of an anthocyanin
molecule(s) that is (are) bound together by a lipid, where such an
interaction results in the increased stability of the anthocyanin
molecule (when compared to the anthocyanin molecule on its own in
solution) under varying conditions such as (but not limited to) pH,
temperature, light intensity. The AVI may also include additional
substances such as phenlypropanoids, proteins, carbohydrates and
inorganic compounds.
[0080] The term "anthocyanins" means glycosides of any of
anthocyanidins, including but not limited to, pelargonindin,
cyanidin, peonidin, delphinidin, petunidin, malvidin,
3-deoxyathocyanindins, and 6- and 8-hydroxyanthocyanidins, and
hydroxylated, acylated, and methylated derivatives thereof.
Preferably the glycosides are glucosides or galactosides.
Preferably the acylation is aromatic acylation.
[0081] Preferably, the anthocyanin has O-glycosylation at one or
more of the 3-, 5-, 7-, 3'- or 5' positions, more preferably the
anthocyanin has O-glycosylation and acylation at one or more of the
3-, 5-, 7-, 3'- or 5' positions, more preferably the anthocyanin is
a 3- or 5-O-glycoside, more preferably the anthocyanin is a
3,5-di-O-glucoside, and most preferably the anthocyanin is a
3,5-di-O-glycoside with acylation. Preferably the acylation is
aromatic acylation.
4.0 Isolation of AVIs
[0082] AVIs may be isolated from plants by various methods. For
example AVIs may be isolated from flower petals using variations of
the method of Markham et al., (2000), and methods described in the
examples section of this specification.
[0083] An example of such an isolation procedure is provided as
follows:
[0084] Healthy petals are excised from fully open flowers and
subsequently washed thoroughly with cold tap water containing
several drops of Tween 20, followed by rinsing with distilled
water.
[0085] The washed petals are then chopped up using an onion chopper
on a plastic board until the petals became a slurry. Small amounts
of phosphate buffer (0.1 M, pH 7.0) containing 10 mM EDTA were
added during the chopping. The petal slurry is then added to a
macerating solution containing 1% cellulase Onozuka R-10 (Yakult
Honsha Co., Higashi-Shinbashi, Minatoku, Tokyo) and 0.05%
Pectolyase Y23. The mix is incubated overnight at room temperature
to release protoplasts containing AVIs.
[0086] The incubated mix is subsequently filtered through 50-.mu.m
cheesecloth and washed by vortexing in 0.1 M phosphate buffer plus
17.53 g/L sodium chloride (wash solution). The AVI-containing
protoplasts are then collected by centrifugation at 100 g for 5
min. The wash step is repeated two more times and the final pellet
is suspended in a minimal volume of wash solution.
[0087] The suspended pellet is then transferred into a new tube
containing 80% Percoll (AMRAD-Pharmacia Biotech, Auckland, New
Zealand). The AVI pellet is harvested from the bottom of the
centrifuge tube after centrifugation at 10,000 g for 10 min. The
pellet is washed twice in wash solution and then stored -80.degree.
C. until use.
[0088] The term "isolated" with reference to AVIs means purified
relative their concentration in intact plant material, or in
macerated non-fractionated plant material. Preferably the isolated
AVIs are at least about 10% pure, more preferably at least about
20% pure, more preferably at least about 30% pure, more preferably
at least about 40% pure, more preferably at least about 50% pure,
more preferably at least about 60% pure, more preferably at least
about 70% pure, more preferably at least about 80% pure, more
preferably at least about 90% pure, more preferably at least about
95% pure, most preferably at least about 99% pure.
5.0 Components of Pigment Compositions
[0089] The term "acceptable carrier" with reference to the pigment
composition of the invention means a component, additional to the
AVI itself, which is acceptable for the composition's application.
Acceptable carriers include but are not limited to solvents,
buffers and preservatives.
[0090] Acceptable carriers for use in the pigment composition for
food product applications are known to those skilled in the art and
include but are not limited to:
Water-Soluble Carriers:
[0091] Gums, Carrageenan, Gum Arabic, Alginates, Ammonin alginate,
Potassium alginate, Sodium alginate, Starches, Maltodextrins,
Sucrose, Dextrose, Fructose, Syrups, Alcohols, Glycerol, Sodium
carbonate, Sodium hydrogen carbonate, Ethyl cellulose,
Microcrystalline cellulose, Cellulose, Powdered Benzyl alcohol,
Ethyl Alcohol (ethanol), Ethyl Acetate, Lactose starch, Citric
Acid, Diethyl Tartrate, Lactic acid ethyl ester, Magnesium
Carbonate, Magnesium Hydrogen Carbonate, Polydextrose, Polyethylene
Glycol, Polyoxyethylene (20) Sorbitan Monolaurate, Polyoxyethylene
(20) Sorbitan Monooleate, Polyoxyethylene (20) Sorbitan
Monopalmitate, Polyoxyethylene (20) Sorbitan Monostearate,
Polyoxyethylene (20) Sorbitan Tristearate, Polyvinylpyrrolidone,
Propan-2-Ol, Propylene glycol, and Silicon Dioxide.
Oil Soluble Carriers:
[0092] Edible fats and oils, Medium chain triglycerides, Bees' wax,
Paraffin, Shellac, Rosin, 6-Palmitoyl-L-ascorbic acid, Amyl
Acetate, Butane-1,3-Diol, Diethylene Glycol Monoethyl Ether,
Isopropyl Myristate, Triacetin, and Ethyl Citrate.
[0093] Acceptable carriers for use in the pigment composition for
cosmetic product applications are known to those skilled in the
art.
[0094] Acceptable carriers for use in the pigment composition for
textile colouring applications are known to those skilled in the
art.
6. Preparation of the Pigment Compositions of the Invention
[0095] Pigment composition comprising AVIs may be prepared by
methods known to those skilled in the art.
[0096] For example, the pigment composition can be sold as an
additive for food or other products and can be prepared in dry,
e.g., powdered, form or as a water or alcohol-based concentrate or
syrup depending on the end use and the proposed method of
addition.
[0097] The pigment composition of the invention may be added to
food products by methods known in the art.
[0098] The pigment composition may be combined with one or more
spray drying aids such as gum arabic and maltodextrins or the
solution may be encapsulated by standard coascervation techniques
in edible materials using gelatin.
[0099] The pigment composition may be added in conjunction with
other materials such as flavouring adjuvants.
[0100] The pigment composition can be incorporated as a solid or an
aqueous solution or syrup at various stages during the manufacture
or processing of the food product.
[0101] The AVIs can be incorporated to food and beverage products
in many ways, such as but not limited to: [0102] direct mixing via
batch (tanks/blenders), using rotational impellers in aqueous
materials. [0103] continuous or inline mixers in aqueous materials.
[0104] dry blending into convenience foods for reconstitution by
the consumer. [0105] coating the exterior surfaces of products
through spraying, enrobing, and pan coating.
[0106] The AVIs and the carriers may need to be pre-dissolved in
water to ensure maximum dispersibility and even mixing during food
and beverage product manufacture. Small amounts of highly
concentrated AVIs may be dispersed in icings (sugar/fat
blends).
[0107] Due to their stable nature, the AVIs can be added to the
early stages of production, ensuring they are distributed evenly,
while maintaining optimal visual appeal.
[0108] AVIs can be used in combination with other food colouring
materials to obtain the desired colour in the finished product.
7.0 Products
[0109] The term "food product" as used herein includes solid foods,
liquid beverages and other edible materials regardless of their
specific form. The term food product includes products that are
chewed without being swallowed. Preferred food products include but
are not limited to dairy products, such as yoghurts.
[0110] The present invention is not limited to use of the pigment
compositions or AVIs in food products. Use of AVIs or
AVI-containing compositions to colour other products such as
cosmetics, topical creams, fabrics, and particularly products such
as clothing and cosmetics that come into human contact, are
included within the scope of the invention.
BRIEF DESCRIPTION OF THE DRAWINGS
[0111] The present invention will be better understood with
reference to the accompanying drawings in which:
[0112] FIG. 1 shows the absorbance spectra of AVI-bound
anthocyanins resuspended in water and pH buffers ranging from
3.0-8.0 over a period of 22 days. The samples were incubated at
room temperature under room lighting conditions
(natural/fluorescent light). T1=1 hour, D1=1 Day, D2=2 days-D7=7
days
[0113] FIG. 2 shows the absorbance spectra of anthocyanins, bound
and unbound to AVIs that were resuspended in water and pH buffers
ranging from 3.0-8.0 over a period of 24 hours. The samples were
incubated at room temperature under room lighting conditions
(natural/fluorescent light).
[0114] FIG. 3 shows lisianthus AVIs resuspended in natural yoghurt
and left at room temperature under room lighting conditions
(natural/fluorescent light) for 24 and 48 hours.
[0115] FIG. 4. NMR spectrum of carnation purified AVIs in DMSO
solvent. The two peaks around chemical shift 1.0 ppm are from
lipids present in AVIs. The major peaks between chemical shifts
2.0-9.0 ppm are from pelargonidin-3,5-di-O-glucoside present in the
AVIs.
[0116] FIG. 5. NMR spectrum of lipids isolated from carnation AVIs.
The lipids were dissolved in a mixture of 50% methanol and 50%
chloroform. The peaks at the chemical shifts of 3.4, 4.7 and 7.6
ppm are due to the solvents and all the rest reflect from the
lipids.
[0117] The invention will now be illustrated with reference to the
following non-limiting examples.
EXAMPLES
Example 1
Isolation of AVIs from Plants
Plant Material
[0118] The lisianthus lines used in this study were lines #54 and
Wakamurasaki (see Deroles et al. 1998 for further details). Fully
open flowers were harvested and washed with distilled water to
remove potential contaminants such as pollen grains. Various petal
regions were excised with a razor blade for further study.
[0119] Lisianthus AVIs are found as one or more large irregular
shapes in the vacuoles of adaxial epidermal cells in the throat of
the flower. The lisianthus AVIs could be removed from cell vacuoles
using narrow glass micropipettes. They remained intact, and adhered
firmly to glass indicating that they are structural rather than
localized concentrations of dissolved anthocyanins. In addition,
electron microscopy images show that the AVI body is not membrane
bound.
Protoplast Generation and Isolation
[0120] Protoplasts were generated from macerated inner petal
material as described by Morgan (1998). Material from 10 flowers
was added to a protoplasting solution containing 1% cellulose
Onozuka R-10 (Yakult Honsha Co., Higashi-Shinbashi, Minatoku,
Tokyo) and 0.05% Pectolyase Y23. The mix was incubated overnight at
room temperature and then filtered through a 50 .mu.m stainless
steel mesh, washed in 1/10 strength VIM macro salts (Binding and
Nehls. 1977) plus 17.53 g/l NaCl (wash solution), and the
protoplasts collected by centrifugation (100.times.g, 5 min). The
pelleted protoplasts were washed twice with wash solution.
AVI Isolation
[0121] To enrich for protoplasts containing the AVIs, isolated
protoplasts were subjected to centrifugation through a 4 step
discontinuous Percoll (AMRAD-Pharmacia Biotech, Auckland, NZ)
gradient (20%, 30%, 50%, 80% v/v, Percoll/wash solution). The
gradient was formed by careful sequential addition of equal volumes
of each gradient solution in descending order. Isolated protoplasts
in wash solution were added to the top of the gradient and the
gradient developed by centrifugation (300.times.g, 5 min in a
swing-out bucket rotor). Intensely pigmented protoplasts containing
AVIs were collected from the 50%/80% boundary and washed twice with
wash solution. The AVI-containing protoplasts were lysed using
sonication and the product was carefully layered onto a second
Percoll gradient that was developed as described above. Isolated
AVIs pelleted at the bottom of the centrifuge tube and were washed
twice with wash solution.
[0122] For use in control experiments anthocyanins were leeched
from AVIs under acidic conditions (90% MeOH/5% Acetic Acid).
Example 2
Stability of AVI-Bound Anthocyanins in a Range of pH
Environments
[0123] AVIs isolated from lisianthus were resuspended in a
McIlvaines buffer (Citric Acid/Sodium Phosphate) array from pH 3.0
to 8.0. The samples were left at room temperature and lighting
conditions (fluorescent/natural light) for up to 7 days to
determine the stability and colourfastness of the AVI-bound
anthocyanins. A spectrum of each sample (350-750 nm) was taken at
daily intervals to determine the hue and intensity of the AVI-bound
anthocyanins over time. In the control experiment, anthocyanins
were extracted from the AVIs and subjected to the same pH
conditions.
[0124] As shown in FIG. 1 the anthocyanins in this experiment have
maintained their colour over a wide pH range and for a significant
period of time. All the other treatments do not change
significantly until 7 days where the samples at more alkaline pH
levels (pH 6-8) begin to fade.
[0125] Anthocyanins were then extracted from the lisianthus AVIs
and resuspended in the pH buffer array used previously. A sample of
intact AVIs was also treated at the same time. Again, the
absorbance spectrum of the AVI-bound anthocyanins does not change
significantly over the pH range, or over a 24-hour period as shown
in FIG. 2. However the same anthocyanins, when released form the
AVI structures behave in a more predictable manner in that they
have significantly different absorbance spectra according to the pH
of the solution and their spectra also changed over time. This
demonstrates clearly that the lisianthus AVI structure effectively
protects bound anthocyanins from the effects of differing pH
environments.
Example 3
Stability of AVI-Bound Anthocyanins in Commercial Natural
Yoghurt
[0126] AVIs from lisianthus were mixed with a commercial natural
yoghurt sample (pH 4.18) obtained from a supermarket in order to
determine their ability to maintain colour in a processed food
environment. The samples were left at room temperature under room
lighting conditions for 24 to 48 hours.
[0127] As shown in FIG. 3, the lisianthus AVIs were able to
maintain their colour over a 48-hour period.
Example 4
Elucidation of AVI Structure/Composition
[0128] Isolation of AVIs from Carnation and Lisianthus
Plant Material
[0129] Carnation (a mauve coloured cultivar of Dianthus
caryophyllus) or Lisianthus (#54 cultivar of Eustoma grandiflorum)
were grown in the glasshouse under the conditions of natural
lighting and day length. Fully expanded flower petals were
collected and washed to remove unwanted contaminants.
[0130] Carnation AVIs are more round-shaped throughout the petal
than the more irregular shaped AVIs of lisianthus. Carnation AVIs
are found throughout the petals. The structure of AVIs was
previously unknown. The applicants hypothesised that although AVIs
do not have surface membranes to contain them, they may have
infrastructural membranes or lipids. The applicants have now shown
that anthocyanins and lipids are the two major components of AVIs
as described below.
[0131] AVIs were isolated as follows:
Protoplast Preparation
[0132] 1) The washed flower petals were mechanically macerated
using an onion chopper or razor-blade to form a slurry.
Subsequently, the slurry was incubated in an enzyme solution
comprising 1% cellulase Onozuka R-10 (Yakult Honsha Co.,
Higashi-Shinbashi, Minatoku, Tokyo) and 0.05% pectolase Y23
(Morgan, 1998). The enzymatic maceration was carried out overnight
at room temperature. [0133] 2) AVI-containing protoplasts were
enriched by filtering the enzymatically macerated slurry through
cheese cloth or metal screens with pore sizes that allow AVI
containing protoplasts to pass. The AVI-containing protoplasts were
then washed with buffers in which any released AVIs are stable, for
example, 0.1 phosphate buffer containing 17.53 g/L sodium chloride.
The AVI-containing protoplasts were then concentrated into a loose
pellet by centrifuging. [0134] 3) The AVI-containing pellet was
then transferred into 80% percoll (AMRAD-Pharmacia Biotech,
Auckland, New Zealand). AVIs were released from the protoplasts by
vortexing or gentle ultrasonic suspension treatment.
Elucidation of the Structure of AVIs
Anthocyanin Analysis
[0135] For anthocyanin analysis, the AVIs, prepared according to
the procedures described above, were extracted with 80% methanol
with 5% acetic acid. The extract was then subjected to LC-MS
analysis. Table 2 below lists the anthocyanins found in the
carnation and lisianthus AVIs.
TABLE-US-00002 TABLE 2 [M + H]+ Lisianthus AVIs (purple)
Cyanidin-3-O-glucoside 449 Delphinidin-3-O-glucoside 465
Cyanidin-3-O-galactoside-5-O-(6-O-p-coumaroylglucoside) 757
Cyanidin-3-O-galactoside-5-O-(6-O-ferulylglucoside) 787
Delphinidin-3-O-galactoside-5-O-(6-O-p-coumaroylglucoside) 773
Delphinidin-3-O-galactoside-5-O-(6-O-ferulylglucoside) 803
Cyanidin-O-coumaroylglucoside 595 Delphinidin-O-coumaroylglucoside
611 Delphinindin-3-O-acetylglucoside-5-O-glucoside(or galactoside)
669 Peonidin-3-O-rhamnogalactoside-5-O-(6-O-p-coumaroylglucoside)
813 Delphinidin-3-O-galactoside-5-O-(6-O-p-coumaroylglucoside) +
896 m/z 123 Cyanidin-3-O-galactoside-5-O-(6-O-p-coumaroylglucoside)
+ 912 m/z 155
Delphinidin-3-O-rhamnogalactoside-5-O-(6-O-p-coumaroyl- 919
glucoside)
Cyanidin-3-di-O-galactoside-5-O-(6-O-p-coumaroylglucoside) 919
Delphinidin-3-O-galactoside-5-O-(6-O-ferulylglucoside) + m/z 123
926 Delphinidin-3-di-O-galactoside-5-O-(6-O-p-coumaroylglucoside)
935 Cyanidin-3-O-galactoside-5-O-(6-O-ferulylglucoside) + m/z 155
942 Delphinidin-3-O-rhamnogalactoside-5-O-(6-O-ferulylglucoside)
949 Cyanidin-3-di-O-galactoside-5-O-(6-O-ferulylglucoside) 949
Delphinidin-3-di-O-galactoside-5-O-(6-O-ferulylglucoside) 965
Pelargonidin-3-O-galactoside-5-O-(6-O-p-coumaroylglucoside) 741
Pelargonidln-3-O-galactoside-5-O-(6-O-ferulylglucoside) 771
Carnation AVIs Pelargonidin 271 Pelargonidin-3-O-glucoside 433
Pelargonidin-3,5-di-O-glucoside 595
[0136] Freeze dried carnation and lisianthus AVIs were separately
dissolved in DMSO-D6 and subjected to NMR analysis. FIG. 4 shows a
NMR spectrum clearly reflecting the presence of pelargonidin
3,5-di-O-glucoside and another major component.
Lipid Analysis
[0137] To confirm the presence of lipids in the AVIs, lipids were
isolated from both carnation and lisianthus AVIs using an organic
solvent: chloroform:methanol:TFA (50:50:0.5 v:v:v). Freeze-dried
AVIs were dissolved with the organic solvent. Subsequently, the
organic solvent containing dissolved AVIs was partitioned against
acidified water (water with 0.5% TFA). The process was repeated
several times until no anthocyanic colour was obvious in the
organic phase. The organic phase containing lipids was collected,
and was subsequently dried under nitrogen to prepare lipids. The
dried lipids were then dissolved in a chloroform (d4) and methanol
to carry out NMR measurement.
[0138] FIG. 5 shows the NMR spectrum of the extracted lipids from
AVIs. Proton Nuclear Magnetic Resonance (.sup.1H NMR) Spectroscopy
of total lipids extracted from carnation AVIs displayed a typical
upfield region of lipids extracted from a biological membrane
sample (Kriat M, et al. 1993). The most prominent .sup.1H NMR peak
at 1.3 ppm is --(CH.sub.2)n in fatty acyl chain. The peak at 0.9
ppm is characteristic of --CH.sub.3 in fatty acyl chain. The peak
at 1.6 ppm represents the bold italic H in the
--CO--CH.sub.2--CH.sub.2; at 2.1 ppm CH.sub.2CH.dbd.CH--; at 2.3
ppm --CH.sub.2--COO--; at 2.8 ppm .dbd.CHCH.sub.2CH--
(polyunsaturated fatty acyl signal); at 3.6 ppm --CH.sub.2--N+; at
5.4 ppm --CH.dbd.CH-- in fatty acyl chain.
[0139] The above examples illustrate practice of the invention. It
will be well understood by those skilled in the art that numerous
variations and modifications may be made without departing from the
spirit and scope of the invention.
REFERENCES
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