Note: Descriptions are shown in the official language in which they were submitted.
CA 02364443 2001-09-05
wo oois2o92 TITLE PCT/US00/05467
POLYMER COMPOSITIONS
This application claims the benefit of British Application No. 9904939.7,
filed
March 5, 1999.
FIELD OF THE INVENTION
The present invention relates to polymer compositions, and in particular to
polymer
compositions comprising a first polymer comprising arabinosyl-substituted (1-
4)-(3-
xylopyranosyl units cross-linked to a second polymer comprising D-galacturonic
acid units.
The polymer compositions may be vegetable gels and they have a wide variety of
uses in the
food and medical industries and in agriculture.
BACKGROUND TO THE INVENTION
The term "hemicellulose" is a term of art used to embrace non-cellulosic, non-
starch
plant polysaccharides. The term therefore embraces inter alia pentosans,
pectins and gums.
Some hemicelluloses are suitable as substrates for oxidative gelation
("gelling
hemicelluloses"): such hemicelluloses often have substituents with phenolic
groups which
are cross-linkable with certain oxidizing agents.
Arabinoxylan and pectin constitute two particularly important classes of
hemicellulose. Arabinoxylans consist predominantly of the pentoses arabinose
and xylose,
and are therefore often classified as pentosans. However, in many cases
hexoses and
hexuronic acid are present as minor constituents, and therefore they may also
be referred to
descriptively as heteroxylans.
The arabinoxylan molecule consists of a linear backbone of (1-4)-~3-
xylopyranosyl
units, to which substituents are attached through 02 and 03 atoms of the
xylosyl residues.
The major substituents are single a-L-arabinofuranosyl residues. Single a-D-
glucoronopyranosyl residues and their 4-O-methyl ethers are also common
substituents.
Arabinoxylan preparations are usually heterogeneous with respect to the ratio
of
xylose to arabinose (i.e., the degree of substitution) and in the pattern of
substitution of the
arabinosyl units along the (1-4)-(3-xylan backbone.
Phenolic acid (including ferulic acid) and acetyl substituents may occur at
intervals
along the arabinoxylan chains. These substituents may have an effect on the
solubility of the
arabinoxylan. They render the arabinoxylan oxidatively cross-linkable to
produce viscous
solutions or gels via their phenolic substituents (referred to herein as
"gelling
arabinoxylans"). Arabinoxylan preparations bearing phenolic (e.g., ferulic
acid) substituents
are referred to herein as "AXF", while those bearing acetyl substituents are
designated
"AXA". Similarly, preparations bearing both phenolic (e.g., ferulic acid) and
acetyl
substituents are hereinafter abbreviated to the designation "AXFA".
Arabinoxylan
preparations having few phenolic (e.g., ferulic acid) substituents are
designated "AX": when
CA 02364443 2001-09-05
WO 00/52092 PCT/US00/05467
the degree of substitution falls below that required for oxidative gelation,
the arabinoxylan is
designated a "non-gelling arabinoxylan" (a term which therefore embraces AX
and AXA).
Pectins constitute another important class of hemicelluloses. As used herein
and
unless otherwise indicated, the term "pectin" is used sensu lato to define
hemicellulose
polymers rich in D-galacturonic acid. Many (but not all) are cell wall
components. The
term "pectin" is also used herein sensu stricto to define the so-called "true
pectins", which
are characterized by the presence of an O-(a-D-galacturonopyranosyl)-(1-2)-L-
rhamnopyranosyl linkage within the molecule.
The pectins may be subcategorized on the basis of their structural complexity.
At
one extreme are "simple pectins", which are galacturonans. At the other
extreme are
"complex pectins" exemplified by rhamnogalacturonan II, which contains at
least 10
different monosaccharide components in the main chain or as a components of
branches.
Pectins of intermediate complexity (herein referred to as "mesocomplex
pectins" contain
alternate rhamnose and galacturonic acid units, while others have branches of
glucoronic
acid linked to galacturonic acid.
Complex and mesocomplex pectins are made up of "smooth" regions (based on
linear homogalacturonan) and "hairy" regions corresponding to the
rhamnogalacturonan
backbone with side-branches of varying length.
Certain pectins (for example, pectins obtainable from representatives of the
plant
family Chenopodiaceae, which include beets (e.g., sugar beet), spinach and
mangelwurzels)
are substituted to some extent with substituents derived from carboxylic acids
(usually
substituted cinnamic acids) containing phenolic groups. Such pectins (which
include
feruloylated pectins) may be oxidatively cross-linked to produce viscous
solutions or gels via
their phenolic substituents. This can be achieved by powerful oxidants (e.g.,
persulfate - see
J.-F. Thibault et alia, in The Chemistry and Technolocy of Pectin, Academic
Press 1991,
Chapter 7, pages 119-133) or a combination of peroxidase and hydrogen peroxide
(see
Thibault et alia, ibidem). FR 2 545 101 Al also describes the gelling of beet
pectins using
an oxidant (e.g., hydrogen peroxide) and an enzyme (peroxidase). Such pectins
are referred
to herein as "gelling pectins". Thus, gelling pectins may be subject to
"oxidative gelation"
(as herein defined).
Sugar beet pectin is especially rich in arabinan. Arabinan contains (3-1,5-
linked
arabinose in the backbone with a-(1->3) or a-(1->2)-linked arabinose residues,
whereas
arabinogalactan contains ~i-1,4-linked galactose in the backbone, with a-(1-
>3) or a-(1->2)
linked arabinose residues. Ferulyl substituents are linked to the arabinose
and/or the
galactose in the arabinan and arabinogalactan side-branches of the
rhamnogalacturonan part.
The "ferulic acid" content varies according to the extraction method, but is
often about 0.6%.
Beet pectins obtained by processes which partially remove arabinose residues
may
exhibit improved gelling properties. Thus, procedures involving mild acid
treatment and/or
2
CA 02364443 2001-09-05
WO 00/52092 PCT/US00/05467
treatment with an a-arabinofuranosidase will improve the gelling properties of
the pectin
(see F. Guillon and J.-F. Thibault, ibidem). Such pectins are hereinafter
referred to as
''treated pectins".
Phenolic acids
The phenolic acids (chiefly ferulic andp-coumaric acids) are common in cell
walls
from cereal grains and have also been detected in barley husks and embryo.
They may be
attached to barley storage proteins and are found in starchy endosperm cell
walls and in the
aleurone layer. The phenolic acids (e.g., ferulic acid) may be associated with
polysaccharides (such as hemicelluloses, e.g., arabinoxylans), where they may
be cross-
linkable by oxidative gelation (see infra). The phenolic aldehydes p-
hydroxybenzaldehyde,
vanillin and syringaldehyde have been identified in cell walls of grasses and
are apparently
linked at their phenolic groups.
Oxidative relation ~ellin~ hemicelluloses and hemicellulose gels
Aqueous extracts of several different types of hemicelluloses are known to
form gels
(or viscous liquids) when treated with certain oxidizing agents. For example,
it has long
been known that certain flour extracts (e.g., wheat and rye flour extracts)
can form gels in
the presence of certain oxidants (e.g., upon the addition of hydrogen
peroxide).
The phenomenon is known in the art as "oxidative gelation", and an extensive
literature exists on the subject of oxidative gelation of wheat flour
extracts. The term
"oxidative cross-linking" is used mutatis mutandis to define the oxidative
coupling of
polymers which is associated with oxidative gelation. The terms "oxidative
gelation" and
"oxidative cross-linking" are used herein in a broad sense to include the case
where viscous
solutions are produced rather than true gels, and the term "gel" is therefore
to be interpreted
loosely to cover viscous liquids. This reflects the fact that oxidative
gelation/cross-linking
are progressive phenomena which may be controlled to vary the degree of cross-
linking/gelation to the extent that hard, brittle gels are formed at one
extreme and slurries or
viscous liquids at the other.
The biochemical basis of the gelling process is not completely or consistently
described in the prior art. According to one model, the gels arise as high
molecular weight
arabinoxylan and protein molecules become inter- and/or infra-linked (via
inter alia phenolic
substituents, for example ferulic acid-derived diferulate bridges): see e.g.,
Hoseney and
Faubion (1981), Cereal Chem., 58:421.
In another model, gel formation and/or viscosity increases arise (at least in
part) from
cross-linking within and/or between macromolecular components of the
hemicellulose
mediated by ferulic acid residues (for example, involving diferulate generated
by oxidative
coupling of the aromatic nucleus of ferulic acid).
It should be noted that, as used herein (and as is usual in the art), the
terms "ferulic
acid" and "ferulate" are used sensu lato encompass ferulyl (often denoted
feruloyl) groups
3
CA 02364443 2001-09-05
WO 00/52092 PCT/US00/05467
(i.e., 4-hydroxy-3-methoxy-cinnamyl groups) and derivatives (particularly
oxidized
derivatives) thereof.
Only a few oxidizing agents are known to have the ability to induce gelation,
and
these include hydrogen peroxide (usually in conjunction with a peroxidase),
ammonium
persulphate and formamidine disulphide.
Most of the work in the area of oxidative gelation has focused on water
soluble
pentosans from wheat flour. In these studies. wheat flour is extracted with
water (usually at
room temperature) to yield gelling arabinoxylans. However, water-insoluble
wheat
pentosans extracted from wheat flours with various concentrations of cold
sodium hydroxide
have also been shown to form gels (Michniewicz et alia, Cereal Chemistry
67(5):434-439
(1990), and oxidative gelation of beet pectins has also been described: see J.-
F. Thibault
et alia, in The Chemistry and Technolo~:y of Pectin, Academic Press 1991,
Chapter 7,
pages 119-133) and FR 2 545 101 Al, discussed earlier.
WO 93/10158 describes the preparation of hemicellulosic material from various
brans and the oxidative gelation of maize-derived hemicelluloses using an
oxidizing system
comprising a peroxide (such as hydrogen peroxide) and an oxygenase (such as a
peroxidase).
The hemicellulosic material for use as a gelling agent is prepared by hot
water or mild alkali
extraction.
WO 96/03440 describes the use of an oxidase (preferably a laccase) for
promoting
oxidative gelation of inter alia arabinoxylans. However, laccase may not be
acceptable for
use in certain food applications, is relatively expensive and the supply is
limited. Moreover,
oxidases such as laccase are relatively weak oxidation-promoters, and the
range of different
gel strengths obtainable by the use of such enzymes is limited. Indeed, it is
possible that the
crosslinking achieved through the use of laccase and other oxidases differs
fundamentally
from that mediated by e.g., hydrogen peroxide, so that the gels may differ
significantly in
structure from those produced by other forms of oxidative gelation.
The gels and viscous fluids derived from gelling hemicelluloses (and in
particular
arabinoxylan and pectin) have long been recognized to have a wide variety of
uses in
industry (particularly the food industry). There is therefore great interest
in the mechanisms
which promote the gelling of these materials and in processes for increasing
the e~ciency of
the gelling process and the quality of the resultant gels.
SUMMARY OF THE INVENTION
It has now surprisingly been discovered that certain arabinoxylan and pectin
polymers interact synergistically when mixtures of these polymers are
oxidatively cross-
linked: the gelling capacity of the mixtures is far greater than the sum of
the contribution of
each polymer alone (on a weight/volume basis). The phenomenon is so marked
that in some
circumstances strong gels can be produced using quantities of mixed polymers
which would
not gel (or produce only very weak gels) if used alone in similar quantities.
The rate of
4
CA 02364443 2001-09-05
WO 00/52092 PCT/US00/05467
formation of a gel by the mixture of polymers has been found to be dependent
on pH, thus
adjustment of pH may be necessary to achieve the desired gelling.
Since the chemical structures of a wide variety of different arabinoxylans and
pectins
have been exhaustively characterised and described in the prior art, the
implications of this
discovery extend beyond applications based on naturally-occurring vegetable
hemicellulose
(e.g., arabinoxylan and/or pectin) extracts: the invention permits the
rational design,
synthesis and formulation in vitro of a novel range of polymer compositions
with
predetermined and highly desirable gelling characteristics.
According to the present invention there is provided a composition comprising
a first
polymer comprising arabinosyl-substituted (1-4)-~3-xylopyranosyl units cross-
linked to a
second polymer comprising D-galacturonic acid units. A closely related aspect
of the
invention provides a composition comprising co-gelled first and second
polymers, the first
polymer comprising arabinosyl-substituted (1-4)-(3-xylopyranosyl units and the
second
polymer comprising D-galacturonic acid units.
The first and/or second polymers need not be present in highly purified form,
though
it is preferred that the first and second polymers be substantially purified.
In the case of first
and second polymers which are derived from natural sources, the polymers need
not be
purified to homogeneity, either with respect to other species of macromolecule
or
carbohydrate present or with respect to other (non-hemicellulosic) components.
Indeed, for
most applications the first and/or second polymers will comprise a
heterogeneous mixture of
different polymers, together with contaminating proteinaceous, carbohydrate,
cellulosic and
fatty materials (often in minor or trace amounts).
When derived or extracted from a natural (often plant or vegetable) source,
the
polymers for use in the invention are preferably substantially isolated. The
term "isolated" is
used herein to indicate that the polymer exists in a physical milieu distinct
from that in which
it occurs in nature. For example, the isolated polymers may be substantially
isolated with
respect to the complex chemical milieu in which they naturally occur. The
absolute level of
purity is not critical, and those skilled in the art can readily determine
appropriate levels of
purity according to the use to which the polymers) are to be put.
In many circumstances, the isolated polymers) will form part of a composition
(for
example a more or less crude extract containing other components), buffer
system or
pharmaceutical excipient, which may for example contain other components (such
as and not
limited to colouring agents, flavouring agents, antimicrobial agents, enzymes,
preservatives
or dispersants).
In other circumstances, the isolated polymers) may be purified to essential
homogeneity. In preferred embodiments, the isolated polymers) of the invention
are
essentially the sole active gelling agents in a given composition.
5
CA 02364443 2001-09-05
WO 00/52092 PCT/L1S00/05467
As explained above. the first and second polymers may be present in a form and
at
relative and/or absolute concentrations such that they produce a synergistic
effect on gel
strength or viscosity.
The first and second polymers may comprise phenolic acid substituents, the
first and
second polymers being crosslinked via the phenolic acid substituents.
Preferably, the
phenolic acid substituents comprise ferulic acid substituents. Particularly
preferred are
compositions wherein the phenolic acid substituents of the first polymer
comprise
a-L-arabinofuranosyl residues (as is the case with naturally-occurring
arabinoxylans).
The cross-links may comprise diferulate bridges, though any other convenient
form
of covalent crosslinking may be employed. Particularly preferred are
compositions in which
the first and second polymers are oxidatively crosslinked.
The first polymer may conveniently comprise an arabinoxylan, preferably a
gelling
arabinoxylan. Particularly preferred is arabinoxylan ferulate. The second
polymer may
conveniently comprise a pectin, preferably a gelling pectin. Particularly
preferred is a
feruloylated pectin.
For many applications, the composition is preferably in the form of a gel or
viscous
fluid. For other applications the composition may be provided in the non-cross-
linked or
non-co-gelled state. The latter is preferred for applications where
gelationlcross-linking is to
be carned out by the end user. In yet other applications, the composition may
be provided as
a gel or viscous fluid in dehydrated form. Also contemplated are such
dehydrated gels or
viscous fluids in rehydrated form.
In another aspect the invention provides a process for preparing a gel or
viscous fluid
comprising the step of oxidatively co-gelling a first polymer comprising
arabinosyl-
substituted (1-4)-(3-xylopyranosyl units and a second polymer comprising D-
galacturonic
acid units. Also contemplated are gels or viscous fluids produced by (or
obtainable by) the
processes of the invention.
The invention also contemplates a pharmaceutical or cosmetic preparation or
medical
device comprising the composition of the invention, the preparation or device
being for
example selected from: a wound plug, wound dressing, controlled release
device, an
encapsulated medicament or drug, a lotion, cream, suppository, pessary, spray,
artificial skin,
protective membrane, a nutraceutical, prosthetic, orthopaedic, ocular insert,
injectant,
lubricant or cell implant matrix, optionally further comprising an antibiotic,
analgesic and/or
anti-inflammatory agent.
The composition of the invention finds application in therapy, prophylaxis or
diagnosis, for example in the treatment of skin lesions (e.g., bums, abrasions
or ulcers).
Also contemplated is a bread improver comprising the composition of the
invention
as well as a foodstuff, dietary fibre source, food ingredient, additive,
lubricant, supplement
or dressing comprising the composition of the invention, for example being
selected from a
6
CA 02364443 2001-09-05
WO 00/52092 PCT/US00/05467
drink (e.g., a stabilized milk-protein drink), yogurt, chocolate, a petfood
(wherein the gel
e.g., acts as a binder), a preserve (e.g., jam or marmalade), a flavour
delivery agent, a
stabilizer, a pectin replacer, a canning gel, fat replacer (e.g., comprising
macerated gel of any
one of the preceding claims), a coating, a glaze, a bait or a gelatin
replacer.
Also contemplated are masking agents comprising the composition of the
invention,
for example for use in masking semiconductor wafers. etching plates or
surfaces to be
painted.
BRIEF DESCRIPTION OF THE FIGURES
Figure 1 depicts the rate of gelation of AXF-sugar beet pectin mixtures at
25°C.
Figure 1 shows G', Pa (elastic modulus) as a function of time, in minutes, for
mixtures of
AXF with sugar beet pectin. 50 ul peroxidase plus 50 ul peroxide to 50 g of
solution. All
solutions 2 wt % unless stated otherwise. 1 wt % pectin did not gel.
Figure 2 shows gelation of AXF, SBP and AXF-SBP (l:l) mixture at 2 wt %, pH 4
and 25°C.
Figure 3 depicts gelation of AXF, SBP and AXF-SBP (l:l) mixture at 2 wt %, pH
5
and 25°C.
DETAILED DESCRIPTION
Polymers for use in the invention
The first polymer for use in the invention may be any polymer comprised of
arabinosyl-substituted (1-4)-(3-xylopyranosyl units. Preferred are
arabinoxylans or
heteroxylans or their synthetic counterparts (i.e., structural analogues of a
naturally-
occurring arabinoxylans/heteroxylans synthesised in vitro by any
chemical/enzymic
synthesis or modification). Particularly preferred are arabinoxylans with
substituents with
phenolic (e.g., ferulic acid) groups which are cross-linkable with certain
oxidizing agents.
These "gelling'' arabinoxylans are particularly preferred for use in the
invention. Thus, of
the arabinoxylans, particularly preferred are AXFA and AXF.
The second polymer for use in the invention may be any polymer comprising
D-galacturonic acid units. Particularly preferred for use in the invention are
gelling (e.g.,
feruloylated) pectins, including the true pectins, simple pectins, complex
pectins and
mesocomplex pectins. Pectins obtainable from representatives of the plant
family
Chenopodiaceae, which include beets (e.g., sugar beet), spinach and
mangelwurzels) are
particularly suitable, and especially preferred is sugar beet pectin (for
example in the form of
sugar beet pulp). Also useful in the invention are treated pectins (as
hereinbefore defined).
Also suitable are synthetic pectins (i.e., structural analogues of naturally-
occurring pectins
synthesised in vitro by any chemical/enzymic synthesis or modification).
The first and/or second polymers may be obtained from a wide range of plant
sources. Thus, suitable starting materials containing hemicellulose for use in
the processes
of the invention (either as starting materials in the fractionation processes
or as sources of
CA 02364443 2001-09-05
WO 00/52092 PCT/US00/05467
hemicellulose per se) typically include plant material of various kinds and
any part or
component thereof.
Plant materials useful as a starting material in the invention include the
leaves and
stalks of woody and nonwoody plants (particularly monocotyledonous plants),
and grassy
species of the family Gramineae. Particularly preferred are gramineous
agricultural residues,
i.e., the portions of grain-bearing grassy plants which remain after
harvesting the seed. Such
residues include straws (e.g., wheat, oat, rice, barley, rye, buckwheat and
flax straws), corn
stalks, corn cobs and corn husks.
Other suitable starting materials include grasses, such as prairie grasses,
gamagrass
and foxtail. Other suitable sources include dicotyledonous plants such as
woody dicots (e.g.,
trees and shrubs) as well as leguminous plants.
Another preferred source are fruits, roots and tubers (used herein in the
botanical
sense). The term "fruit" includes the ripened plant ovary (or group thereof)
containing the
seeds, together with any adjacent parts that may be fused with it at maturity.
The term
"fruif' also embraces simple dry fruits (follicles, legumes, capsules,
achenes, grains, samaras
and nuts (including chestnuts, water chestnuts, horsechestnuts etc.)), simple
fleshy fruits
(berries, drupes, false berries and pomes), aggregate fruits and multiple
fruits. The term
"fruit" is also intended to embrace any residual or modified leaf and flower
parts which
contain or are attached to the fruit (such as a bract). Encompassed within
this meaning of
fruit are cereal grains and other seeds.
Also contemplated for use as starting materials are fruit components,
including bran,
seed hulls and calms, including malt calms. "Bran" is a component of cereals
and is defined
as a fraction obtained during the processing of cereal grain seeds and
comprises the
lignocellulosic seed coat as separate from the flour or meal. Other suitable
component parts
suitable as starting materials include flours and meals (particularly cereal
flours and meals,
and including nonwoody seed hulls, such as the bracts of oats and rice).
The term "root" is intended to define the usually underground portion of a
plant body
that functions as an organ of absorption, aeration and/or food storage or as a
means of
anchorage or support. It differs from the stem in lacking nodes, buds and
leaves. The term
"tuber" is defined as a much enlarged portion of subterranean stem (stolon)
provided with
buds on the sides and tips.
Preferred lignocellulosic starting materials include waste stream components
from
commercial processing of crop materials such as various beets and pulps
thereof (including
sugar beet pulp), citrus fruit pulp, wood pulp, fruit rinds, nonwoody seed
hulls and cereal
bran. Suitable cereal sources include maize, barley, wheat, oats, rice, other
sources include
pulses (e.g., soya), legumes and fruit.
CA 02364443 2001-09-05
WO 00/52092 PCT/US00/05467
Other suitable starting materials include pollen, bark, wood shavings, aquatic
plants,
marine plants (including algae), exudates, cultured tissue. synthetic gums,
pectins and
mucilages.
Particularly preferred as a starting material for the first polymer is
testaceous plant
material, for example waste testaceous plant material (preferably containing
at least about
20% of arabinoxylan and/or glucoronoarabinoxylan).
The starting material may be treated directly in its field-harvested state or
(more
usually) subject to some form of pre-processing. Typical pre-processing steps
include
chopping, grinding, cleaning, washing, screening, sieving, etc.
Preferably, the starting material is in a substantially ground form. It may be
air
classified or sieved (for example to reduce the level of starch).
Alternatively, or in addition,
the starting material may be treated with enzymes to remove starch (e.g.,
alpha- and/or beta-
amylase). The starting material may also be pre-digested with a carbohydrase
enzyme to
remove (3-glucan.
Suitable washing treatments include washing with hot water or acid (e.g., at a
pH of
3-6, e.g., about 5). This at least partially separates protein. Other pre-
treatments include
protease treatment.
Post-extraction processin~/isolation
Once extracted and prior to oxidative gelation, the polymers) may be further
processed to concentrate, purify or simply isolate them from the unextracted
residue.
Other post-extraction treatments include supplementing the extracted polymers)
with
an oxidase (e.g., glucose oxidase) supplement, optionally together with a
peroxidase (e.g.,
horse radish peroxidase) and/or an oxidase substrate (e.g., glucose)
supplement. This
supplementing step is carned out when gelation is to be carried out
subsequently by in situ
generation of hydrogen oxide by redox enzymes.
Particularly preferred are post-extraction processes which avoid the use of
alcohol
precipitation, so avoiding the costs associated with this step.
Preferred processing steps include any of centrifugation, filtration (e.g.,
ultrafiltration
or filtration of vega clay), precipitation (e.g., isoelectric precipitation),
chromatography (e.g.,
silica hydrogel and/or ion exchange chromatography). Particularly preferred is
ultrafiltration
or concentration by spray-, drum- or freeze-drying, or vacuum rotary drying.
Other
treatments include desalting treatments, for example dialysis or tangential
flow
ultrafiltration.
Although not preferred, alcohol (e.g., IMS, methanol, ethanol or iso-propanol)
precipitation, for example with up to 60-70% v/v alcohol, may be employed.
However,
particularly preferred is direct spray or freeze drying followed by drying, in
the absence of
an alcohol precipitation step.
9
CA 02364443 2001-09-05
WO 00/52092 PCT/US00/05467
Any of the aforementioned processes may be applied directly to the extracted
polymer(s). The extract may be dried, either before or after oxidative
gelation. Dried
preparations may be supplemented with carriers or dispersants, such as
glucose.
Oxidative eelation
Any of a variety of known oxidative gelation process can be used to gel the
polymers
of the invention. Suitable agents include hydrogen peroxide (usually in
conjunction with a
peroxidase), ammonium persulphate and formamidine disulphide.
The oxidative gelation may also be accomplished enzymically, for example as
described in WO 96/03440 in which an oxidase (preferably a laccase) is used to
promote
oxidative gelation of inter alia arabinoxylans.
Other enzymic approaches include promoting the generation of hydrogen peroxide
in situ by redox enzymes. The redox enzymes preferably comprise an oxidase
(e.g., glucose
oxidase) and a peroxidase (e.g., horse radish peroxidase), which are
preferably present as
supplements in the hemicellulosic material.
Alternatively, gelation may be achieved as described in WO 93/10158, which
describes an oxidizing system comprising a peroxide (such as hydrogen
peroxide) and an
oxygenase (such as a peroxidase).
Gelation of the first and second polymers of the invention is carried out at a
gelling
pH, that is a pH at which coupling of the polymers takes place to form a gel
or viscous fluid.
A suitable pH is 9 or below, for example 8 or below. The gelling pH may be
from 4 to 8.
Preferably the first and second polymers are co-gelled at a pH of 5 or below.
The desired
gelling pH may be achieved by adjusting the pH of the biopolymer solution
using small
amounts of dilute alkali or acid, for example sodium hydroxide or hydrochloric
acid.
Applications
The compositions of the invention (i.e., the gels, dehydrated gels, rehydrated
dehydrated gels, gelling (but un-gelled) compositions and viscous liquids of
the invention)
find a variety of applications in various therapeutic, surgical, prophylactic,
diagnostic and
cosmetic (e.g., skin care) applications.
For example, the aforementioned materials may be formulated as a
pharmaceutical or
cosmetic preparation or medical device, for example selected from: a wound
plug, wound
dressing, wound debriding system, controlled release device, an encapsulated
medicament or
drug, a lotion, cream (e.g., face cream), suppository, pessary, spray,
artificial skin, protective
membrane, a nutraceutical, prosthetic, orthopaedic, ocular insert, injectant,
lubricant or cell
implant matrix. They may be particularly useful as agents which maintain the
integrity of
the gut wall lining, and as agents for coating the Iuminal wall of the
gastrointestinal tract.
They may therefore find particular application in animal feeds and in the
treatment of
gastrointestinal disorders.
CA 02364443 2001-09-05
WO 00/52092 PCT/US00/05467
In such embodiments the compositions of the invention may further comprise an
antibiotic. electrolyte, cell, tissue. cell extract, pigment, dye.
radioisotope, label, imaging
agent, enzyme, co-factor, hormone, cytokine, vaccine, growth factor, protein
(e.g., a
therapeutic protein), allergen, hapten or antigen (for e.g., sensitivity
testing), antibody, oil,
analgesic and/or anti inflammatory agent (e.g., NSAID).
Thus, the above-listed materials find application in therapy, surgery,
prophylaxis or
diagnosis, for example in the treatment of surface (e.g., skin or membrane
lesions, e.g.,
burns, abrasions or ulcers). In a particularly preferred embodiment, the
invention
contemplates a wound dressing comprising the above listed materials of the
invention, for
example in the form of a spray. Such wound dressings are particularly useful
for the
treatment of bums, where their great moisture retaining properties help to
prevent the wound
drying out.
Particularly preferred for such application is a self gelling liquid
comprising the
composition of the invention supplemented with glucose and peroxidase and/or
oxidase
enzymes which gels on contact with oxygen in the air. Such compositions can be
provided
in the form of oxygen-free liquids in airtight containers which can be sprayed
onto the skin,
whereupon the liquid gels after exposure to the air. Such composition may
advantageously
be formulated so as to produce a slight excess of hydrogen peroxide on
exposure to oxygen,
so that a sterilizing, antibacterial, bacteriostatic and/or cleansing effect
is obtained which
helps promote healing.
The invention also contemplates water absorbent nappies, diapers, incontinence
pads,
sanitary towels, tampons and panty liners comprising the above-listed
materials, as well as
domestic and industrial cleaning or liquid (e.g., water) recovery operations
(e.g., in the oil
industry).
Alternatively, the gels of the invention can be provided in the form of
hydrated or
dehydrated sheets or pellicles for application to various internal or external
surfaces of the
body, for example during abdominal surgery to prevent adhesions.
Other applications include enzyme immobilizing systems, brewing adjuncts and
bread improvers.
The materials listed above also find application as a foodstuff, dietary fibre
source,
food ingredient, additive, lubricant, supplement or food dressing. Such
products are
preferably selected from crumb, alginate replacer, cottage cheeses, aerosol
toppings, frozen
yoghurts, milk shakes, ice cream, low calorie products such as dressings and
jellies, batters,
cake mixes, frozen chips, binders, gravies, pastas, noodles, doughs, pizza
toppings, sauces,
mayonnaise, jam, preserve, pickles, relish, fruit drinks, a clouding agent in
drinks, syrups,
toppings and confectionary (e.g., soft centres), petfood (wherein the gel
e.g., acts as a
binder), a flavour delivery agent, a canning gel, fat replacer (e.g.,
comprising macerated gel),
a coating, a glaze, a bait, a binder in meat and meat analogue products (for
example
11
CA 02364443 2001-09-05
WO 00/52092 PCT/US00/05467
vegetarian products), an edible adhesive, a gelatin replacer or dairy product
or ingredient
(e.g., a yoghurt supplement).
When used as a fat replacer the gel of the invention is preferably macerated
to
optimize its mouthfeel and fat mimetic properties.
The invention will now be further illustrated by way of specific Examples,
which are
purely illustrative and not intended to limit the scope of the invention in
any way.
EXAMPLE 1
Production of arabinoxylan ferulate
101 of sodium acetate buffer (pH 5, 0.02 M) were pre-equilibrated at
50°C and 10 ml
of liquid protease (ProfixT"") was added.
1 kg of fine wheat bran was added to this enzyme solution, and the suspension
mixed
vigorously for 60 min, maintaining the temperature at 50°C. The bran
residue was then
washed over a 200 p,m sieve, and rinsed with 3 1 of hot water. The washings
were discarded
and the bran residue recovered.
The washed bran residue was then resuspended in 5 1 of sodium acetate buffer
(pH 5,
0.02 M) at 60°C and mixed continuously, maintaining the temperature at
60°C. 25 g of
KOH pellets were then added, and mixing continued for 60 min at
60°C.
After 60 min, the mixture was neutralized to pH 7 with acetic acid and
filtered to
recover liquid. The mixture was then left to stand while a precipitate forms.
Alternatively,
the mixture may be centrifuged. A clear, dark golden brown supernatant is
recovered.
The pH of the supernatant was then brought to pH 4.8 with acetic acid and
1.5 volumes of IMS added. Further acetic acid is added to maintain the pH at
4.8.
Polysaccharides are then recovered by centrifugation and solvent exchange, and
the
polysaccharides then dried with acetone.
Gelation
Solutions of hydrocolloids were prepared by gradually adding dry powder to
water
with stirring until completely dispersed. Once prepared, solutions were stored
at 4°C until
required (two days maximum). AXF or sugar beet pectin (supplied by Copenhagen
Pectin)
were placed in plastic containers to give a total solution weight of 50 g. No
adjustments
were made for pH or ionic environment. Gelation was commenced by adding
peroxidase
followed by peroxide solution. As soon as the peroxide was added the container
was quickly
shaken to mix the reactants and a portion of the sample placed on the
rheometer. There was
a delay of approximately 1 minute between the start of the reaction and first
measurement.
Gelation was followed in oscillatory dynamic mode using a strain of 1 % and
frequency of
1 rads-1. Parallel plate geometry was used with 50 mm diameter plates and a
gap of 1 mm.
Measurements were taken every 30 s for 45 min. 60 min or 120 min. The
temperature was
maintained at 25°C. In some instances gelled systems were prepared as
above and stored at
ambient temperature overnight for visual assessment the next day.
12
CA 02364443 2001-09-05
WO 00/52092 PCT/US00/05467
Results and discussion
The minimum concentration for gelation of the AXF alone was approximately
0.25 wt %. Figure 1 shows G' (elastic modulus) as a function of time, in
minutes, for
mixtures of AXF with sugar beet pectin. Sugar beet pectin alone at 2 wt % gave
the smallest
increase in G' and did not gel at all at 1 wt %. However, the rate of reaction
of sugar beet
pectin was faster than that of AXF and most of the increase in G' was complete
within 5 min.
The addition of AXF to sugar beet pectin resulted in an increase in G'; for
example, G'
(60 min) for 2 wt % pectin was 97 Pa and for AXF:pectin 0.25:0.75 (2 wt %
total) was
514 Pa. The highest values of G' (after 60 min) were for AXFaugar beet pectin
mixtures of
the ratios 0.9:0.1, 0.8:0.2 and 0.75:0.25. Gelation of all mixtures containing
sugar beet
pectin was faster than for AXF alone.
To investigate whether the synergistic effect of sugar beet pectin-AXF
mixtures was
due to effects other than cross-linking through ferulic acid residues, the
reaction was done
with a mixture of pectin with non-gelling arabinoxylan (AX). The synergistic
effect was not
apparent with this form of arabinoxylan, showing that the increase in G' for
the gelation of
sugar beet pectin is not a salt or non-electrolyte effect.
EXAMPLE 2
Effect of pH on the rate of gel formation
The rate of gel formation, ie. increase in the elastic modulus, (Gi) as a
function of
time, was measured following the adjustment of the pH of solutions of AXF,
sugar-beet
pectin (SBP) and AXF-SBP (l:l) by the addition of small amounts of dilute NaOH
or HCI.
In all cases the final concentration of biopolymer was 2 wt %.
Figures 2 and 3 show gel formation for AXF, SBP and AXF-SBP (1:1) mixtures at
pH 4 and 5 respectively. The 4th line (AXF-SBP-calc) is the average of the AXF
and SBP
curves. Both Figures 2 and 3 show that the measured curve deviates from the
predicted
curve; measured values of G~ being greater than predicted, demonstrating a
synergistic
effect.
Various modifications of the invention in addition to those shown and
described herein
will be apparent to those skilled in the art from the foregoing description.
Such
modifications are also intended to fall within the scope of the appended
claims.
The disclosure of each reference set forth above is incorporated herein by
reference in
its entirety.
13
