U.S. patent application number 11/908107 was filed with the patent office on 2008-07-10 for high pressure processing of metal ion lactoferrin.
This patent application is currently assigned to Fonterra-Co-operative Group Limited. Invention is credited to Timothy Joseph Carroll, David Francis Elgar, Miguel Alejandro Gonzalez-Martin, Kay Patricia Palmano, Hasmukh Ambalal Patel.
Application Number | 20080166466 11/908107 |
Document ID | / |
Family ID | 36953610 |
Filed Date | 2008-07-10 |
United States Patent
Application |
20080166466 |
Kind Code |
A1 |
Palmano; Kay Patricia ; et
al. |
July 10, 2008 |
High Pressure Processing of Metal Ion Lactoferrin
Abstract
The present invention relates to a method of treating a
composition comprising metal ion lactoferrin and in particular to a
method of pressure treating a composition comprising metal ion
lactoferrin to prevent the growth of at least one unwanted
microorganism while retaining a desired level of metal ion
binding.
Inventors: |
Palmano; Kay Patricia;
(Palmerston North, NZ) ; Carroll; Timothy Joseph;
(Palmerston North, NZ) ; Patel; Hasmukh Ambalal;
(Palmerston North, NZ) ; Gonzalez-Martin; Miguel
Alejandro; (Rellingen, DE) ; Elgar; David
Francis; (Palmerston North, NZ) |
Correspondence
Address: |
KNOBBE MARTENS OLSON & BEAR LLP
2040 MAIN STREET, FOURTEENTH FLOOR
IRVINE
CA
92614
US
|
Assignee: |
Fonterra-Co-operative Group
Limited
Auckland
NZ
|
Family ID: |
36953610 |
Appl. No.: |
11/908107 |
Filed: |
March 8, 2006 |
PCT Filed: |
March 8, 2006 |
PCT NO: |
PCT/NZ2006/000038 |
371 Date: |
February 6, 2008 |
Current U.S.
Class: |
426/573 ;
426/522; 426/580; 426/583 |
Current CPC
Class: |
A23C 3/00 20130101; A23C
9/1322 20130101; A23L 33/19 20160801; A23V 2002/00 20130101; A23V
2002/00 20130101; A23V 2200/306 20130101; A61P 29/00 20180101; A23V
2200/308 20130101; A23V 2200/324 20130101; A23C 2210/15 20130101;
A23V 2250/54248 20130101; A23L 2/66 20130101; A61P 19/10 20180101;
A61P 7/06 20180101; A01N 63/10 20200101; C07K 14/79 20130101; A61P
19/00 20180101; A23L 3/0155 20130101; A61P 37/02 20180101; A23L
5/30 20160801; A61P 19/02 20180101; A23C 3/08 20130101; A23L 33/165
20160801; A61K 35/20 20130101; A61L 2/0011 20130101; A61P 37/04
20180101; A61P 9/00 20180101; A61P 35/00 20180101 |
Class at
Publication: |
426/573 ;
426/522; 426/580; 426/583 |
International
Class: |
A23L 1/05 20060101
A23L001/05; A23L 3/00 20060101 A23L003/00; A23C 9/154 20060101
A23C009/154; A23C 21/00 20060101 A23C021/00 |
Foreign Application Data
Date |
Code |
Application Number |
Mar 8, 2005 |
NZ |
538671 |
Dec 23, 2005 |
NZ |
544408 |
Claims
1. (canceled)
2. (canceled)
3. A method of treating a composition to maintain or increase its
keeping quality comprising (a) providing a composition comprising
metal ion lactoferrin or a metal ion functional variant or fragment
thereof and having a pH of from about 3.0 to 8.0, and (b)
subjecting the composition to a pressure treatment at a treatment
pressure of from about 350 MPa to about 1000 MPa to prevent the
growth of unwanted microorganisms that may be present in the
composition and retain at least a desired level of metal ions bound
to the lactoferrin or functional variant or fragment thereof.
4. A method of treating a composition to maintain or increase its
keeping quality comprising (a) providing a composition comprising
metal ion lactoferrin or a metal ion functional variant or fragment
thereof and one or more unwanted microorganisms and having a pH of
from about 3.0 to about 8.0, and (b) selecting a treatment pressure
of from about 350 MPa to 1000 MPa that will prevent the growth of
the unwanted microorganisms and retain at least a desired level of
metal ions bound to the lactoferrin or functional variant or
fragment thereof, and (c) subjecting the composition to a pressure
treatment at the treatment pressure.
5. The method of claim 3 wherein the composition consists
essentially of metal ion lactoferrin or a metal ion functional
variant or fragment thereof.
6. The method of claim 3 wherein the composition comprises about
0.1 mg/ml to 1000 mg/ml of metal ion lactoferrin or a metal ion
functional variant or fragment thereof or a mixture of metal ion
lactoferrin and at least one metal ion functional variant or
fragment thereof.
7. The method of claim 3 wherein the composition comprises about 1
mg/ml to 1000 mg/ml of metal ion lactoferrin or a metal ion
functional variant or fragment thereof or a mixture of metal ion
lactoferrin and at least one metal ion functional variant or
fragment thereof.
8. The method of claim 3 wherein the metal ion is selected from the
group consisting of aluminium ions, calcium ions, copper ions,
chromium ions, cobalt ions, gold ions, iron ions, manganese ions,
magnesium ions, platinum ions, ruthenium ions, selenium ions, zinc
ions, and mixtures thereof.
9. The method of any one of claim 8 wherein the metal ion is
iron.
10. (canceled)
11. (canceled)
12. The method of claim 3 wherein the metal ion lactoferrin or a
metal ion functional variant or fragment thereof is at least about
5% metal ion saturated.
13. The method of claim 3 wherein the metal ion lactoferrin or a
metal ion functional variant or fragment thereof is at least about
10% metal ion saturated.
14. The method of claim 3 wherein the metal ion lactoferrin or a
metal ion functional variant or fragment thereof is at least about
20% metal ion saturated.
15. The method of claim 3 wherein the metal ion lactoferrin or a
metal ion functional variant or fragment thereof is at least about
50% metal ion saturated.
16. The method of claim 3 wherein the metal ion lactoferrin or a
metal ion functional variant or fragment thereof is at least about
100% metal ion saturated.
17-35. (canceled)
36. The method of claim 3 wherein the composition comprises at
least one of the following: recombined or fresh whole milk,
recombined or fresh skim milk, reconstituted whole or skim milk
powder, skim milk concentrate, skim milk isolate, whole or skim
milk powder, skim milk retentate, concentrated milk, buttermilk,
ultrafiltered milk retentate, milk protein concentrate (MPC), milk
protein isolate (MPI), calcium depleted milk protein concentrate,
calcium depleted milk protein isolate, low fat milk, low fat milk
protein concentrate, low fat milk protein isolate, colostrum, a
colostrum fraction, colostrum protein concentrate (CPC), colostrum
milk protein concentrate, colostrum milk protein isolate, colostrum
whey, colostrum whey protein concentrate, colostrum whey protein
isolate, an immunoglobulin fraction from colostrum, whey, whey
protein concentrate (WPC), whey protein isolate (WPI), sweet whey,
lactic acid whey, mineral acid whey, reconstituted whey powder,
hyperimmune milk, hyperimmune milk protein concentrate, hyperimmune
milk protein isolate, hyperimmune whey, hyperimmune whey protein
concentrate, hyperimmune whey protein isolate, hyperimmune
colostrum, hyperimmune colostrum milk protein concentrate,
hyperimmune colostrum milk protein isolate, hyperimmune colostrum
whey, hyperimmune colostrum whey protein concentrate, hyperimmune
colostrum whey protein isolate, a composition derived from any milk
or colostrum processing stream, a composition derived from the
retentate or permeate obtained by ultrafiltration or
microfiltration of any milk or colostrum processing stream, or a
composition derived from the breakthrough or adsorbed fractions
obtained by chromatographic separation of any milk or colostrum
processing stream, full or partial hydrolysates thereof and
mixtures thereof.
37. The method of claim 3 wherein the composition is a beverage, an
acidified beverage, a neutral beverage, a carbonated beverage, a
yogurt or a jelly.
38. A composition treated according to the method of claim 3.
39. The composition of claim 38, wherein the composition is one of:
a beverage, a yogurt, and a jelly.
40-44. (canceled)
45. A pressure-treated composition comprising about 0.1 mg/ml to
1000 mg/ml of metal ion lactoferrin or a metal ion functional
variant or fragment thereof and less than about 50,000 cfu/ml of
microorganisms.
46. The pressure-treated composition of claim 45, wherein the
composition is a jelly, a yoghurt or a beverage.
47-48. (canceled)
49. The pressure-treated composition of claim 45 comprising about 1
mg/ml to 1000 mg/ml of metal ion lactoferrin or a metal ion
functional variant or fragment thereof and less than about 50,000
cfu/ml of microorganisms.
50-53. (canceled)
54. The composition of claim 45 wherein the metal ion lactoferrin
or a metal ion functional variant or fragment thereof is at least
about 20% metal ion saturated.
55. The composition of claim 45 wherein the metal ion lactoferrin
or a metal ion functional variant or fragment thereof is at least
about 40% metal ion saturated.
56. The composition of claim 45 wherein the metal ion lactoferrin
or a metal ion functional variant or fragment thereof is at least
about 60% metal ion saturated.
57-63. (canceled)
64. The composition of claim 39, wherein the beverage is an
acidified beverage, a neutral beverage or a carbonated beverage.
Description
FIELD OF INVENTION
[0001] The present invention relates to a high pressure processing
of metal ion lactoferrin compositions and in particular to a method
of pressure treating a composition comprising metal ion lactoferrin
to maintain or increase its keeping quality while retaining a
desired level of metal ion binding.
BACKGROUND
[0002] The delivery of bioactive components (proteins, lipids or
hydrolysates thereof, for example) in food or other ingestible
products is constrained by the need to provide a safe product with
a useful shelf life while retaining bioactivity. Products with a
useful shelf life are said to have a good keeping quality and are
less prone to spoilage.
[0003] Delivery of bioactive components is desirable at least
because such components are physiologically active when ingested
and can have positive health benefits, including but not limited to
bone health, immune benefits, anti-inflammatory activity, heart
health and efficacy in cancer treatment.
[0004] Traditional means of ensuring a useful keeping quality have
a negative impact on the bioactivity of food products and the like.
In particular, thermal processing is not generally suitable for the
production of commercially sterile bioactive products. For example,
an analysis of immunoglobulin proteins in commercial dairy products
revealed that although between 60% and 75% of the immunoglobulins
are retained through pasteurization, levels in UHT or canned
(evaporated) milk are negligible (Li-Chan et al, 1995). Commercial
sterility in acid foods may be achieved by employing a
lower-temperature heating than that used in canning, but the
sensitivity of immunoglobulins to denaturing under heating is
exacerbated by acidification (Dominguez et al, 2001).
[0005] There are many processes in the manufacture of bioactive
products, ingredients and foods that may result in a partial or
complete loss of bioactivity. In the case of dairy-based
ingredients and foods, processes that involve heating steps that
may affect bioactivity include thermal pasteurization,
homogenisation, thermalisation, evaporation and drying. In the case
of food processing, examples of heating steps that may affect
bioactivity include heat treatments preceding fermentation,
UHT-treatments, retorting, hot filling and hot packing. A bioactive
component will typically be subjected to one or more of these
heating steps during the manufacture of a food. This is
particularly true of dairy-based products where processing always
includes an initial pasteurization step and typically includes
further heating steps prior to packaging and sale. Korhonen et al
(1998) report that heating to temperatures in the range 60.degree.
C. to 90.degree. C. denatures proteins therefore reducing the
activity of bioactive proteins.
[0006] Drying of products produced using pasteurized milk may be
used to improve keeping quality with losses of up to 40% of
immunoglobulins (Li-Chan, 1995), but commercial applications are
then limited to direct consumption (for example, tablets) or fresh
products (for example, yoghurt) where the dried bioactive
ingredient is not subsequently heated again. Losses due to drying
and heating may be compensated for by supplementing intermediate or
final products with the bioactive component of interest but this
can increase the cost to the end consumer.
[0007] Pressure treatment with pressures above about 350 Ma has
been employed to achieve commercially-useful improvements in
keeping quality for meat, vegetable and fruit-based products (such
as cooked ham, avocado products and juices respectively). However,
Huppertz et al (2002) report that high pressure denatures whey
proteins in milk. Additionally, Korhonen et al (1998) report that
pressure treatments at pressures of about 500 MPa and above
irreversibly denature proteins in most cases. Felipe et al (1997)
report that appreciable levels of immunoglobulin denaturation occur
in goat's milk at pressures of 500 MPa.
[0008] Masuda et al (2001) report that pressures of 400 MPa and
above may not be used to improve the keeping quality of bovine
colostrum because such pressures denature the immunoglobulin
protein.
[0009] Tonello et al (1992) report that pressures of 200 MPa
applied for 2 hours may be used to retain at least 85% of the
immunoglobulin activity, although the microbial load of colostrum
is reduced by less than 2-log cycles. The same process at
63.degree. C. can reduce the microbial load below limits of
detection (more than 7-log cycles), but at least 50% of the
immunoglobulin activity is lost.
[0010] Thermal treatment is the currently recognised method for
commercial sterilisation of lactoferrin solutions for various
applications, at specific pH (Tomita et al., 1994) and for specific
purposes (for example, as a drink or beverage; see Tanaka, et al.,
1991). Although lactoferrin naturally loses some iron at lower
(acidic) pH values by virtue of a decrease in affinity for iron
(Baker et al., 2002), this loss does not become significant until
below pH 4.0 (Groves, 1960; Bluard-Deconinck et al., 1978; Tweedie
et al., 1994; Parry & Brown, 1974). However, recommended heat
treatments for various important applications such as beverages (pH
3.8-4.0, 85.degree. C. for 10 min) and yoghurts (pH 4.6-5.0,
90.degree. C. for 10 min) result in loss of iron (or oriented iron
binding) above and beyond that incurred by pH lowering alone.
Moreover, the ability of lactoferrin to re-bind iron may be
significantly reduced. At higher pH values that favour
stoichiometric iron-binding, lactoferrin is less thermally stable
(Steijns & van Hooijdonk, 2000) and at these values iron is
also lost upon heat treatment, with accompanying loss of
iron-binding ability. Thus, thermal processing may limit the use of
iron lactoferrin in various applications, not only those at lower
pH (for example, yoghurt, jellies, acid beverages) but also those
across a broader pH range (for example, smoothies and other
applications at neutral pH).
[0011] The need exists for a process that can provide a
commercially useful keeping quality for a bioactive product such as
a food or other ingestible product while retaining the bioactivity
of at least one bioactive component.
[0012] Therefore it is an object of this invention to provide an
improved or alternative method of preventing the growth of unwanted
microorganisms in a composition comprising metal ion lactoferrin
while retaining a desired level of metal ion binding or to at least
provide the public with a useful choice.
SUMMARY OF INVENTION
[0013] Accordingly, in one aspect the present invention relates to
a method of treating a composition to maintain or increase its
keeping quality, the method comprising [0014] (a) providing a
composition comprising metal ion lactoferrin or a metal ion
functional variant or fragment thereof, and [0015] (b) subjecting
the composition to a pressure treatment at a treatment pressure
that will prevent the growth of at least one unwanted
microorganism.
[0016] In another aspect the present invention relates to a method
of treating a composition to maintain or increase its keeping
quality, the method comprising [0017] (a) providing a composition
comprising metal ion lactoferrin or a metal ion functional variant
or fragment thereof, [0018] (b) selecting a treatment pressure that
will prevent the growth of at least one unwanted microorganism, and
[0019] (c) subjecting the composition to a pressure treatment at
the treatment pressure.
[0020] In another aspect the present invention relates to a method
of treating a composition to maintain or increase its keeping
quality comprising [0021] (a) providing a composition comprising
metal ion lactoferrin or a metal ion functional variant or fragment
thereof and having a pH of from about 3.0 to about 8.0 or higher,
and [0022] (b) subjecting the composition to a pressure treatment
at a treatment pressure of from about 350 MPa to about 1000
MPa.
[0023] In another aspect the present invention relates to a method
of treating a composition to maintain or increase its keeping
quality, the method comprising [0024] (a) providing a composition
comprising metal ion lactoferrin or a metal ion functional variant
or fragment thereof and one or more unwanted microorganisms and
having a pH of from about 3.0 to about 8.0 or higher and, [0025]
(b) selecting a treatment pressure of from about 350 MPa to about
1000 MPa that will prevent the growth of at least one unwanted
microorganism and retain at least a desired level of metal ions
bound to the lactoferrin or functional variant or fragment thereof,
and [0026] (c) subjecting the composition to a pressure treatment
at the treatment pressure.
[0027] The following embodiments may relate to any of the aspects
described above.
[0028] In one embodiment the composition comprises at least about
0.1 mg/mil, preferably about 0.1, 0.5, 1, 2, 3, 4, 5, 10, 20, 30,
40, 50, 60, 70, 80, 90, 100, 200, 200, 400, 500, 600, 700, 800,
900, or 1000 mg/ml of metal ion lactoferrin or a metal ion
functional variant or fragment thereof, and useful ranges may be
selected between any of these values (for example, from about 0.1
to 1000 mg/ml, about 1 to 1000 mg/ml, about 2 to 1000 mg/ml, about
3 to 1000 mg/ml, about 4 to 1000 mg/ml, about 5 to 1000 mg/ml,
about 10 to 1000 mg/ml, about 0.1 to 100 mg/ml, about 1 to 100
mg/ml, about 2 to 100 mg/ml, about 3 to 100 mg/mil, about 4 to 100
mg/ml, about 5 to 100 mg/ml, and about 10 to 100 mg/ml).
[0029] In another embodiment the composition or product consists
essentially of or consists of metal ion lactoferrin or a metal ion
functional variant or fragment thereof.
[0030] In a further embodiment the composition or product
comprises, consists essentially of or consists of at least about
0.01, 0.05, 0.1, 0.5, 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55,
60, 65, 70, 75, 80, 85, 90, 95, 99, 99.5 or 100% by weight metal
ion lactoferrin or a metal ion functional variant or fragment
thereof and useful ranges may be selected between any of these
values (for example, from about 0.01 to about 100%, about 0.05 to
about 100%, about 0.1 to about 100%, about 0.5 to about 100%, about
1 to about 100%, about 5 to about 100%, about 10 to about 100%,
about 15 to about 100%, about 20 to about 100%, about 25 to about
100%, about 30 to about 100%, about 35 to about 100%, about 40 to
about 100%, about 45 to about 100%, about 50 to about 100%, about
55 to about 100%, about 60 to about 100%, about 65 to about 100%,
about 70 to about 100%, about 75 to about 100%, about 80 to about
100%, about 85 to about 100%, about 90 to about 100%, about 95 to
about 100%, about 99 to about 100%, from about 0.01 to about 0.05%,
from about 0.01 to about 0.1%, from about 0.01 to about 0.5%, from
about 0.01 to about 1%, from about 0.01 to about 5%, from about
0.01 to about 10%, from about 0.01 to about 15%, from about 0.01 to
about 20%, from about 0.01 to about 25%, from about 0.01 to about
30%, from about 0.01 to about 35%, from about 0.01 to about 40%,
from about 0.01 to about 45%, from about 0.01 to about 50%, from
about 0.01 to about 55%, from about 0.01 to about 60%, from about
0.01 to about 65%, from about 0.01 to about 70%, from about 0.01 to
about 75%, from about 0.01 to about 80%, from about 0.01 to about
85%, from about 0.01 to about 90%, from about 0.01 to about 95%,
from about 0.01 to about 99%, and from about 0.01 to about
99.9%).
[0031] In one embodiment the composition or product has been
enriched with a composition consisting essentially of or consisting
of metal ion lactoferrin or a metal ion functional variant or
fragment thereof or a mixture thereof. That is, a composition
consisting essentially of or consisting of metal ion lactoferrin or
a metal ion functional variant or fragment thereof or a mixture
thereof is added to the composition or product to increase the
concentration of metal ion lactoferrin or a metal ion functional
variant or fragment thereof or a mixture thereof.
[0032] In one embodiment the composition comprises one or more
unwanted microorganisms, such as one or more bacteria (including
one or more probiotic bacteria), one or more fungi, one or more
molds, one or more yeasts, or one or more algae, or a mixture
thereof. It should be understood that in one embodiment the methods
useful herein are preferably intended to prevent the growth of at
least one unwanted microorganism. However, often the method may be
carried out prophylactically and there may not actually be any
unwanted organisms present. In such cases, the method is carried
out to maintain or improve keeping quality or to comply with food
safety requirements, current good manufacturing practice or
regulatory requirements. Regardless of the presence or absence of
an unwanted microorganism, a pressure treatment method useful
herein retains at least a desired level of activity of at least one
bioactive component and is useful to maintain or increase keeping
quality.
[0033] In one embodiment the method prevents the growth of at least
one unwanted microorganism while retaining at least a desired level
of metal ion saturation. That is, the method prevents the growth of
an unwanted microorganism without substantially affecting the
degree of metal ion saturation of any lactoferrin polypeptides or
functional variants or fragments thereof that are present.
[0034] In one embodiment the metal ion is an ion selected from the
group comprising aluminium, calcium, copper, chromium, cobalt,
gold, iron, manganese, magnesium, platinum, ruthenium, selenium and
zinc ions. Preferably the metal ion is an iron ion.
[0035] In one embodiment the lactoferrin is selected from the group
comprising sheep, goat, pig, mouse, water buffalo, camel, yak,
horse, donkey, llama, bovine and human lactoferrin, and mixtures
thereof. Preferably the lactoferrin is bovine lactoferrin.
[0036] In one embodiment the lactoferrin is recombinant, synthetic
or natural lactoferrin, or mixtures thereof.
[0037] In one embodiment the metal ion lactoferrin or a metal ion
saturated functional variant or fragment thereof is at least about
5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85,
90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 99.5 or 100% metal ion
saturated and useful ranges may be selected between any of the
foregoing values (for example, from about 5 to about 100%, about 10
to about 100%, about 15 to about 100%, about 20 to about 100%,
about 25 to about 100%, about 30 to about 100%, about 35 to about
100%, about 40 to about 100%, about 45 to about 100%, about 50 to
about 100%, about 55 to about 100%, about 60 to about 100%, about
65 to about 100%, about 70 to about 100%, about 75 to about 100%,
about 80 to about 100%, about 85 to about 100%, about 90 to about
100%, about 95 to about 100% and about 99 to about 100%).
[0038] In one embodiment the metal ion lactoferrin or a metal ion
functional variant or fragment thereof is at least about 105, 110,
115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175,
180, 185, 190, 195 or 200% metal ion saturated and useful ranges
may be selected between any of the foregoing values (for example,
about 105 to about 150%).
[0039] In one embodiment a composition or product provides a
population of lactoferrin polypeptides or functional variants or
fragments thereof wherein at least about 5, 10, 15, 20, 25, 30, 35,
40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96,
97, 98, 99, 99.5 or 100% of the available metal ion binding pockets
in the population are bound to a metal ion, preferably an iron ion,
and useful ranges may be selected between any of the foregoing
values (for example, about 5 to about 100%, about 10 to about 100%,
about 15 to about 100%, about 20 to about 100%, about 25 to about
100%, about 30 to about 100%, about 35 to about 100%, about 40 to
about 100%, about 45 to about 100%, about 50 to about 100%, about
55 to about 100%, about 60 to about 100%, about 65 to about 100%,
about 70 to about 100%, about 75 to about 100%, about 80 to about
100%, about 85 to about 100%, about 90 to about 100%, about 95 to
about 100% and about 99 to about 100%).
[0040] In one embodiment a composition or product provides a
population of lactoferrin polypeptides or functional variants or
fragments thereof wherein about 100% of the available metal ion
binding pockets in the population are bound to a metal ion,
preferably an iron ion, and additional metal ions are bound to the
lactoferrin molecules in non specific binding sites so that the
lactoferrin is 105, 110, 115, 120, 125, 130, 135, 140, 145, 150,
155, 160, 165, 170, 175, 180, 185, 190, 195 or 200% metal ion on a
stoichiometric basis.
[0041] Useful metal ion saturation ranges include about 25 to about
200%, about 30 to about 200%, about 35 to about 200%, about 40 to
about 200%, about 45 to about 200%, about 50 to about 200%, about
55 to about 200%, about 60 to about 200%, about 65 to about 200%,
about 70 to about 200%, about 75 to about 200%, about 80 to about
200%, about 85 to about 200%, about 90 to about 200%, about 95 to
about 200% and about 100 to about 200% metal ion saturation.
[0042] In one embodiment, the post-treatment metal ion saturation
value is selected from a value or range listed above.
[0043] In one embodiment the metal ion lactoferrin or metal ion
functional fragment of variant thereof retains at least about 40,
45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 99 or 100% of the metal
ions bound.
[0044] In one embodiment the method comprises administration of a
mixture of metal ion lactoferrin and at least one metal ion
functional variant or fragment thereof.
[0045] In one embodiment the treatment pressure is selected from at
least about 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450,
460, 470, 480, 490, 500, 510, 520, 530, 540, 550, 560, 570, 580,
590, 600, 610, 620, 630, 640, 650, 660, 670, 680, 690, 700, 750,
800, 850, 900, 950 and 1000 MPa or greater, and useful ranges may
be selected between any of these values (for example, from about
350 to about 400 MPa, about 350 to about 450 MPa, about 350 to
about 500 MPa, about 350 to about 550 MPa, about 350 to about 600
MPa, about 350 to about 650 MPa, about 350 to about 700 MPa, about
350 to about 750 MPa, about 350 to about 800 MPa, about 350 to
about 850 MPa, about 350 to about 900 MPa, about 350 to about 950
MPa and about 350 to about 1000 MPa, about 400 to about 1000 MPa,
about 450 to about 1000 MPa, about 500 to about 1000 MPa, about 550
to about 1000 MPa, about 600 to about 1000 MPa, about 650 to about
1000 MPa, about 700 to about 1000 MPa, about 750 to about 1000 MPa,
about 800 to about 1000 MPa, about 850 to about 1000 MPa, about 900
to about 1000 MPa, about 950 to about 1000 MPa, about 500 to about
550 MPa, about 500 to about 600 MPa, about 500 to about 650 MPa,
about 500 to about 700 MPa, about 500 to about 750 MPa, about 500
to about 800 MPa, about 550 to about 800 MPa, about 600 to about
800 MPa, about 650 to about 800 MPa, about 700 to about 800 MPa,
about 750 to about 800 MPa, about 400 to about 800 MPa, about 400
to about 750 MPa, about 400 to about 700 MPa, about 400 to about
650 MPa, about 400 to about 600 MPa, about 450 to about 800 MPa,
about 450 to about 750 MPa, about 450 to about 700 MPa, about 450
to about 650 MPa, about 450 to about 600 MPa, about 500 to about
800 MPa, about 500 to about 750 MPa, about 500 to about 700 MPa,
about 500 to about 650 MPa, about 500 to about 600 MPa, about 525
to about 675 MPa, about 550 to about 650 MPa and about 575 to about
625 MPa). Preferably the treatment pressure is at least about 350,
400, 450, 500 or 600 MPa.
[0046] In another embodiment the pH of the composition when it is
subjected to the pressure treatment is at least about 3.0, 3.1,
3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4,
4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7,
5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0,
7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9 or 8.0 or greater, and
useful ranges may be selected between any of these values (for
example, from about pH 3.0 to about 4.5, about pH 3.0 to about 5.0,
about pH 3.0 to about 5.5, about pH 3.0 to about 6.0, about pH 3.0
to about 6.5, about pH 3.0 to about 7.0, about pH 3.0 to about 7.5,
about pH 3.0 to about 8.0, about pH 3.1 to about 4.9, about pH 3.2
to about 4.8, about pH 3.3 to about 4.7, about pH 3.4 to about 4.6,
about pH 3.5 to about 4.5, about pH 3.6 to about 4.4, about pH 3.7
to about 4.3, about pH 3.8 to about 4.2, about pH 3.9 to about 4.1,
about pH 3.1 to about 8.0, about pH 3.2 to about 8.0, about pH 3.3
to about 8.0, about pH 3.4 to about 8.0, about pH 3.5 to about 8.0,
about pH 3.6 to about 8.0, about pH 3.7 to about 8.0, about pH 3.8
to about 8.0, about pH 3.9 to about 8.0, about pH 4.0 to about 8.0,
about pH 4.5 to about 8.0, about pH 5.0 to about 8.0, about pH 5.5
to about 8.0, about pH 6.0 to about 8.0, about pH 6.5 to about 8.0,
and about pH 7.0 to about 8.0). Alternatively, in still another
embodiment, the pH of the composition is adjusted before pressure
treatment to a pH or within a pH range listed above.
[0047] In one embodiment the method is conducted at ambient
temperature. Preferably the pressure treatment is conducted at a
temperature of at least about 0, 4, 5, 10, 15, 20, 25, 30, 35, 40,
45, 50, 55 or 60 degrees Celsius, and useful ranges may be selected
between any of these forgoing values (for example, from about 5 to
about 40 degrees Celsius).
[0048] In one embodiment the treatment pressure may be applied for
a treatment time of about 1 second to about 30 minutes. Preferably
the treatment time is selected from about 1, 5, 10, 20, 30, 60, 90,
120, 150, 180, 210, 240, 270 or 300 seconds or about 0.5, 1, 1.5,
2, 2.5, 3, 3.5, 4, 4.5, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40,
45, 50, 55 or 60 minutes, and useful ranges may be selected between
any of these values (for example, from about 1 to about 10 minutes,
25 from about 1 to about 5 minutes or from about 2 to about 4
minutes).
[0049] In another embodiment the treatment pressure is held
substantially constant for the treatment time. In another
embodiment the pressure is increased from ambient pressure (usually
atmospheric pressure) to the treatment pressure and then returned
to ambient pressure within the treatment time. Ambient pressure
will usually be atmospheric pressure.
[0050] It should be understood that in one embodiment a treatment
time of a period listed above is the time taken to increase the
pressure from atmospheric pressure to the treatment pressure and
then return the pressure to atmospheric pressure. Accordingly, in
one embodiment a treatment time of 1 minute means that the pressure
is increased from atmospheric pressure to the treatment pressure
and then returned to atmospheric pressure within 1 minute.
[0051] It should be understood that in another embodiment a
treatment time of a period listed above is the time that the
pressure is held at the treatment pressure (the "hold time").
Accordingly, in one embodiment a treatment time of 1 minute means
that the pressure is held at the treatment pressure for 1 minute.
Therefore, a treatment time of 0 (or "no hold") in this embodiment
means that the pressure is raised to the treatment pressure but not
held, and the pressure is then returned to atmospheric pressure. In
this embodiment, preferred treatment times include 0 (no hold),
0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8,
8.5, 9, 9.5 and 10 minutes. Preferably the pressure is held for
about 0, 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5 or 5 minutes, or for
about 0, 0.5, 1, 1.5, 2, 2.5 or 3 minutes, or for about 0
minutes.
[0052] In one alternative embodiment the total treatment time is
less than about 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6,
6.5, 7, 7.5, 8, 8.5, 9, 9.5 or 10 minutes. That is, the time taken
for the pressure to be raised from and returned to ambient pressure
(usually atmospheric pressure) is less than about 0.5, 1, 1.5, 2,
2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5 or 10
minutes. Preferably less than about 8 minutes, preferably less than
about 7 minutes, preferably less than about 6 minutes or preferably
less than about 5 minutes.
[0053] In another embodiment the composition may be subjected to
additional pressure treatments. The treatment pressure may be
changed from one treatment pressure to another, without first
returning to atmospheric pressure. Each pressure treatment may be
conducted for a separate treatment time. Accordingly, in one
embodiment the pressure is increased from ambient pressure to a
first treatment pressure for a first treatment time and then the
pressure is changed to a second treatment pressure for a second
treatment time. Preferably the first treatment time is longer than
the second treatment time. Preferably the first treatment is
shorter than the second treatment time. In yet another embodiment
the pressure is increased to a first treatment pressure and then
changed to a second treatment pressure within the treatment
time.
[0054] In one embodiment the composition is subjected to the
treatment pressure before incorporation into a product. In another
embodiment the composition is subjected to the treatment pressure
after incorporation into a product. Accordingly, the method further
comprises subjecting the composition to the treatment pressure
before or after incorporating the composition into a product.
[0055] In another embodiment the composition is subjected to the
treatment pressure before packaging. In another embodiment the
composition is subjected to the treatment pressure after packaging.
Accordingly, the method further comprises subjecting the
composition to the treatment pressure before or after
packaging.
[0056] In one embodiment the treatment pressure is about 600 MPa
and the hold time is about 0, 1, 2 or 3 minutes. In another
preferred embodiment the treatment pressure is about 400 MPa and
the hold time is less than about 30 minutes.
[0057] In one preferred embodiment the treatment pressure is about
350 to about 650 MPa and the hold time is about 0 minutes to about
5 minutes. Preferably the composition is yoghurt.
[0058] In one preferred embodiment the treatment pressure is about
350 to about 650 MPa and the hold time is about 0 minutes to about
5 minutes. Preferably the composition a beverage.
[0059] In one preferred embodiment the treatment pressure is about
350 to about 650 MPa and the hold time is about 0 minutes to about
5 minutes. Preferably the composition a fermented drink.
[0060] In one preferred embodiment the treatment pressure is about
350 to about 650 MPa and the hold time is about 0 minutes to about
5 minutes. Preferably the composition an acid drink, a liquid
concentrate (including gels) or yoghurt.
[0061] In one preferred embodiment the treatment pressure is about
350 to about 700 MPa, the hold time is about 0 minutes to about 30
minutes, the pH of the composition is about pH 3.0 to about pH 8.0
and the composition is a beverage (including an acidified beverage,
a neutral beverage or a carbonated beverage), a yoghurt or
ajelly.
[0062] In one embodiment the composition comprises dairy protein or
a dairy ingredient. Preferably the dairy protein composition or the
dairy ingredient is recombined or fresh whole milk, recombined or
fresh skim milk, reconstituted whole or skim milk powder, skim milk
concentrate, skim milk isolate, whole or skim milk powder, skim
milk retentate, concentrated milk, buttermilk, ultrafiltered milk
retentate, milk protein concentrate (MPC), milk protein isolate
(MPI), calcium depleted milk protein concentrate, calcium depleted
milk protein isolate, low fat milk, low fat milk protein
concentrate, low fat milk protein isolate, colostrum, a colostrum
fraction, colostrum protein concentrate (CPC), colostrum milk
protein concentrate, colostrum milk protein isolate, colostrum
whey, colostrum whey protein concentrate, colostrum whey protein
isolate, an immunoglobulin fraction from colostrum, whey, whey
protein concentrate (WPC), whey protein isolate (WPI), sweet whey,
lactic acid whey, mineral acid whey, reconstituted whey powder,
hyperimmune milk, hyperimmune milk protein concentrate, hyperimmune
milk protein isolate, hyperimmune whey, hyperimmune whey protein
concentrate, hyperimmune whey protein isolate, hyperimmune
colostrum, hyperimmune colostrum milk protein concentrate,
hyperimmune colostrum milk protein isolate, hyperimmune colostrum
whey, hyperimmune colostrum whey protein concentrate, hyperimmune
colostrum whey protein isolate, a composition derived from any milk
or colostrum processing stream, a composition derived from the
retentate or permeate obtained by ultrafiltration or
microfiltration of any milk or colostrum processing stream, or a
composition derived from the breakthrough or adsorbed fractions
obtained by chromatographic separation of any milk or colostrum
processing stream, or a full or partial hydrolysates of any of
these compositions, or a mixture thereof.
[0063] Preferably the dairy ingredient is from a cow, sheep, goat,
pig, mouse, water buffalo, camel, yak, horse, donkey, llama or
human source or mixtures thereof.
[0064] In another embodiment the composition or product further
comprises a component selected from lysozyme, immunoglobulins,
glycomacropeptide, a composition comprising at least about 1% w/w
immunoglobulins, a product made by inoculating an animal to
increase antibody levels, immune milk, and mixtures thereof.
[0065] In another embodiment the composition or product further
comprises a component selected from non-polar lipids and polar
lipids such as phospholipids, sphingolipids, gangliosides and
ceramides, and mixtures thereof.
[0066] In another embodiment the composition or product further
comprises at least about 1% w/w immunoglobulins wherein the
immunoglobulins comprise one or more of IgA, IgD, IgE, IgG or
IgM.
[0067] In one embodiment the composition or product is a food
product. Preferably the food product is an acidic beverage or a
carbonated beverage. Preferably the food product is a yoghurt
including set, stirred, flavoured, fruit and probiotic yoghurts,
fromage frais, petit suisse, quarg, fermented food or drink,
acidified drink or milk product. Preferably the food product is a
jelly.
[0068] In one embodiment the composition further comprises a
probiotic microorganism selected from the group consisting of
Lactobacillus, Streptococcus, Lactococcus, Leuconostoc,
Pediococcus, Bifidobacteriurn, Propionibacterium, Enterococcus or
Bacillus, or a mixture thereof.
[0069] In one embodiment the composition further comprises a
probiotic microorganism selected from the group consisting of
Lactobacillus acidophilus, Lactobacillus delbrueckii subsp.
bulgaricus, Lactobacillus casei, Lactobacillus crispatus,
Lactobacillus johnsonii, Lactobacillus plantarum, Lactobacillus
reuteri, Lactobacillus rhamnosus, Lactobacillus salivarius,
Bifidiobacteriun bifidum, Bifidiobacterium breve, Bifidobacterium
infantis, Bifidiobacterium animalis subsp. lactis, Bifidobacterium
longum, or Streptococcus thermophilus, or a mixture thereof.
[0070] In one embodiment the composition further comprises a
probiotic microorganism selected from the group consisting of
Lactobacillus rhamnosus HN001 (AGAL NM 97/09514), Bifidiobacterium
animalis subsp. lactis HN019 (AGAL NM 97/09513), Lactobacillus
acidophilus HN017 (AGAL NM 97/09515), Lactobacillus rhamnosus HN067
(AGAL NM 97/01925) (all of which are described in U.S. Pat. No.
6,379,663), Lactobacillus johnsonii NCC533 (La1) (CNCM I-1225),
Lactobacillus rhamnosus GG (ATCC 53103), Lactobacillus casei
Shirota (FERM-P4751), Lactobacillus acidophilus NCFM (ATCC 700396),
Lactobacillus plantarum 299v (DSMZ 9843), Lactobacillus casei
DN114001 (CNCM I-1518), Lactobacillus salivarius UCC4331 (NCIMB
40829), Bifidiobacteriun animalis subsp. lactis BB12 (ATCC 27536
and DSMZ 10140), or Bifidobacterium infantis 35624 (NCIMB 41003),
or a mixture thereof. Preferred unwanted organisms or probiotic
microorganisms include Lactobacillus rhamnosus HN001,
Bifidiobacterium animalis subsp. lactis HN019, Lactobacillus
acidophilus HN017, or Lactobacillus rhamnosus HN067, or a mixture
thereof.
[0071] In one embodiment the probiotic microorganism is an
inactivated probiotic microorganism. An inactivated probiotic
microorganism may be non-viable, non-viable but still metabolically
active, or dead.
[0072] In another aspect the present invention relates to a
composition or product treated or produced according to a method of
the invention.
[0073] In another aspect the present invention relates to use of a
composition or product treated or produced according to a method of
the invention in the manufacture of a food, drink, food additive,
drink additive, dietary supplement, nutritional product, medical
food, nutraceutical, medicament or pharmaceutical.
[0074] In one embodiment the use is for stimulating skeletal
growth, inhibiting bone resorption, stimulating chondrocyte
proliferation, stimulating osteoblast proliferation, inhibiting
osteoclast development, or treating or preventing a skeletal, joint
or cartilage disorder.
[0075] In one embodiment the use is for inhibiting tumour formation
in a subject, inducing apoptosis in a subject, inducing apoptosis
of tumour cells in a subject, inhibiting angiogenesis in a subject,
inhibiting tumour angiogenesis in a subject, maintaining or
improving one or both of the white blood cell count and red blood
cell count in a subject, stimulating the immune system in a
subject, increasing the production of Th1 and Th2 cytokines within
a tumor in a subject, increasing the production of Th1 and Th2
cytokines within the intestine of a subject, increasing the level
of Th1 and Th2 cytokines in the systemic circulation of a subject,
increasing an anti-tumour immune response in a subject, increasing
the responsiveness of a subject to a cancer therapy, or increasing
the responsiveness of a tumour in a subject to a cancer
therapy.
[0076] In one embodiment the use is for treating or preventing
iron-deficiency anaemia or the iron-deficiency associated with
pregnancy, for increasing haemoglobin count in anaemia sufferers or
for gut renewal therapies for malabsorption.
[0077] In another aspect the present invention relates to a method
of stimulating skeletal growth, inhibiting bone resorption,
stimulating chondrocyte proliferation, stimulating osteoblast
proliferation, inhibiting osteoclast development, or treating or
preventing a skeletal, joint or cartilage disorder, the method
comprising administration of a composition or product of the
invention to a subject in need thereof.
[0078] In another aspect the present invention relates to a method
of inhibiting tumour formation in a subject, inducing apoptosis in
a subject, inducing apoptosis of tumour cells in a subject,
inhibiting angiogenesis in a subject, inhibiting tumour
angiogenesis in a subject, maintaining or improving one or both of
the white blood cell count and red blood cell count in a subject,
stimulating the immune system in a subject, increasing the
production of Th1 and Th2 cytokines within a tumor in a subject,
increasing the production of Th1 and Th2 cytokines within the
intestine of a subject, increasing the level of Th1 and Th2
cytokines in the systemic circulation of a subject, increasing an
anti-tumour immune response in a subject, increasing the
responsiveness of a subject to a cancer therapy, or increasing the
responsiveness of a tumour in a subject to a cancer therapy, the
method comprising administration of a composition or product of the
invention to a subject in need thereof.
[0079] In another aspect the present invention relates to a method
of treating or preventing iron-deficiency anaemia or the
iron-deficiency associated with pregnancy, for increasing
haemoglobin count in anaemia sufferers or for gut renewal therapies
for malabsorption, the method comprising administration of a
composition or product of the invention to a subject in need
thereof.
[0080] Other aspects relate to a pressure-treated composition
comprising about 0.1 mg/ml to 1000 mg/ml of metal ion lactoferrin
or a metal ion functional variant or fragment thereof and less than
about 50,000 cfu/ml of microorganisms; a pressure-treated jelly
comprising about 0.1 mg/ml to 1000 mg/ml of metal ion lactoferrin
or a metal ion functional variant or fragment thereof and less than
about 50,000 cfu/ml of microorganisms; a pressure-treated yoghurt
comprising about 0.1 mg/ml to 1000 mg/ml of metal ion lactoferrin
or a metal ion functional variant or fragment thereof and less than
about 50,000 cfu/ml of microorganisms; a pressure-treated beverage
comprising about 0.1 mg/ml to 1000 mg/ml of metal ion lactoferrin
or a metal ion functional variant or fragment thereof and less than
about 50,000 cfu/ml of microorganisms; a pressure-treated
composition comprising about 1 mg/ml to 1000 mg/ml of metal ion
lactoferrin or a metal ion functional variant or fragment thereof
and less than about 50,000 cfu/ml of microorganisms; a
pressure-treated jelly comprising about 1 mg/ml to 1000 mg/ml of
metal ion lactoferrin or a metal ion functional variant or fragment
thereof and less than about 50,000 cfu/ml of microorganisms; a
pressure-treated yoghurt comprising about 1 mg/ml to 1000 mg/ml of
metal ion lactoferrin or a metal ion functional variant or fragment
thereof and less than about 50,000 cfu/ml of microorganisms; or a
pressure-treated beverage comprising about 1 mg/ml to 1000 mg/ml of
metal ion lactoferrin or a metal ion functional variant or fragment
thereof and less than about 50,000 cfu/ml of microorganisms.
[0081] The entire disclosures of all applications, patents and
publications, cited above and below, if any, are hereby
incorporated by reference.
[0082] It is intended that reference to a range of numbers
disclosed herein (for example, 1 to 10) also incorporates reference
to all rational numbers within that range (for example, 1, 1.1, 2,
3, 3.9, 4, 5, 6, 6.5, 7, 8, 9 and 10) and also any range of
rational numbers within that range (for example, 2 to 8, 1.5 to 5.5
and 3.1 to 4.7) and, therefore, all sub-ranges of all ranges
expressly disclosed herein are hereby expressly disclosed. These
are only examples of what is specifically intended and all possible
combinations of numerical values between the lowest value and the
highest value enumerated are to be considered to be expressly
stated in this application in a similar manner.
[0083] The invention may also be said broadly to consist in the
parts, elements and features referred to or indicated in the
specification of the application, individually or collectively, in
any or all combinations of two or more of said parts, elements or
features, and where specific integers are mentioned herein that
have known equivalents in the art to which the invention relates,
such known equivalents are deemed to be incorporated herein as if
individually set forth.
BRIEF DESCRIPTION OF THE DRAWINGS
[0084] FIG. 1 is a flow chart summarizing the manufacture of
control iron lactoferrin (FeLF) yoghurt, heat treated FeLF yoghurt
and pressure treated (HPP) FeLF yoghurt.
[0085] FIG. 2 is a flow chart summarizing the manufacture of a
standard (heat treated) acidic drink, an unprocessed (control)
acidic drink and a pressure treated acidic drink.
[0086] FIG. 3 is a flow chart summarizing the manufacture of
control (untreated) FeLF jelly and pressure treated FeLF jelly.
[0087] FIG. 4 is a flow chart summarizing the manufacture of a
control (untreated) FeLF carbonated beverage and pressure treated
LF carbonated beverage.
DETAILED DESCRIPTION OF THE INVENTION
1. Definitions
[0088] The term "comprising" as used in this specification and
claims means "consisting at least in part of". When interpreting
statements in this specification and claims that include that term,
the features, prefaced by that term in each statement, all need to
be present but other features can also be present. Related terms
such as "comprise" and "comprised" are to be interpreted in the
same manner.
[0089] The terms "enhance the immune system" and "stimulate the
immune system" (and different tenses of these terms) refer to the
ability of metal ion lactoferrin to stimulate the generation of
antigen-specific cytolytic activity (the activity of immune cells,
particularly cytotoxic T-lymphocytes) and/or NK cell activity,
improve the cellular immune response to antigens (through the
activity of at least cytotoxic T-lymphocytes), improve immune
protection (by at least restoring the activity of cytotoxic
T-lymphocytes and/or NK cells and enhancing cytokine production),
restore immune protection (by at least restoring or stimulating the
activity of cytotoxic T-lymphocytes and/or NK cell activity and
enhancing cytokine production) or generate pro-inflammatory and
immunoregulatory mediators (Th1 and Th2 cytokines).
[0090] The term "functional fragment" is intended to mean a
naturally occurring or non-naturally occurring portion of a
lactoferrin polypeptide that has one or two metal ion binding
pockets and is able to bind a metal ion in at least one pocket.
Useful lactoferrin fragments include truncated lactoferrin
polypeptides, metal ion binding hydrolysates of lactoferrin,
fragments that comprise the N-lobe binding pocket (including but
not limited to N-lobe sequences), fragments that comprise the
C-lobe binding pocket (including but not limited to C-lobe
sequences), and metal ion binding fragments generated (by
artificial or natural processes) and identified by known techniques
as discussed below.
[0091] The term "functional variant" is intended to mean a variant
of a lactoferrin polypeptide that is able to bind two metal ions
per lactoferrin molecule.
[0092] The term "glycosylated" when used in relation to a
lactoferrin polypeptide, functional variant or fragment is intended
to mean that the lactoferrin is fully or partially glycosylated
with naturally occurring or non-naturally occurring human or bovine
glycosyl groups. Glycosylated and aglycosyl forms of lactoferrin
are known (see Pierce, et al. (1991); Metz-Boutigue, et al. (1984);
van Veen, et al. (2004)).
[0093] The term "increasing the responsiveness of a subject" is
intended to mean that a subject exhibits a greater reduction in the
rate of tumour growth, in tumour size, or in clinical symptoms of
disease than a subject who is not subjected to a method of the
invention.
[0094] The term "increasing the sensitivity of a tumour" is
intended to mean that a tumour exhibits a greater reduction in the
rate of tumour growth, in tumour size, or is eradicated whereas a
tumour that is not subjected to a method of the invention will not
exhibit these effects.
[0095] The term "inhibiting tumour formation" is intended to mean
that tumours do not form, or that tumours form but do not establish
or grow, or that tumours form but remain small, benign and do not
become cancerous or metastasize, or that tumours grow more slowly.
Tumour formation may be monitored through CT scans and tumor
markers where available.
[0096] The term "inhibiting tumour growth" is intended to mean that
tumours do not form in a subject treated according to the
invention, or that one or more tumours that may be present in a
subject treated according to the invention do not grow in size or
become cancerous or metastasize, or that one or more tumours
present in a subject treated according to the invention reduce in
size (preferably by at least about 20, 30, 40, 50, 60, 70, 80, 90
or 100% by volume) or that one or more tumours present in a subject
treated according to the invention are eradicated. Tumour size may
be monitored through CT scans and tumor markers where
available.
[0097] The terms "iron-lactoferrin" and "iron-saturated
lactoferrin" (otherwise referred to as "FeLF") as used herein are
intended to refer to a population of partially or fully iron
saturated lactoferrin polypeptides or functional variants or
fragments thereof providing a population of iron binding pockets
where at least about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60,
65, 70, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 99.5,
99.9 or 100% of the metal ion binding pockets present in the
population have an iron ion bound.
[0098] The term "hold time" refers to a preferred embodiment where
the treatment time is the time that the pressure is held at the
treatment pressure. For example, a hold time of 1 minute means that
the pressure is held at the treatment pressure for 1 minute. A hold
time of 0 minutes (or "no hold") means that the pressure is raised
to the treatment pressure but not held, and is then returned to
ambient (usually atmospheric) pressure. In this embodiment,
preferred treatment times include 0 (no hold), 0.5, 1, 1.5, 2, 2.5,
3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5 and 10
minutes. It should be understood that although the pressure is not
intentionally held at the treatment pressure, there may be a very
short holding period (possibly several milliseconds) due to the
nature of the equipment used. This very short holding period is
unlikely to substantially affect the working of the method.
[0099] The term "keeping quality" as used herein is intended to
mean the ability of a composition to resist the growth of unwanted
microorganisms over time. Compositions that are not treated with
heat or an acceptable alternative such as provided herein are
unlikely to have a commercially acceptable keeping quality.
Reference to maintaining keeping quality is intended to mean that a
method of the invention is at least as effective as a heat treated
control at extending the shelf-life of a pressure treated
composition. Reference to increasing or increased, or improving or
improved keeping quality is intended to mean that the ability of
the composition to resist the growth of unwanted microorganisms
over time is enhanced compared to an untreated composition.
Enhanced keeping quality preferably leads to, for example,
properties such as an extended shelf-life and an enhanced ability
to withstand temperature variation. Temperature variation (such as
removal from cold store) can induce growth of any residual
bacteria.
[0100] Preferably the keeping quality is assessed with reference to
the Aerobic Plate Count (APC). APC is a bacterial enumeration
procedure used to estimate bacterial density in a sample and is
otherwise known as Total Plate Count, Standard Plate Count or Total
Viable Count. Samples are collected, blended, diluted, and plated
in an agar medium suitable for detecting the bacteria studied (for
example, food or dairy contaminants such as Escherichia coli,
Staphylococcus aureus, Salinonellae, Shigellae, coliforms, yeasts
and molds, mesophilic spores, thermophilic spores). The APC result
is the number of colony forming units in one millilitre (cfu/ml) of
sample that is plated and incubated for 72 hrs at 32.degree. C. An
APC of 50,000 cfu/ml or less is highly preferred for fresh dairy
products that are not intended to contain viable cultures. Products
having an APC of 50,000 cfu/ml or more are unlikely to have an
acceptable keeping quality, unless the organism present is one that
is particularly suited to the product--examples of this latter
class of products include yoghurts or fermented products where a
viable culture is desirable.
[0101] Accordingly, in one embodiment, a preferred method is one
wherein the aerobic plate count (APC) after treatment is less than
or equal to about 100,000, 75,000, 50,000, 25,000, 10,000, 5,000,
1,000, 100, or 10 colony forming units per millilitre (cfu/ml).
Preferably the APC is less than about 50,000 cfu/ml.
[0102] The term "lactoferrin" refers to any non-glycosylated or
glycosylated wild-type lactoferrin amino acid sequence including
homologous lactoferrin sequences such as those described below. A
lactoferrin polypeptide has two metal ion binding pockets and so
can bind metal ions in a stoichiometric ratio of 2 metal ions per
lactoferrin molecule. One metal ion binding pocket is present in
the N-terminal lobe (N-lobe) of lactoferrin and the other pocket is
present in the C-terminal lobe (C-lobe) (Moore et, al, 1997).
Verified sequences of bovine and human lactotransferrins
(lactoferrin precursors), lactoferrins and peptides therein can be
found in Swiss-Prot
(http://au.expasy.org/cgi-bin/sprot-search-ful). Indicative
lactoferrin polypeptides include the bovine lactotransferrin
precursor accession number P24627, bovine lactoferrin, the human
lactotransferrin precursor accession number P02788 and human
lactoferrin.
[0103] The terms "metal ion saturation" and "metal ion binding" are
intended to refer to binding of a metal ion in an iron binding
pocket of a lactoferrin polypeptide or a functional variant or
fragment thereof.
[0104] The terms "metal ion lactoferrin" and "metal ion saturated
lactoferrin" are intended to refer to a population of partially or
fully metal ion saturated lactoferrin polypeptides that provide a
population of metal ion binding pockets where at least about 5%,
preferably at least about 25% of the metal ion binding pockets
present in the population have a metal ion bound. It should also be
understood that the population may contain polypeptides of
different species; for example, some molecules binding no ion and
others each binding one or two ions. In cases where different metal
ions are used, some molecules may bind an iron ion and others a
different ion.
[0105] Equally, the terms "metal ion lactoferrin fragment" and
"metal ion saturated lactoferrin fragment" are intended to refer to
a population of partially or fully metal ion saturated lactoferrin
polypeptide fragments that provide a population of metal ion
binding pockets where at least about 5%, preferably at least about
25% of the metal ion binding pockets present in the population have
a metal ion bound.
[0106] The present invention may employ a mixture of partially or
fully metal ion saturated lactoferrin polypeptides and lactoferrin
fragments. In such an embodiment, the population of metal ion
binding pockets is made up of two pockets for every lactoferrin
polypeptide and one or two pockets for every lactoferrin fragment,
depending on the nature of the fragments.
[0107] The degree of saturation may be determined by
spectrophotometric analysis (Brock & Arzabe, 1976; Bates et al,
1967; Bates et al, 1973). It should be understood that there may be
metal ion exchange between lactoferrin polypeptides. Metal ion
saturated lactoferrin may be prepared by any useful method. In one
embodiment, iron saturated lactoferrin may be prepared by the
method of Law, et al (1977). In another embodiment, iron saturated
lactoferrin may be prepared by the method of Kawakami et al (1993).
Metal ion saturated lactoferrin may be prepared by binding metal
ions to the metal ion binding sites in lactoferrin, including the
metal ion binding pockets such as the Fe binding pockets and other
non-specific binding sites on the lactoferrin molecule or
lactoferrin fragment. In a preferred embodiment, metal ions are
only bound by metal ion binding pockets and minimal or no
non-specific binding occurs, unless it is during metal ion exchange
between binding pockets.
[0108] In one embodiment at least about 5, 10, 15, 20, 25, 30, 35,
40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96,
97, 98, 99, 99.5, 99.9 or 100% of the metal ion binding pockets
present in the population of lactoferrin molecules have a metal ion
bound and useful ranges may be selected between any of the
foregoing values (for example, about 5 to about 100%, about 10 to
about 100%, about 15 to about 100%, about 20 to about 100%, about
25 to about 100%, about 30 to about 100%, about 35 to about 100%,
about 40 to about 100%, about 45 to about 100%, about 50 to about
100%, about 55 to about 100%, about 60 to about 100%, about 65 to
about 100%, about 70 to about 100%, about 75 to about 100%, about
80 to about 100%, about 85 to about 100%, about 90 to about 100%,
about 95 to about 100% and about 99 to about 100%). In one
embodiment the metal ion saturated lactoferrin is super-saturated
lactoferrin.
[0109] The term "oral administration" includes oral, buccal,
enteral and intra-gastric administration.
[0110] The terms "oriented metal ion binding" and "oriented iron
binding" refer to the stereospecific nature of metal ion binding by
lactoferrin or functional fragments when the metal ion (such as
iron) is coordinated correctly in the iron-binding pocket, is not
partially bound within the pocket, nor non-specifically surface
bound. There may be conditions where the metal ion is bound in the
iron binding pocket but without full coordination, and this may
show in the spectral properties. Oriented iron binding is
illustrated by a UV-VIS spectrum having a single iron-lactoferrin
maxima at 465 nm. In one embodiment oriented metal ion binding is
preferred. In another embodiment non-oriented metal ion binding is
acceptable.
[0111] The term "parenteral administration" includes but is not
limited to topical (including administration to any dermal,
epidermal or mucosal surface), subcutaneous, intravenous,
intraperitoneal, intramuscular and intratumoral (including any
direct administration to a tumour) administration.
[0112] The term "pharmaceutically acceptable carrier" is intended
to refer to a carrier including but not limited to an excipient,
diluent or auxiliary that can be administered to a subject as a
component of a composition of the invention. Preferred carriers do
not reduce the activity of the composition and are not toxic when
administered in doses sufficient to deliver an effective amount of
a lactoferrin polypeptide or functional variant or fragment
thereof. The formulations can be administered orally, nasally or
parenterally.
[0113] The term "pressure treatment" refers to ultra high-pressure
(UHP) treatment. Such a treatment is generally accepted as using a
pressure of at least 100 MPa. This is also known as "high pressure"
treatment, "high hydrostatic pressure" (HHP) or "high pressure
processing" (HPP). Products that have been "pressure treated" are
those that have been subjected to UHP treatment; namely, pressure
treatment at a pressure of at least 100 MPa, preferably pressure
treatment at a pressure of at least about 350, 400, 450, 500, 600,
700 or 800 MPa (or otherwise within this range as described
above).
[0114] Reference to retaining a "desired level" of metal ion
saturation or metal ion binding is intended to mean that the level
of metal ion saturation of the lactoferrin polypeptides or
functional variants or fragments thereof present in a composition
is not substantially reduced when the composition is treated
according to a method described herein. Preferably the level of
metal ion saturation following treatment is at least about 40% of
the metal ion saturation level before treatment. That is,
preferably at least about 40, 45, 50, 55, 60, 65, 70, 75, 80, 85,
90, 95, 99 or 100% of the metal ions bound by lactoferrin
polypeptides or variants or fragments thereof are retained. For
example, for a composition comprising 100% metal ion saturated
lactoferrin, preferably the level of metal ion saturation is at
least about 40% following treatment, preferably at least about 40,
45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 99 or 100%, and
preferred ranges may be selected between any of these values (for
example, from about 40 to 100%, about 45 to 100%, about 50 to 100%,
about 55 to 100%, about 60 to 100%, about 65 to 100%, about 70 to
100%, about 75 to 100%, about 80 to 100%, about 85 to 100%, about
90 to 100%, about 95 to 100%, and about 99 to 100%).
[0115] The term "subject" is intended to refer to an animal,
preferably a mammal, more preferably a mammalian companion animal
or a human. Preferred companion animals include cats, dogs and
horses.
[0116] The term "super-saturated lactoferrin" refers to a
population of lactoferrin polypeptides or functional fragments
providing a population of metal ion binding pockets where
sufficient metal ions are available to fill 100% of the binding
pockets and additional metal ions are present and bound by
non-specific binding sites on the lactoferrin polypeptide or
lactoferrin fragment. In other words, a stoichiometric excess of
metal ions is provided. Preferably no free metal ions are present
in a composition of the invention comprising super-saturated
lactoferrin, although metal ion exchange between binding pockets,
between non-specific binding sites and between binding pockets and
non-specific binding sites may occur. Preferably super-saturated
lactoferrin does not form insoluble aggregates. In one embodiment
the super-saturated lactoferrin is at least about 105, 110, 115,
120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180,
185, 190, 195 or 200% metal ion saturated, preferably iron
saturated.
[0117] The term "treat" and its derivatives should be interpreted
in their broadest possible context. The term should not be taken to
imply that a subject is treated until total recovery. Accordingly,
"treat" broadly includes amelioration and/or prevention of the
onset of the symptoms or severity of a particular condition. The
term "treat" also broadly includes the maintenance of good health
for sensitive individuals and building stamina for disease
prevention.
[0118] The term "unwanted microorganisms" refers to all
microorganisms that may grow in a composition before pressure
treatment. While such growth is undesirable, the presence of the
microorganisms in states which do not grow, including non-viable,
attenuated or dead microorganisms, may be of no consequence or may
be desirable.
[0119] Reference to preventing the growth of unwanted
microorganisms is intended to mean that the growth of
microorganisms such as bacteria (including probiotic bacteria),
fungi, molds, yeasts and algae is substantially reduced, delayed or
eliminated. The microorganisms need not be responsible for spoilage
or be pathogens. The growth of unwanted microorganisms can be
assessed by visual inspection or by employing standard techniques
that are known in the art, including but not limited to microscopy,
staining, PCR, cell sorting and the like (see Lund, et al., 2000).
Preferably the APC of the composition after pressure treatment is
less than or equal to about 100,000, 75,000, 50,000, 25,000,
10,000, 5,000, 1,000, 100, or 10 cfu/ml, preferably less than about
50,000 cfu/ml
[0120] As indicated above, compositions that are not heat treated
or pressure treated are unlikely to have a commercially acceptable
keeping quality. That is, the keeping quality of untreated
compositions is usually unacceptable because no steps are taken to
prevent the growth of unwanted microorganisms. Where such steps are
taken, the keeping quality will be improved. A post-treatment
aerobic plate count (APC) less than or equal to about 100,000,
75,000, 50,000, 25,000, 10,000, 5,000, 1,000, 100, or 10 colony
forming units per millilitre (cfu/ml), preferably less than about
50,000 cfu/ml, will reduce, delay or eliminate the ability of any
organisms present to have impact on keeping quality. In combination
with low pH or refrigeration (storage at temperatures less than
about 10.degree. C., preferably less than about 4.degree. C.) or
both, their ability to affect keeping quality will be further
reduced, delayed or eliminated.
[0121] In embodiments employing probiotic microorganisms, probiotic
microorganism growth is unwanted but the presence of probiotic
microorganisms is desirable. Accordingly, reference to preventing
the growth of an unwanted microorganism in such embodiments is
intended to mean that the growth of the probiotic microorganism is
substantially reduced, delayed or eliminated. Preferably the
pressure treatment will attenuate the probiotic microorganism, or
more preferably, kill the probiotic microorganism, while retaining
at least a desired level of probiotic activity.
[0122] In one embodiment the probiotic microorganism is an
inactivated probiotic microorganism yet retains at least a desired
level of probiotic activity. An inactivated probiotic microorganism
may be non-viable, non-viable but still metabolically active, or
dead, yet in all cases retain at least a desired level of probiotic
activity.
[0123] In another embodiment the probiotic microorganism is an
inactivated probiotic microorganism before pressure treatment.
Pressure treatment in this embodiment prevents the growth of other
unwanted microorganisms while retaining probiotic activity of the
probiotic factors present.
[0124] The term "variant" refers to a naturally occurring (an
allelic variant, for example) or non-naturally occurring (an
artificially generated mutant, for example) lactoferrin polypeptide
or lactoferrin fragment that varies from the predominant wild-type
amino acid sequence of a lactoferrin polypeptide of a given species
(such as those listed below) or fragment thereof by the addition,
deletion or substitution of one or more amino acids.
[0125] Generally, polypeptide sequence variant possesses
qualitative biological activity in common when assayed according to
the examples below. Further, these polypeptide sequence variants
may share at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%,
90%, 95%, 96%, 97%, 98% or 99% sequence identity. Also included
within the meaning of the term "variant" are homologues of
lactoferrin polypeptides. A homologue is typically a polypeptide
from a different species but sharing substantially the same
biological function or activity as the corresponding polypeptide
disclosed herein.
[0126] Preferred variant polypeptides preferably have at least
about 70, 75, 80, 85, 90, 95 or 99% identity, preferably at least
about 90, 95 or 99% identity to bovine or human lactoferrin.
Variant fragments preferably have at least about 70, 75, 80, 85,
90, 95 or 99% identity, preferably at least about 90, 95 or 99%
identity to a fragment described herein, including but not limited
to fragments of human or bovine lactoferrin. Identity can be
determined by comparing a candidate amino acid sequence to a
sequence described herein, such as a lactoferrin polypeptide or
fragment thereof using the BLAST suite of programs (version 2.2.12;
28 Aug. 2005) (Tatusova, et al. (1999); McGinnis, et al. (2004))
that is publicly available from NCBI
(ftp://ftp.ncbi.nih.gov/blast/).
[0127] Conservative substitutions of one or several amino acids of
a lactoferrin polypeptide sequence without significantly altering
its biological activity are also useful. A skilled artisan will be
aware of methods for making phenotypically silent amino acid
substitutions (see for example Bowie et al., (1990)).
2. Lactoferrin Polypeptides
[0128] In addition to the useful lactoferrin polypeptides and
fragments listed above, examples of lactoferrin amino acid and mRNA
sequences that have been reported and are useful in methods of the
invention include but are not limited to the amino acid (Accession
Number NP.sub.--002334) and mRNA (Accession Number NM.sub.--002343)
sequences of human lactoferrin; the amino acid (Accession Numbers
NP.sub.--851341 and CAA38572) and mRNA (Accession Numbers X54801
and NM.sub.--180998) sequences of bovine lactoferrin; the amino
acid (Accession Numbers JC2323, CAA55517 and AAA97958) and mRNA
(Accession Number U53857) sequences of goat lactoferrin; the amino
acid (Accession Number CAA09407) and mRNA (Accession Number
AJ010930) sequences of horse lactoferrin; the amino acid (Accession
Numbers NP.sub.--999527, AAL40161 and AAP70487) and mRNA (Accession
Number NM.sub.--214362) sequences of pig lactoferrin; the amino
acid (Accession Number NP.sub.--032548) and mRNA (Accession Number
NM.sub.--008522) sequences of mouse lactoferrin; the amino acid
(Accession Number CAA06441) and mRNA (Accession Number AJ005203)
sequences of water buffalo lactoferrin; and the amino acid
(Accession Number CAB53387) and mRNA (Accession Number AJ131674)
sequences of camel lactoferrin. These sequences may be used
according to the invention in wild type or variant form.
Polypeptides encoded by these sequences may be isolated from a
natural source, produced as recombinant proteins or produced by
organic synthesis, using known techniques.
[0129] Methods for generating useful polypeptides and variants are
known in the art and discussed below. Useful recombinant
lactoferrin polypeptides and fragments and methods of producing
them are reported in US patent specifications U.S. Pat. No.
5,571,691, U.S. Pat. No. 5,571,697, U.S. Pat. No. 5,571,896, U.S.
Pat. No. 5,766,939, U.S. Pat. No. 5,849,881, U.S. Pat. No.
5,849,885, U.S. Pat. No. 5,861,491, U.S. Pat. No. 5,919,913, U.S.
Pat. No. 5,955,316, U.S. Pat. No. 6,066,469, U.S. Pat. No.
6,080,599, U.S. Pat. No. 6,100,054, U.S. Pat. No. 6,111,081, U.S.
Pat. No. 6,228,614, U.S. Pat. No. 6,277,817, U.S. Pat. No.
6,333,311, U.S. Pat. No. 6,455,687, U.S. Pat. No. 6,569,831, U.S.
Pat. No. 6,635,447, US 2005-0064546 and US 2005-0114911.
[0130] Useful variants also include bovine lactoferrin variants
bLf-a and bLf-b (Tsuji, et al. (1989); Yoshida, et al. (1991)).
Further useful variants include glycosylated and aglycosyl forms of
lactoferrin (Pierce, et al. (1991); Metz-Boutigue, et al. (1984);
van Veen, et al. (2004)) and glycosylation mutants (having variant
points of glycosylation or variant glycosyl side chains).
[0131] Useful fragments include the N-lobe and C-lobe fragments
(Baker, et al., 2002) and any other lactoferrin polypeptides that
retain a lactoferrin binding pocket, such as truncated lactoferrin
polypeptides.
[0132] Useful truncated lactoferrin polypeptides include
polypeptides truncated by about 1 to about 300 amino acids,
preferably about 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60,
65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135,
140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200,
205, 210, 215, 220, 225, 230, 235, 240, 245, 250, 255, 260, 265,
270, 275, 280, 285, 290, 295 or 300 amino acids or more, and
including polypeptides truncated at the N-terminus, at the
C-terminus or at both the N-terminus and C-terminus, provided that
the truncated polypeptide retains at least one of the N-lobe or the
C-lobe metal ion binding pockets. It is reported that residues Asp
60, Tyr 92, Tyr 192, H is 253 of bovine lactoferrin are the amino
acid metal ion ligands in the N-lobe. It is reported that residues
Asp 395, Tyr 433, Tyr 526, H is 595 of bovine lactoferrin are the
amino acid metal ion ligands in the C-lobe (Karthikeyan, et al.,
1999). Any fragment that contains these residues may be able to
form a metal ion binding pocket.
[0133] Candidate variants or fragments of lactoferrin for use
according to the present invention may be generated by techniques
including but not limited to techniques for mutating wild type
proteins (see Sambrook, et al. (1989) and elsewhere for a
discussion of such techniques) such as but not limited to
site-directed mutagenesis of wild type lactoferrin and expression
of the resulting polynucleotides; techniques for generating
expressible polynucleotide fragments such as PCR using a pool of
random or selected primers; techniques for full or partial
proteolysis or hydrolysis of wild type or variant lactoferrin
polypeptides; and techniques for chemical synthesis of
polypeptides. Variants or fragments of lactoferrin may be prepared
by expression as recombinant molecules from lactoferrin DNA or RNA,
or variants or fragments thereof. Nucleic acid sequences encoding
variants or fragments of lactoferrin may be inserted into a
suitable vector for expression in a cell, including eukaryotic
cells such as but not limited to Aspergillus or bacterial cells
such as but not limited to E. coli. Lactoferrin variants or
fragments may be prepared using known PCR techniques including but
not limited to error-prone PCR and DNA shuffling. Error-prone PCR
is a process for performing PCR under conditions where the copying
fidelity of the DNA polymerase is low, such that a high rate of
point mutations is obtained along the entire length of the PCR
product (Leung, et al. (1989); Cadwell, et al. (1992)). DNA
shuffling refers to forced homologous recombination between DNA
molecules of different but highly related DNA sequence in vitro,
caused by random fragmentation of the DNA molecule based on
sequence homology, followed by fixation of the crossover by primer
extension in a PCR reaction (Stemmer (1994)). Suitable lactoferrin
nucleic acid sequences for use in such methods include those listed
above or may be generated by known methods including, for example,
reverse transcription-PCR (RT-PCR) of tissue RNA isolates. Suitable
primers for RT-PCR may be designed with reference to the mRNA
sequences listed above. Commercial kits are available for RT-PCR
(for example, Cells-to-cDNA.TM. kits from Ambion, USA).
[0134] Variants or fragments of lactoferrin may also be generated
by known synthetic methods (see Kimmerlin, et al., 2005, for
example). Alternatively, lactoferrin polypeptides or functional
variants or fragments thereof can be produced by well established
synthetic Fmoc chemistry as described for human kaliocin-1 and the
lactoferricin derived peptide by Viejo-Diaz et al., (2003); and
bovine lactoferricin peptide as described by Nguyen et al., (2005);
and lactoferrampin and shorter fragments as described by van der
Kraan et al., (2004).
[0135] Metal ion binding variants or fragments of lactoferrin may
be obtained by known techniques for isolating metal binding
polypeptides including but not limited to metal affinity
chromatography, for example. Candidate variants or fragments of
lactoferrin may be contacted with free or immobilised metal ions,
such as Fe.sup.3+ and purified in a suitable fashion. For example,
candidate variants or fragments may be contacted at neutral pH with
a metal ion immobilised by chelation to a chromatography matrix
comprising iminodiacetic acid or tris(carboxymethyl)ethylenediamine
ligands. Bound variants or fragments may be eluted from the
supporting matrix and collected by reducing the pH and ionic
strength of the buffer employed. Metal bound variants or fragments
may be prepared according to the methods described herein.
[0136] Functional variants, fragments and hydrolysates of
lactoferrin may be obtained by selecting variants, fragments and
hydrolysates of lactoferrin and assessing their efficacy assays
chosen to test the desired efficacy.
[0137] In one embodiment the lactoferrin is any mammalian
lactoferrin including but not limited to sheep, goat, pig, mouse,
water buffalo, camel, yak, horse, donkey, llama, bovine or human
lactoferrin. Preferably the lactoferrin is bovine lactoferrin.
[0138] In another embodiment the lactoferrin is any recombinant
mammalian lactoferrin including but not limited to recombinant
sheep, goat, pig, mouse, water buffalo, camel, yak, horse, donkey,
llama, bovine or human lactoferrin. Preferably the lactoferrin is
recombinant bovine lactoferrin. Recombinant lactoferrin may be
produced by expression in cell free expression systems or in
transgenic animals, plants, fungi or bacteria, or other useful
species. Alternatively, lactoferrin may be produced using known
organic synthetic methods.
[0139] In yet another embodiment the lactoferrin is isolated from
milk, preferably sheep, goat, pig, mouse, water buffalo, camel,
yak, horse, donkey, llama, bovine or human milk. Preferably the
lactoferrin is isolated from milk by cation exchange chromatography
followed by ultrafiltration and diafiltration.
3. Isolation of Lactoferrin from Milk
[0140] The following is an exemplary procedure for isolating
lactoferrin from bovine milk.
[0141] Fresh skim milk (7 L, pH 6.5) is passed through a 300 ml
column of S Sepharose Fast Flow equilibrated in milli Q water, at a
flow rate of 5 ml/min and at 4.degree. C. Unbound protein is washed
through with 2.5 bed volumes of water and bound protein eluted
stepwise with approximately 2.5 bed volumes each of 0.1 M, 0.35 M,
and 1.0 M sodium chloride. Lactoferrin eluting as a discreet pink
band in 1 M sodium chloride is collected as a single fraction and
dialysed against milli Q water followed by freeze-drying. The
freeze-dried powder is dissolved in 25 mM sodium phosphate buffer,
pH 6.5 and subjected to chromatography on S Sepharose Fast Flow
with a sodium chloride gradient to 1 M in the above buffer and at a
flow rate of 3 ml/min. Fractions containing lactoferrin of
sufficient purity as determined by gel electrophoresis and reversed
phase HPLC are combined, dialyzed and freeze-dried. Final
purification of lactoferrin is accomplished by gel filtration on
Sephacryl 300 in 80 mM dipotassium phosphate, pH 8.6, containing
0.15 M potassium chloride. Selected fractions are combined,
dialyzed against milli Q water, and freeze-dried. The purity of
this preparation is greater than 95% as indicated by HPLC analysis
and by the spectral ratio values (280 nm/465 nm) of 19 or less for
the iron saturated form of lactoferrin.
4. Metal Ion Saturation or Depletion of Lactoferrin
[0142] Iron saturation is achieved by addition of a 2:1 molar
excess of 5 mM ferric nitrilotriacetate (Foley and Bates (1987)) to
a 1% solution of the purified lactoferrin in 50 mM Tris, pH 7.8
containing 10 mM sodium bicarbonate. Excess ferric
nitrilotriacetate is removed by dialysis against 100 volumes of
milli Q water (twice renewed) for a total of 20 hours at
4.degree.C. The iron-loaded (holo-) lactoferrin may then be
freeze-dried. Varying degrees of iron saturation may be obtained by
providing less of the metal ion donor.
[0143] Iron-depleted (apo-) lactoferrin is prepared by dialysis of
a 1% solution of the highly purified lactoferrin sample in water
against 30 volumes of 0.1 M citric acid, pH 2.3, containing 500
mg/L disodium EDTA, for 30 h at 4.degree. C. (Masson and Heremans
(1966)). Citrate and EDTA are then removed by dialysis against 30
volumes of milli Q water (once renewed) and the resulting
colourless solution may be freeze-dried.
[0144] A lactoferrin polypeptide can contain an iron ion (as in a
naturally occurring lactoferrin polypeptide) or a non-iron metal
ion (for example, a copper ion, a chromium ion, a cobalt ion, a
manganese ion, a zinc ion, or a magnesium ion). For instance,
lactoferrin isolated from bovine milk can be depleted of iron and
then loaded with another type of metal ion. For example, copper
loading can be achieved according to the same method for iron
loading described above. For loading lactoferrin with other metal
ions, the method of Ainscough, et al. (1979) can be used.
[0145] In one embodiment the metal ion is an ion selected from
aluminium, calcium, copper, chromium, cobalt, gold, iron,
manganese, magnesium, platinum, ruthenium, selenium, or zinc ions,
or a mixture thereof. Preferably the metal ion is an iron ion.
[0146] In a preparation of a composition for use according to the
invention, a lactoferrin polypeptide or metal ion binding
lactoferrin fragment can be of a single species, or of different
species. For instance, the polypeptides or fragments can each
contain a different number of metal ions or a different species of
metal ions; or the lengths of the polypeptides can vary, for
example, some are full-length polypeptides and some are fragments,
and the fragments can each represent a particular portion of a
full-length polypeptide. Such a preparation can be obtained from a
natural source or by mixing different lactoferrin polypeptide
species. For example, a mixture of lactoferrin polypeptides of
different lengths can be prepared by proteinase digestion (complete
or partial) of full-length lactoferrin polypeptides. The degree of
digestion can be controlled according to methods well known in the
art, for example, by manipulating the amount of proteinase or the
time of incubation, and described below. A full digestion produces
a mixture of various fragments of full-length lactoferrin
polypeptides; a partial digestion produces a mixture of full-length
lactoferrin polypeptides and various fragments.
5. Preparation of Lactoferrin Fragments or Lactoferrin
Hydrolysates
[0147] Hydrolysates containing candidate functional fragments can
be prepared by selecting suitable enzymes with known specificity of
cleavage, such as trypsin or chymotrypsin, and controlling/limiting
proteolysis by pH, temperature, time of incubation and enzyme to
substrate ratio. Refinement of such isolated peptides can be made
using specific endopeptidases. As an example, bovine lactoferricin
can be produced by cleavage of bovine lactoferrin with pepsin at pH
2.0 for 45 min at 37.degree. C. (Facon & Skura, 1996), or at pH
2.5, 37.degree. C. for 4 h using enzyme at 3% (w/w of substrate)
(Tomita et al., 1994). The peptide can then be isolated by reversed
phase HPLC (Tomita et al., 1994) or hydrophobic interaction
chromatography (Tomita e al., 2002).
[0148] Alternatively, lactoferrin peptides can be produced by well
established synthetic Fmoc chemistry as described for human
kaliocin-1 and the lactoferricin derived peptide by Viejo-Diaz et
al., (2003); and bovine lactoferricin peptide as described by
Nguyen et al., (2005); and lactoferrampin and shorter fragments as
described by van der Kraan et al., (2004).
[0149] In general, SDS-PAGE may be used to estimate the degree of
hydrolysis by comparison of the hydrolysate to a molecular weight
standard. Size exclusion chromatography may be used to separate
various species within a hydrolysate and to estimate a molecular
weight distribution profile.
[0150] In a preferred hydrolytic method, bovine lactoferrin was
dissolved to 20 mg/mL in 50 mM Tris pH 8.0, 5 mM CaCl2. Trypsin
(Sigma T8642, TPCK treated, Type XII from bovine pancreas, 11700
U/mg protein) was added at an enzyme substrate ratio of 1:50 w/w
and the mixture incubated at 25.degree. C. for 3 h. The reaction
was stopped by the addition of PMSF to 1 mM final concentration and
extent of digestion monitored by SDS-PAGE. The tryptic digest (4
mL) was applied to gel filtration on Sephacryl S300 (Amersham G E)
(90 cm.times.2.6 cm column) in 50 mM Tris, 0.15 MNaCl pH 8.0.
Suitable fractions containing the major fragments of bovine
lactoferrin (Legrand et al., 1984) were then subjected to cation
exchange chromatography on S Sepharose fast Flow (Amersham G E) (15
cm.times.1.6 cm column) using sodium phosphate buffer pH 6.5 and a
salt gradient to 1 M NaCl. Final separation of the C lobe and N+C
lobes was achieved by further gel filtration on Sephacryl S300 as
above but using 10% v/v acetic acid as eluent (Mata et al., 1994).
The identity of the dialysed (versus milli-Q water) and
freeze-dried fragments was confirmed by SDS-PAGE and Edman
N-terminal sequencing.
[0151] In another method, a tryptic digest as above was separated
by RP-HPLC on a Vydac C18 column as in Superti et al., (2001) and
the high mass fragments corresponding to C-lobe and N-lobe
fragments recovered. Identity was confirmed by MALDI MS.
[0152] In one embodiment hydrolysates useful herein contain one or
more metal ion binding fragments.
6. Pressure Treatment of Metal Ion Saturated Lactoferrin
[0153] The present inventors have shown that it is possible to
pressure treat compositions or products comprising metal ion
lactoferrin under conditions which achieve a commercially useful
keeping quality while maintaining at least a desired level of metal
ion binding.
[0154] The inventors have found that the use of high pressure to
either commercially sterilise or extend the shelf-life of iron
loaded lactoferrin solutions (or products containing iron
lactoferrin) allows retention of the iron specifically bound to the
protein at the treatment pH. Pressure treatment is also generally
far less damaging to the protein structure, and thus the
iron-binding ability of lactoferrin (and therefore capacity to
rebind iron) is relatively unimpaired.
[0155] The Examples below show pressure treatment has much less of
an effect than heat treatment on the iron binding of lactoferrin
that is about 15% and about 100% iron saturated. The conditions
assessed are accepted conditions for commercially useful pressure
and heat treatment; that is, conditions that are accepted and
approved for sterilisation or improving keeping quality.
[0156] Fully or partially iron saturated lactoferrin has been
proposed for treating iron-deficiency anaemia (Bethell & Huang,
2004; Huang et al., 2004), the iron-deficiency associated with
pregnancy (Valenti et al., 2005), and as an agent for increasing
haemoglobin count in anaemia sufferers. As there may be an
association between low dietary iron intake and decreased bone
mineral density in postmenopausal women (Medeiros et al., 2002),
and because lactoferrin has been shown to be a bone anabolic agent
(Cornish et al., 2004), the iron loaded form may also be useful in
treating bone disorders such as osteoporosis. Additionally,
iron-lactoferrin could have application in gut renewal therapies
for malabsorption since the iron loaded forms of both human and
bovine lactoferrin proliferate enterocyte cell lines in culture
(Oguchi et al., 1995).
[0157] Several patent specifications report the use of
iron-saturated lactoferrin for iron-supplementation of beverages,
food products and feeds (see Sakurai et al., 2000; Dugas et al.,
2001; Dousako et al, 1991; Tomita et al., 1992; and Tanaka et al.,
1991) and for use as medicaments (see Nitsche, 1991).
[0158] As well as iron-lactoferrin, use of other metal ions may be
beneficial. For example, chromium-lactoferrin has been reported
(Ainscough et al., 1979) as has the use of chromium-lactoferrin as
a therapeutic agent in alleviating symptoms associated with
diabetes (Chiang & Mao, 2002).
[0159] In one embodiment treatment according to the invention
allows use of high pressure to commercially sterilise (by
eliminating the growth of unwanted microorganisms) metal ion
lactoferrin compositions where the pH of the composition or product
is about pH 3.0 to 8.0, preferably less than or equal to pH
4.6.
[0160] In another embodiment treatment according to the invention
allow use of high pressure to extend the shelf-life (by
substantially reducing or delaying the growth of unwanted
microorganisms) of metal ion lactoferrin compositions where the pH
of the composition or product is about pH 3.0 to 8.0, preferably
greater than or equal to pH 4.6 or at a neutral pH.
[0161] In yet another embodiment, it may be possible to achieve
commercial sterilisation or to improve shelf-life of compositions
or products containing metal ion lactoferrin at a selected pH in
the range of pH 3.0 and 8.0 using a suitable combination of pH and
carbon dioxide. Carbon dioxide in combination with pH may give an
effect equal to that at a lower pH (i.e. effective growth
prevention and retention of a desirable level of metal ion
binding). Commercial sterilisation or improved shelf-life may
thereof be obtained through use of carbon dioxide and pH control
(e.g. in carbonated beverages).
[0162] By way of example, compositions or products processed
according to the invention may be delivered in pressure-treated
products or ingredients; added to products or ingredients which are
not subsequently heat-treated; or added to products or ingredients
which are subsequently pressure-treated.
[0163] Accordingly, the present invention relates to methods of
treating or producing lactoferrin-containing compositions and
products as described above.
[0164] While not intended to be limiting, a pressure treatment
useful in a method according to the invention preferably comprises
the following steps: [0165] (i) placing a composition or product to
be pressure treated into the chamber of a pressure vessel and
sealing the chamber; [0166] (ii) raising the pressure in the
chamber to a predetermined set pressure (the "treatment pressure");
[0167] (iii) holding the chamber at this pressure for a
predetermined time, including less than one minute (the "hold
time"); [0168] (iv) releasing the pressure from the chamber; and
[0169] (v) removing the pressure treated composition or
product.
[0170] Such a protocol may be followed using batch or continuous
processing equipment.
[0171] It should be understood that the pressure treatment may
result in temperature fluctuations in the composition or product
during treatment. As such, references to preferred temperatures
during pressure treatment refer to the temperature of the
composition or product before the pressure is raised.
[0172] One method of identifying a suitable treatment pressure
according to the present invention is to select a composition or
product and subject it to a treatment pressure suitable for
controlling an unwanted microorganism. A suitable treatment
pressure is a pressure of at least about 100 MPa. If necessary, the
composition or product may be inoculated with an unwanted organism
for the purposes of assessment.
[0173] Another method of identifying a suitable treatment pressure
according to the present invention is to select a composition or
product and subject it to a treatment pressure that does not
substantially affect the metal ion binding of lactoferrin or a
functional variant or fragment thereof.
[0174] The growth of any unwanted microorganisms or the metal ion
binding of lactoferrin or both may then be assessed as described
herein. See for example, Lund, et al. (2000) for methods of
assessing microorganism growth.
7. Dairy Products and Ingredients
[0175] A composition or product of the invention may be or may
include a dairy product or ingredient.
[0176] Milk is not only a complete source of nutrients for the
neonate, but also provides a rich source of physiologically
bioactive components and as such has been referred to as `natures`
pharmacy`. In addition to providing complete nutrition, milk also
has vital roles in the development, protection and repair of the
young. Healthy adults usually only require the nutritional benefits
of milk, but in conditions of chronic ailments the bioactivities
derived from milk have more to offer in terms of both prevention
and treatment of illness. Preferably the milk is sheep, goat, pig,
mouse, water buffalo, camel, yak, horse, donkey, llama, bovine or
human milk, and most preferably bovine milk. Dairy protein is known
to be immuno-stimulatory.
[0177] Colostrum is the pre-milk produced immediately after birth
before standard milk production begins. Prime colostrum from cows
is obtained within the first six hours after calving but colostrum
can be collected within the first two days following calving. Prime
colostrum typically contains more than twice the milk solids and
four times the protein found in milk from the same cow obtained
after about forty-eight hours later. The concentrations of
digestive enzymes, immunoglobulins (including IgA, IgD, IgE, IgG
and IgM), cytokines, interferons, growth factors, glycoproteins,
proline-rich peptides and vitamins A, D, E and K are all higher in
prime colostrum compared to standard milk. Colostrum milk protein
concentrates (MPC) and colostrum whey protein concentrates (WPC)
may be prepared as described by Elfstrand et al., 2002.
[0178] Milk and colostrum derivatives and methods of their
manufacture are known in the art. Such derivatives are generally
obtained by a combination centrifugation (for fat removal), casein
precipitation (with acid or enzymes), filtration (to remove
lactose, minerals and water, or optionally to remove proteins),
chromatography (to purify protein components) and include
recombined or fresh whole milk, recombined or fresh skim milk,
reconstituted whole or skim milk powder, skim milk concentrate,
skim milk isolate, whole or skim milk powder, skim milk retentate,
concentrated milk, buttermilk, ultrafiltered milk retentate, milk
protein concentrate (MPC), milk protein isolate (MPI), calcium
depleted milk protein concentrate, calcium depleted milk protein
isolate, low fat milk, low fat milk protein concentrate, low fat
milk protein isolate, colostrum, a colostrum fraction, colostrum
protein concentrate (CPC), colostrum milk protein concentrate,
colostrum milk protein isolate, colostrum whey, colostrum whey
protein concentrate, colostrum whey protein isolate, an
immunoglobulin fraction from colostrum, whey, whey protein
concentrate (WPC), whey protein isolate (WPI), sweet whey, lactic
acid whey, mineral acid whey, reconstituted whey powder, a
composition derived from any milk or colostrum processing stream, a
composition derived from the retentate or permeate obtained by
ultrafiltration or microfiltration of any milk or colostrum
processing stream, or a composition derived from the breakthrough
or adsorbed fractions obtained by chromatographic separation of any
milk or colostrum processing stream, or a full or partial
hydrolysate of any of these compositions, and or a mixture thereof.
See for example the Dairy Processing Handbook (Tetra Pak Processing
Systems, Lund, Sweden, 1995). Other such derivatives include the
dairy protein compositions and dairy ingredients described
above.
[0179] The proteins found in milk include immunoglobulins
(including IgA, IgD, IgE, IgG and IgM), growth factors, bovine
serum albumin (BSA), alpha-lactalbumin, beta-lactoglobulin and a
large number of caseins, all of which are phosphoproteins. These
proteins, with the exception of casein, are also present in whey.
Milk is known to contain a variety of mitogenic proteins and
proteins which may be involved directly in bone remodeling. Growth
factors (IGF--Insulin-like Growth Factor, TGF--Transforming Growth
Factor etc), immunoglobulins (including IgA, IgD, IgE, IgG and
IgM), BSA and some beta lactoglobulin are recovered from milk or
whey by cation exchange chromatography. Some growth factors are
recovered as neutral proteins. Caseinoglycomacropeptide (CGMP) is
an acidic protein fraction recoverable by anion exchange.
Osteopontin is a highly phosphorylated and glycosylated protein
found in all body fluids (including milk).
[0180] CGMP is a peptide released from kappa-casein during the
rennet-mediated casein coagulation step (through the action of
chymosin) of the cheese making process and is found in the whey
fraction which is known as Sweet Whey or Cheese Whey. CGMP is
sometimes referred to simply as GMP (glycomacropeptide). Cheese
whey proteins consist of 15% to 20% CGMP. CGMP has been put forward
as one of the bone health promoting components of milk, as reported
in WO 00/49885.
[0181] Lactic acid whey is produced by fermentation with lactic
acid bacteria or direct addition of lactic acid during the
manufacture of caseinate or cottage and ricotta cheeses. Mineral
acid whey is produced by addition of mineral acids during caseinate
manufacture. Lactic acid whey and mineral acid whey do not contain
CGMP. The basis of these two processes is to lower pH to about 4.6
to cause casein to precipitate as opposed to using the action of
chymosin to cause precipitation. Therefore any milk products that
have not been exposed to chymosin will not contain CGMP.
[0182] Whey is a by-product of cheese or casein manufacture, and
the protein products derived from whey may be classified on the
basis of their protein content, including whey protein concentrates
(WPC) containing at least 30% protein, to whey protein isolates
(WPI) containing at least 90% protein (Huffman, 1996; IDF, 1998).
Membrane ultrafiltration and diafiltration is typically used in the
manufacture of such products to concentrate and purify the whey
protein to 25-35% solids before drying, and the protein concentrate
derived from the membrane filtration step is known in the art as
retentate. Whey protein is a collective term encompassing several
individual proteins (including but not limited to
alpha-lactalbumin, beta-lactoglobulin, proteose peptones,
inumunoglobulins (including IgA, IgD, IgE, IgG and IgM),
glycomacropeptide, growth factors (such as TGF .beta.1 and TGF
.beta.2), bovine serum albumin, lactoferrin, and lactoperoxidase)
and in the present invention may include whey protein collectively
or fractions thereof. Methods suitable for the commercial
production of whey are described by Zadow (1992) and Sienkiewicz et
al (1990). Methods for producing WPCs and WPIs are known in the art
and discussed in the US Dairy Export Council Reference Manual for
U.S. Whey and Lactose Products, Chapter 7: Whey
Products--Definition, Composition, Functions; Page, J., Meyer, D.,
Haines, B., Lagrange, V., and Kenney, A. (Eds), American Dairy
Products Institute, Elmhurst, Ill., USA, (June 2004) (also
available on-line at
http://www.usdec.org/files/pdfs/US08D.sub.--04.pdf). See also the
Dairy Processing Handbook (Tetra Pak Processing Systems, Lund,
Sweden, 1995). Whey protein is known to be immunostimulatory.
[0183] Hyperimmune milk and hyperimmune colostrum are made by
immunizing pregnant milk producing mammals with antigens from
pathogens to raise specific antibodies in the colostrum and milk
(see Korhonen, et al., 2000 for a review of such methods). Protein
concentrates of hyperimmune products may be prepared according to
the known methods referenced above. Hyperimmune milk and
hyperimmune colostrum are known to be immuno-stimulatory (Korhonen,
et al., 2000). Hyperimmune milk and hyperimmune colostrum may be
processed like ordinary milk and colostrum to produce derivatives
such as hyperimmune milk protein concentrate, hyperimmune milk
protein isolate, hyperimmune whey, hyperimmune whey protein
concentrate, hyperimmune whey protein isolate, hyperimmune
colostrum, hyperimmune colostrum milk protein concentrate,
hyperimmune colostrum milk protein isolate, hyperimmune colostrum
whey, hyperimmune colostrum whey protein concentrate, or
hyperimmune colostrum whey protein isolate, or a mixture
thereof.
[0184] By way of example, compositions or products processed
according to the invention may be delivered in pressure-treated
products or ingredients; added to products or ingredients which are
not subsequently heat-treated; or added to products or ingredients
which are subsequently pressure-treated.
[0185] In order to reduce unwanted effects on proteins present in
compositions treated according to the invention, it may be
desirable in some embodiments where the composition is a liquid to
add a stabiliser. For example, to stabilise any casein present in
the composition. Accordingly, in one embodiment the composition
further comprises a stabiliser such as a gum selected from locust
bean gum, guar gum, xanthan gum, cassia gum, konjac flour,
beta-glucan, tara gum, gum arabic, gellan gum,
carboxymethylcellulose, methylcellulose, hydroxypropyl
methylcellulose, tragacanth gum, karaya gum, gum acacia, chitosan,
arabinoglactins, alginate, pectin, carrageenan, or psyllium or a
mixture thereof. Preferably the stabiliser is pectin or
carboxymethylcellulose (CMC). Preferably a composition to be
treated according to the methods of the invention comprises about
0.01, 0.05, 0.1, 0.15, 0.2, 0.25, 0.3, 0.35% w/v or more of a
stabiliser, as required.
[0186] To stabilise bioactive components at about neutral pH, in
some embodiments it may be desirable to include in the composition
one or more hydrophobic ligands. The presence of a hydrophobic
ligand allows a bioactive component to be pressure treated at a pH
of about 7.0, preferably at a pH of about 5.0 to 8.0, while
retaining a higher level of activity than would be readily obtained
in the absence of the ligand. Without wishing to be bound by
theory, it is believed these ligands bind hydrophobic pockets in
the bioactive components, reducing their sensitivity to
denaturation during pressure treatment. Accordingly, in some
embodiments it may be desirable that the composition further
comprises one or more hydrophobic ligands selected from palmitic
acid, myristic acid, linoleic acid, conjugated linoleic acid (CLA),
one or more phospholipids, one or more phosphatidylcholines, one or
more sphingomyelins, one or more gangliosides, butyric acid, one or
more omega-3 fatty acids (including but not limited to
eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA)), one or
more phytosterols, one or more phytosterol esters, one or more
phytosterol acetates, one or more omega-6 fatty acids (including
but not limited to fish oil), fat soluble hydrophobic vitamins
(including vitamin A [retinol] and vitamin D), lycopene, or sodium
dodecyl sulphate, or a mixture thereof. Preferably the pH of the
composition in this embodiment is from about 5.0 to 8.0.
[0187] Examples of product formulations useful herein include
beverages (including acidified beverages and carbonated beverages),
yoghurts and jellies. Such products may be formulated as described
below and assessed as described above and in the examples.
[0188] Various aspects of the invention will now be illustrated in
non-limiting ways by reference to the following examples.
EXAMPLES
1. Lactoferrin Solutions
[0189] Lactoferrin (.about.15% iron saturation--native saturation
levels) was obtained from Fonterra Co-operative Group limited.
[0190] Iron-saturated lactoferrin (.about.100%) was made by
addition of a 2:1 molar excess of 5 mM ferric nitrilotriacetate
(Foley and Bates, 1987) to a 1% solution of lactoferrin in 50 mM
Tris-HCl, pH 7.8 containing 10 mM sodium bicarbonate. Excess
reagent was removed by dialysis against 100 volumes of Milli Q
water (twice renewed) for a total of 20 hours at 4.degree. C. The
iron-loaded (holo-) lactoferrin was then freeze-dried.
[0191] Lactoferrin solutions for heat and pressure treatment were
made to 6% (w/v) in MilliQ water and the pH adjusted with 3M NaOH
or 3 M HCl to the required value. After initial pH adjustment, the
solutions were allowed to equilibrate overnight and the pH of the
solutions finally re-adjusted to the required pH.
2. Pressure Treatment
[0192] The solutions were transferred into Beckman Polyallomer
Quick-seal.TM. centrifuge tubes (13 mm internal diameter, 51 mm
high; Beckman Instruments, Inc., Spinco division, Palo Alto, Calif.
94304) and the tubes heat-sealed. The sample tubes were then
treated in a high-pressure unit at 600 MPa at ambient temperature
for 5, 15 and 30 min. After depressurisation, the sample tubes were
cut open and the pressure-treated samples and the untreated
(control) were analysed using various techniques.
3. Heat Treatment
[0193] Aliquots (2 ml) of lactoferrin solution were placed in 8 ml
Wheaton glass screw-capped vials and the cap firmly fitted to
ensure a tight seal. Vials in each sample series were preincubated
in a water bath at 37.degree. C. for 5 min. The vials were quickly
transferred to an oil bath set to the desired temperature and
rocked and the time counted from this point. Sample vials were
removed at the appropriate time points and immediately put on ice,
ensuring complete immersion of the vial for rapid cooling. Samples
were stored at 4.degree. C. until analysis.
4. Analyses
[0194] Qualitative and quantitative changes in the samples arising
from heat or pressure processing were assessed by a range of
techniques as below.
4.1 PAGE Analysis
[0195] Reducing and non-reducing polyacrylamide gel electrophoresis
(PAGE) was performed as described by Manderson et al. (1998) using
15% gels.
4.2 HPLC
[0196] Samples were diluted appropriately in MilliQ water and
analysed by reversed-phase HPLC as described in Palmano & Elgar
(2002). Quantitation was by reference to a standard curve
constructed using high purity bovine lactoferrin.
4.3 ELISA
[0197] The ELISA assay was performed on appropriately diluted
samples using Bethyl kit following the manufacturer's
instruction.
4.4 Immunoaffinity
[0198] Samples were appropriately diluted in Hepes buffer and
analysed using a BiaCore instrument as described in Indyk &
Filonzi (2005). Quantitation was by reference to a standard curve
constructed using high purity bovine lactoferrin.
4.5 Iron Binding
[0199] The level of iron saturation in samples was assessed by
absorbance at 465 nm. Spectrophotometric titration using ferric
nitrilotriacetate (FeNTA) (Bates et al. 1967; Brock & Arzabe,
1976) was used to assess the iron binding capacity of lactoferrin
in samples.
Example 1
[0200] A 6% (w/v) native (.about.15% iron saturated) lactoferrin
solution prepared as described above was translucent and a burnt
orange colour. A solution of apo-lactoferrin (.about.0% iron
saturated) was clear. Heat treatment of native lactoferrin over the
pH range 3.8-7.0 showed that mild conditions (for example,
75.degree. C. for 5 min) induced some colour change in the test
solutions indicating loss of iron or oriented iron-binding in
lactoferrin, particularly at low pH (Table 1). Higher temperatures
(85.degree. C. to 90.degree. C. for 5 min) resulted in virtually
complete loss of colour in all solutions over the pH range. At pH
7.0, a thick precipitate formed indicating extensive denaturation
of lactoferrin.
TABLE-US-00001 TABLE 1 Effect of heat treatment on native
lactoferrin as a function of pH pH: Treatment Conditions: 3.3 3.8
4.0 5.2 7.0 Control clear ++ ++ +++ +++ 75.degree. C./5 min clear
almost + ++ ++ clear 85.degree. C./5 min clear clear clear clear
thick ppt 90.degree. C./5 min clear clear clear clear thick ppt "+"
symbols indicate colour depth on visual inspection. Fewer "+"
symbols indicate less iron binding.
[0201] In contrast, pressure treatment at 600 MPa for up to 45 min
resulted in no colour change or turbidity in any of the samples
except those at pH 3.8 and 4.0, where colour became more closely
aligned with the solutions at higher pH (Table 2), indicating some
re-adjustment in oriented iron binding at these lower pHs. This
colour change at certain pH may have been due to pressure-induced
`solubilisation` of carbon dioxide with consequent re-equilibration
to bicarbonate, which would furnish carbonate ions for iron
binding.
TABLE-US-00002 TABLE 2 Effect of pressure treatment on native
lactoferrin as a function of pH pH: Treatment Conditions: 3.3 3.8
4.0 5.2 7.0 Control clear ++ ++ +++ +++ 600 MPa/5 min clear +++ +++
+++ +++ 600 MPa/10 min clear +++ +++ +++ +++ 600 MPa/30 min clear
+++ +++ +++ +++ 600 MPa/45 min clear +++ +++ +++ +++ "+" symbols
indicate colour depth on visual inspection. Fewer "+" symbols
indicate less iron binding or less oriented binding.
Example 2
[0202] A 6% (w/v) 100% iron saturated lactoferrin solution prepared
as described above was translucent and a dark burgundy colour. Heat
treatment of fully iron-saturated lactoferrin (.about.100%) at pH
3.8, 4.8 and 7.0 (85.degree. C. for 10 min and 90.degree. C. for 10
min) resulted in some loss of colour (at pH 3.8 and 4.8) indicating
a loss of bound iron and complete aggregation and loss of soluble
lactoferrin in the case of the pH 7.0 sample (data not shown).
[0203] In contrast, pressure treatment at 600 MPa for up to 30 min
resulted in no visible colour change for any of the solutions
(Table 3).
TABLE-US-00003 TABLE 3 Effect of pressure and heat treatment on
100% iron saturated lactoferrin as a function of pH pH: Treatment
Conditions: 3.8 4.8 Control burgundy burgundy 600 MPa/5 min
burgundy burgundy 600 MPa/30 min burgundy burgundy 85.degree. C./10
min orange-red orange-red 95.degree. C./10 min orange-red
orange-red
Example 3
[0204] Samples of a fully iron-saturated lactoferrin solution
(.about.100%) were adjusted to pH 7.0, 4.8 or 3.8 and subjected to
high pressure at 600 MPa for 5 min or 30 min or heat treatment at
85.degree. C. for 10 min or 95.degree. C. for 10 min. The integrity
of lactoferrin, bound iron and iron-binding capacity were
determined for each sample. Untreated samples at each pH served as
controls. In the case where there were precipitates or insoluble
aggregates formed as a result of treatment, the samples were
centrifuged at 8000.times.g for 30 min, at 10.degree. C. and the
supernatant taken for analysis.
[0205] Table 4 shows the results from HPLC. Quantitation was by
integration of peaks, to give total soluble lactoferrin as a
percentage compared to an untreated control. Peak shape was used as
a qualitative index of denaturation.
TABLE-US-00004 TABLE 4 Effect of Heat and Pressure on
Iron-saturated Lactoferrin assessed with RP-HPLC pH 3.8 pH 4.8 pH
7.0 Control 100 100 100 600 MPa .times. 5 min 93 98 102 600 MPa
.times. 30 min 103 92 92 85.degree. C. .times. 10 min 92 82 0.3
90.degree. C. .times. 10 min 90 76 0.2 Results are expressed as a %
of the control at each pH
[0206] For the pressure treated samples there was little change in
peak shape or amount of lactoferrin over the pH range studied,
whereas for the heat treated samples, there were visible changes in
peak shape and greater loss of lactoferrin at each pH. This was
most dramatic at pH 7.0 where there was complete denaturation of
the lactoferrin.
[0207] Table 5 shows the results from BiaCore analysis.
TABLE-US-00005 TABLE 5 Effect of Heat and Pressure on
Iron-saturated Lactoferrin assessed by BiaCore analysis pH 3.8 pH
4.8 pH 7.0 Control 100 100 100 600 mPa .times. 5 min 104 127 99.2
600 mPa .times. 30 min 99.7 93.5 102.9 85.degree. C. .times. 10 min
68.4 63.5 0.43 90.degree. C. .times. 10 min 65.3 65.9 0.28 Results
are expressed as a % of the control at each pH
[0208] Tables 4 and 5 show pressure treatment substantially
retained iron saturated lactoferrin levels at pH 3.8, 4.8 and 7.0
whereas heat treatment resulted in a loss of iron bound lactoferrin
at all pHs, especially neutral pH.
[0209] Samples were analysed by non-reducing and reducing SDS-PAGE
(data not shown). In all cases, lactoferrin in the pressure treated
samples showed no change from the untreated controls. In contrast,
the heat treated samples all showed some change in lactoferrin. For
example, at lower pH values (3.8 & 4.8) the non-reducing gels
show aggregated material which did not penetrate into the gel. This
material was virtually absent after reduction indicating that
aggregation was due to heat-induced polymerisation. At pH 7 almost
no measurable lactoferrin remained in heat treated samples.
[0210] The control solutions of iron-saturated lactoferrin showed
spectral differences which were dependent on the pH (data not
shown). The spectrum of the parent iron-saturated lactoferrin (pH
8.0, unadjusted) was typical of those reported for iron-bound
transferrins, with the absorption maxima for iron-binding at 465 nm
(Bates & Schlabach, 1973). Notably, the pressure treated
samples at each pH gave spectra which were virtually identical to
those for the untreated pH controls. The amount of bound iron
assessed from the spectra (absorbance at 465 nm in the presence of
bicarbonate and Tris buffer pH 8.0) was the same as for the parent
solution. Hence pressure treatment resulted in no change to iron
bound, or oriented iron-binding at each pH.
[0211] In contrast, the spectra obtained from heat-treated samples
showed definite differences from controls (data not shown). At pH
3.8, the heat treated samples gave spectra showing decreased
absorbance at 465 nm, indicating loss of iron-bound lactoferrin,
whereas at pH 4.8 the heat treated samples gave spectral profiles
which were different from the control (no spectra could be recorded
for the pH 7.0 samples due to aggregation of the sample as
described above). This indicates that some irreversible changes had
occurred due to the heating regimes and appropriate iron-binding
had been compromised. In all cases above, addition of further iron
as FeNTA resulted in no change in the spectra observed.
Example 4
[0212] Samples of a 6% w/v fully iron-saturated (.about.100%)
lactoferrin solution were adjusted to pH 3.3, 3.8, 4.2, 5.0, 6.0,
7.0 or 8.0 and inoculated with coliforms and yeast and moulds.
Samples were subjected to pressure treatment at 600 MPa with no
hold, 3 minutes or 15 minutes, or heat treated at 85.degree. C./10
min or 90.degree. C./10 min. Microbial analysis of an untreated
control showed a yeasts and moulds count of 7.0.times.10.sup.5
cfu/ml whereas no yeasts and moulds were detectable in all pressure
treated lactoferrin samples at every pH. Data is shown for samples
at pH 5.0 to 8.0 in Table 6. HPLC analysis (Palmano & Elgar,
2000) on all samples showed that pressure treatment at each pH had
no effect on quantifiable lactoferrin (relative to the unprocessed
control) and absorbance measurement at 465 nm showed that at pH 5.0
and greater, iron saturation was essentially 100%. RP-HPLC is a
measurement of total lactoferrin content. Absorbance at 465 nm can
be used to measure the amount of fully iron saturated lactoferrin
(FeLF) present in the solution. Where the RP-HPLC measurement and
the measurement derived from 465 nm are approximately the same
(mg/ml), the concentration of approximately fully (100%) saturated
lactoferrin the in solution is indicated.
TABLE-US-00006 TABLE 6 Microbial analysis of FeLF solutions (6%
w/v) adjusted at various pH and pressure treated (600 MPa for
different holding times) or heat treated (as shown) FeLF Yeast
& FeLF (mg/ml) Sample Coliforms moulds (mg/ml) Absorbance
description pH (cfu/ml) (cfu/ml) RP-HPLC at 465 nm Unprocessed 7.0
ND 7.0 .times. 10.sup.5 53.0 52.7 control No hold 5.0 ND ND 52.6
52.5 3 min ND ND 54.2 51.2 15 min ND ND 51.4 51.5 85.degree. C./10
min NP NP 52.2 ** 90.degree. C./10 min NP NP 48.8 ** No hold 6.0 ND
ND 51.7 52.1 3 min ND ND 54.6 50.1 15 min ND ND 52.8 52.3
85.degree. C./10 min NP NP 5.2 7.62 90.degree. C./10 min NP NP 1.33
** No hold 7.0 ND ND 52.6 51.0 3 min ND ND 53.5 52.8 15 min ND ND
51.7 52.0 85.degree. C./10 min NP NP NQ ** 90.degree. C./10 min NP
NP NQ ** No hold 8.0 ND ND 52.2 49.5 3 min ND ND 50.5 53.2 15 min
ND ND 52.8 55.0 85.degree. C./10 min NP NP NQ ** 90.degree. C./10
min NP NP NQ ** ND = Not found or not detected; NQ = not
quantifiable because peak shape indicated extensive denaturation
and aggregation of lactoferrin; NP = test not performed; **
indicates absorbance not able to be assessed due to irregularities
in the spectrum associated with denaturation.
Example 5
[0213] Fully iron saturated (.about.100%) lactoferrin (prepared as
described above) was incorporated into yoghourt (prepared according
to the method outlined in FIG. 1), a beverage (prepared according
to the method outlined in FIG. 2), or jelly (prepared according to
the method outlined in FIG. 3). Heat treated, pressure treated and
control samples were prepared as described in the Figures.
Lactoferrin was extracted from these samples for analysis of
integrity, bound iron and iron-binding ability.
[0214] Briefly, samples of yoghourt (.about.100 g) were centrifuged
at 8000.times.g, at 10.degree. C. for 30 minutes. The supernatant
whey was removed, diluted 1:1 with 0.1M disodium phosphate buffer,
pH 6.5, and the pH adjusted to pH 6.5 with NaOH. The precipitate
formed was removed by centrifugation as above. The resulting
supernatant (.about.100-110 mL) was loaded on to a column
(1.6.times.20 cm) of S Sepharose Big Beads (SPBB) at 2 mL/min.
[0215] For beverage samples, 100 mL beverage was diluted to 400 mL
with milliQ water and the pH adjusted to 6.5 with NaOH. A sample of
diluted beverage (100 mL) was filtered through a 0.45 .mu.m PVDF
membrane (Millipore) and 80 mL applied to a column (1.6.times.20
cm) of S Sepharose Big Beads (SPBB) at 2 mL/min.
[0216] For jelly samples, 50 g jelly was diluted to 200 mL with
milliQ water, with stirring to homogenise. A sample of diluted
jelly (.about.100 mL) was filtered through a 0.45 .mu.m PVDF
membrane (Millipore) and the filtrate applied to a column
(1.6.times.20 cm) of SPBB at 2 mL/min.
[0217] Separate columns for each sample were treated as follows.
Unbound material was washed through with 0.1M disodium phosphate
buffer, pH 6.5. Bound protein was firstly eluted with 0.45 M NaCl
(until absorbance at 280 nm & 214 nm had reached baseline) and
then with 1.5 M NaCl. Analysis of the fractions by reversed-phase
HPLC (Palmano & Elgar, 2000) showed that virtually all the
lactoferrin was captured in the 1.5 M NaCl eluted fractions, and
was at least 93% purity for the yoghurt or beverage samples or
82-88% purity for the jelly samples. The 1.5 M NaCl eluted
fractions for each sample were dialysed against milliQ water,
freeze-dried and stored at -30.degree. C. prior to analysis.
Results--Iron Binding
[0218] The amount of lactoferrin in the yoghourts, beverages and
jellies was assayed at .about.14 days after manufacture by
reversed-phase HPLC, and by immunoassay using BiaCore (Indyk &
Filonzi, 2005) analysis. Degree of iron saturation in the extracted
lactoferrins was measured by spectrophotometric titration. Results
are given in Table 7.
TABLE-US-00007 TABLE 7 Lf (mg/g) Lf (mg/g) (RP- Fe Saturation
Sample (BiaCore) HPLC) (%) Yoghourt control 3.38 3.91 12 Yoghourt
HPP 2.99 4.05 17 Yoghourt Heat treated 0.0004 0.079 ND Beverage
control 2.45 3.42 78 Beverage HPP 1.9 3.10 83 Beverage Heat treated
0.23 ND ND Jelly control 3.72 4.701 23 Jelly HPP 3.36 4.069 61 ND =
Not determined; HPP = pressure treated.
[0219] The above results show that high pressure treatment of the
application samples maintained the integrity of iron-lactoferrin,
whereas heat treatment was severely detrimental to the protein. In
all cases measured, iron saturation in the pressure treated samples
was approximately equal to or greater than that in the
corresponding control, indicating that pressure treatment can
maintain iron saturation status and even improve iron binding
status in particular applications. Lactoferrin concentration and
integrity in the heat treated samples was too residual to warrant
extraction of lactoferrin. The low saturation observed in the
yoghurts may be due to iron-scavenging by microorganisms combined
with low pH. In all cases, the extracted lactoferrin retained full
iron binding bioactivity, being able to bind iron in excess of 90%
saturation (data not shown).
Results--Microbiology
[0220] Microbial analysis of the beverages, jellies and yoghurts
was conducted within 1 to 2 weeks and is shown in Tables 8 to 10.
The results indicate that pressure treatment was effective in
reducing microbial counts.
TABLE-US-00008 TABLE 8 Microbiology of FeLF yoghurts Pressure
Control Heat treated treated Test Description (cfu/ml) (cfu/ml)
(cfu/ml) Total count acid tolerant (.degree. C.) 60 <10 10 B.
Cereus Confirmed count <10 EST <10 EST <10 EST Coliform
counts by direct plating <1 EST <1 EST <1 EST Confirmed
coagulase +ve <10 EST <10 EST <10 EST staphylococci Yeast
and molds counts <1 EST <1 EST <1 EST Aerobic Plate count
130 <10 EST 60 EST Clostridia perfringens counts <10 EST
<10 EST <10 EST Mesophilic spores 20 EST 10 EST 10 EST
Thermophiles 50 EST 30 EST 30 EST E. Coli detect LSTMUG ND ND ND E.
Coli detect LSTMUG ND ND ND Listeria ABSENT ABSENT ABSENT
Salmonella ABSENT ABSENT ABSENT ND = not detected.
TABLE-US-00009 TABLE 9 Microbiology of FeLF acid drinks Pressure
Control Heat treated treated Test Description (cfu/ml) (cfu/ml)
(cfu/ml) Total count acid tolerant (.degree. C.) 2.8 .times.
10.sup.3 <10 10 B. Cereus Confirmed count <10 EST <10 EST
<10 EST Coliform counts by direct plating <1 EST <1 EST
<1 EST Confirmed coagulase +ve <10 EST <10 EST <10 EST
staphylococci Yeast and molds counts <1 EST <1 EST <1 EST
Aerobic Plate count 640 <10 EST 60 EST Clostridia perfringens
counts <10 EST <10 EST <10 EST Mesophilic spores <10
EST <10 EST <10 EST Thermophiles 10 EST <10 EST <10 EST
E. Coli detect LSTMUG ND ND ND E. Coli detect LSTMUG ND ND ND
Listeria ABSENT ABSENT ABSENT Salmonella ABSENT ABSENT ABSENT ND =
not detected.
TABLE-US-00010 TABLE 10 Microbiology of FeLF jellies Control
Pressure treated Test description (cfu/ml) (cfu/ml) Total count
acid tolerant (.degree. C.) 500 molds <10 B. Cereus Confirmed
count <10 EST <10 EST Coliform counts by direct plating <1
EST <1 EST Confirmed coagulase +ve staphylococci <10 EST
<10 EST Yeast and molds counts <1 EST <1 EST Aerobic Plate
count <10 EST <10 EST Clostridia perfringens counts <10
EST <10 EST Mesophilic spores <10 EST <10 EST Thermophiles
<10 EST <10 EST E. Coli detect LSTMUG ND ND E. Coli detect
LSTMUG ND ND Listeria ABSENT ABSENT Salmonella ABSENT ABSENT ND =
not detected.
Example 6
[0221] Carbonated beverages prepared as outlined in FIG. 4
containing iron-saturated freeze-dried lactoferrin powder at 4
mg/ml. The beverages were challenged with coliforms and yeast and
moulds before pressure treatment. The beverages were left untreated
or were pressure treated. Lactoferrin content in the beverages was
analysed by HPLC (Palmano & Elgar, 2000) and ELISA. Results
show that pressure treatment had little effect on quantifiable
lactoferrin but it reduced coliform and yeast and mould and APC
counts by more than 5 logs (Table 11).
TABLE-US-00011 TABLE 11 Microbial analysis of FeLF carbonated drink
formulated at different pH and challenged with coliforms and yeast
and moulds Yeast & FeLF FeLF Sample Coliforms mould APC (mg/mL)
(mg/mL) Description (cfu/mL) (cfu/mL) (cfu/mL) (HPLC) (ELISA) pH
4.5 8.0 .times. 10.sup.5 <10.sup.5 1.5 .times. 10.sup.6 3.74
2.49 Unprocessed pH 4.5 600 ND ND ND 3.81 1.83 MPa/3 min pH 6.0 1.6
.times. 10.sup.6 1.0 .times. 10.sup.5 1.7 .times. 10.sup.5 3.79
2.36 Unprocessed pH 6.0 600 ND ND ND 3.74 2.12 MPa/3 min ND = Not
detected
INDUSTRIAL APPLICATION
[0222] The methods of the present invention have utility in
preparing metal ion lactoferrin compositions for the food and
health industries. The compositions and products produced according
to the invention have a number of health benefits and therapeutic
applications, as described above.
[0223] Those persons skilled in the art will understand that the
above description is provided by way of illustration only and that
the invention is not limited thereto.
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