U.S. patent application number 09/946876 was filed with the patent office on 2002-09-05 for method of removing isoflavones and phytates.
Invention is credited to Daab-Krzykowski, Andre, Johns, Paul W., Mazer, Terrence B., Suh, John D..
Application Number | 20020122871 09/946876 |
Document ID | / |
Family ID | 7665219 |
Filed Date | 2002-09-05 |
United States Patent
Application |
20020122871 |
Kind Code |
A1 |
Johns, Paul W. ; et
al. |
September 5, 2002 |
Method of removing isoflavones and phytates
Abstract
Methods for sequentially removing isoflavones and phytates from
an active surface by utilizing an aqueous medium for isoflavone
removal and an aqueous medium for phytate removal. The aqueous
medium for isoflavone removal contains at least one alcohol and at
least one acid. The aqueous medium for phytate removal is either a
relatively stronger acidic solution, a basic solution, or with some
active surfaces may be an aqueous solution of pH 2-7, which is
essentially free of alcohol and organic solvents. The use of the
methods disclosed allows sequential isolation of isoflavones and
phytates, compounds which may then be utilized in various foods for
human consumption.
Inventors: |
Johns, Paul W.; (Columbus,
OH) ; Daab-Krzykowski, Andre; (Columbus, OH) ;
Mazer, Terrence B.; (Reynoldsburg, OH) ; Suh, John
D.; (Gahanna, OH) |
Correspondence
Address: |
ROSS PRODUCTS DIVISION OF ABBOTT LABORATORIES
DEPARTMENT 108140-DS/1
625 CLEVELAND AVENUE
COLUMBUS
OH
43215-1724
US
|
Family ID: |
7665219 |
Appl. No.: |
09/946876 |
Filed: |
September 5, 2001 |
Current U.S.
Class: |
426/656 |
Current CPC
Class: |
F04C 14/08 20130101;
F04C 15/0061 20130101; F04C 2/14 20130101; F02M 37/06 20130101;
F16H 15/42 20130101 |
Class at
Publication: |
426/656 |
International
Class: |
A23J 001/00 |
Foreign Application Data
Date |
Code |
Application Number |
Nov 30, 2000 |
DE |
100 59 422.0 |
Claims
What is claimed is:
1. A method for the sequential removal of isoflavones and phytates
from one, or more, active surfaces, comprising the steps of: a)
providing active surfaces comprising an anion exchange resin; b)
contacting said active surfaces with an aqueous medium for
isoflavone removal; and c) contacting said active surfaces with an
aqueous medium for phytate removal.
2. A method as defined in claim 1, wherein said active surfaces are
contained within a column which has at least one inlet and at least
one outlet, said inlet located lower in the column than said
outlet, such that said aqueous medium for isoflavone may be
contacted with said active surfaces and a first eluate collected,
and then said aqueous medium for phytate removal may be contacted
with said active surfaces and a second eluate collected.
3. A method as defined in claim 2, wherein said first eluate
contains less than 1% (w/v) of phytate.
4. A method as defined in claim 2, wherein said method is conducted
at room temperature.
5. A method as defined in claim 2, wherein said method is conducted
at a temperature from 90.degree. to 120.degree. F.
6. A method as defined in claim 2, wherein said aqueous medium for
isoflavone removal comprises: a) 1-40% (w/w) of at least one acid
selected from the group consisting of acetic acid, citric acid,
malic acid, malonic acid, lactic acid, and mixtures thereof; and b)
10-90% (v/v) of at least one alcohol selected from the group
consisting of methanol, ethanol, propanol, butanol, and mixtures
thereof.
7. A method as defined in claim 2, wherein said aqueous medium for
isoflavone removal comprises 50-70% v/v of at least one alcohol
selected from the group consisting of methanol, ethanol, propanol,
butanol, and mixtures thereof.
8. A method as defined in claim 2, wherein said aqueous medium for
isoflavone removal comprises: (a) at least one acid selected from
the group consisting of acetic acid, citric acid, hydrochloric
acid, sulfuric acid, nitric acid, phosphoric acid, succinic acid,
malic acid, malonic acid, tartaric acid, lactic acid, pyruvic acid,
fumaric acid and mixtures thereof; (b) 10-90% (v/v) of at least one
alcohol selected from the group consisting of methanol, ethanol,
propanol, butanol, and mixtures thereof; and has a pH ranging from
1.5 to 3.5.
9. A method as defined in claim 2, wherein said aqueous medium for
phytate removal has a pH less than 1.
10. A method as defined in claim 2, wherein said aqueous medium for
phytate removal comprises at least one acid selected from the group
consisting of acetic acid, citric acid, hydrochloric acid, sulfuric
acid, nitric acid, phosphoric acid, succinic acid, malic acid,
malonic acid, tartaric acid, lactic acid, pyruvic acid, fumaric
acid, and mixtures thereof, and has a pH less than 1.
11. A method as defined in claim 2, wherein said aqueous medium for
phytate removal has a pH between 13 and 14.
12. A method as defined in claim 2, wherein said aqueous medium for
isoflavone removal comprises at least two separate aqueous
solutions.
13. A method for the sequential removal of isoflavones and phytates
active surfaces, comprising the steps of: a) providing active
surfaces comprising alkylsilane bonded phase media; b) contacting
said active surfaces with an aqueous medium for isoflavone removal;
and c) contacting said active surfaces with an aqueous medium for
phytate removal.
14. A method as defined in claim 13, wherein said alkylsilane
bonded phase medium is contacted with an aqueous medium for phytate
removal which comprises an aqueous solution with a pH of 2 to 7,
essentially free of alcohol and organic solvents, and said
alkylsilane bonded phase medium is then contacted with an aqueous
medium for isoflavone removal.
15. A food containing isoflavones isolated by a method as defined
in claim 2.
16. A food containing phytates isolated by a method as defined in
claim 2.
17. A method for sequentially isolating isoflavones and phytates
from plant materials comprising: a) providing at least one anion
exchange resin; b) providing a slurry of plant protein that
contains isoflavones and phytic acid; c) placing the anion exchange
resin in a structure which has at least one inlet and at least one
outlet, and said inlet is located lower in the structure than the
outlet; d) passing the slurry into the structure and over the resin
by entering through the inlet and exiting through the outlet; e)
contacting said resin with an aqueous medium for isoflavone removal
and collecting a first eluate; and (f) then contacting the resin
with an aqueous medium for phytate removal and collecting a second
eluate.
Description
FIELD OF THE INVENTION
[0001] The invention relates to methods for sequentially removing
isoflavones and phytates from active surfaces.
BACKGROUND
[0002] Plant proteins are frequently utilized as protein sources in
food for human consumption such as nutritional formulas or cereals,
but are often purified prior to such use. Purification may be
utilized to remove compounds such as phytoestrogens or plant
estrogens, manganese or nucleotides. Phytoestrogens are plant
substances that are structurally and functionally similar to the
gonadal steroid, 17 .beta.-estradiol, that produce estrogenic
effects. There are three main groups of nonsteroidal dietary
estrogens: (1) isoflavones, (2) coumestans, and (3) mycoestrogens
(fungal). A review of phytoestrogens and their effects in mammals
is reported by Kaldas and Hughes in "Reproductive and General
Metabolic Effects of Phytoestrogens in Mammals," Reproductive
Toxicology, vol. 3, pp. 81-89, 1989. As used herein, the term
"isoflavones" is equivalent to the term "phytoestrogens" as the
term is defined in the Kaldas et al. article. Thus, isoflavones
include flavonones, flavonols, flavones, isoflavones, aurones,
chalcones, dihydrochalcones, anthocyanins, leucoanthocyanins,
leucoanthocyanidins, anthocyanidins, anhydroflavenols, catechins
and chemical derivatives of these groups.
[0003] Research has suggested that isoflavones may inhibit the
growth of human cancer cells. See e.g., Setchell, K. D. R., and
Welch, M. B., F. Chrom., 386 (1987), pp. 315-323; High Performance
Liquid Chromatographic Analysis of Phytoestrogens in Soy Protein
Preparations with Ultraviolet, Electrochemical and Thermospray Mass
Spectrometric Detection, McLachlan, J. A., ed. Estrogens in the
Environment, New York: Elsevier Press, 1985, pp. 69-85; and
Setchell, et al., "Nonsteroidal Estrogens of Dietary Origin:
Possible Roles in Hormone Dependent Disease," Am. F. Clin. Nutr.,
1984, 40, pp.569-578. There is also some indirect, demographic
support for an isoflavone mediated reduction in cancers of hormone
responsive tissues based on observations that women in countries
consuming vegetarian diets have a lower incidence of breast cancer
compared to women in meat-eating countries. Adlercreutz et al.,
"Determination of Urinary Lignans and Phytoestrogen Metabolites,
Potential Antiestrogens and Anticarcinogens, in Urine of Women on
Various Habitual Diets," Steroid. Biochem., 1986, 25, pp. 791-797.
Isoflavones have also been suggested to have antiviral and
fungicidal properties. And, they have been implicated in the
reduction of serum cholesterol in humans, positive immunological
effects and activity as an antioxidant. Isoflavones may also be
useful as an alleviator of vasomotor symptoms in menopausal women,
and have been used historically in Chinese medicine to treat "hot
flashes."
[0004] Plant proteins also contain significant amounts of phytates,
accounting for as much as 85% of the total phosphorus in certain
plants. One phytate is phytic acid. Phytic acid is also known as
inositol hexaphosphate. As used herein, the term "phytates" means
phytic acid and its isomers, the salts and derivatives of phytic
acid and its isomers, and/or partially dephosphorylated isomers of
phytic acid, and salts and derivatives of partially
dephosphorylated isomers of phytic acid. Phytic acid serves several
physiological functions and influences the functional and
nutritional properties of cereals and vegetables by its ability to
complex with both proteins and essential minerals. See, e.g.,
Ceryan, M., CRC Crit. Rev. Food Sci., Nutr., vol. 13, 297, 1980 and
Graf, E., F. Am. Oil Chem. Soc., vol. 60, 1861, 1983. Phytic acid
is also reported to be effective in preventing cancer. See, eg.,
Reddy, B. S. et al., Cancer Res., vol. 60, no. 17, 2000, pp.
4792-4797; Shamsuddin, A. M. and Vucenik, I., Anticancer Res., vol.
19, no. 5A, 1999, pp. 3671-3674. Such properties have prompted
research into methods for removal of phytates from plant sources,
such as soy proteins. U.S. Pat. No. 5,213,835 to Nardelli et al.,
discloses a process for removing phosphorus from milk and whey
proteins.
[0005] Purification of plant proteins and/or isolation of compounds
such as isoflavones and phytates may be achieved by use of such
methods as ion exchange technology. Such methods are disclosed, for
example, in U.S. Pat. Nos. 5,985,338, 5,804,234, and 600,020,471,
which are herein incorporated-by-reference. In such methods,
purification of the plant proteins or removal of phytoestrogens or
plant estrogens, manganese or nucleotides is effected by passing an
aqueous slurry of an isoflavone containing material over an active
surface such as an anion exchange resin, thereby binding such
compound or compounds to the active surface. As those of skill in
the art can appreciate, exchange resins and active surfaces have a
finite capacity, but may be regenerated to an active state after
exhaustion or near-exhaustion. After a certain amount of use,
reconditioning of the active surface, or ion exchange resin will be
useful. Such regeneration or reconditioning generally comprises
removal of the bound compound or compounds from the active
surface.
[0006] U.S. Pat. No. 5,804,234 to Suh et al., discloses a method
for regenerating or reconditioning exchange resins (or removing the
bound compounds) after contact with plant protein by contact with a
salt solution comprising 6% NaOH, 1% HCl and 1.5% NaHCO3.
[0007] U.S. Pat. No. 6,020,471 to Johns et al., discloses a method
for rinsing or releasing bound isoflavones from an ion exchange
resin by contact with an aqueous alcohol solution.
[0008] U.S. Pat. No. 6,146,668 to Kelly et al., discloses a
non-chromatographic approach to recovering isoflavones from plant
material, and requires the use of organic solvents (e.g., ethyl
acetate, hexane, acetone). The residual levels of such organic
solvent would constitute a safety concern in the utilization of the
recovered isoflavones in foods for human consumption. Kelly et al.
makes no provision for phytate removal.
[0009] U.S. Pat. No. 6,171,638 to Gugger et al., discloses an ion
exchange process for separation and purification of isoflavones,
utilizing aqueous alcohol, but makes no provision for phytate
removal.
[0010] U.S. Pat. No. 5,789,581 to Matsuura et al., discloses a
process for obtaining certain isoflavones which comprises the use
of an aqueous alcohol solution as an eluant.
[0011] U.S. Pat. No. 5,670,632 to Chaihorsky, discloses a process
for recovering isoflavones from a soy extract which comprises the
use of a highly polar sulfonic acid cationic exchange resin as an
adsorbent and the use of an acidic alkanol containing 1 to 3 carbon
atoms to facilitate desorption of the isoflavone 7-glycosides. The
solution utilized in desorption is prepared with a concentrated
alcohol (i.e., 96%) to yield a solution of 86% aqueous alcohol. The
patent makes no provision for the recovery of phytates.
[0012] U.S. Pat. No. 5,506,211 to Barnes et al., discloses the use
of a particular isoflavone (genistein) as an inhibitor of
osteoclasts. Barnes et al. describes the aqueous extraction of
isoflavones using 80% aqueous methanol.
[0013] U.S. Pat. No. 4,428,876 to Iwamura discloses a process for
isolating saponins and flavonoids from leguminous plants which
comprises the use of a polar solvent (such as methanol or aqueous
methanol) to elute the saponins and flavonoids adsorbed on a
resin.
SUMMARY
[0014] Isoflavones and phytates can be sequentially removed from an
active surface by utilizing a method whereby the active surface(s)
are contacted with an aqueous medium for isoflavone removal and
separately contacted with an aqueous medium for phytate removal.
The aqueous medium for isoflavone removal comprises an acidic
aqueous alcohol solution. The aqueous medium for phytate removal
comprises either a relatively stronger aqueous acid solution or an
aqueous basic solution. Alternatively, for use with some active
surfaces, the aqueous medium for phytate removal may comprise an
aqueous solution with a pH between about 2 and about 7 which is
essentially free of alcohol and organic solvents. By this method,
isoflavones and phytates are separately and sequentially removed
and may be recovered. The recovered isoflavones and phytates may be
utilized in products for human consumption, such as nutritional
products or cereals.
DETAILED DESCRIPTION
[0015] Methods are disclosed for sequentially removing isoflavones
and phytates from active surface(s) comprising contacting the
active surface with an aqueous medium for isoflavone removal and
separately with an aqueous medium for phytate removal. The aqueous
medium for isoflavone removal comprises an acidic aqueous alcohol
solution. The aqueous medium for phytate removal comprises either a
relatively stronger (i.e., lower pH) aqueous acid solution or an
aqueous basic solution, or with some active surfaces may comprise
an aqueous solution with a pH between about 2 and about 7 which is
essentially free of alcohol and organic solvents.
[0016] The acid utilized in the aqueous medium for isoflavone may
be selected from various acids, including, but not limited to,
acetic acid, citric acid, hydrochloric acid, sulfuric acid, nitric
acid, phosphoric acid, succinic acid, malic acid, malonic acid,
tartaric acid, lactic acid, pyruvic acid, fumaric acid and mixtures
thereof. Table I provides a comparison of isoflavone recovery with
various acids utilized in the aqueous medium for isoflavone
removal. The alcohol(s) utilized in the aqueous medium for
isoflavone removal may be selected from various alcohols,
including, but not limited to, methanol, ethanol, propanol and
butanol, and mixtures thereof. Various relative amounts of acid and
alcohol will be appropriate for use in the aqueous medium for
isoflavone removal, depending upon factors such as the strength of
the acid and the intended end use of the compound isolated. The
amount of alcohol present in the aqueous medium for isoflavone
removal may be between about 10 and about 90% (v/v). More
preferably, the amount of alcohol present is between about 50 and
about 70% (v/v). Table II provides a comparison of isoflavone
recovery utilizing citric acid in the aqueous medium for isoflavone
removal along with varying concentrations of alcohol. The amount of
acid present in the aqueous medium for isoflavone removal will vary
according to the strength of the acid (pKa) and concentration of
acid utilized in preparing the medium, but generally should be an
amount sufficient for the pH of the aqueous medium to be between
about 1.5 and about 3.5. The amount of acid required to bring the
aqueous medium for isoflavone removal to within such a pH range can
easily be calculated by one of ordinary skill in the art. Generally
amounts between about 0.1 and about 40% (w/w) are sufficient. As an
example, when glacial acetic acid is utilized in the aqueous medium
for isoflavone removal, it is preferably present in an amount from
about 5 to about 40% (v/v), and more preferably in an amount from
about 20 to about 30% (v/v). Table III provides a comparison of
isoflavone recovery utilizing an aqueous medium for isoflavone
removal with varying amounts of ethanol and glacial acetic acid.
Table IV provides a comparison of isoflavone recovery utilizing an
aqueous medium for isoflavone removal with 60% ethanol and varying
amounts of glacial acetic acid. When citric acid is utilized in the
aqueous medium for isoflavone removal, it is preferably present in
an amount from about 10 to about 40 grams/liter, and more
preferably in an amount from about 20 to about 30 grams/liter. A
combination of more than one acid may also be utilized in the
aqueous medium for isoflavone removal. For example, a suitable
aqueous medium for isoflavone removal could contain approximately
10% glacial acetic acid and approximately 10 g/liter citric acid,
along with approximately 60% reagent alcohol (reagent alcohol is
denatured ethanol; the terms reagent alcohol and ethanol are used
interchangeably herein). Table V illustrates comparative recovery
of isoflavones utilizing a combination of more than one acid in the
aqueous medium for isoflavone removal.
[0017] In another embodiment, the aqueous medium for isoflavone
removal comprises more than one aqueous solution. For example, a
first aqueous solution and a second aqueous solution may be used.
The first aqueous solution may comprise an acid aqueous solution
(with a pH between about 2 and about 7) essentially free of
alcohol, and the second aqueous solution may comprise an aqueous
alcohol solution essentially free of added acid. The method of
contacting comprises first contacting the active surfaces with the
first aqueous solution, and subsequently contacting the active
surfaces with the second aqueous solution. The first aqueous
solution contains no alcohol or organic solvent, but may contain at
least one acid selected from the group consisting of citric acid,
hydrochloric acid, sulfuric acid, nitric acid, phosphoric acid,
succinic acid, malic acid, malonic acid, tartaric acid, lactic
acid, pyruvic acid, fumaric acid and mixtures thereof. The pH of
the first aqueous solution ranges from about 2 to about 7. More
preferably, the pH of the first aqueous solution ranges from about
3 to about 6. The second aqueous solution may comprise at least one
alcohol selected from the group consisting of methanol, ethanol,
propanol, butanol, and mixtures thereof, but is essentially free of
added acid. This second aqueous solution preferably comprises about
10 to about 90% (v/v) alcohol, more preferably from about 50 to
about 80% (v/v) alcohol.
[0018] Generally, the aqueous medium for phytate removal comprises
either a relatively stronger aqueous acid solution or an aqueous
base solution. Alternatively, as discussed below, when used with
some active surfaces, the aqueous medium for phytate removal may
comprise an aqueous solution with a pH of about 2 to about 7 which
is essentially free of alcohol and organic solvents. The acid
utilized in the aqueous medium for phytate removal may be selected
from various acids, including, but not limited to acetic acid,
citric acid, hydrochloric acid, sulfuric acid, nitric acid,
phosphoric acid, succinic acid, malic acid, malonic acid, tartaric
acid, lactic acid, pyruvic acid, fumaric acid, and mixtures
thereof. Table VI provides a comparison of phytic acid removal with
various acids utilized in the aqueous medium for phytate removal.
When the aqueous medium for phytate removal comprises an aqueous
base solution, the base may be selected from various bases
including, but not limited to, sodium hydroxide, potassium
hydroxide, ammonium hydroxide, lithium hydroxide, sodium carbonate,
and mixtures thereof. For pH values in the acidic range (i.e., less
than 7), the relative amount of phytates removed from an active
surface increases with decreasing pH, see Table VII, which
illustrates phytic acid and isoflavone recovery at various pH
values (hydrochloric acid concentrations). The amount of acid or
base utilized in the aqueous medium for phytate removal will vary
according to the strength of the acid (pKa) or base (pKb), and
concentration of acid or base utilized in preparing the medium, but
generally should be an amount sufficient for the pH of the aqueous
medium for phytate removal to be less than about 1, more preferably
about 0.1 to about 1.0 for an acid solution or alternatively about
13 to about 14 for a basic solution. The amount of acid or base
required to bring the aqueous medium for phytate removal to within
such a pH range can easily be calculated by one of ordinary skill
in the art. As an example, when hydrochloric acid is utilized in
the aqueous medium for phytate removal, the amount present is
preferably sufficient so that the medium is about 0.2 to about 2 M
HCl, and more preferably about 0.4 to about 0.8 M HCl. When a base
is utilized in the aqueous medium for phytate removal, it is
preferably present in an amount from about 2 to about 10% (w/w),
and more preferably in an amount from about 4 to about 6%
(w/w).
[0019] The particular pH that is chosen for both the aqueous medium
for isoflavone removal and the aqueous medium for phytate removal
may be varied according to the use which the isolated isoflavones
and phytates will be put. Additionally, the amount of time that the
aqueous medium for isoflavone removal and the aqueous medium for
phytate removal are in contact with the active surfaces and the
amount of each medium that is utilized may be varied. Generally,
the total amount of isoflavone or phytate recovered increases as
the contact time with the active surfaces increases and as the
amount of medium that is utilized increases. However, such
increased recovery must be balanced with other factors, including
the cost of the protein (isoflavone and phytate source), disposal
or treatment costs for additional solvents generated when increased
volumes of media are utilized, costs for concentrating the
isoflavones or phytates, and life time and cost of the active
surfaces. In the examples provided below and in the tables provided
herein, various contact times and volumes of media are utilized.
Volumes of media are expressed herein as bed volumes or column
volumes with contact times expressed in terms of bed volumes or
column volumes per hour or per minute.
[0020] The active surface from which the isoflavones and phytates
are removed and isolated is preferably an anion exchange resin.
Suitable anion exchange resins are macroporous resins, preferably a
Type I or Type II macroporous resin. For anion exchange
chromatography, the anion exchange resin is selected from weak base
anion exchange resins, strong base anion exchange resins and
mixtures thereof. Representative examples include Amberlite.RTM.
RA95, IRA-910 and IRA-900 (available from Rohn and Haas Company),
Dowex-22 and MSA-1(available from Dow Chemical), and Purolite A510
and A500 (available from Purolite Company). As used herein, the
term resin is meant to include gels, which those skilled in the art
would understand to be useful in the process described herein.
Representative gels include Amberlite.RTM. IRA 410 (Type II gel,
strong base anion) (available from Rohm and Haas) and IRA 402 (Type
II gel, strong base anion exchange, not macroporous). The anion
exchange resin may be contained within a column which has at least
one inlet and at least one outlet, with the inlet located lower in
the column structure than the outlet. Such a setup allows for a
slurry of plant protein to be passed through the column and over
the resin by entering the column through the inlet and exiting
through the outlet. After the desired amount of plant protein
slurry has been passed through the column, the resin is then
contacted with the aqueous medium for isoflavone removal, prepared
in the manner described above and the eluate is collected. After a
desired amount of isoflavone eluate has been collected, the aqueous
medium for phytate removal, prepared in the manner described above,
may be passed through the column and contacted with the resin. The
second eluate which is collected will contain phytates. Table VIII
provides a comparison of the amounts of isoflavones and phytic acid
sequentially recovered utilizing various combinations of aqueous
medium for isoflavone removal and aqueous medium for phytate
removal.
[0021] The active surface may also comprise an alkylsilane bonded
phase medium. Alkylsilane bonded phase media are chromatography
column packings made by chemically bonding an alkylsilane (e.g.,
octadecylsilane [ODS or C18] or octylsilane [C8] or butylsilane
[C4]) to the surface of silica. When such are utilized, they are
preferably contained within a column which has at least one inlet
and at least one outlet, with the inlet located higher in the
structure than the outlet. A representative example is Sep-Pak C18,
an octadecylsilane, 55-105 micrometer diameter, 125 A pore size,
12% carbon (available from Waters Corporation). When the active
surface comprises an alkylsilane bonded phase medium, the aqueous
medium for phytate removal comprises an aqueous solution with a pH
between about 2 and about 7 which is essentially free of alcohol
and organic solvents. As used herein, the term organic solvents
includes solvents such as ethanol, methanol, propanol, isopropanol,
acetone, dimethylsulfoxide (DMSO), dimethylformamide (DMF),
tetrahydrofuran (THF), dichloromethane, and etc. Suitable solutions
for phytate removal for use with an alkylsilane phase bonded medium
include, but are not limited to, water and various buffers with an
appropriate pH. The aqueous medium for phytate removal may comprise
a sufficient amount of an acid selected from the group consisting
of acetic acid, citric acid, hydrochloric acid, sulfuric acid,
nitric acid, phosphoric acid, succinic acid, malic acid, malonic
acid, tartaric acid, lactic acid, pyruvic acid, fumaric acid and
mixtures thereof in an amount sufficient to the solution to have a
pH between about 2 and about 7, and is essentially free of alcohol
and organic solvents. The aqueous medium for isoflavone removal for
use with an alkylsilane bonded phase medium comprises an acidic
aqueous alcohol solution, as generally described above. The acid
utilized in the aqueous medium for isoflavone may be selected from
various acids, including, but not limited to, acetic acid, citric
acid, hydrochloric acid, sulfuric acid, nitric acid, phosphoric
acid, succinic acid, malic acid, malonic acid, tartaric acid,
lactic acid, pyruvic acid, fumaric acid and mixtures thereof. The
alcohol(s) utilized in the aqueous medium for isoflavone removal
may be selected from various alcohols, including, but not limited
to, methanol, ethanol, propanol and butanol, and mixtures thereof.
Various relative amounts of acid and alcohol will be appropriate
for use in the aqueous medium for isoflavone removal, depending
upon factors such as the strength of the acid and the intended end
use of the compound isolated. The amount of alcohol present in the
aqueous medium for isoflavone removal may be between about 10 and
about 90% (v/v). More preferably, when utilized with an alkylsilane
bonded phase medium, the amount of alcohol present is between about
50 and about 80% (v/v). As discussed above, the amount of acid
present in the aqueous medium for isoflavone removal will vary
according to the strength of the acid (pKa) and concentration of
acid utilized in preparing the medium, but when utilized with an
alkylsilane bonded phase medium generally should be an amount
sufficient for the pH of the aqueous medium to be between about 2
and about 7. The amount of acid required to bring the aqueous
medium for isoflavone removal to within such a pH range can easily
be calculated by one of ordinary skill in the art. Generally
amounts between about 0.1 and about 40% (w/w) are sufficient. As an
example, when glacial acetic acid is utilized in the aqueous medium
for isoflavone removal, it is preferably present in an amount from
about 5 to about 40% (v/v), and more preferably in an amount from
about 20 to about 30% (v/v). When citric acid is utilized in the
aqueous medium for isoflavone removal, it is preferably present in
an amount from about 10 to about 40 grams/liter, and more
preferably in an amount from about 20 to about 30 grams/liter. A
combination of more than one acid may also be utilized in the
aqueous medium for isoflavone removal. Additionally, when the
active surface comprises an alkylsilane bonded phase medium, the
medium will be contacted first with the aqueous medium for phytate
removal, and then separately contacted with an aqueous medium for
isoflavone removal.
[0022] Additionally and alternatively, the active surfaces may
comprise both anion exchange resin and alkylsilane bonded phase
medium. When such are utilized, they are preferably contained
within a column which has at least one inlet and at least one
outlet, with the inlet located lower in the structure than the
outlet. Preferably, the alkylsilane bonded phase medium is
positioned closer to the inlet than said anion exchange resin, so
that when an aqueous medium is passed through the column it
contacts the alkylsilane bonded phase medium before it contacts the
anion exchange resin. Aqueous media for isoflavone removal and for
phytate removal, as described generally above may be utilized. More
specifically, the aqueous medium for isoflavone removal comprises
an acidic aqueous alcohol solution and the aqueous medium for
phytate removal comprises either a relatively stronger (i.e., lower
pH) aqueous acid solution or an aqueous basic solution. In an
application where both anion exchange resin and alkylsilane bonded
phase medium are utilized as the active surfaces, the pH of the
aqueous medium for isoflavone removal is preferably between about 2
and 3.5.
[0023] Various mixtures of plant protein may be utilized for
isolation of isoflavones and phytates. Commercially available
mixtures may be utilized or custom blended mixtures may be
prepared. One commercially available plant protein mixture is a soy
protein isolate known as Ardex F.RTM. (available from Archer
Daniels Midland, Inc.). Other materials that may be used to supply
the source of isoflavones and phytates include any material that
contains a detectable level of isoflavones and phytates. Such
materials include protein obtainable from soybeans, corn, wheat,
peas, beans, cottonseed, peanuts, carrots, alfalfa, algae,
potatoes, apples, barley, bluegrass, clovers, coffee, garlic, hops,
marijuana, oats, orchard grass, parsley, rice, rye, sage, sesame,
yeast, fungus, hydrolyzates thereof, and mixtures thereof.
[0024] Methods described herein may be conducted at room
temperature, or may be conducted at elevated temperatures, such as
at about 90.degree. to about 120.degree. F. For purposes of this
invention, room temperature is defined as between about 60.degree.
F. and about 80.degree. F. The methods may also be conducted at
other temperatures. There is no preference on temperature, although
as one of ordinary skill in the art will recognize, lower and upper
limits will necessarily be restrained by the freezing points and
boiling points of the various solutions utilized.
[0025] Processes whereby active surfaces, such as an anion exchange
resin or an alkylsilane bonded phase medium, are utilized to purify
plant proteins will be readily recognizable to those of ordinary
skill in the art. Examples of such processes and details regarding
such processes are provided in U.S. Pat. Nos. 5,985,338, 5,804,234,
and 600,020,471. The methods claimed in this patent are not
intended to be limited to methods for sequentially removing
isoflavones and phytic acid from surfaces disclosed or described in
those patents.
EXAMPLES
[0026] Isoflavones and phytates can be sequentially isolated by
methods within the scope of the claims by the following procedures.
These examples are being presented as illustrations and should not
be interpreted as limiting in any way.
Example A
[0027] Method Utilizing Various Eluants.
[0028] IEX Resin Preparation: 22 grams of IRA-910 anion exchange
resin were weighed into a 250 milliliter beaker. Using laboratory
water (i.e., purified water (distilled, deionized, demineralized),
the resin was transferred into a 2.5.times.10 cm glass open column
(Bio-Rad catalog no. 737-2511) equipped with a 2-way stopcock
(Bio-Rad catalog no. 732-8102). (The terms laboratory water,
purified water and deionized water are used interchangeably
herein.) The following solutions were then passed through the
resin: 45 milliliters of 6% NaOH in approximately 20 minutes;
followed by 90 milliliters of laboratory water in approximately 20
minutes; followed by 100 milliliters of 1% HCl (0.12 M HCl) in
approximately 30 minutes; 180 milliliters of laboratory water in
approximately 40 minutes; 55 milliliters of 1.4% NaHCO.sub.3 in
approximately 30 minutes; and 360 milliliters of laboratory water
in approximately 80 minutes.
[0029] IEX Column Preparation: 22.0 grams of Commodity 1922 (PTI
soy protein isolate) were thoroughly suspended in 250 milliliters
of laboratory water in a 400 milliliter beaker. A 1.0-1.5 gram
sample was removed for isoflavone determination. 22 grams of
prepared resin (from procedure above) were added to the soy protein
isolate suspension and stirred gently for 180 minutes. The resin
was allowed to settle to the bottom of the beaker, and another 2.00
milliliter aliquot sample was removed for isoflavone determination.
The soy protein isolate suspension was decanted and discarded. The
resin was rinsed with 4.times.250 milliliters of laboratory water,
with decanting and discarding after each addition. Laboratory water
was used to pour equal amounts of resin (approximately 7 grams)
into each of three 2.5.times.10 cm glass open columns equipped with
a 2-way stopcock. Each column was rinsed with 20 milliliters of
laboratory water.
[0030] Three eluants were utilized, one for each column, as listed
in Table I. The first eluant was 0.48 M HCl in 60% ethanol. The
second eluant was 20% glacial acetic acid in 60% ethanol. The third
eluant was 20 g/L citric acid in 60% ethanol. Each eluant was
passed through one of the columns at a rate of 3.6 bed volumes per
hour. A total of 14 bed volumes of eluant were passed through each
column. Two fractions were collected, each consisting of the eluant
from 7 bed volumes. The amounts of isoflavones recovered are listed
in Table I.
[0031] Isoflavones were determined using gradient elution reverse
phase HPLC. 5.00 milliliters of each fraction were diluted to 25
milliliters with laboratory water, and tested for isoflavones by
the HPLC system described below:
HPLC System for Isoflavone Determination
[0032] HPLC Column: Waters Nova-Pak C18, 3.9.times.150 mm, 4 um,
60A, Waters #86344
[0033] Mobile Phase A: 920 mL 0.02M KH.sub.2PO.sub.4, 80 mL
acetonitrile; pH 3.1 with H.sub.3PO.sub.4
[0034] Mobile Phase B: 400 mL 0.02M KH.sub.2PO.sub.4, 600 mL
acetonitrile; pH 3.1 with H.sub.3PO.sub.4
[0035] Flow Rate: 0.6 mL/minute
[0036] Column Temperature: 40.degree. C.
[0037] Detection: UV at 262 nm and 250 nm
[0038] Injection: 20 uL
[0039] Run Time: 60 minutes
[0040] Elution Program:
1 Time (minutes) 0 5 40 42 45 48 60 Mobile Phase B (%) 0 0 42 100
100 0 end
Example B
[0041] Method Utilizing Citric Acid and Various Amounts of
Ethanol.
[0042] IEX resin and columns were prepared as described above for
Example A except that the soy protein isolate and resin suspension
was stirred for 2 hours, and 5 columns were prepared, and each
column contained 4.4 grams of resin. For this example, five eluants
containing citric acid and varying amounts of ethanol were prepared
and utilized. All eluants contained 20 g/L citric acid. The amounts
of ethanol utilized in the five eluants were: 0% (v/v), 10% (v/v),
20% (v/v), 60% (v/v) and 90% (v/v).
[0043] As in Example A, each eluant was passed through one of the
columns at a rate of 3.6 bed volumes per hour. A total of 14 bed
volumes of eluant were passed through each column. Each eluate was
tested for isoflavones according to the method described above in
Example A. The amounts of isoflavones recovered are listed in Table
II.
Example C
[0044] Method Utilizing Various Amounts of Glacial Acetic acid,
Ethanol and Water.
[0045] IEX Resin Preparation: The IEX Resin was prepared as
described in Example A above.
[0046] IEX Column Preparation: 4.0 grams of soy protein isolate
(PTI # C7H-XTO-9001) were thoroughly suspended in 200 milliliters
of laboratory water in a 400 milliliter beaker. A 2.00 milliliter
aliquot sample was removed for isoflavone determination ("column
feed"). 20 grams of prepared resin (from procedure above) were
added to the soy protein isolate suspension and stirred at
approximately 400 rpm for 60 minutes. The resin was allowed to
settle to the bottom of the beaker, and another 2.00 milliliter
aliquot sample was removed for isoflavone determination ("unbound
by IEX resin"). The soy protein isolate suspension was decanted and
discarded. The resin was rinsed with 2.times.200 milliliters of
laboratory water, with decanting and discarding after each
addition. Laboratory water was used to resuspend the resin, and
equal amounts of resin (approximately 3.3 grams) were poured into
each of six 20 milliliters columns. Each column was rinsed with 10
milliliters of laboratory water. 15.0 milliliters of eluant was
transferred by pipet into the column, and eluted at a flow rate of
approximately 1 column volume per minute. A total of 5 column
volumes of eluate were passed through each column. The eluate was
tested for isoflavones according to the method described above in
Example A.
[0047] Various eluants were prepared and utilized as listed in
Table III. The eluants contained varying amounts of ethanol, water
and glacial acetic acid. The amounts of isoflavones removed are
listed in Table III.
Example D
[0048] Method Utilizing Ethanol and Varying Amounts of Glacial
Acetic Acid.
[0049] IEX resin and columns were prepared as described above for
Example B. For this example, five eluants containing ethanol and
varying amounts of glacial acetic acid were prepared and utilized.
All eluants contained 60% ethanol (v/v). The concentrations of
glacial acetic acid present in the five eluants were: 0% (v/v), 10%
(v/v), 20% (v/v), 30% (v/v), and 40% (v/v).
[0050] Each eluant was passed through one of the columns at a rate
of approximately 10 column volumes per hour. A total of 10 bed
volumes of eluant were passed through each column. The eluates were
collected and each eluate was tested for isoflavones according to
the method described above in Example A. The amounts of isoflavones
recovered are listed in Table IV.
Example
[0051] Method Utilizing Various Eluants.
[0052] IEX Resin and columns were prepared as described above for
Example A.
[0053] For this example, three eluants were prepared and utilized.
The first eluant contained 20 g/L citric acid in 60% ethanol. The
second contained 20% glacial acetic acid in 60% ethanol. The third
contained 10 g/L citric acid and 10% glacial acetic acid in 60%
ethanol. Each eluant was passed through one of the columns at a
rate of approximately 3.6 bed volumes per hour. A total of 14 bed
volumes was passed through each column. The eluates were collected
and tested for isoflavones. The amounts of isoflavones recovered
are listed in Table V.
Example F
[0054] Method Utilizing Various Eluants.
[0055] IEX Resin and columns were prepared as described above in
Example A, except that 6 columns were prepared, each column
containing 3.6 grams of resin.
[0056] For this example, six eluants were prepared and utilized.
The first contained 20% glacial acetic acid in 60% ethanol. The
second contained 20% glacial acetic acid in water. The third
contained 20 g/L citric acid in 60% ethanol. The fourth contained
20 g/L citric acid in water. The fifth contained 0.48 M HCl in 60%
ethanol. The sixth contained 0.48 M HCl in water. Each eluant was
passed through one of the columns at a rate of approximately 3.6
bed volumes per hour. A total of 14 bed volumes was passed through
each column. The eluates were collected and each eluate was tested
for phytic acid. The amounts of phytic acid recovered are listed in
Table VI.
[0057] Phytic Acid was determined by the following procedure. For
ethanolic eluate samples, 7.00 milliliters of the eluate was
evaported to dryness with compressed N.sub.2, and the residue
resuspended in 7.00 milliliters of 0.02 M NaMalonate (pH 2.5). For
aqueous eluate samples, the pH was adjusted to 2.5 with NaOH. 3.00
milliliters of eluate sample was transferred by pipet into each of
two 1-dram vials. A phytase suspension was prepared by throughly
suspending 36 mg of phyrate (Sigma catalog no. P-9792; crude, from
Aspergillus ficum) in 4 milliliters of 0.02 M NaMalonate (pH 2.5).
To the first vial was added 400 microliters of 0.02 M NaMalonate;
this was the phorphorus control. To the second vial was added 400
microliters of the phytate suspension; this was the phytase digest.
The vials were capped, mixed well, and incubated at 40.degree. C.
for 120 minutes. The vials were removed from the water bath. The
sample suspensions were filtered through a 0.45 micrometer Acrodisc
syringe filter (Gelman P/N 4497). The filtrate was tested for
inorganic phosphorus, using Sigma's colorimetric Inorganic
Phosphorus test kit (Sigma catalog no. 670-A) and a
spectrophotometer. Eluate phytic acid was calculated by subtracting
the non-phytic acid phosphorus (as measured in the phosphorus
control) from the phytase digest phosphorus and by using the
appropriate dilution factor and molecular weights for phosphorus
(30.97) and for phytic acid (660.0).
Example G
[0058] Method Utilizing Ethanol and Varying Amounts of HCl.
[0059] IEX Resin Preparation: The IEX Resin was prepared as
described in Example A above.
[0060] IEX Column Preparation: 22 grams of Commodity 1922 (PTI soy
protein isolate "PP1610") were thoroughly suspended in 250
milliliters of laboratory water in a 400 milliliter beaker. A
1.0-1.5 gram sample was removed for isoflavone determination. 22
grams of prepared resin (from procedure above) were added to the
soy protein isolate suspension and stirred gently for 120 minutes.
The resin was allowed to settle to the bottom of the beaker, and
another 2.00 milliliters aliquot sample was removed for isoflavone
determination. The soy protein isolate suspension was decanted and
discarded. The resin was rinsed with 250 milliliters of laboratory
water and decanted and the supernatant discarded. Rinsing was
repeated until the resin was thoroughly rinsed of visible soy
protein isolate solids. An equal quantity of resin (approximately
4.4 grams) was transferred into each of five 2.5.times.10 cm glass
open columns (Bio-Rad # 737-2511) equipped with a 2-way stopcock
(Bio-Rad #732-8102). Each column was rinsed with 50 milliliters of
laboratory water. At this point, elution of isoflavones and/or
phytates with desired eluants may begin.
[0061] For this example, five eluants were prepared and utilized.
All contained 60 milliliters of ethanol and 40 milliliters of HCl
in varying concentrations. HCl concentrations were 1.25 N, 1.00 N,
0.75 N, 0.50 N and 0.25 N. The entire volume of each eluant was
passed through one of the columns at a rate of approximately 25
milliliters per hour. Each eluant was collected and analyzed for
isoflavones, phytic acid and protein. The amounts of isoflavones,
phytic acid and protein recovered are listed in Table VII.
[0062] Phytic acid was determined by pipetting 8.00 milliliters of
column eluate into a 30 milliliter beaker. Ethanol was evaporated
with a stream of compressed nitrogen. 15.0 milliliters of 0.05 M
malonic acid were added to the beaker. The pH was adjusted to 2.5
with 2 N NaOH. The sample was diluted to 25 milliliters with 0.05 M
malonic acid. 10.0 milliliters was pipetted into each of two vials.
5 mg of phytase (EC 3.1.3.8, Sigma # P-9792, 3.5U/mg solid) was
added to one of the two vials. Both vials were incubated at
37.degree. C. for fourteen hours. The vials were cooled to room
temperature and 10.0 milliliters of 20% trichloroacetic acid (w/v)
was added to each vial. The vials were capped, mixed well and 3-4
milliliters were filtered through a 0.45 micrometer membrane
(Gelman Acrodisc, P/N 4497). 1.00 milliliters of filtrate was
tested for inorganic phosphorus by Sigma Test Kit 670-A (inorganic
phosphorus; colormetric endpoint method; Sigma 1999 Catalog).
[0063] Protein was determined by pipetting 5.00 milliliters of
column eluate into a 2 dram vial. The sample was evaporated to
dryness with a stream of compressed nitrogen. The residue was
suspended in 2 milliliters of 6 M HCl and transferred into a 2
milliliter ampule. The ampule was nitrogen-blanketed, flame-sealed
and heated at 110.degree. C. for 22 hours. The ampule was cooled to
room temperature and the sample evaporated to dryness. The residue
was reconstituted in 2.00 milliliters of Beckman Na-S buffer and
tested for amino acids on a Beckman Model 6300 Automated Amino Acid
Analyzer (i.e., by ion exchange chromatography, post-column
ninhydrin derivatization, and visible absorbance detection).
Example H
[0064] Method Utilizing Sequential Eluants.
[0065] IEX Resin and columns were prepared as described above in
Example A.
[0066] For this example, three different sequential eluants were
utilized, with varying amounts of each sequential eluant passed
through the particular column. In the first column, three different
eluants were utilized sequentially. The first eluant was 20%
glacial acetic acid in 60% ethanol; 14 bed volumes of this eluant
were utilized. The second eluant was 20% glacial acetic acid in
water; 14 bed volumes of this eluant were utilized. The third
eluant was 0.48 M HCl in water; two fractions of 20 bed volumes
each were utilized.
[0067] In the second column, three different eluants were utilized
sequentially. The first eluant was 20 g/L citric acid in 60%
ethanol; 14 bed volumes of this eluant were utilized. The second
eluant was 20 g/L citric acid in water; 14 bed volumes of this
eluant were utilized. The third eluant was 0.48 M HCl in water; two
fractions of 20 bed volumes each were utilized.
[0068] In the third column, two different eluants were utilized
sequentially. The first eluant was 0.48 M HCl in 60% ethanol; 14
bed volumes of this eluant were utilized. The second eluant was
0.48 M HCl in water; two fractions were utilized, the first of 14
bed volumes and the second of 20 bed volumes.
[0069] All eluants were passed through the columns at approximately
3.6 bed volumes per hour. Eluates were collected and isoflavones
and phytic acid measured by the procedures described above
(Examples A and F). The amounts of isoflavones and phytic acid
recovered are listed in Table VIII.
Example I
[0070] Method Utilizing an Alkylsilane Active Surface with Various
Eluants.
[0071] Seven cartridges, each packed with 100 mg of octadecylsilane
(C18) bonded phase medium, were prepared by conditioning each with
20 bed volumes of methanol, and then rinsing each with 20 bed
volumes of laboratory water. Onto each cartridge was loaded 1.00
milliliters of a 1% (w/w) slurry of soy protein isolate in
laboratory water.
[0072] In this experiment, seven different eluants were prepared
and utilized. The eluants were as follows: water; 20% glacial
acetic acid in water; 20 g/L citric acid in water; ethanol; 20%
glacial acetic acid in 60% ethanol; 20 g/L citric acid in 60%
ethanol; and 10% glacial acetic acid and 10 g/L citric acid in 60%
ethanol. Ten bed volumes of eluant were passed through each
cartridge at a rate of four bed volumes per minute. Eluates were
collected, and isoflavones measured by the procedure described
above (Example A). The isoflavone recoveries are listed in Table
IX.
Example J
[0073] Method Utilizing Glacial Acetic Acid and Ethanol in the
Aqueous Medium for Isoflavone Removal and Hydrochloric Acid in the
Aqueous Medium for Phytate Removal.
[0074] From a well-stirred slurry of soy protein isolate (2.0 g of
soy protein isolate in 100 milliliters of laboratory water) are
removed 2 milliliters for control testing of the isoflavone and
phytate contents. To the remainder of the suspension are added 10.0
g of anion exchange resin (IRA-910 in the chloride form). The whole
is stirred magnetically for about 60 minutes, whereupon the
suspension is decanted from the anion exchange resin beads. The
latter are rapidly rinsed twice with laboratory water (100
milliliters) by decanting, and then slurried with a further 100
milliliters of laboratory water and transferred to a glass column
chromatography tube (2.5.times.10 cm) equipped with a support frit
and an outlet valve. The resin is rinsed with a further 100
milliliters of water, the eluant flow rate being adjusted to 0.5
column volumes/minute. Two hundred milliliters of an eluant (the
aqueous medium for isoflavone removal) composed of ethanol, glacial
acetic acid, and laboratory water in the ratio by volume of 3:1:1
are passed through the resin bed at 0.5 column volumes/minute. The
column eluate is collected and tested for isoflavones by gradient
elution reverse phase HPLC. Two hundred milliliters of an eluant
(the aqueous medium for phytate removal) composed of 0.48M
hydrochloric acid in deionized water are passed through the resin
bed at 0.5 column volumes/minute. The column eluate is collected
and tested for phytic acid by an enzymatic/colorimetric method (the
enzyme phytase is used to selectively release phosphate from phytic
acid, and the phosphate is then quantified as inorganic phosphorus
by the phosphomolybdate colorimetric method).
Example K
[0075] Method Utilizing Citric Acid and Ethanol in the Aqueous
Medium for Isoflavone Removal and Sulfuric Acid in the Aqueous
Medium for Phytate Removal.
[0076] The procedure is followed as set forth in Example 1 except
that the aqueous medium for isoflavone in Example J is replaced
with 20 g/L citric acid in 60% (v/v) ethanol, and the aqueous
medium for phytate removal is replaced with 0.24M sulfuric acid in
deionized water.
Example L
[0077] Method Utilizing Hydrochloric Acid and Ethanol in the
Aqueous Medium for Isoflavone Removal and Sodium Hydroxide in the
Aqueous Medium for Phytate Removal.
[0078] The procedure is followed as set forth in Example J, except
that the aqueous medium for isoflavone removal in Example J is
replaced with 0.05M hydrochloric acid in 60% (v/v) ethanol, and the
aqueous medium for isoflavone is replaced with 5% (w/v) sodium
hydroxide in deionized water. The flow rate (for both aqueous
media) is also changed to 2 column volumes/minute.
Example M
[0079] Method Utilizing C18 Bonded Phase Medium.
[0080] From a well-stirred slurry of soy protein isolate (2.0 g of
soy protein isolate in 100 milliliters of laboratory water) is
removed 2 milliliters for control testing of the isoflavone and
phytate contents. A cartridge containing 100 mg of a C18 bonded
phase medium is conditioned with 2 column volumes of ethanol and
two column volumes of laboratory water. One milliliter of the soy
protein isolate suspension is passed through the C18 bonded phase
medium at a flow rate of 1 column volume/minute. The column is
rinsed with one milliliter of laboratory water. The column eluate
(i.e., from the load+the rinse) is collected and tested for phytic
acid by an enzymatic/colorimetric method (as described above). Two
milliliters of an eluant composed of 20 g/L of citric acid in 60%
ethanol are passed through the cartridge at a flow rate of 1 column
volume/minute. The column eluate is collected and tested for
isoflavones by gradient elution reverse phase HPLC.
[0081] Particular embodiments have been described above that fall
within the scope of the invention as set forth in the claims. These
embodiments are not intended to limit the scope of the invention to
the specific forms disclosed. The invention is intended to cover
all modifications and alternative forms falling within the spirit
and scope of the invention.
2TABLE I Isoflavone Recovery Comparison Isoflavone Recovery (% of
IEX Bound) Fraction 1 Fraction 2 Total IEX* Eluant Composition (7
BV)** (7 BV)** (14 BV)** 0.48 M HCl in 60% ethanol 46 18 64 20%
glacial acetic acid in 60% 46 16 62 ethanol 20 g/L citric acid in
60% ethanol 36 26 62 *IEX = ion exchange resin **BV = bed volumes
of eluant; flow rate = 3.6 bed volumes per hour
[0082]
3TABLE II Isoflavone Recovery vs. Eluant Alcohol Concentration -
Isoflavone Elution from IRA-910 IEX Column. Isoflavone Recovery (%
IEX Column Eluant Composition** of IEX Bound) 20 g/L citric acid
monohydrate in 0% ethanol (v/v) 7 20 g/L citric acid monohydrate in
10% ethanol (v/v) 12 20 g/L citric acid monohydrate in 20% ethanol
(v/v) 19 20 g/L citric acid monohydrate in 60% ethanol (v/v) 64 20
g/L citric acid monohydrate in 90% ethanol (v/v) 49 *all eluants
were prepared with laboratory water and isoflavones were eluted
with 14 bed volumes at a rate of 3.6 bed volumes per hour.
[0083]
4TABLE III Isoflavone Removal vs. Eluant Composition Isoflavones*
Removed by 5 CV** at 1 Eluant Composition CV/minute (% of
(percentages are volume basis) IEX Bound) 50% ethanol, 20% glacial
acetic acid, 30% water 46.6 60% ethanol, 20% glacial acetic acid,
20% water 49.4 70% ethanol, 20% glacial acetic acid, 10% water 38.3
80% ethanol, 20% glacial acetic acid, 0% water 18.4 60% ethanol,
30% glacial acetic acid, 10% water 41.0 60% ethanol, 40% glacial
acetic acid, 0% water 39.7 *Isoflavones measured were daidzein and
genistein. **column volumes
[0084]
5TABLE IV Isoflavone Recovery vs. Eluant Acetic Acid Isoflavones in
10 Isoflavone Eluant Composition CV* Recovery** 60% ethanol, 0%
glacial acetic acid 5.0 mg/L 9.3% 60% ethanol, 10% glacial acetic
acid 24.7 mg/L 46.2% 60% ethanol, 20% glacial acetic acid 29.3 mg/L
54.8% 60% ethanol, 30% glacial acetic acid 28.8 mg/L 53.95% 60%
ethanol, 40% glacial acetic acid 20.6 mg/L 38.4% *10 column volumes
of eluant at a rate of approximately 10 column volumes per hour.
**recovery of total aglycones bound by the IRA-910 IEX resin
[0085]
6TABLE V Isoflavone Recovery from IRA-910 IEX Column Isoflavone
Recovery (% IEX Column Eluant Composition* of IEX Bound) 20 g/L
citric acid monohydrate in 60% ethanol 55 20% glacial acetic acid
in 60% ethanol 56 10 g/L citric acid in l0% glacial acetic acid in
53 60% ethanol *isoflavones were eluted with 14 bed volumes at a
rate of 3.6 bed volumes per hour.
[0086]
7TABLE VI Phytic Acid Removal Comparison mg of Phytic Acid removed
IEX Eluant Composition from IEX Column* 20% glacial acetic acid in
60% ethanol <1 20% glacial acetic acid in water <1 20 g/L
citric acid in 60% ethanol <1 20 g/L citric acid in water <1
0.48 M HCl in 60% ethanol 19 0.48 M HCl in water 30 *mg phytic acid
removed by 14 bed volumes of eluant at a rate of 3.6 bed volumes
per hour.
[0087]
8TABLE VII IEX Elution vs. Hydrochloric Acid Concentration IEX
Eluant HCl Conc. pH* Isoflavones Phytic Acid Protein A 0.5 N 0.30
857 .mu.g 22.7 mg 11.6 mg B 0.4 N 0.40 903 .mu.g 15.8 mg 11.9 mg C
0.3 N 0.52 820 .mu.g 9.35 mg 10.2 mg D 0.2 N 0.70 904 .mu.g 5.61 mg
10.9 mg E 0.1 N 1.00 805 .mu.g 2.53 mg 9.5 mg *pH values are
calculated as the negative log the hydrogen ion concentration.
"isoflavones" = aglycone sum of daidzein, glycitein, and genistein
compounds "protein" = (dehydrated) amino acid total after acid
hydrolysis
[0088]
9TABLE VIII Sequential Elution of Isoflavones and Phytic Acid IEX
Isoflavone Phytic Col- # of Recovery (% Acid umn Eluant Composition
BV* of IEX Bound) (mg) A 20% glacial acetic acid in 60% 14 62 <1
ethanol 20% glacial acetic acid in water 14 -- <1 0.48 M HCl in
water 20 -- 50 0.48 M HCl in water 20 -- 24 B 20 g/L citric acid
monohydrate 14 62 <1 in 60% ethanol 20 g/L citric acid
monohydrate 14 -- <1 in water 0.48 M HCl in water 20 -- 47 0.48
M HCl in water 20 -- 22 C 0.48 M HCl in 60% ethanol 14 64 19 0.48 M
HCl in water 14 -- 30 0.48 M HCl in water 20 -- 20 *BV = bed
volumes of eluant at a rate of approximately 3.6 bed volumes per
hour.
[0089]
10TABLE IX Isoflavone Recovery from C18 Bonded Phase Medium -
Eluant Recovery Comparison Isoflavone Recovery Eluant (10 bed
volumes) (% of C18 Bound) Water <1% 20% glacial acetic acid in
water 30% 20 g/L citric acid in water <1% ethanol 96% 20%
glacial acetic acid in 60% ethanol 89% 20 g/L citric acid in 60%
ethanol 92% 10% glacial acetic acid, 10 g/L citric acid in 60% 95%
ethanol
* * * * *