U.S. patent application number 10/510766 was filed with the patent office on 2005-11-10 for canola protein isolate compositions.
Invention is credited to Logie, James, Milanova, Radka.
Application Number | 20050249828 10/510766 |
Document ID | / |
Family ID | 29254469 |
Filed Date | 2005-11-10 |
United States Patent
Application |
20050249828 |
Kind Code |
A1 |
Logie, James ; et
al. |
November 10, 2005 |
Canola protein isolate compositions
Abstract
A new canola protein isolate is provided along with a new canola
protein. The new canola protein isolate is obtained from the
supernatant from the production of a canola protein micellar mass
and contains a predominance of 2S protein. The canola protein
isolate derived from PMM contains a predominance of a 7S protein.
Compositions of the canola protein isolate are provided.
Inventors: |
Logie, James; (Winnipeg
Manitoba, CA) ; Milanova, Radka; (Manitoba,
CA) |
Correspondence
Address: |
Michael I Stewart
Sim & McBurney
6th Floor
330 University Avenue
Toronto Ontario
M5G 1R7
CA
|
Family ID: |
29254469 |
Appl. No.: |
10/510766 |
Filed: |
April 29, 2005 |
PCT Filed: |
April 15, 2003 |
PCT NO: |
PCT/CA03/00557 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
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60372165 |
Apr 15, 2002 |
|
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60430687 |
Dec 4, 2002 |
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Current U.S.
Class: |
424/755 ;
514/21.2; 530/370 |
Current CPC
Class: |
A23J 1/14 20130101; A61K
38/168 20130101; A61K 36/31 20130101; A23J 3/14 20130101; C07K
14/415 20130101 |
Class at
Publication: |
424/755 ;
514/002; 530/370 |
International
Class: |
A61K 038/16; A61K
035/78; C07K 014/415 |
Claims
What we claim is:
1. A canola protein isolate having a protein content of at least
about 90 wt % on a dry weight basis and at a Kjeldahl nitrogen
conversion of N.times.6.25 and which exhibits a protein profile
which is: about 60 to about 95% wt % of 2S protein, about 5 to
about 40 wt % of 7S protein, and 0 to about 5 wt % of 12S
protein.
2. The isolate of claim 1 which exhibits a protein profile which
is: about 70 to about 95 wt % of 2S protein, about 5 to about 30 wt
% of 7S protein, and 0 to about 2 wt % of 12S protein.
3. The isolate of claim 2 having a protein content of at least
about 100 wt %.
4. A canola protein isolate composition comprising: (1) a first
canola protein isolate having a protein content of at least about
90 wt % on a dry weight basis and at a Kjeldahl nitrogen conversion
of N.times.6.25 and which exhibits a protein profile which is:
about 60 to about 98 wt % of 7S protein, about 1 to about 15 wt %
of 12S protein, 0 to about 25 wt % of 2S protein, and (2) a second
canola protein isolate having a protein content of at least about
90 wt % on a dry weight basis and a Kjeldahl nitrogen conversion of
N.times.6.25 and which exhibits a protein profile which is: about
60 to about 95 wt % of 2S protein about 5 to about 40 wt % of 7S
protein 0 to about 5 wt % of 12S protein.
5. The composition of claim 4 wherein said first canola protein
isolate and said second canola protein isolate are present in said
composition in a weight ratio of about 5:95 to about 95:5.
6. The composition of claim 5 wherein said first canola protein
isolate exhibits a protein profile which is: about 88 to about 98
wt % of 7S protein, about 1 to about 10 wt % of 12S protein, and 0
to about 6 wt % of 2S protein.
7. The composition of claim 6 wherein said first canola protein
isolate has a protein content of at least about 100 wt %.
8. The composition of claim 5 wherein the second canola protein
isolate exhibits a protein profile which is: about 70 to about 95
wt % of 2S protein, about 5 to about 30 wt % of 7S protein, and 0
to about 2 wt % of 12S protein.
9. The composition of claim 8 wherein the second canola protein
isolate has a protein content of at least about 100 wt %.
10. An isolated and purified 7S protein of canola.
11. The protein of claim 10 having a molecular weight, as
determined by MALDI-MS, of about 145 kDa.
12. The protein of claim 11 which is comprised of three subunits,
each sized approximately 413 amino acids.
13. The protein of claim 12 having an amino acid analysis
substantially as set forth in Table VII or Table VIII.
14. The protein of claim 10 which is isolated and purified from a
canola protein micellar mass.
15. The protein of claim 14 wherein said protein micellar mass is
formed by diluting a concentrated saline solution of canola protein
extracted from canola oil seed meal under mildly acidic
conditions.
16. A composition, comprising a mixture of an isolated and purified
7S protein of canola protein and at least one additional isolated
and purified canola protein extracted from the group consisting of
the 2S and 12S canola proteins.
17. A method of isolating and purifying an individual canola
protein from a canola protein isolate containing a mixture of at
least two different canola proteins selected from the group
consisting of the 2S, 7S and 12S proteins, which comprises:
providing said canola protein isolate having a protein contents of
at least about 90 wt % on a dry weight basis and at a Kjeldahl
nitrogen conversion of N.times.6.25, solubilizing the canola
protein isolate, subjecting the resulting protein solution to
preparative high pressure liquid chromatography or equivalent
procedure separating proteins on the basis of molecular size,
collecting fractions of eluant containing the individual selected
canola protein, subjecting the selected fractions to
ultrafiltration to reduce the volume of eluant, dialyzing the
concentrated eluant to remove solubilizing material, and drying the
dialyzate to recover the individual canola protein.
18. The method of claim 17 wherein said canola protein isolate is
one which exhibits a protein profile which is: about 60 to about 98
wt % of 7S protein, about 1 to about 15 wt % of 12S protein, and 0
to about 25 wt % of 2S protein.
19. The method of claim 18 wherein said canola protein exhibits a
protein profile which is: about 88 to about 98 wt % of 7S protein,
about 1 to about 10 wt % of 12S protein, and 0 to about 6 wt % of
2S protein.
20. The method of claim 18 wherein said canola protein isolate is
processed to isolate and purify the 7S and/or 12S proteins.
21. The method of claim 17 wherein said canola protein isolate is
one which exhibits a protein profile which is: about 60 to about 95
wt % of 2S protein, about 5 to about 30 wt % of 7S protein, 0 to
about 2 wt % of 12S protein.
22. The method of claim 21 wherein said canola protein isolate
exhibits a protein profile which is: about 70 to about 95 wt % of
2S protein, about 5 to about 30 wt % of 7S protein, 0 to about 2 wt
% of 12S protein.
23. The method of claim 21 wherein said canola protein isolate is
processed to isolate and purify the 2S protein.
Description
REFERENCE TO RELATED APPLICATIONS
[0001] This application claims priority pursuant to 35 USC 119(e)
from copending U.S. Patent Applications Nos. 60/372,165 filed Apr.
15, 2002 and 60/430,687 filed Dec. 4, 2002.
FIELD OF INVENTION
[0002] The present invention relates to canola protein isolate
compositions and individual protein components of such
compositions.
BACKGROUND TO THE INVENTION
[0003] Canola protein isolates can be formed from canola oil seed
meal. In copending U.S. Patent Applications Nos. 60/288,415 filed
May 4, 2001, 60/326,987 filed Oct. 5, 2001, 60/331,066 filed Nov.
7, 2001, 60/333,494 filed Nov. 26, 2001, 60/374,801 filed Apr. 24,
2002 and 10/137,391 filed May 3, 2002 all assigned to the assignee
hereof and the disclosures of which are incorporated herein by
reference, there is described a method of making canola protein
isolates from canola oil seed meal, such isolates having at least
100 wt % protein content (N.times.6.25). The procedure involves a
multiple step process comprising extracting canola oil seed meal
using a salt solution, separating the resulting aqueous protein
solution from residual oil seed meal, increasing the protein
concentration of the aqueous solution to at least about 200 g/L
while maintaining the ionic strength substantially constant by
using a selective membrane technique, diluting the resulting
concentrated protein solution into chilled water to cause the
formation of protein micelles, settling the protein micelles to
form an amorphous, sticky, gelatinous gluten-like protein micellar
mass (PMM), and recovering the protein micellar mass from
supernatant having a protein content of at least about 100 wt % as
determined by Kjeldahl nitrogen (N).times.6.25. As used herein,
protein content is determined on a dry weight basis. The recovered
PMM may be dried.
[0004] In one embodiment of the process described above and as
specifically described in Applications Nos. 60/326,987, 60/331,066,
60/333,494, 60/374,801 and 10/137,391, the supernatant from the PMM
settling step is processed to recover a protein isolate comprising
dried protein from the wet PMM and supernatant. This procedure may
be effected by initially concentrating the supernatant using
ultrafiltration membranes, mixing the concentrated supernatant with
the wet PMM and drying the mixture. The resulting canola protein
isolate has a high purity of at least about 90 wt % of protein
(N.times.6.25), preferably at least about 100 wt % protein
(N.times.6.25).
[0005] In another embodiment of the process described above and as
specifically described in Applications Nos. 60/333,494, 60/374,801
and 10/137,391, the supernatant from the PMM settling step is
processed to recover a protein from the supernatant. This procedure
may be effected by initially concentrating the supernatant using
ultrafiltration membranes and drying the concentrate. The resulting
canola protein isolate has a high purity of at least about 90 wt %
protein (N.times.6.25), preferably at least about 100 wt % protein
(N.times.6.25).
[0006] The procedures described in the aforementioned US Patent
Applications are essentially batch procedures. In copending U.S.
Patent Applications Nos. 60/331,646 filed Nov. 20, 2001, 60/383,809
filed May 30, 2002 and 10/298,678 filed Nov. 19, 2002. assigned to
the assignee hereof and the disclosures of which are incorporated
herein by reference, there is described a continuous process for
making canola protein isolates. In accordance therewith, canola oil
seed meal is continuously mixed with a salt solution, the mixture
is conveyed through a pipe while extracting protein from the canola
oil seed meal to form an aqueous protein solution, the aqueous
protein solution is continuously separated from residual canola oil
seed meal, the aqueous protein solution is continuously conveyed
through a selective membrane operation to increase the protein
content of the aqueous protein solution to at least about 200 g/L
while maintaining the ionic strength substantially constant, the
resulting concentrated protein solution is continuously mixed with
chilled water to cause the formation of protein micelles, and the
protein micelles are continuously permitted to settle while the
supernatant is continuously overflowed until the desired amount of
PMM has accumulated in the settling vessel. The PMM is removed from
the settling vessel and may be dried. The PMM has a protein content
of at least about 90 wt % as determined by Kjeldahl nitrogen
(N).times.6.25, preferably at least about 100 wt %
(N.times.6.25).
[0007] As described in the aforementioned U.S. Patent Applications
Nos. 60/326,987, 60/331,066, 60/333,494, 60/333,494, 60/374,801 and
10/137,391, the overflowed supernatant may be processed to recover
canola protein isolate therefrom.
[0008] Canola seed is known to contain about 10 to about 30 wt %
proteins and several different protein components have been
identified. These proteins are distinguished by different
sedimentation coefficients (S). These known and identified proteins
include a 12S globulin, known as cruciferin, and a 2S storage
protein, known as napin.
[0009] Canola is also known as rapeseed or oil seed rape.
SUMMARY OF INVENTION
[0010] In addition to the 12S and 2S proteins, we have found that
the procedures described above for the isolation of canola protein
isolate produces significant quantities of a 7S protein, which
appears to be a new protein. Accordingly, in one aspect of the
invention, there is provided an isolated and purified 7S protein of
canola.
[0011] We have also now found that the relative proportions of the
12S, 7S and 2S proteins differ as between protein micellar
mass-derived canola protein isolate and supernatant-derived canola
protein isolate, prepared using the above-described procedures. In
particular, it has been found that PMM-derived canola protein
isolate having a protein content of at least about 90 wt %
(N.times.6.25), preferably at least about 100 wt %, has a protein
component content of about 60 to about 98 wt % of 7S protein, about
1 to about 15 wt % of 12S protein and 0 to about 25 wt % of 2S
protein. By way of contrast, the supernatant-derived canola protein
isolate having a protein content of at least about 90 wt %
(N.times.6.25), preferably at least about 100 wt %, has a protein
component content of 0 to about 5 wt % of 12S protein, about 5 to
about 40 wt % of the 7S protein and about 60 to about 95 wt % of 2S
protein.
[0012] The PMM-derived canola protein isolate preferably has a
protein component content of about 88 to about 98 wt % of 7S
protein, about 1 to about 10 wt % of 12S protein and 0 to about 6
wt % of 2S protein while the supernatant-derived canola protein
isolate preferably has a protein component content of about 70 to
about 95 wt % of 2S protein, about 5 to about 30 wt % of 7S protein
and 0 to about 2 wt % of 12S protein.
[0013] The protein component profiles, therefore, are very
different for the two canola protein isolates. In the case of the
PMM-derived canola protein isolate, the predominant protein species
is the 7S protein while in the case of the supernatant-derived
canola protein isolate, the predominant species is the 2S protein.
These differences lead to different behaviour in environments where
the canola protein isolates are employed.
[0014] The different protein content profiles enable canola protein
isolate compositions to be provided wherein mixtures of the two
different canola protein isolates are combined in any desired
proportion for a specific application to provide any desired
2S/7S/12S protein profile as between that of the PMM-derived
isolate and that of the supernatant-derived isolate.
[0015] The supernatant-derived isolate is a novel canola protein
isolate. Accordingly, in another aspect of the present invention,
there is provided a canola protein isolate having a protein content
of at least about 90 wt %, preferably at least about 100 wt %, on a
dry weight basis and at a Kjeldahl nitrogen conversion of
N.times.6.25 and which exhibits a protein profile which is about 60
to about 95 wt % of 2S protein, about 5 to about 40 wt % of 7S
protein and 0 to about 5 wt % of 12S protein. Preferably, the
supernatant-derived canola protein isolate preferably exhibits a
protein profile which is about 70 to about 95 wt % of 2S protein,
about 5 to about 30 wt % of 7S protein and 0 to about 2 wt % of 12S
protein.
[0016] The combination of the PMM-derived canola protein isolate
product in any desired proportion with any desired proportion of
the supernatant-derived canola protein isolate product to provide a
composition containing desired proportions of 2S, 7S and 12S canola
protein is a novel canola protein isolate composition. Accordingly,
in another aspect, the present invention provides a canola protein
isolate composition comprising (1) a first canola protein isolate
having a protein content of at least about 90 wt %, preferably at
least about 100 wt %, on a dry weight basis and at a Kjeldahl
nitrogen conversion of N.times.6.25 and which exhibits a protein
profile which is about 60 to about 98 wt % of 7S protein, about 1
to about 15 wt % of 12S protein and 0 to about 25 wt % of 2S
protein, and (2) a second canola protein isolate having a protein
content of at least about 90 wt %, preferably at least about 100 wt
%, on a dry weight basis and a Kjeldahl nitrogen conversion of
N.times.6.25 and which exhibits a protein profile which is about 60
to about 95 wt % of 2S protein, about 5 to about 40 wt % of 7S
protein and 0 to about 5 wt % of 12S protein.
[0017] The first canola protein isolate preferably exhibits a
protein profile which is about 88 to about 98 wt % of 7S protein,
about 1 to about 10 wt % of 12S protein and 0 to about 6 wt % of 2S
protein. The second canola protein isolate preferably exhibits the
preferred protein profile of the novel canola protein isolate
described herein.
[0018] Variation in the proportions of the 12S/7S/2S proteins in a
specific PMM-derived canola protein isolate and/or a specific
supernatant-derived canola protein isolate may be achieved by
varying the process conditions used to derive the respective canola
protein isolates from canola oil seed meal.
[0019] The individual components of the canola protein isolate have
been isolated, purified and characterized. The individual proteins
may be separated from the respective PMM- and supernatant-derived
canola protein isolates produced according to the procedures
described above by any conventional procedure whereby protein
mixtures may be separated based on size differences, preferably by
preparative high pressure liquid chromatography (HPLC), followed by
ultrafiltration to reduce the volume of eluant and dialysis to
reduce residual solubilizing salt content. The dialyzed material
may be dried to remove the pure protein.
[0020] The procedure for the isolating and purifying an individual
canola proteins from a canola protein isolate containing a mixture
of at least two different canola proteins selected from the group
consisting of the 2S, 7S and 12S proteins, including the
PMM-derived canola protein isolate and the supernatant-derived
canola protein isolate described herein, constitutes a further
aspect of the invention.
[0021] The 2S protein usually is isolated and purified from the
supernatant-derived canola protein isolate while the 7S and 12S
proteins usually are isolated and purified from the protein
micellar mass. The 2S protein has a molecular weight, as determined
by MALDI-MS, of about 14 kDa, the 7S protein has a molecular
weight, as determined by MALDI-MS, of about 145 kDa and the 12S
protein has a molecular weight, as determined by MALDI-MS, of about
290 kDa.
[0022] The 7S and 12S proteins have a very similar amino acid
profile and are made up of numbers of the same subunits each
containing approximately 413 amino acids. The 7S protein contains
approximately 1240 amino acids while the 12S protein contains
approximately 2480 amino acids. The 2S protein has a quite
different amino acid profile from the 7S and 12S proteins and
contains approximately 120 amino acids. The amino acid profiles
obtained are set forth below in the Examples.
[0023] The provision of the individual isolated and purified 2S, 7S
and 12S canola proteins enables there to be produced compositions
of the 7S protein with different proportions of the 12S and/or 2S
proteins in order to provide compositions which may have unique
functionalities in the utilization of the purified proteins. Such
compositions constitute another aspect of the present
invention.
[0024] The canola protein isolates produced according to the
process herein may be used in conventional applications of protein
isolates, such as, protein fortification of processed foods,
emulsification of oils, body formers in baked goods and foaming
agents in products which entrap gases. In addition, the canola
protein isolates may be formed into protein fibers, useful in meat
analogs, may be used as an egg white substitute or extender in food
products where egg white is used as a binder. The canola protein
isolate may be used as nutritional supplements. Other uses of the
canola protein isolate are in pet foods, animal feed and in
industrial and cosmetic applications and in personal care products.
The individual isolated and purified proteins may be put to similar
use.
BRIEF DESCRIPTION OF DRAWINGS
[0025] FIG. 1 is a schematic flow sheet of a procedure for
producing canola protein isolates of differing protein profiles
according to one embodiment of the invention;
[0026] FIG. 2 is a schematic flow sheet of a continuous procedure
for producing canola protein isolates of differing protein profiles
according to another embodiment of the invention;
[0027] FIGS. 3 to 5 are analytical HPLC chromatograms of extracts
from low temperature meal; and
[0028] FIGS. 6 and 7 contain overlap of seven consecutive
preparative HPLC runs for the collection of individual 2S, 7S and
12S canola proteins.
GENERAL DESCRIPTION OF INVENTION
[0029] The respective PMM-derived canola protein isolate and
supernatant-derived canola protein isolate may be isolated from
canola oil seed meal by either a batch process or a continuous
process or a semi-continuous process as generally described in the
aforementioned United States patent applications.
[0030] The initial step of the process of providing the canola
protein isolates involves solubilizing proteinaceous material from
canola oil seed meal. The proteinaceous material recovered from
canola seed meal may be the protein naturally occurring in canola
seed or other oil seed or the proteinaceous material may be a
protein modified by genetic manipulation but possessing
characteristic hydrophobic and polar properties of the natural
protein. The canola meal may be any canola meal resulting from the
removal of canola oil from canola oil seed with varying levels of
non-denatured protein, resulting, for example, from hot hexane
extraction or cold oil extrusion methods. The removal of canola oil
from canola oil seed usually is effected as a separate operation
from the protein isolate recovery procedure described herein.
[0031] Protein solubilization is effected most efficiently by using
a food grade salt solution since the presence of the salt enhances
the removal of soluble protein from the oil seed meal. Where the
canola protein isolate is intended for non-food uses,
non-food-grade chemicals may be used. The salt usually is sodium
chloride, although other salts, such as, potassium chloride, may be
used. The salt solution has an ionic strength of at least about
0.10, preferably at least about 0.15, to enable solubilization of
significant quantities of protein to be effected. As the ionic
strength of the salt solution increases, the degree of
solubilization of protein in the oil seed meal initially increases
until a maximum value is achieved. Any subsequent increase in ionic
strength does not increase the total protein solubilized. The ionic
strength of the food grade salt solution which causes maximum
protein solubilization varies depending on the salt concerned and
the oil seed meal chosen.
[0032] In view of the greater degree of dilution required for
protein precipitation with increasing ionic strengths, it is
usually preferred to utilize an ionic strength value less than
about 0.8, and more preferably a value of about 0.15 to about
0.6.
[0033] In a batch process, the salt solubilization of the protein
is effected at a temperature of at least about 5.degree. C. and
preferably up to about 35.degree. C., preferably accompanied by
agitation to decrease the solubilization time, which is usually
about 10 to about 60 minutes. It is preferred to effect the
solubilization to extract substantially as much protein from the
oil seed meal as is practicable, so as to provide an overall high
product yield.
[0034] The lower temperature limit of about 5.degree. C. is chosen
since solubilization is impractically slow below this temperature
while the upper preferred temperature limit of about 35.degree. C.
is chosen since the process becomes uneconomic at higher
temperature levels in a batch mode.
[0035] In a continuous process, the extraction of the protein from
the canola oil seed meal is carried out in any manner consistent
with effecting a continuous extraction of protein from the canola
oil seed meal. In one embodiment, the canola oil seed meal is
continuously mixed with a food grade salt solution and the mixture
is conveyed through a pipe or conduit having a length and at a flow
rate for a residence time sufficient to effect the desired
extraction in accordance with the parameters described herein. In
such continuous procedure, the salt solubilization step is effected
rapidly, in a time of up to about 10 minutes, preferably to effect
solubilization to extract substantially as much protein from the
canola oil seed meal as is practicable. The solubilization in the
continuous procedure preferably is effect at elevated temperatures,
preferably above about 35.degree. C., generally up to about
65.degree. C.
[0036] The aqueous food grade salt solution and the canola oil seed
meal have a natural pH of about 5 to about 6.8 to enable a protein
isolate to be formed by the micellar route, as described in more
detail below.
[0037] At and close to the limits of the pH range, protein isolate
formation occurs only partly through the micelle route and in lower
yields than attainable elsewhere in the pH range. For these
reasons, mildly acidic pH values of about 5.3 to about 6.2 are
preferred.
[0038] The pH of the salt solution may be adjusted to any desired
value within the range of about 5 to about 6.8 for use in the
extraction step by the use of any convenient acid, usually
hydrochloric acid, or alkali, usually sodium hydroxide, as
required.
[0039] The concentration of oil seed meal in the food grade salt
solution during the solubilization step may vary widely. Typical
concentration values are about 5 to about 15% w/v.
[0040] The protein extraction step with the aqueous salt solution
has the additional effect of solubilizing fats which may be present
in the canola meal, which then results in the fats being present in
the aqueous phase.
[0041] The protein solution resulting from the extraction step
generally has a protein concentration of about 5 to about 40 g/L,
preferably about 10 to about 30 g/L.
[0042] The aqueous phase resulting from the extraction step then
may be separated from the residual canola meal, in any convenient
manner, such as by employing vacuum filtration, followed by
centrifugation and/or filtration to remove residual meal. The
separated residual meal may be dried for disposal.
[0043] The colour of the final canola protein isolate can be
improved in terms of light colour and less intense yellow by the
mixing of powdered activated carbon or other pigment adsorbing
agent with the separated aqueous protein solution and subsequently
removing the adsorbent, conveniently by filtration, to provide a
protein solution. Diafiltration also may be used for pigment
removal.
[0044] Such pigment removal step may be carried out under any
convenient conditions, generally at the ambient temperature of the
separated aqueous protein solution, employing any suitable pigment
adsorbing agent. For powdered activated carbon, an amount of about
0.025% to about 5% w/v, preferably about.0.05% to about 2% w/v, is
employed.
[0045] Where the canola seed meal contains significant quantities
of fat, as described in U.S. Pat. Nos. 5,844,086 and 6,005,076,
assigned to the assignee hereof and the disclosures of which are
incorporated herein by reference, then the defatting steps
described therein may be effected on the separated aqueous protein
solution and on the concentrated aqueous protein solution discussed
below. When the colour improvement step is carried out, such step
may be effected after the first defatting step.
[0046] As an alternative to extracting the oil seed meal with an
aqueous salt solution, such extraction may be made using water
alone, although the utilization of water alone tends to extract
less protein from the oil seed meal than the aqueous salt solution.
Where such alternative is employed, then the salt, in the
concentrations discussed above, may be added to the protein
solution after separation from the residual oil seed meal in order
to maintain the protein in solution during the concentration step
described below. When a colour removal step and/or a first fat
removal step is carried out, the salt generally is added after
completion of such operations.
[0047] Another alternative procedure is to extract the oil seed
meal with the food grade salt solution at a relatively high pH
value above about 6.8, generally up to about 9.9. The pH of the
food grade salt solution, may be adjusted in pH to the desired
alkaline value by the use of any convenient food-grade alkali, such
as aqueous sodium hydroxide solution. Alternatively, the oil seed
meal may be extracted with the salt solution at a relatively low pH
below about pH 5, generally down to about pH 3. Where such
alternative is employed, the aqueous phase resulting from the oil
seed meal extraction step then is separated from the residual
canola meal, in any convenient manner, such as by employing vacuum
filtration, followed by centrifugation and/or filtration to remove
residual meal. The separated residual meal may be dried for
disposal.
[0048] The aqueous protein solution resulting from the high or low
pH extraction step then is pH adjusted to the range of about 5 to
about 6.8, preferably about 5.3 to about 6.2, as discussed above,
prior to further processing as discussed below. Such pH adjustment
may be effected using any convenient acid, such as hydrochloric
acid, or alkali, such as sodium hydroxide, as appropriate.
[0049] The aqueous protein solution then is concentrated to
increase the protein concentration thereof while maintaining the
ionic strength thereof substantially constant. Such concentration
generally is effected to provide a concentrated protein solution
having a protein concentration of at least about 200 g/L,
preferably at least about 250 g/L.
[0050] The concentration step may be effected in any convenient
manner consistent with batch or continuous operation, such as by
employing any convenient selective membrane technique, such as
ultrafiltration or diafiltration, using membranes, such as
hollow-fibre membranes or spiral-wound membranes, with a suitable
molecular weight cut-off, such as about 3000 to about 50,000
daltons, having regard to differing membrane materials and
configurations, and, for continuous operation, dimensioned to
permit the desired degree of concentration as the aqueous protein
solution passes through the membranes.
[0051] The concentration step may be effected at any convenient
temperature, generally about 20.degree. to about 60.degree. C., and
for the period of time to effect the desired degree of
concentration. The temperature and other conditions used to some
degree depend upon the membrane equipment used to effect the
concentration and the desired protein concentration of the
solution.
[0052] The concentrating of the protein solution to a concentration
above about 200 g/L in this step not only increases the process
yield to levels above about 40% in terms of the proportion of
extracted protein which is recovered as dried protein isolate,
preferably above about 80%, but also decreases the salt
concentration of the final protein isolate after drying. The
ability to control the salt concentration of the isolate is
important in applications of the isolate where variations in salt
concentrations affect the functional and sensory properties in a
specific food application.
[0053] As is well known, ultrafiltration and similar selective
membrane techniques permit low molecular weight species to pass
therethrough while preventing higher molecular weight species from
so doing. The low molecular weight species include not only the
ionic species of the salt but also low molecular weight materials
extracted from the source material, such as, carbohydrates,
pigments and anti-nutritional factors, as well as any low molecular
weight forms of the protein. The molecular weight cut-off of the
membrane is usually chosen to ensure retention of a significant
proportion of the protein in the solution, while permitting
contaminants to pass through having regard to the different
membrane materials and configurations.
[0054] Depending on the temperature employed in the concentration
step, the concentrated protein solution may be warmed to a
temperature of at least about 200, and up to about 60.degree. C,
preferably about 25.degree. to about 40.degree. C., to decrease the
viscosity of the concentrated protein solution to facilitate
performance of the subsequent dilution step and micelle formation.
The concentrated protein solution should not be heated beyond a
temperature above which the temperature of the concentrated protein
solution does not permit micelle formation on dilution by chilled
water. The concentrated protein solution may be subject to a
further defatting operation, if required, as described in U.S. Pat.
Nos. 5,844,086 and 6,005,076.
[0055] The concentrated protein solution resulting from the
concentration step and optional defatting step then is diluted to
effect micelle formation by mixing the concentrated protein
solution with chilled water having the volume required to achieve
the degree of dilution desired. Depending on the proportion of
canola protein desired to be obtained by the micelle route and the
proportion from the supernatant, the degree of dilution of the
concentrated protein solution may be varied. With higher dilution
levels, in general, a greater proportion of the canola protein
remains in the aqueous phase.
[0056] When it is desired to provide the greatest proportion of the
protein by the micelle route, the concentrated protein solution is
diluted by about 15 fold or less, preferably about 10 fold or
less.
[0057] The chilled water with which the concentrated protein
solution is mixed has a temperature of less than about 15.degree.
C., generally about 3.degree. to about 15.degree. C., preferably
less than about 10.degree. C., since improved yields of protein
isolate in the form of protein micellar mass are attained with
these colder temperatures at the dilution factors used.
[0058] In a batch operation, the batch of concentrated protein
solution is added to a static body of chilled water having the
desired volume, as discussed above. The dilution of the
concentrated protein solution and consequential decrease in ionic
strength causes the formation of a cloud-like mass of highly
associated protein molecules in the form of discrete protein
droplets in micellar form. In the batch procedure, the protein
micelles are allowed to settle in the body of chilled water to form
an aggregated, coalesced, dense, amorphous sticky gluten-like
protein micellar mass (PMM). The settling may be assisted, such as
by centrifugation. Such induced settling decreases the liquid
content of the protein micellar mass, thereby decreasing the
moisture content generally from about 70% by weight to about 95% by
weight to a value of generally about 50% by weight to about 80% by
weight of the total micellar mass. Decreasing the moisture content
of the micellar mass in this way also decreases the occluded salt
content of the micellar mass, and hence the salt content of dried
isolate.
[0059] Alternatively, the dilution operation may be carried out
continuously by continuously passing the concentrated protein
solution to one inlet of a T-shaped pipe, while the diluting water
is fed to the other inlet of the T-shaped pipe, permitting mixing
in the pipe. The diluting water is fed into the T-shaped pipe at a
rate sufficient to achieve the desired degree of dilution.
[0060] The mixing of the concentrated protein solution and the
diluting water in the pipe initiates the formation of protein
micelles and the mixture is continuously fed from the outlet from
the T-shaped pipe into a settling vessel, from which, when full,
supernatant is permitted to overflow. The mixture preferably is fed
into the body of liquid in the settling vessel in a manner which
minimizes turbulence within the body of liquid.
[0061] In the continuous procedure, the protein micelles are
allowed to settle in the settling vessel to form an aggregated,
coalesced, dense, amorphous, sticky, gluten-like protein micellar
mass (PMM) and the procedure is continued until a desired quantity
of the PMM has accumulated in the bottom of the settling vessel,
whereupon the accumulated PMM is removed from the settling
vessel.
[0062] The combination of process parameters of concentrating of
the protein solution to a protein content of at least about 200 g/L
and the use of a dilution factor less than about 15, result in
higher yields, often significantly higher yields, in terms of
recovery of protein in the form of protein micellar mass from the
original meal extract, and much purer isolates in terms of protein
content than achieved using any of the known prior art protein
isolate forming procedures discussed in the aforementioned US
patents.
[0063] By the utilization of a continuous process for the recovery
of canola protein isolate as compared to the batch process, the
initial protein extraction step can be significantly reduced in
time for the same level of protein extraction and significantly
higher temperatures can be employed in the extraction step. In
addition, in a continuous operation, there is less chance of
contamination than in a batch procedure, leading to higher product
quality and the process can be carried out in more compact
equipment.
[0064] The settled isolate is separated from the residual aqueous
phase or supernatant, such as by decantation of the residual
aqueous phase from the settled mass or by centrifugation. The PMM
may be used in the wet form or may be dried, by any convenient
technique, such as spray drying, freeze drying or vacuum drum
drying, to a dry form. The dry PMM has a high protein content, in
excess of about 90 wt % protein, preferably at least about 100 wt %
protein (calculated as Kjeldahl N.times.6.25), and is substantially
undenatured (as determined by differential scanning calorimetry).
The dry PMM isolated from fatty oil seed meal also has a low
residual fat content, when the procedures of U.S. Pat. Nos.
5,844,086 and 6,005,076 are employed, which may be below about 1 wt
%.
[0065] The PMM-derived canola protein isolate predominantly
consists of the 7S canola protein with minor quantities of the 12S
canola protein and optionally small quantities of the 2S canola
protein. In general, the PMN contains:
[0066] about 60 to about 98 wt % of 7S protein
[0067] about 1 to about 15 wt % of 12S protein
[0068] 0 to about 25 wt % of 2S protein
[0069] Preferably, the PMM contains:
[0070] about 88 to about 98 wt % of 7S protein
[0071] about 1 to about 10 wt % of 12S protein
[0072] 0 to about 6 wt % of 2S protein
[0073] The supernatant from the PMM formation and settling step
contains significant amounts of canola protein, not precipitated in
the dilution step, and is processed to recover canola protein
isolate therefrom. The supernatant from the dilution step,
following removal of the PMM, is concentrated to increase the
protein concentration thereof. Such concentration is effected using
any convenient selective membrane technique, such as
ultrafiltration using membranes with a suitable molecular weight
cut-off permitting low molecular weight species, including the salt
and other non-proteinaceous low molecular weight materials
extracted from the protein source material, to pass through the
membrane, while retaining canola protein in the solution.
Ultrafiltration membranes having a molecular weight cut-off of
about 3000 to 10,000 daltons, having regard to differing membrane
materials and configuration, may be used. Concentration of the
supernatant in this way also reduces the volume of liquid required
to be dried to recover the protein. The supernatant generally is
concentrated to a protein concentration of about 100 to about 400
g/L, preferably about 200 to about 300 g/L, prior to drying. Such
concentration operation may be carried out in a batch mode or in a
continuous operation, as described above for the protein solution
concentration step.
[0074] The concentrated supernatant may be dried by any convenient
technique, such as spray drying, freeze drying or vacuum drum
drying, to a dry form to provide a further canola protein isolate.
Such further canola protein isolate has a high protein content, in
excess of about 90 wt %, preferably at least about 100 wt % protein
(calculated as Kjeldahl N.times.6.25) and is substantially
undenatured (as determined by differential. scanning
calorimetry).
[0075] The dried supernatant predominantly consists of the 2S
canola protein with minor quantities of the 7S canola protein and
optionally small quantities of the 12S canola protein. In general,
the supernatant-derived canola protein isolate contains:
[0076] about 60 to about 95 wt % of 2S protein
[0077] about 5 to about 40 wt % of 7S protein
[0078] 0 to about 5 wt % of 12S protein
[0079] The supernatant-derived canola protein isolate preferably
contains:
[0080] about 70 to about 95 wt % of 2S protein
[0081] about 5 to about 30 wt % of 7S protein
[0082] 0 to about 2 wt % of 12S protein
[0083] If desired, at least a portion of the wet PMM may be
combined with at least a portion of the concentrated supernatant
prior to drying the combined protein streams by any convenient
technique to provide a combined canola protein isolate composition
according to one invention. The relative proportions of the
proteinaceous materials mixed together may be chosen to provide a
resulting canola protein isolate composition having a desired
profile of 2S/7S/12S proteins. Alternatively, the dried protein
isolates may be combined in any desired proportions to provide any
desired specific 2S/7S/12S protein profiles in the mixture and
thereby provide a composition according to the invention. The
combined canola protein isolate composition has a high protein
content, in excess of about 90 wt %, preferably at least about 100
wt %, (calculated as Kjeldahl N.times.6.25) and is substantially
undenatured (as determined by differential scanning
calorimetry).
[0084] In another alternative procedure, where a portion only of
the concentrated supernatant is mixed with a part only of the PMM
and the resulting mixture dried, the remainder of the concentrated
supernatant may be dried as may any of the remainder of the PMM.
Further, dried PMM and dried supernatant also may be dry mixed in
any desired relative proportions, as discussed above.
[0085] By operating in this manner, a number of canola protein
isolates may be recovered, in the form of dried PMM, dried
supernatant and dried mixtures of various proportions by weight of
PMM-derived canola protein isolate and supernatant-derived canola
protein isolate, generally from about 5:95 to about 95:5 by weight,
which may be desirable for attaining differing functional and
nutritional properties based on the differing proportions of
2S/7S/12S proteins in the compositions.
[0086] As an alternative to dilution of the concentrated protein
solution into chilled water and processing of the resulting
precipitate and supernatant as described above, protein may be
recovered from the concentrated protein solution by dialyzing the
concentrated protein solution to reduce the salt content thereof.
The reduction of the salt content of the concentrated protein
solution results in the formation of protein micelles in the
dialysis tubing. Following dialysis, the protein micelles may be
permitted to settle, collected and dried, as discussed above. The
supernatant from the protein micelle settling step may be
processed, as discussed above, to recover further protein
therefrom. Alternatively, the contents of the dialysis tubing may
be directly dried. The latter alternative procedure is useful where
small laboratory scale quantities of protein are desired.
[0087] The relative quantities of the respective proteins in any
given protein isolate may be determined by any convenient
analytical techniques, such as an analytical separation technique.
The most common of these techniques uses selective media in a
column that permits separation based on size. For gel permeation
chromatography (GPC) applications, spherical gel-like materials are
used. Where pressure is used, as in high pressure liquid
chromatography (HPLC), then a rigid media is used. The latter
technique also is known as size exclusion chromatography (SEC). The
results obtained using such techniques on samples of canola protein
isolate prepared as described herein are contained in the Examples
below.
[0088] The procedure of mass spectroscopy (MS) may be used to
identify and analyze protein samples, including the 2S, 7S and 12S
proteins of canola described herein. In electron spray ionization
(ESI) mass spectroscopy, the protein is bombarded with low energy
electrons and charged fragments or species formed from the
collision are detected. In particular, a matrix assisted laser
desorption ionization (MALDI) mass spectrometer may be used wherein
there is laser vaporization of a dry sample of material for
analysis that contains a specific molecular matrix. The matrix
contains a compatible, small molecular weight organic molecule that
protects the biopolymer under analysis sufficiently to reduce the
number of ionized species produced from the laser impact
energy.
[0089] Data derived from protein isolate samples as described
herein suggests that the 2S protein is not stable to electron
bombardment by ESI-MS and readily fragments into numerous
polypeptide fragments, which include 4 kDa, 10 kDa and 21 kDa
species. Using MALDI-MS, however, produced an intact 2S fraction
with a molecular mass of close to 14,000 daltons.
[0090] The 7S protein in the PMM-derived protein isolate also
produced a large number of sub-10 kDa fragments, suggesting
molecular instability to electron bombardment in ESI-MS. In
addition, a 21 kDa species was identified in analysis of 7S protein
from PMM-derived canola protein isolate, but this species was
absent in the 7S protein from supernatant-derived canola protein
isolate.
[0091] ESI-MS analysis of the 7S protein from PMM-derived canola
protein isolate also indicated six protein molecular masses ranging
from 126 to 166 kDa while the 7S protein from supernatant-derived
canola protein isolate indicated one large species at 167 kDa.
[0092] MALDI-MS analysis results described herein indicate that the
2S protein has a molecular mass between 13,960 and 14,250 da and is
quite resistant to mild acid treatment. The 7S and 12S proteins are
composed of the same building blocks with a basic subunit of 48,200
to 48,400 da. The 7S protein has a molecular mass of three subunits
or about 145,000 da molecular mass while the 12S protein contains 6
subunits with a combined molecular mass of about 290,000 da. Acid
hydrolysis of the 7S and 12S proteins did not produce 2S protein
and produced almost identical results, confirming that these two
globular proteins are obtained from the same subunit.
[0093] The individual 2S, 7S and 12S proteins may be isolated and
purified from the respective protein isolates by any conventional
procedure, including analytical high pressure liquid chromatography
(HPLC) for smaller quantities of protein and preparative HPLC for
larger quantities. Other procedures achieving composition on the
basis of molecular mass may be used. The 2S protein generally is
isolated and purified from the supernatant-derived canola protein
isolate while the 7S and 12S proteins generally are isolated and
purified from the PMM-derived canola protein isolate. The canola
protein isolate is solubilized, such as by using saline, and then
passed through the HPLC column. The HPLC separated proteins
contained in fractions of eluant containing one of the proteins,
usually are subjected to ultrafiltration to reduce the volume of
eluant followed by protein isolate dialysis to reduce the residual
salt content in the proteins. The dialyzed material may be dried to
provide dried isolated and purified individual canola protein.
DESCRIPTION OF PREFERRED EMBODIMENTS
[0094] Referring to FIG. 1, there is illustrated schematically a
flow sheet of a batch process for the preparation of canola protein
isolates. Canola oil seed meal and aqueous extraction medium are
fed by line 10 to an extraction vessel 12 wherein the oil seed meal
is extracted and an aqueous protein solution is formed. The slurry
of aqueous protein solution and residual oil seed meal is passed by
line 14 to a vacuum filter belt 16 for separation of the residual
oil seed meal which is removed by line 18. The aqueous protein
solution then is passed by line 20 to a clarification operation 22
wherein the aqueous protein solution is centrifuged and filtered to
remove fines, which are recovered by line 24.
[0095] The clarified aqueous protein solution is pumped by line 26
through ultrafiltration membrane 28 to produce a concentrated
protein solution as the retentate in line 30 with the permeate
being recovered by line 32. The concentrated protein solution is
passed into a precipitation vessel 34 containing cold water fed by
line 36. Protein micellar mass formed in the precipitation vessel
34 is removed by line 38 and passed through a spray dryer 40 to
provide dry canola protein isolate 42.
[0096] Supernatant from the precipitation vessel 34 is removed by
line 44 and pumped through ultrafiltration membranes 46 to produce
a concentrated protein solution as the retentate in line 48 with
the permeate being removed by line 50. The concentrated protein
solution is passed through a spray dryer 52 to provide further dry
canola protein isolate 54.
[0097] As an alternative, the concentrated protein solution in line
4S may be passed by line 56 to mix with the protein micellar mass
before the mixture then is dried in spray dryer 40.
[0098] Referring to FIG. 2, there is illustrated schematically a
flow sheet of a continuous process for the preparation of canola
protein isolates. Canola oil seed meal and aqueous extraction
medium are fed by lines 110 and 112 respectively to a blender 114
wherein the oil seed meal and aqueous extraction medium are mixed
and the mixture is passed by line 116 to a mixing pipe 118. In the
mixing pipe 118, the oil seed meal is extracted and an aqueous
protein solution is formed. The slurry of aqueous protein solution
and residual oilseed meal is passed by line 120 to a vacuum filter
belt 122 for separation of the residual oil seed meal which is
removed by line 124. The aqueous protein solution then is passed by
line 126 to a clarification operation 128 wherein the aqueous
protein solution is centrifuged and filtered to remove fines, which
are recovered by line 130.
[0099] The clarified aqueous protein solution is pumped by line 132
through ultrafiltration membranes 134 sized to provide the desired
degree of concentration of the aqueous protein solution to produce
a concentrated protein solution as the retentate in line 136 with
the permeate being recovered by line 138. The concentrated protein
solution is passed into the inlet of a mixing tee 140, with cold
water being fed thereto by line 142 in a volume sufficient to
achieve the desired degree of dilution. The resulting solution is
fed by line 144 to a settling tank 146 to permit the protein
micellar mass to settle. Protein micellar mass settled in the
settling vessel 146 is removed by line 148 from time to time and
passed through a spray dryer 150 to provide dry canola protein.
isolate 152.
[0100] Supernatant from the settling tank is removed by line 154
and pumped through ultrafiltration membranes 152 to produce a
concentrated protein solution as the retentate in line 158 with the
permeate being removed by line 160. The concentrated protein
solution is passed through a spray dryer 162 to provide further dry
canola protein isolate 164.
[0101] As an alternative, the concentrated protein solution in line
158 may be passed by line 166 to mix with the protein micellar mass
before the mixture then is dried in spray dryer 150.
EXAMPLES
Example 1
[0102] This Example illustrates the procedure adopted to provide
the canola protein isolates of the invention.
[0103] `a` kg of commercial canola meal was added to `b` L of 0.15
M NaCl solution at ambient temperature, agitated for 30 minutes to
provide an aqueous protein solution having a protein content of `c`
g/L. The residual canola meal was removed and washed on a vacuum
filter belt. The resulting protein solution was clarified by
centrifugation to produce `d` L of a clarified protein solution
having a protein content of `e` g/L.
[0104] A `f` L aliquot of the protein extract solution was reduced
in volume to `g` L by concentration on an ultrafiltration system
using `h` dalton molecular weight cut-off membranes. The resulting
concentrated protein solution had a protein content of `i` g/L.
[0105] The concentrated solution at `j` .degree. C. was diluted `k`
into 4.degree. C. water. A white cloud formed immediately and was
allowed to settle. The upper diluting water was removed and the
precipitated, viscous, sticky mass (PMM) was recovered from the
bottom of the vessel in a yield of `1` wt % of the extracted
protein. The dried PMM-derived protein was found to have a protein
content of `m`% (N.times.6.25) d.b. (Percentage nitrogen values
were determined using a Leco FP528 Nitrigen Detelminator). The
product was given designation `n".
[0106] The parameters `a` to `n` for five PMM-derived canola
protein isolates are given in the following Table I:
1TABLE I BW-AL017-B14- BW-AL017-B20- BW-AL017- BW-AL021-I24-
BW-AL021-I30- n 02A-C300 02A-C300 D29-C300 02A-C300 02A-C300 a 150
150 150 150 150 b 1000 1000 1000 1000 1000 c 22.0 22.6 20.2 29.3
30.0 d 1000 1040 1040 1080 1080 e 15.4 15.3 14.6 20.8 19.2 f 600
500 1040 1080 1080 g 18 17 44 47.5 48.0 h 3000 3000 5000 5000 5000
i 289 236 225 311 218 j 31 32 30 24 19 k 1:15 1:15 1:15 1:15 1:15 l
19 22 29 35 20 m 105.8 102.9 103.2 105.4 103.0
[0107] The removed diluting water was reduced in volume by
ultrafiltration using a `o` dalton molecular weight cut-off
membrane to a protein concentration of `p` g/L. The concentrate was
dried. With the additional protein recovered from the supernatant,
the overall protein recovery was `q` wt %. The dried protein which
was formed had a protein content of `r` wt % (N.times.6.25)
d.b.
[0108] The product was given designation `s`. The parameters `o` to
`s` for five supernatant-derived canola protein isolates are given
in the following Table II:
2TABLE II BW-AL017-B14- BW-AL017-B20- BW-AL017- BW-AL021-I24-
BW-AL021-I30- s 02A-C200 02A-C200 D29-C200 02A-C200 02A-C200 o 3000
3000 5000 5000 5000 p 60.8 57.8 121.8 78.0 68.1 q 31.0 33.0 45 49
33 r 97.8 103.6 100.8 103.7 97.8
Example 2
[0109] This Example illustrates analysis of the PMM-derived and
supernatant-derived canola protein isolates of Example 1.
[0110] A Gradi-Frac (Pharmacia Arnersham) protein separator having
100 cm column and Sephacryl 300 HR media (Sephacryl 300 HR is a
dextran polymer, cross-linked with methylene bis-acrylamide that
permits fractionation of globular proteins with 10,000 to 1,500,000
dalton size) was run with a series of standards of protein origin
to determine the residence time (RT) of each component, as measured
at A280 nm, at an elution flow rate of 1.0 mL/min. Saline solution,
pH adjusted and containing sodium azide as an antibacterial agent,
was used as the column solvent. Eluant was collected in test tubes
on an autosample rack with each test tube holding 5 mL of liquid.
Relative protein fractions were calculated by peak area, obtained
by multiplying half-base times peak height at maximum.
[0111] An analytical Varian high pressure liquid chromatography
column (HPLC), using a 300 .times.7.8 mm BioSep S3000 Size
Exclusion Chromatography (SEC) column containing hydrophilic-bonded
silica rigid support media, 5-micron diameter, 290-Angstrom pore
size, capable of separating globular proteins from 5,000 to 700,000
dalton size, was run using the same BioRad protein standards as
with the Gradi-Frac system. The BioRad proteins cover a range from
17,000 daltons (myoglobulin) to 670,000 daltons (thyroglobulin)
with Vitamin B12 added as a low molecular mass marker at 1,350
daltons. Each component is measured at 280 nm at an elution
flowrate of 1.0 mL/min. Saline solution, pH adjusted and containing
sodium azide as an antibacterial agent, was used as the column
solvent and to dissolve dry samples. Eluant was discarded after UV
detection as no more than 50 microliters of sample are required per
run. The HPLC Prostar system automatically calculated retention
times and peak areas and printed out a summary report.
[0112] Samples of the PMM-derived and supernatant-derived canola
protein isolates, prepared as described in Example 1 for lots
BW-AL-017-B14-02A-C300 and BW-AL017-B20-02A-C300, were run on each
column. The peak area counts were converted to percentage for each
peak. All peaks on different runs were taken into calculation and
then the three major protein fractions, 12S, 7S and 2S, were
recalculated separately.
[0113] In addition, standard curves, derived from the BioRad
standard, were used to calculate approximate molecular weights for
the three protein fractions, 12S, 7S and 2S, for runs made using
both GPC and HPLC. The Gradi-Frac system contains more variability
than the HPLC system which results from the larger sample size (1
milliliter versus 25 microliters) and column diameter, the manual
calculation of the Gradi-Frac results and the run time differences
(25 minutes for the HPLC versus 5 hours for the Gradi-Frac). In
addition, the Gradi-Frac system uses volume as the measure of
protein retention while HPLC uses time.
[0114] The variation in the data shown in the Tables III and IV
below reflects the differences indicated above. The HPLC system
statistically is better (lower variability) than the Gradi-Frac
system. The Gradi-Frac system does have the advantage of providing
individual sample fractions that can be further tested (for
example, using mass spectrometry), unlike the HPLC system.
[0115] The results obtained are set forth in the following Table
III for the PMM-derived canola protein isolate (CPI) and Table IV
for the supernatant-derived canola protein isolate (CPI):
3TABLE III Molecular Weight calculations based on BioRad GPC
Standards containing animal proteins. HPLC GPC Protein Ratios
PMM-Derived CPI Calculated Protein Fractions: % of all Peaks
2,7,12S Others Molecular Weights Protein % % % % % % % of all % of
all in kilo-Daltons Type: Meal: Run#: 12S 7S 2S 12S 7S 2S Peaks
Peaks 12S 7S 2S HPLC-SEC PMM AL017 B14 6% 94% 0.1% 6% 92% 0% 98% 2%
341 147 12.1 PMM AL017 B20 6% 94% 0.1% 6% 92% 0% 98% 2% 342 146
11.5 Average 6% 94% 0% 6% 92% 0% 98% 2% 342 146 11.8 s.d. 0% 0% 0%
0% 0% 0% 0% 0% 1 1 0.4 Gradi-Frac GPC PMM AL017 B14 8% 82% 9% 8%
82% 9% 99% 1% 354 152 8.7 PMM AL017 B20 9% 89% 2% 9% 88% 2% 99% 1%
403 163 8.6 Average 9% 86% 6% 8% 85% 6% 99% 1% 378 158 8.6 s.d. 0%
5% 5% 0% 5% 5% 0% 0% 35 8 0.0
[0116]
4TABLE IV Molecular Weight calculations based on BioRad GPC
Standards containing animal proteins. Supernatant- HPLC GPC Protein
Ratios derived CPI Calculated Protein Fractions: % of all Peaks
2,7,12S Others Molecular Weights Protein % % % % % % % of all % of
all in kilo-Daltons Type: Meal: Run#: 12S 7S 2S 12S 7S 2S Peaks
Peaks 12S 7S 2S HPLC-SEC Supernatant AL017 B14 0% 25% 75% 0% 25%
75% 99% 1% none 126 13.9 Supernatant AL017 B20 0% 24% 76% 0% 24%
76% 99% 1% none 135 15.2 Average 0% 24% 76% 0% 24% 75% 99% 1% 130
14.5 s.d. 0% 1% 1% 0% 1% 1% 0% 0% 6 1.0 Gradi-Frac GPC Supernatant
AL017 B14 0% 37% 63% 0% 29% 50% 79% 21% none 147 10.6 Supernatant
AL017 B20 0% 37% 63% 0% 27% 46% 73% 27% none 152 11.0 Average 0%
37% 63% 0% 28% 48% 76% 24% 150 10.8 s.d. 0% 0% 0% 0% 1% 3% 4% 4% 4
0.3
[0117] As may be seen from the data presented in Table III, the
PMM-derived samples contained a large amount of 7S protein with
minor percentages of 2S and 12S proteins.
[0118] As may be seen from Table IV, 12S protein is substantially
absent from the supernatant-derived canola protein isolate while
the predominant protein was the 2S protein, with quantities of 7S
protein being present.
[0119] The calculated molecular weights for the HPLC runs were
lower for the 12S and 7S proteins but higher for the 2S protein.
This apparent discrepancy may be due, in part, to difficulties in
accurately pinpointing the peak maximums for the GPC runs, which
were all done by hand.
[0120] The GPC calculated weight of 385,000 dalton for 12S is
higher than reported in the literature for canola cruciferin, which
range from 300,000 to 310,000 da. The GPC and HPLC calculated
molecular weights for 7S are similar and close to the reported
value of about 150,000 da. The HPLC estimate for 2S is higher than
the GPC average and both less than the reported value for napin of
14,000 da.
[0121] As may be seen from the above data, the PMM-derived canola
protein isolate in the samples tested contains the 7S fraction in
amounts ranging from 82 to 89% (by GPC) and 94% (by HPLC). As
mentioned above, the HPLC procedure is faster and is considered to
be more accurate than GPC. Based on the three protein fraction
areas, it is considered that the PM-derived samples contain:
[0122] about 88 to about 98 wt % of 7S protein
[0123] about 1 to about 10 wt % of 12S protein
[0124] 0 to about 6 wt % of 2S protein
[0125] Similarly, for the supernatant-derived samples, based on the
three protein fraction areas, it is considered that these samples
contain:
[0126] about 70 to about 95 wt % of 2S protein
[0127] about 5 to about 30 wt % of 7S protein
[0128] 0 to about 2 wt % of 12S protein
[0129] The term "Protein Fraction" as used above is defined as the
area of a peak at HPLC (or GPC) Retention Time that generates
calculated molecular weights in the ranges discussed above,
according to acceptable GPC/HPLC protein standards, such as
available from BioRad. These peaks may contain other components,
but as long as they lie in an acceptable range of the target
molecular weights, their presence would be irrelevant.
[0130] An acceptable target of molecular weights, based on the
information contained in this Example, would appear to be:
[0131] 12S: 300,000 to 360,000 da
[0132] 7S: 125,000 to 160,000 da
[0133] 2S: 9,000 to 15000 da
Example 3
[0134] This Example shows the effects of certain parameters on
protein extraction.
[0135] In a first set of experiments, 50 g samples of canola oil
seed meal which had been low temperature toasted (LT meal) at
100.degree. C. to remove residual solvent were added to 500 mL
samples of 0.05 M or 0.10 M NaCl solution at room temperature
(20.degree. C.) and stirred for 15 minutes. The slurry was
centrifuged at 5000.times.g for 10 minutes to extract and spent
meal.
[0136] In a second set of experiments, 500 mL of water with no salt
added was first heated to 60.degree. C. on a hot plate stirrer and
then 50 g of canola oil seed meal which had been low temperature
toasted at 100.degree. C. to remove residual solvent were added and
stirred for 15 minutes while the temperature was maintained. The
extract was separated from the spent meal by centrifugation at
5000.times.g for 10 minutes.
[0137] The protein concentration of the various aqueous protein
solutions obtained in these experiments were determined and appear
in the following Table V:
5TABLE V Protein Concentrations in Extracts (wt %) 0.05 M saline
0.10 M saline 60.degree. C. water LT meal 1.11 1.44 0.98
[0138] The protein extractability from the meals was determined
from the protein concentration data of Table V and this data is
presented in Table VI:
6TABLE VI Protein Extractability (wt %)* 0.05 M saline 0.10 M
saline 60.degree. C. water LT meal 28.6 37.4 25.5 *Defined as
percentage of the amount of protein extracted of the total amount
of protein in the meal.
[0139] Samples of extracts prepared as described in this Example
were run on each of the HPLC and SEC columns described in Example
2. The peak area counts were converted to percentage for each peak.
All peaks on different runs were taken into calculation and then
the three major protein fractions, 12S, 7S and 2S, were
recalculated separately. The results obtained are shown in the
graphical data of FIGS. 2 to 4.
[0140] Each chromatogram showed a distinct peak representing 7S
canola protein fraction and a small bump of 12S canola protein
fraction. The peak for the 2S canola protein fraction was present
among peaks for other components of the extract. The peaks in the
lower molecular weight end of the chromatogram were not properly
identified, but likely correspond to non-protein nitrogenous
compounds, such as short peptides and free amino acids, as well as
other meal components, such as phenolic compounds, glucosinolates
and phytates.
Example 4
[0141] This Example illustrates analysis of canola protein isolate
samples.
[0142] Gel permeation chromatography as described in Example 2 was
performed on samples of spray dried canola protein isolate samples
from lots BW-AL017-D29-C200 and BW-AL017-D29-C300, prepared as
described in Example 1, which produced four protein fractions that
were labelled 2S, >2S, 7S and 12S, with the 2S and possibly
>2S fractions representing small, water soluble albumins and the
7S and 12S protein representing the more hydrophobic, less-water
soluble globulins.
[0143] These fractions and mild acid-treated samples were analyzed
by MALDI mass spectroscopy and produced the following results:
[0144] 2S Fraction:
[0145] Analysis using MALDI-MS indicated a molecular mass of close
to 14,000 daltons. A narrow band peak was observed with the top of
the peak splintered, with the major spike lying between 13,960 da
and 14,250 da, a difference of 2 to 3 amino acids, which could be
indication of different protein isozymes.
[0146] Acid treatment of the 2S fraction in 5% acetic acid for 24
hours did not reveal any smaller polypeptide species to a low limit
cut-off of 8,000 da, indicating a resistance of the 2S protein to
mild acidification.
[0147] >2S Fraction:
[0148] The MS scans of this material indicated a small peak at
about 13,950 to 13,990 da, attributable to 2S protein, and a major
peak at 13,380 to 13,410 da of unknown origin. The fraction also
contained a significant but less intense peak at between 17,160 and
17,260 da, also of unknown origin.
[0149] Mild acid treatment of the latter fraction with 5% acetic
acid for 24 hours reduced only the major peak at 13,400 da, with
some minor components appearing at about 7,000 to 8,000 da,
possibly hydrolysis products. The 2S peak did not appear
diminished.
[0150] 7S Fraction:
[0151] The MS scans of this material showed the most significant
peak to occur at between 48,150 and 49,950 da with the major spike
at about 48,400+/-100 da. A much less intense and broader peak
appeared with two spikes at approximately 95,940 da and 97,510 da
respectively, which indicates the presence of a small quantity of
protein containing two subunits. The analyses carried out in
Example 2 suggest a molecular mass of about 150,000 da for the 7S
protein while the MALDI analysis herein indicates a slightly lower
molecular mass of about 145,000 da.
[0152] A minor component appeared at 13,420 da, in the area of the
major >2S fraction peak, but not at 14,000 da. A major triplet
of peak appeared at between 16,975 and 17,905 da. These peaks are
probably attributable to a major polypeptide in the protein subunit
and was also present in the >2S fraction but not the 2S
fraction.
[0153] Acid treatment of the 7S fraction with 1% acetic acid for 24
hours left the major species intact at 48,310-da with a lesser peak
at 45,750 da (possible loss of a small polypeptide). New peaks
appeared at 16,310 da, 22,710 da and 24,070 da and likely represent
hydrolysis products.
[0154] Further acid treatment of the 7S fraction was effected using
20% formic acid. Scans were taken after one hour of treatment and
again after three days exposure to the formic acid.
[0155] A new species appeared at about 26,300 da after the one hour
exposure and the other peaks were approximately where noted for the
5% acetic acid treatment. Exposure for three days led to a drop in
the peak intensity for the major subunit (48,260 da) with a rise in
the major peaks at 16,315 da and 17,850 da.
[0156] Scans below 9,000 da revealed a characteristic pattern of
six peaks at 4,490 da, 5,720 da, 6,530 da, 7,030 da and 7,330 da,
although the peak at 4,490 da increased significantly by the third
day.
[0157] 12S Fraction
[0158] MALDI-MS analysis of the 12S fraction produced a major peak
centered at between 48,150 and 48,200 da, representing the major
protein subunit. The major downfield peak occurred at 94,960 da
with other lesser peaks at 64,620 da, 77,540 da, 126,990 da and
143,280 da. The large molecular mass peaks were not well defined
and can only approximate the true molecular masses. The major
downfield peak is close to two times the subunit molecular mass
while the broad peak at 143,280 da is close to three times the
subunit mass.
[0159] Other significant peaks appear at about 13,400 da, 16,400
da, 17,900 da and 29,000 to 29,700 da, which are probably some of
the polypeptides that make up to protein subunits.
[0160] Acid treatment of the 12S fraction with 5% acetic acid for
24 hours produced a profile nearly identical to that for the 7S
fraction and described above. Treatment with 20% formic acid after
three days showed a loss of the major subunit at 48,100 da and a
rise in the 17,900 da species.
[0161] Scans below 9,000 da were almost identical to the 7S
hydrolysis after one hour in 20% formic acid, with six major peaks
at 4,480 da, 5,730 da, 6,510 da, 6,650 da, 7,020 da and 7,310 da.
The peak at 4,480 da did not increase significantly by the third
day of formic acid treatment unlike the results for the 7S
fraction.
[0162] Having regard to the results of the MALDI-MS analysis
reported in this Example, it can be concluded that:
[0163] (a) the 2S protein has a molecular mass of between 13,960
and 14,250 da, in line with published results of close to 14,000 da
and is quite resistant to mild acid treatment.
[0164] (b) The 7S and 12S proteins are composed of the same
building blocks with a basic subunit in the 48,200 to 48,400 da
range. The 7S protein has a molecular mass of three subunits or
about 145,000 da and the 12S protein contains 6 subunits with a
combined molecular mass of about 290,000 da. These values are lower
than those obtained by the GPL and HPLC SEC molecular mass
calculations in Example 2, but those results are based on animal
standards.
[0165] (c) Acid hydrolyses of the 7S and 12S proteins do not
produce the 2S protein.
[0166] (d) Acid hydrolysis of the two globular proteins gave almost
identical results, confirming that the two proteins were obtained
from the same subunit.
Example 5
[0167] This Example illustrates preparative high pressure liquid
chromatography (HPLC).
[0168] A Varian Preparative high pressure liquid chromatography
system (prep-HPLC), using a 300 .times.21.20 mm Phenomenex BioSep
S3000 size Exclusion chromatography (SEC) main column, containing
hydrophilic-bonded silica rigid support media, 5-micron diameter,
290-Angstrom pore size, was used for the fractionation of the
canola proteins. A disposable pre-column, containing the same
packing and with a size of 60 .times.21.20 mm, was attached ahead
of the main column.
[0169] Each analyte was monitored at 280 nm at an elution rate
between 6 mL/minute and 8 mL/minute. The upper pressure limit of
1,000 psi on the column was not exceeded at these flow rates.
Saline solution, containing sodium azide as an antibacterial agent,
was used as the column mobile phase and was also used to dissolve
the dry canola protein samples. Eluant was collected in a Varian
Model 701 Fraction collector. Run times ranged from 12 minutes to
15 minutes depending on the sample.
[0170] The sample injection volume was between 1.0 and 1.5 mL for a
sample concentration of between 2.0% and 3.0% by dry weight of
protein isolate, or 20 mg to 45 mg solids per injection. The
capacity of the column can be exceeded beyond 1.5 mL, dependent on
sample type and concentration. Sample preparations were stirred for
a minimum of 30 minutes before centrifugation at 10,000 rpm for 20
minutes. The supernatant was then vacuum-filtered through a minimum
of a 0.45-micron membrane disk.
[0171] Although the system can be run continuously over a 24-hour
period, this was not usually done as a cleaning procedure,
involving acetonitrile in water, was required daily to remove
impurities from the column. Even so, 80 to 100 runs can usually be
made each day, with between 0.5 and 1.2 grams of analyte
collected.
[0172] Samples were prepared from PMM-derived and
supernatant-derived canola protein isolates, including the
following: BW-AL-017-D29-02A-C200, BW-AL021-I24-02A-C200,
BW-AL021-I30-02A-C200, BW-AL-017-D29-02A-C300,
BW-AL021-I24-02A-C300 and BW-AL021-I30-02A-C300, prepared as
described in Example 1.
[0173] PMM (C300) samples were used for the collection of 7S and
12S proteins, while supernatant-derived (C200) samples were
primarily used to collect 2S. Fraction aliquots were tested on the
Varian Analytical SEC-HPLC for purity. The eluant protein levels
were typically less than 0.3 wt % (N.times.6.25) or less than 2.0
Absorbance Units at 280 nm, and the salt content was normally
between 0.5 wt % and 0.7 wt % NaCl as determined by
conductivity.
Example 6
[0174] This Example illustrates ultrafiltration and dialysis of the
fractions collected in Example 5.
[0175] Ultra-filtration, followed by dialysis, are both effected to
reduce the large eluant volume and the high salt-to-protein ratio.
The dilute eluant from the Varian Preparative HPLC system described
in Example 5 was concentrated in two Amicon Series 8000
Ultra-filtration stirred cell units, each with a rated maximum
capacity of 400 mL of volume.
[0176] Each unit was fitted with a 76 mm diameter membrane with
selective molecular size cut-off. For 2S protein, membranes used
included: Ultracel Amicon YM1 UF disc with regenerated cellulose,
1,000 NMWL (Nominal Molecular Weight Limit), #13342, and Ultracel
Anicon YM10 UF discs of regenerated cellulose, 10,000 NMWL,
#13642.
[0177] For the larger 7S and 12S globular proteins, higher NMWL
membranes were used: Biomax PBQK UF discs of polyethylene sulfone
(PES), 50,000 NMWL, #PBQK 07610, or Biomax PBHK UF discs of PES,
100,000 NMWL, #PBHK 07610.
[0178] Higher NMWL membranes produced slight losses in protein
retention, called the Retentate, but also removed
lower-molecular-weight impurities that are weakly associated with
the protein. Higher NMWL cut-offs also reduced the UF operating
times. Typical run times for 1,200 ml of HPLC sample on the two UF
units were 4 to 5 hours.
[0179] In a typical run, sample was added to the 325 mL line in
each unit. With stirring, the levels rise to about 375 mL. The
units were sealed and pressure was applied to 60 psi, while the
samples were stirred. When the Retentate inside the units dropped
to about 100 mL, the pressure was released and additional sample
was added to each unit. This process continued until the entire
sample was added. The final Retentate was concentrated to between
75 mL and 100 mL inside each cell.
[0180] The eluant, called the Permeate, was removed from each unit
and measured for salinity by conductivity, and for relative protein
content by absorbance at 280 nm using a 1-cm cell on a UV-Visible
UltraSpec 1000E Spectrophotometer. A mass balance was calculated to
provide information on the efficiency of the UF membranes. UF
Permeate and final Retentate samples were tested by analytical
SEC-HPLC, as described in Example 2, for purity.
[0181] The Ultra-Filtration Retentate was poured into Spectra/Por 7
Dialysis membranes, 1,000 MWCO (Molecular Weight Cut-Off), 24.2 mm
diameter, #132104. Each piece of tubing was cut to about 300 mm.
The filled membranes were then placed into sealed 4-liter bottles
containing RO water. The RO water was replaced twice before the
dialyzed samples were removed from the tubes and placed in pans for
freezing at -65.degree. C. Final salinities were typically below
0.1 wt % based on conductivity.
[0182] The frozen samples were then placed into the Virtis SRCX-15
Freeze Dryer. Drying times were variable, dependent upon water
loading. The dried samples were removed from the pans and weighed
prior to distribution for analyses.
Example 7
[0183] This Example illustrates amino acid analysis.
[0184] Individual ultra-filtered, dialyzed and freeze-dried canola
proteins 2S, 7S and 12S, prepared as described in Examples 5 and 6,
have been analyzed for amino acid content. The 2S sample was
derived from canola protein isolate AL021-I30-02A C200 while the 7S
and 12S proteins were derived from canola protein isolate
AL017-D29-02A C300.
[0185] The amino acid analysis is set forth in the following Table
VII:
7TABLE VII g/100 g dry matter AL021- AL017- AL017- Amino Acid
I30-02A D29-02A D29-02A MW(1) Amino Acid 2S 7S 12S 133.1 Aspartic
3.18 11.60 3.02 119.1 Threonine 2.70 3.34 1.00 105.1 Serine 3.84
4.52 1.23 204.2 Tryptophan 1.16 1.42 0.40 146.1 Glutamic 25.10
21.50 5.91 75.1 Glycine 3.91 5.44 1.45 89.1 Alanine 3.67 4.47 1.18
121.1 Cystine 4.13 1.19 0.34 117.1 Valine 4.02 5.92 1.55 149.2
Methionine 2.55 1.74 0.41 131.2 Isoleucine 2.89 5.12 1.31 131.2
Leucine 6.13 8.70 2.31 181.2 Tyrosine 1.14 2.67 0.70 165.2
Phenylalanine 2.43 5.01 1.28 155.2 Histidine 2.58 1.60 0.46 146.2
Lysine 5.92 3.17 0.88 174.2 Arginine 5.99 8.02 2.13 115.1 Proline
9.02 5.83 1.68 Sum: 90.36 101.26 27.24 Avg. aa MW(1) 134.61 134.86
134.77 Anhydrous MW(2) 116.59 116.85 116.75 Note: (1)Molecular
Weight of "free" amino acids. (2)Weight Average Molecular Weight of
polymeric amino acids.
[0186] The values presented in Table VII represent amino acids on
the basis of grams per 100 grams dry weight. The 12S sample
contained residual salt, even after ultrafiltration, accounting for
the low total amino acid weight in the samples in comparison to the
7S and 2S samples. The data was adjusted to the basis of 100 grams
of amino acid and the revised data is shown in the following Table
VIII:
8TABLE VIII Amino Acid Summary: g/100 g Amino Acids Hydro
phobicity: AL021- AL017- AL017- Gibb's: I30-02A D29-02A D29-02A
kJ/100 g: Amino Acid 2S 7S 12S -- Aspartic* 3.5 11.5 11.1 1.40
Threonine.sup.e 3.0 3.3 3.7 -- Serine 4.2 4.5 4.5 6.96
Tryptophan.sup.e 1.3 1.4 1.5 -- Glutamic* 27.8 21.2 21.7 -- Glycine
4.3 5.4 5.3 2.35 Alanine 4.1 4.4 4.3 3.45 Cystine.sup.e 4.6 1.2 1.2
5.35 Valine.sup.e 4.4 5.8 5.7 3.64 Methionine.sup.e 2.8 1.7 1.5
9.56 Isoleucine.sup.e 3.2 5.1 4.8 7.32 Leucine.sup.e 6.8 8.6 8.5
5.30 Tyrosine 1.3 2.6 2.6 6.33 Phenylalanine.sup.e 2.7 4.9 4.7 1.35
Histidine.sup.e 2.9 1.6 1.7 -- Lysine.sup.e 6.6 3.1 3.2 --
Arginine.sup.e 6.6 7.9 7.8 9.44 Proline 10.0 5.8 6.2 Sum: 100.0
100.0 100.0 Sum essential aa: 44.8 44.7 44.3 % Hydrophobic aa: 46.9
46.4 46.3 Hydroph. kJ/100 g: 274.5 279.3 277.7 .sup.e= 11 essential
amino acids aa = amino acids *Glutamic acid and aspartic acid are
mostly amidated to glutamine and asparagine
[0187] As may be seen from Table VIII, the albumin 2S protein
differs from the globular 7S and 12S proteins in relative levels of
the different amino acids. The key differences arise for Aspartic,
Glutamic, Cysteine, Lysine and Proline, although differences are
also apparent for most of the amino acids listed. By comparison,
the 7S and 12S profiles are almost identical, and would certainly
fall within the standard variation for this test. The Glutanic acid
quantities also include glutamine, and Aspartic acid includes
Asparagine. Both Glutamine and Asparagine are de-aminated during
acid hydrolysis, leading to detection of the amino acid
analyses.
[0188] Table VII includes the molecular weights for the individual
amino acids. Combined with the individual quantities, the average
molecular weights for the "free" amino acids for 2S, 7S and 12S are
shown, and are all just under 135-da. The anhydrous Weight Averaged
Molecular Weights are also shown since the proteins are biopolymers
of anhydrous amino acids, (each minus a water molecule, excluding
one terminal amino acid per polypeptide). In spite of the
differences between the two protein types, the average polymeric
amino acid molecular weights are almost identical at about 117-da.
This means that 2S, with a MALDI-MS determined molecular weight of
14-kDa, consists of approximately 120 amino acids, 7S, with a
MALDI-MS molecular weight of 145-kDa, would contain approximately
1,240 amino acids, and 12S would contain double this number, or
2,480 amino acids. Each 7S/12S subunit would contain about 413
amino acids.
[0189] Table VIII also indicates the essential amino acids, which
cannot be synthesized by humans. The overall content of the eleven
essential amino acids is very similar for the two types of protein.
The weaknesses in one amino acid are balanced by strengths in other
essential amino acids. Lysine is found in 2S at about twice the
levels in 7S and 12S. However, the globular proteins have higher
overall levels in the aromatic essential amino acids Tyrosine and
Phenylalanine, and are generally higher in the aliphatic
hydrophobic essential amino acids, with the exceptions of
Methionine and Cysteine.
[0190] The hydrophobicity of the amino acids in Table VIII is
determined according to Gibb's Free Energy values, expressed as KJ
per 100 grams amino acid. The Gibb's Free Energy values are listed
in Food Chemistry, 3.sup.rd ed., edited by Owen Fennema, Marcel
Dekker, New York, 1996, p 330. The two types of protein do not
differ substantially in either weight sums or energy sums for
hydrophobicity, but hydrophobicity is also dependent upon the
structural orientation of the polypeptides and not just the
chemistry of the individual amino acids. Overall, the globular
proteins have an Isoelectric Point near neutral pH (7.2) while 2S
has an Isoelectric Point above pH 9.0 according to the review of
rapeseed proteins by Mieth et al. (Table IX below). 2S is very
small in molecular weight, while 7S and 12S are considerably larger
with more potentially hydrophobic interior regions that would
resist water hydration.
[0191] The amino acid composition can be converted into amino acid
residues by using an estimate of the number of amino acid residues
in the biopolymer. As previously discussed, 2S contains
approximately 120 amino acid residues, while 7S contains about
1,240 and 12S about 2,480 residues, based on MALDI-MS analyses.
[0192] The following Table IX converts the amino acid analyses
results presented above to amino acids for 2S and compares the
results with published amino acid residue ranges.
9TABLE IX Comparison of 2S Amino Acid Residues with Literature
Values Values Based on amino acid residues: AL021-I30-02A 2S @ 2S
Liter. 2S Liter. Amino Acid 2S 120 aa: Range.sup.1: Range.sup.2:
Aspartic* 3.4 4 2-5 2 Threonine.sup.e 3.2 4 3-4 4 Serine 5.2 6 4-7
6-7 Tryptophan.sup.e 0.8 1 1-2 1 Glutamic* 24.6 29 27-32 29-34
Glycine 7.4 9 6-10 7-9 Alanine 5.9 7 6-7 6-8 Cystine.sup.e 4.9 6 8
7-8 Valine.sup.e 4.9 6 5-7 6-7 Methionine.sup.e 2.4 3 1-3 2-3
Isoleucine.sup.e 3.2 4 4-7 4 Leucine.sup.e 6.7 8 7-9 8-9 Tyrosine
0.9 1 1-2 1 Phenylalanine.sup.e 2.1 3 3-5 2-3 Histidine.sup.e 2.4 3
3-4 3-4 Lysine.sup.e 5.8 7 5-9 6-9 Arginine.sup.e 4.9 6 5-8 4-6
Proline 11.2 13 13-14 12-15 Sum: 100.0 120 114-126 110-133 .sup.e=
11 essential amino acids aa = amino acids MALDI-MS data indicate 2S
has MW of about 14,000-da, or 120 polymeric amino acids *Glutamic
acid and aspartic acid are mostly amidated. .sup.1Monsalve et al.,
J. Experimental Botany, Vol. 41, No. 222, pp. 89-94, January 1990.
.sup.2Mieth et al., Die Nahrung, 27, 7, 1983, pp.675-697.
[0193] The conversions agree very well with the published results.
The more recent works by Monsalve et al. (1990, per Table IX), plus
work by Gehrig et al., Proc. Natl. Acad. Sci. USA, Vol. 93, pp.
3647-3652, April, 1996, and by Ericson et al., J. Biological Chem.,
Vol. 261, No. 31, pp. 14576-14581, November 1986 give detailed
information about 2S and the precursor molecule pro-Napin,
including actual amino acid sequencing.
[0194] The individual amino acids lie within the ranges from
published papers indicated on Table IX. The listed ranges are due
to variations within rapeseed varieties and may also be due to
minor hydrolytic losses in the experimental work reported.
[0195] Although less is published on cruciferin (12S), what is
known about the globular canola proteins is in good agreement with
our current findings. The amino acid analysis data was converted to
amino acid residues in Table X and compared with published ranges
for 12S proteins.
10TABLE X Comparison of 7S/12S Amino Acid Residues with Literature
Values Burcon Burcon AL017- AL017- Literature Review D29-02A
D29-02A 7S @ 12S @ Range of residues Amino Acid 7S 12S 1240 aa:
2480 aa: 12S .sup.1 Aspartic* 11.09 10.73 137 266 235-270 Threonine
3.57 3.97 44 98 Serine 5.47 5.53 68 137 Tryptophan 0.88 0.93 11 23
Glutamic* 18.72 19.12 232 474 434-531 Glycine 9.22 9.13 114 226
Alanine 6.38 6.26 79 155 Cystine 1.25 1.33 16 33 14-37 Valine 6.43
6.26 80 155 Methionine 1.48 1.30 18 32 13-44 Isoleucine 4.96 4.72
62 117 Leucine 8.44 8.32 105 206 Tyrosine 1.87 1.83 23 45
Phenylalanine 3.86 3.66 48 91 Histidine 1.31 1.40 16 35 45-47
Lysine 2.76 2.85 34 71 78-96 Arginine 5.86 5.78 73 143 83-143
Proline 6.44 6.90 80 171 Sum: 100.0 100.00 1240 2480 The average
(anhydrous) MW for 7S and 12S is calculated to be 116.8 daltons.
MALDI = MS results indicate a MW of 145-kDa for 7S and 290-kDa for
12S protein (Example 4). Therefore, 7S would consist of
approximately 1,240 amino acid residues and 12S would contain
double, or approximately 2,480 residues. .sup.1 Mieth et al., Die
Nahrung, 27, 7, 1983, pp. 678-697.
[0196] The published information comes from the review of rapeseed
proteins by Mieth et al., 1983. This reference lists the range of
specific amino acid residues. The data presented herein is
generally in line with these ranges as shown.
[0197] The authors (Mieth et al.) refer to the 12S molecule
contains 6 major sub-units, each consisting of two polypeptides
with molecular masses of about 18,000-da and 31,000-da. This
information is also in line with what the MALDI-MS data presented
in Example 4.
SUMMARY OF DISCLOSURE
[0198] In summary of this disclosure, the present invention
provides novel canola protein isolate compositions having a unique
profile of 2S, 7S and 12S proteins as well as individual isolated
and purified proteins. Modifications are possible within the scope
of the invention.
* * * * *