U.S. patent application number 16/489383 was filed with the patent office on 2020-01-16 for preparation of acid soluble pulse protein hydrolyzates with little or no astringency and pulse protein hydrolyzates of improved .
This patent application is currently assigned to Burcon NutraScience (MB) Corp.. The applicant listed for this patent is BURCON NUTRASCIENCE (MB) CORP.. Invention is credited to Brandy GOSNELL, Sarah MEDINA, Martin SCHWEIZER, Kevin SEGALL, Randy WILLARDSEN.
Application Number | 20200015496 16/489383 |
Document ID | / |
Family ID | 63370597 |
Filed Date | 2020-01-16 |
United States Patent
Application |
20200015496 |
Kind Code |
A1 |
SCHWEIZER; Martin ; et
al. |
January 16, 2020 |
PREPARATION OF ACID SOLUBLE PULSE PROTEIN HYDROLYZATES WITH LITTLE
OR NO ASTRINGENCY AND PULSE PROTEIN HYDROLYZATES OF IMPROVED AMINO
ACID SCORE
Abstract
The invention relates to a method of processing a pulse protein
material, which comprises effecting hydrolysis of the pulse protein
material, optionally adjusting the pH, then separating to form a
soluble fraction and processing the soluble fraction to provide a
pulse protein hydrolyzate which is substantially completely soluble
throughout the pH range of about 2 to about 7 and which provides
little or no astringency when an acidic beverage containing the
pulse protein hydrolyzate is consumed and a solid residue, and
processing the solid residue to provide a second pulse protein
hydrolyzate having an improved Amino Acid Score, which is improved
compared to the substrate pulse protein material.
Inventors: |
SCHWEIZER; Martin;
(Winnipeg, CA) ; GOSNELL; Brandy; (Winnipeg,
CA) ; WILLARDSEN; Randy; (Roseville, CA) ;
MEDINA; Sarah; (Winnipeg, CA) ; SEGALL; Kevin;
(Winnipeg, CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
BURCON NUTRASCIENCE (MB) CORP. |
Winnipeg |
|
CA |
|
|
Assignee: |
Burcon NutraScience (MB)
Corp.
Winnipeg
MB
|
Family ID: |
63370597 |
Appl. No.: |
16/489383 |
Filed: |
March 5, 2018 |
PCT Filed: |
March 5, 2018 |
PCT NO: |
PCT/CA2018/050255 |
371 Date: |
August 28, 2019 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
62466581 |
Mar 3, 2017 |
|
|
|
Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A23J 3/30 20130101; A23V
2300/28 20130101; A23J 1/14 20130101; A23L 2/39 20130101; A23J 3/14
20130101; A23L 33/18 20160801; A23L 2/66 20130101; A23V 2002/00
20130101; A23L 5/55 20160801; A23J 3/346 20130101; A23J 3/34
20130101; A23V 2250/548 20130101; A23L 33/185 20160801 |
International
Class: |
A23J 3/34 20060101
A23J003/34; A23J 3/14 20060101 A23J003/14; A23L 2/39 20060101
A23L002/39; A23L 2/66 20060101 A23L002/66; A23L 33/18 20060101
A23L033/18; A23L 33/185 20060101 A23L033/185; A23L 5/00 20060101
A23L005/00 |
Claims
1. A method of processing a pulse protein material, which comprises
effecting hydrolysis of the pulse protein material, optionally
adjusting the pH, then separating to form a soluble fraction and
processing the soluble fraction to provide a pulse protein
hydrolyzate which is substantially completely soluble throughout
the pH range of about 2 to about 7 and which provides little or no
astringency when an acidic beverage containing the pulse protein
hydrolyzate is consumed and a solid residue, and processing the
solid residue to provide a second pulse protein hydrolyzate having
an improved Amino Acid Score, which is improved compared to the
substrate pulse protein material.
2. The method of claim 1, wherein the pulse protein material is a
neutral dry powder, having a protein content of at least about 60
wt % (N.times.6.25) on a dry weight basis (d.b.) and a natural pH
in aqueous solution of about 6.0 to about 8.0.
3. The method of claim 2, wherein the neutral dry powder is
rehydrated to provide a protein solution, the protein solution is
optionally adjusted in pH within the range of about 6.0 to about
8.0, the protein solution is treated with a proteolytic enzyme to
effect hydrolysis of the pulse protein material, the enzymatically
treated pulse protein is heat treated to inactivate the enzyme, the
pH of the resulting solution is adjusted to an acid pH value, the
acidified solution is centrifuged to separate the soluble fraction
(centrate) from residual solids, the centrate is concentrated and
optionally diafiltered on a membrane filtrate system to decrease
the content of salt and/or other impurities in the centrate, and
the retentate is dried to provide a pulse protein hydrolyzate,
having a protein content of at least about 60 wt % (N.times.6.25)
on a dry weight basis (d.b.) and which is acid soluble and provides
little or no astringency in acidic solution or, alternatively, the
heat treatment step is effected after the acidification step and
prior to the centrifugation step.
4. The method of claim 3, wherein the residual solids separated
from the centrate are processed by drying or washed and then dried
to provide a second pulse protein hydrolyzate having a protein
content of at least about 60 wt % (N.times.6.25) d.b. and an
improved Amino Acid Score as compared the substrate pulse protein
material.
5. The method of claim 3, wherein the residual solids separated
from the centrate are adjusted in pH to about 6.0 to about 8.0 and
then dried or the residual solids are washed and then adjusted in
pH to a pH of about 6.0 to about 8.0 prior to the drying step or
the residual solids are neutralized during the washing step by
adjusting the mixture of solids and wash water to a pH of about 6.0
to about 8.0 using a food grade alkaline solution and then
collecting the solids by centrifugation and drying to provide a
second pulse protein hydrolyzate having a protein content of at
least about 60 wt % (N.times.6.25) d.b. and an improved Amino Acid
Score as compared to the substrate pulse protein source.
6. The method of claim 2, wherein the neutral dry powder pulse
protein product is rehydrated to prepare a pulse protein solution,
the protein solution is optionally adjusted in pH within the range
of about 6.0 to about 8.0, the protein solution is treated with a
proteolytic enzyme, the enzymatically treated pulse protein product
is heated to inactivate the enzyme, the resulting solution is
centrifuged to separate the soluble fraction (centrate) from
residual solids, the centrate is concentrated and optionally
diafiltered on a membrane filtration system to decrease the content
of salt and/or other impurities in the centrate and the retentate
is dried to produce a pulse protein hydrolyzate having a protein
content of at least 60 wt % (N.times.6.25) d.b. and which is acid
soluble and provides little or no astringency in acidic
solution.
7. The method of claim 6, wherein the residual solids separated
from the centrate are directly dried or washed and then dried to
provide a second pulse protein hydrolyzate having a protein content
of at least about 60 wt % (N.times.6.25) d.b. and improved Amino
Acid Score as compared to the substrate pulse protein.
8. A method of claim 1, wherein the pulse protein material is a low
pH dry powder having a protein content of at least about 60 wt %
d.b. and a natural pH in aqueous solution of about 1.5 to about
4.0.
9. The method of claim 8, wherein the low pH dry powder is
rehydrated to provide a protein solution, the protein solution is
adjusted in pH to a range of about 6.0 to about 8.0, the protein
solution is treated with a proteolytic enzyme to effect hydrolysis
of the pulse protein solution, the enzymatically treated pulse
protein is heat treated to inactivate the enzyme, the pH of the
resulting solution is adjusted to an acid pH value, the acidified
solution is centrifuged to separate the soluble fraction (centrate)
from residual solids, the centrate is concentrated and optionally
diafiltered on a membrane filtration system to decrease the content
of salt and/or other impurities in the centrate and the retentate
is dried to provide a pulse protein hydrolyzate having a protein
content of at least about 60 wt % (N.times.6.25) d.b. and which is
acid soluble and provides little or no astringency in acidic
solution or, alternatively, the heat treatment step is effected
after the acidification step and prior to the centrifugation
step.
10. The method of claim 9, wherein the residual solids separated
from the centrate are processed by drying or washed and then dried
to provide a second pulse protein hydrolyzate having a protein
content of at least about 60 wt % (N.times.6.25) d.b. and an
improved Amino Acid Score as compared the substrate pulse protein
material.
11. The method of claim 9, wherein the residual solids separated
from the centrate are adjusted in pH to about 6.0 to about 8.0 and
then dried or the residual solids are washed and then adjusted in
pH to a pH of about 6.0 to about 8.0 prior to the drying step or
the residual solids are neutralized during the washing step by
adjusting the mixture of solids and wash water to a pH of about 6.0
to about 8.0 using a food grade alkaline solution and then
collecting the solids by centrifugation and drying to provide a
second pulse protein hydrolyzate having a protein content of at
least about 60 wt % (N.times.6.25) d.b. and an improved Amino Acid
Score as compared to the substrate pulse protein source.
12. The method of claim 8, wherein the low pH dry powder is
rehydrated to provide a protein solution, the protein solution is
adjusted in pH to a range of about 6.0 to about 8.0, the protein
solution is treated with a proteolytic enzyme to effect hydrolysis
of the pulse protein solution, the enzymatically treated pulse
protein is heat treated to inactivate the enzyme, the heat treated
solution is centrifuged to separate the soluble fraction (centrate)
from residual solids, the centrate is concentrated and optionally
diafiltered on a membrane filtration system to decrease the content
of salt and/or other impurities in the centrate and the retentate
is dried to provide a pulse protein hydrolyzate having a protein
content of at least about 60 wt % (N.times.6.25) d.b. and which is
acid soluble and provides little or no astringency in acidic
solution.
13. The method of claim 12, wherein the residual solids separated
from the centrate are directly dried or washed and then dried to
provide a second pulse protein hydrolyzate having a protein content
of at least about 60 wt % (N.times.6.25) d.b. and improved Amino
Acid Score as compared to the substrate pulse protein.
14. The method of claim 8, wherein the low pH dry powder is
rehydrated to prepare a pulse protein solution, the protein
solution is optionally adjusted in pH within the range of about 1.5
to about 4.0, the protein solution is treated with a proteolytic
enzyme, the enzymatically treated pulse protein product is heated
to inactivate the enzyme, the resulting solution is centrifuged to
separate the soluble fraction (centrate) from the residual solids,
the centrate is concentrated and optionally diafiltered on a
membrane filtration system to decrease the content of salt and/or
other impurities in the centrate and the retentate is dried to
produce a pulse protein hydrolyzate having a protein content of at
least 60 wt % (N.times.6.25) d.b. and which is acid soluble and
provides little or no astringency in acidic solution.
15. The method of claim 14, wherein the residual solids separated
from the centrate are directly dried or washed and then dried to
provide a second pulse protein hydrolyzate having a protein content
of at least about 60 wt % (N.times.6.25) d.b. and improved Amino
Acid Score as compared to the substrate pulse protein.
16. The method of claim 14, wherein the residual solids separated
from the centrate are adjusted in pH to about 6.0 to about 8.0 and
then dried or the residual solids are washed and then adjusted in
pH to a pH of about 6.0 to about 8.0 prior to the drying step or
the residual solids are neutralized during the washing step by
adjusting the mixture of solids and wash water to a pH of about 6.0
to about 8.0 using a food grade alkaline solution and then
collecting the solids by centrifugation and drying to provide a
second pulse protein hydrolyzate having a protein content of at
least about 60 wt % (N.times.6.25) d.b. and an improved Amino Acid
Score as compared to the substrate pulse protein source.
17. The method of claim 1, wherein the pulse protein material is
pulse 810A or pulse 810N.
18. The method of claim 1 wherein the pulse protein material is
formed by extracting a pulse protein source with water to form an
aqueous pulse protein solution at least partially separating the
aqueous pulse protein solution from residual pulse protein source,
adjusting the pH of the aqueous pulse protein solution to a pH of
about 1.5 to about 3.4 to solubilize the bulk of the protein and
form an acidified pulse protein solution and separating the
acidified pulse protein solution from the acid insoluble solid
material and optionally concentrating, diafiltering and diluting
the acidified pulse protein solution.
19. The method of claim 18, wherein the optionally diluted pulse
protein solution is adjusted to a pH of about 6.0 to about 8.0 then
treated with proteloytic enzyme to effect hydrolysis of the pulse
protein material, the enzymatically treated pulse protein solution
is heat treated to inactivate the enzyme, the pH of the resulting
solution is adjusted to an acid pH value, the acidified solution is
centrifuged to separate the soluble fraction (centrate) from
residual solids, the centrate is concentrated and optionally
diafiltered on a membrane filtrate system to decrease the content
of salt and/or other impurities in the centrate and the retentate
is dried to provide a pulse protein hydrolyzate having a protein
content of at least about 60 wt. % (N.times.6.25) on a dry weight
basis (d.b.) and which is acid soluble and provides little or no
astringency in acid solution or alternatively the heat treatment
step is effected after the acidification step and prior to the
centrifugation step.
20. The method of claim 19, wherein the residual solids separated
from the centrate are processed by drying or washed and then dried
to provide a second pulse protein hydrolyzate having a protein
content of at least about 60 wt % (N.times.6.25) d.b. and an
improved Amino Acid Score as compared to the substrate pulse
protein source.
21. The method of claim 19, wherein the residual solids separated
from the centrate are adjusted in pH to about 6.0 to about 8.0 and
then dried, or the residual solids are washed and then adjusted in
pH to about 6.0 to about 8.0 prior to the drying step, or the
residual solids are neutralized during the washing step by
adjusting the mixture of solids and wash water to a pH of about 6.0
to about 8.0 using a food grade alkaline solution and then
collecting the solids by centrifugation and drying to provide a
second pulse protein hydrolyzate having a protein content of at
least about 60 wt % (N.times.6.25) d.b. and an improved Amino Acid
Score as compared to the substrate pulse protein source.
22. The method of claim 19, wherein the optionally diluted pulse
protein solution is adjusted to a pH of about 6.0 to about 8.0 then
treated with proteolytic enzyme to effect hydrolysis of the pulse
protein material, the enzymatically treated pulse protein solution
is heated to inactivate the enzyme, the resulting solution is
centrifuged to separate a soluble fraction (centrate) from residual
solids, the centrate is concentrated and optionally diafiltered on
a membrane filtration system to decrease the content of salt and/or
other impurities in the centrate and the retentate is dried to
produce an acid soluble low astringency pulse protein hydrolyzate
having a protein content of at least about 60 wt % (N.times.6.25)
d.b.
23. The method of claim 22, wherein the residual solids separated
from the centrate are directly dried or washed and then dried to
provide a second pulse protein hydrolyzate having a protein content
of about 60 wt % (N.times.6.25) d.b. and an improved Amino Acid
Score as compared to the substrate pulse protein.
24. The method of claim 18, wherein the optionally diluted protein
solution is processed at the initial low pH value or optionally
after pH adjustment to the range of about 1.5 to about 4.0 by
treating with a proteolytic enzyme, the enzymatically treated pulse
protein solution is heated to inactivate the enzyme, the resulting
solution is centrifuged to separate the soluble fraction (centrate)
from residual solids, the centrate is concentrated and optionally
diafiltered on a membrane filtration system to decrease the content
of salt and/or other impurities in the centrate and the retentate
is dried to produce a pulse protein hydrolyzate having a protein
content of at least about 60 wt % (N.times.6.25) d.b. and which is
acid soluble and provides little or no astringency in acidic
solution.
25. The method of claim 24, wherein the residual solids separated
from the centrate are directly dried or washed and then dried to
provide a second pulse protein hydrolyzate having a protein content
of at least about 60 wt % (N.times.6.25) d.b. and an improved Amino
Acid Score as compared to the substrate protein.
26. The method of claim 24, wherein the residual solids separated
from the centrate are adjusted in pH to about 6.0 to about 8.0 and
then dried, or the residual solids are washed and then adjusted in
pH to about 6.0 to about 8.0 prior to the drying step, or the
residual solids are neutralized during the washing step by
adjusting the mixture of solids and wash water to a pH of about 6.0
to about 8.0 using a food grade alkaline solution and then
collecting the solids by centrifugation and drying to provide a
second pulse protein hydrolyzate having a protein content of at
least about 60 wt % (N.times.6.25) d.b. and an improved Amino Acid
Score as compared to the substrate pulse protein source
27. A pulse protein hydrolyzate having a protein content of at
least about 60 wt % (N.times.6.25) d.b., which is substantially
completely soluble throughout the pH range of about 2 to about 7
and provides little or no astringency when an acidic beverage
containing the pulse protein hydrolyzate is consumed.
28. The pulse protein hydrolyzate of claim 27, which is derived
from a commercial, dry powder pulse protein product having a
protein content greater than about 65 wt % d.b. and a natural pH in
solution of about 6 to about 8, a commercial dry powder pulse
protein product having a protein content greater than about 65 wt %
d.b. and a natural pH in solution of about 1.5 to about 4.0, pulse
810A or pulse 810N.
29. A pulse protein hydrolyzate having an Amino Acid Score which is
improved compared to the substrate pulse protein from which the
hydrolyzate is derived.
30. The pulse protein hydrolyzate of claim 29, which is derived
from a commercial, dry powder pulse protein product having a
protein content greater than about 65 wt % d.b. and a natural pH in
solution of about 6 to about 8, a commercial dry powder pulse
protein product having a protein content greater than about 65 wt %
d.b. and a natural pH in solution of about 1.5 to about 4.0, pulse
810Aor pulse 810N.
31. A pulse protein hydrolyzate having a molecular weight profile
as follows: >100,000 Da--0 to 14 wt % 15,000 to 100,000 Da--4 to
30 wt % 5,000 to 15,000 Da--20 to 29 wt % 1,000 to 5,000 Da--27 to
54 wt % <1,000 Da--8 to 23 wt %.
32. A method of processing a pulse protein material, which
comprises effecting hydrolysis of the pulse protein material,
optionally adjusting the pH, then separating to form a soluble
fraction and processing the soluble fraction to provide a pulse
protein hydrolyzate which is substantially completely soluble
throughout the pH range of about 2 to about 7 and which provides
little or no astringency when an acidic beverage containing the
pulse protein hydrolyzate is consumed.
Description
REFERENCE TO RELATED APPLICATION
[0001] This application is a US National Stage under 35 USC 371 of
International PCT application No. PCT/CA2018/050255 and claims
priority under 35 USC 119(e) from U.S. Provisional Patent
Application No. 62/466,581 filed Mar. 3, 2017.
FIELD OF INVENTION
[0002] The invention relates to the utilization of enzyme
hydrolysis to produce pulse protein hydrolyzates with little or no
astringency in acidic solution as well as pulse protein
hydrolyzates having a modified amino acid profile providing a
higher Amino Acid Score.
BACKGROUND TO THE INVENTION
[0003] There is significant commercial interest in preparing acidic
beverages containing pulse protein. Ideally the pulse protein
product should be completely soluble in the beverage so that
stabilizers are not required to suspend the protein in solution.
The pulse protein should also be heat stable in the acid beverage
to facilitate commercial beverage processing (e.g. hot fill
processing). Clarity of the pulse protein product in solution is
also desirable as it allows maximum flexibility in designing the
cloud level of the beverage. That is, the beverage could be clear
or clouding agents could be added to provide the appropriate level
of haze.
[0004] U.S. patent application Ser. No. 13/103,528 filed May 9,
2011 (US Patent Publication No. 2011/0274797 published Nov. 10,
2011), Ser. No. 13/556,357 filed Jul. 24, 2012 (US Patent
Publication No. 2013/00189408 published Jul. 25, 2013), Ser. No.
13/642,003 filed Jan. 7, 2013 (US Patent Publication No.
2013/0129901 published May 23, 2013) and Ser. No. 15/041,193 filed
Feb. 11, 2016 (US Patent Publication No. 2016/0227833 published
Aug. 11, 2016 ("YP701")), assigned to the assignee hereof and the
disclosures of which are incorporated herein by reference, describe
a pulse protein product which is water soluble at low pH, producing
heat stable solutions, but use of which is limited by an astringent
sensation it introduces in the mouth when consumed. The astringent
sensation is unpleasant and undesirably limits the amount of
protein product that can be formulated into an acid beverage.
[0005] U.S. patent application Ser. No. 14/290,415 filed May 29,
2014 (US Patent Publication No. 2014/0356510 published Dec. 4,
2014) entitled "Production of Pulse Protein Product with Reduced
Astringency", assigned to the assignee hereof and the disclosure of
which is incorporated herein by reference, describes precipitation
and membrane technologies that allow the preparation of pulse
protein products which are water soluble at low pH, produce heat
stable solutions and provide a reduced astringent sensation when
consumed. However, such low astringent products are produced in a
low yield.
[0006] The astringency of acidic beverages containing pulse (or
other) proteins is believed to be related to the protein becoming
insoluble in the mouth. One possible explanation of the cause of
this insolubility is that saliva proteins may bind and precipitate
the proteins that were dissolved in the acidic beverage. Another
theory is that the protein insolubility may arise from the
combination of the acidic protein solution and saliva resulting in
a pH in the mouth at which the protein is poorly soluble. It is
known that protein solubility can be increased by using enzymes to
hydrolyze proteins into smaller units.
[0007] Proteins are comprised of amino acids. Certain of these
amino acids, known as essential amino acids, cannot be synthesized
to meet the needs of the human body and so must be acquired through
the diet. A significant contributor to protein quality is the
content of essential amino acids in the protein. The
bioavailability of these amino acids is another important factor.
The content of essential amino acids in a protein forms the basis
of the measurement known as the Amino Acid Score (AAS). Amino Acid
Score is assessed by comparing the essential amino acid content of
a given protein to a reference pattern of essential amino acids.
The content of each essential amino acid (mg/g protein) is divided
by the content of the same essential amino acid in the reference
pattern (mg/g protein). The lowest resulting value, obtained for
the most limiting essential amino acid, is considered the Amino
Acid Score (AAS) (Report of Joint FAO/WHO Expert Consultation
(1991) Protein Quality Evaluation, FAO Food and Nutrition Paper 51;
Schaarfsma, G. 2000. J. Nutr., 130: 1865S). A protein that supplies
all the essential amino acids in the same proportions as the
reference pattern would have an Amino Acid Score of 1.0. Proteins
having high Amino Acid Scores are valued by food manufacturers.
Traditionally, the Amino Acid Scores of pea protein have been
limited by the concentration of sulfur containing amino acids. A
pea protein product with an improved Amino Acid Score would be of
commercial value. It should be noted that the Amino Acid Score of a
protein is often factored into a PDCAAS value, where the AAS is
corrected by a protein digestibility factor, so as to account for
the bioavailability. The current invention is concerned only with
the Amino Acid Score.
SUMMARY OF THE INVENTION
[0008] In accordance with the present invention, pulse proteins are
hydrolyzed, and the resulting solution is optionally adjusted in pH
and then fractionated into a soluble fraction and a residual solids
fraction. The soluble fraction is further processed to provide a
pulse protein hydrolyzate which is substantially completely soluble
throughout the pH range of about 2 to about 7 and provides little
or no astringency when an acidic beverage containing the pulse
protein hydrolyzate is consumed. The insoluble residue of the
hydrolysis treatment is further processed to provide a second pulse
protein hydrolyzate, having an Amino Acid Score that is improved
compared to the substrate protein and preferably above, at or close
to 1.0. Note that the term "protein hydrolyzate" is used herein to
describe the product of a process that involves a protein
hydrolysis step and it is not intended to infer anything about the
extent of hydrolysis in the final product.
[0009] In accordance with one aspect of the present invention,
there is provided a method of processing a pulse protein material,
which comprises effecting protein hydrolysis of the pulse protein
material, optionally adjusting the pH, then separating to form a
soluble fraction and processing the soluble fraction to provide a
pulse protein hydrolyzate which is substantially completely soluble
throughout the pH range of about 2 to about 7 and which introduces
little or no astringency when an acidic beverage containing the
pulse protein hydrolyzate is consumed and a solid residue, and
processing the solid residue to provide a second pulse protein
hydrolyzate having an improved Amino Acid Score, which is improved
compared to the substrate pulse protein material.
[0010] In accordance with another aspect of the present invention,
there is provided a pulse protein hydrolyzate having a protein
content of at least about 60 wt % (N.times.6.25) d.b., which is
substantially completely soluble throughout the pH range of about 2
to about 7 and introduces little or no astringency when an acidic
beverage containing the pulse protein hydrolyzate is consumed.
[0011] In accordance with a further aspect of the present
invention, there is provided a pulse protein hydrolyzate having an
Amino Acid Score which is improved compared to the substrate pulse
protein from which the hydrolyzate is derived.
[0012] In accordance with an additional aspect of the present
invention, there is provided a method of processing a pulse protein
material, which comprises effecting protein hydrolysis of the pulse
protein material, optionally adjusting the pH, then separating to
form a soluble fraction and processing the soluble fraction to
provide a pulse protein hydrolyzate which is substantially
completely soluble throughout the pH range of about 2 to about 7
and which introduces little or no astringency when an acidic
beverage containing the pulse protein hydrolyzate is consumed.
[0013] In accordance with yet another aspect of the present
invention, there is provided a pulse protein hydrolyzate having a
molecular weight profile as follows:
[0014] >100,000 Da--0 to 14 wt %
[0015] 15,000 to 100,000 Da--4 to 30 wt %
[0016] 5000 to 15,000 Da--20 to 29 wt %
[0017] 1,000 to 5,000 Da--27 to 54 wt %
[0018] <1,000 Da--8 to 23 wt %.
[0019] As would be known to one skilled in the art, the protein
content of a protein hydrolyzate may be determined by the content
of nitrogen in the hydrolyzate multiplied by a conversion factor
(generally 6.25) and expressed as a percentage on a dry weight
basis.
GENERAL DESCRIPTION OF THE INVENTION
[0020] The utilization of a procedure involving protein hydrolysis
of a pulse protein substrate and further processing of the soluble
portion of the hydrolyzate to provide a first pulse protein
hydrolyzate having little or no astringency in acidic solution and
further processing of the insoluble portion of the hydrolyzate to
provide a second pulse protein hydrolyzate with an improved Amino
Acid Score may be implemented on a number of pulse protein
substrates.
[0021] 1) Starting from dry powder (neutral)--A dry powder pulse
protein product that may be used is the 810N pulse protein product
described in copending U.S. patent application Ser. No. 14/811,052
filed Jul. 28, 2015 (US Patent Publication No. 2016/0050956
published Feb. 25, 2016) assigned to the assignee hereof and
incorporated herein by reference. This product having a protein
content of greater than about 60 wt % d.b. and a natural pH in
solution that is neutral or near neutral (pH about 6.0 to about
8.0). Alternatively, dry powder, commercial pulse protein products
having a protein content of greater than about 65 wt % d.b. and a
natural pH in solution that is neutral or near neutral (pH about
6.0 to about 8.0) may be used.
[0022] The procedure for treatment of the neutral dry powder
product involves rehydration of the protein powder to provide a
protein solution, optional adjustment of the solution pH within the
range of about 6.0 to about 8.0, treatment of the protein solution
with proteolytic enzyme, heat treatment of the enzymatically
treated material to inactivate the enzyme, adjustment of the pH of
the resulting solution to an acid value, such as about pH 2 to
about pH 4, centrifugation to separate centrate (soluble fraction)
from residual solids, concentration and optional diafiltration of
the centrate on a membrane filtration system to decrease the
content of salt and/or other impurities in the centrate and drying
the retentate to provide a pulse protein hydrolyzate having a
protein content greater than about 60 wt % (N.times.6.25) d.b. on a
dry weight basis and which is acid soluble and provides little or
no astringency when tasted in acidic solution. This pulse protein
hydrolyzate has an acidic natural pH in solution, which facilitates
the formulation of acidic beverage products. Alternatively, the
heat treatment step may be effected after the acidification step
but prior to the centrifugation step.
[0023] The residual solids separated from the soluble fraction have
an improved Amino Acid Score compared to the substrate protein and
may be further processed to provide a second pulse protein
hydrolyzate. The separated residual solids, which have an acidic
pH, may be directly dried or washed and then dried to provide a
pulse protein hydrolyzate having a protein content greater than
about 60 wt % (N.times.6.25) d.b. Alternatively, the residual
solids may be adjusted in pH to about 6.0 to about 8.0 and then
dried. As a further alternative, the residual solids may be washed
then adjusted in pH to about 6.0 to about 8.0 prior to the drying
step. As a further alternative, the solids may be neutralized
during the washing step by adjusting the mixture of solids and wash
water to a pH of about 6.0 to about 8.0 using food grade alkali
solution, then collecting the solids by centrifugation and drying
the solids to provide a pulse protein hydrolyzate having a protein
content of at least about 60 wt % (N.times.6.25) d.b.
[0024] The neutral dry powder pulse protein product starting
material may also be treated without an acidification step. Such a
procedure involves rehydration of the protein powder to provide a
protein solution, optional adjustment of the solution pH within the
range of about 6.0 to about 8.0, treatment of the protein solution
with proteolytic enzyme, heat treatment of the enzymatically
treated material to inactivate the enzyme, centrifugation to
separate centrate (soluble fraction) from residual solids,
concentration and optional diafiltration of the centrate on a
membrane filtration system to decrease the content of salt and/or
other impurities in the centrate and drying the retentate to
provide a pulse protein hydrolyzate having a protein content
greater than about 60 wt % (N.times.6.25) d.b. on a dry weight
basis and which is acid soluble and provides little or no
astringency when tasted in acidic solution.
[0025] The residual solids separated from the centrate, have an
improved Amino Acid Score compared to the substrate protein and may
be directly dried or washed then dried to provide a second pulse
protein hydrolyzate having a protein content greater than about 60
wt % (N.times.6.25) d.b.
[0026] 2) Starting from dry powder (low pH)--A dry powder pulse
protein product that may be used is the 810A pulse protein product
described in the aforementioned U.S. patent application Ser. No.
14/811,052. This product has a protein content of greater than
about 60 wt % d.b. and a low natural pH in solution (pH about 1.5
to about 4.0). Alternatively, dry, commercial pulse protein
products having a protein content of greater than about 65 wt %
d.b. and a low natural pH in solution (pH about 1.5 to about 4.0)
may be used.
[0027] One procedure for treatment of the low pH dry powder pulse
protein product involves an initial rehydration of the protein
powder to form a protein solution and adjustment of the pH of the
protein solution to the neutral range (pH about 6.0 to about 8.0).
These steps are followed by the steps described above for the
solutions prepared from neutral dry powder pulse protein product,
namely either treatment with proteolytic enzyme, heat treatment to
inactivate the enzyme (alternatively heat treatment may be effected
after acidification), adjustment of pH to an acid value, such as
about pH 2 to about pH 4, centrifugation to effect a solid/liquid
separation, concentration and optional diafiltration of the
centrate, and drying the retentate to provide a pulse protein
hydrolyzate having a protein content of at least about 60 wt %
(N.times.6.25) d.b. and which is acid soluble and provides little
or no astringency when tasted in acidic solution, or alternatively,
similar treatment without the acidification step. When the
acidification step is employed, the resulting pulse protein
hydrolyzate has an acidic natural pH in solution, which facilitates
the formulation of acidic beverage products. As when neutral dry
powder is the starting material, the residual solids, which have an
improved Amino Acid Score compared to the substrate protein, may be
further processed to form a second pulse protein hydrolyzate. The
residual solids may be directly dried or washed and then dried to
provide a pulse protein hydrolyzate having a protein content of at
least about 60 wt % (N.times.6.25) d.b. When the process with the
acidification step is employed, the residual solids may be adjusted
in pH to about 6.0 to about 8.0 and then dried. As a further
alternative for the process employing the acidification step, the
washed solids may be adjusted in pH to about 6.0 to about 8.0 after
the washing step and prior to the drying step. As a further
alternative for the process employing the acidification step, the
solids may be neutralized during the washing step by adjusting the
mixture of solids and wash water to a pH of about 6.0 to about 8.0
using food grade alkali solution, then collecting the solids by
centrifugation and drying the solids to provide a pulse protein
hydrolyzate having a protein content of at least about 60 wt %
(N.times.6.25) d.b.
[0028] As alternative to conducting the enzyme treatment in the
neutral pH range, the low pH protein powder may be rehydrated to
form a protein solution and then the proteolytic enzyme treatment
applied without pH adjustment of the protein solution or after
optional adjustment of the solution pH within the range of about
1.5 to about 4.0. The enzyme treatment is followed by heat
treatment to inactivate the enzyme, centrifugation to effect a
solid/liquid separation, concentration and optional diafiltration
of the centrate, and drying the retentate to provide a pulse
protein hydrolyzate having a protein content of at least about 60
wt % (N.times.6.25) d.b. and which is acid soluble and provides
little or no astringency when tasted in acidic solution. This pulse
protein hydrolyzate has an acidic natural pH in solution, which
facilitates the formulation of acidic beverage products. The
residual solids, which have an improved Amino Acid Score compared
to the substrate protein may be further processed to form a second
pulse protein hydrolyzate. The residual solids, which have an
acidic pH, may be directly dried or washed and then dried to
provide a pulse protein hydrolyzate having a protein content of at
least about 60 wt % (N.times.6.25) d.b. Alternatively, the residual
solids may be adjusted in pH to about 6.0 to about 8.0 and then
dried. As a further alternative, the washed solids may be adjusted
in pH to about 6.0 to about 8.0 after the washing step and prior to
the drying step. As a further alternative, the solids may be
neutralized during the washing step by adjusting the mixture of
solids and wash water to a pH of about 6.0 to about 8.0 using food
grade alkali solution, then collecting the solids by centrifugation
and drying the solids to provide a pulse protein hydrolyzate having
a protein content of at least about 60 wt % (N x 6.25) d.b.
[0029] 3) Enzyme treatment in pulse 810 process.times.The pulse 810
process involves membrane processing of a low pH protein solution,
which may be dried to form 810A or neutralized and dried to form
810N.
[0030] The procedure for enzyme treatment in the pulse 810 process
involves the preparation of a partially or fully concentrated
acidic protein solution by the procedure described in the
aforementioned pulse 810 US patent application followed by an
optional dilution step. The optionally diluted protein solution may
be adjusted to a pH of about 6.0 to about 8.0 and then enzyme
hydrolyzed or the enzyme hydrolysis may be conducted at the initial
low pH value or optionally after pH adjustment within the range of
about 1.5 to about 4.0. When the optionally diluted protein
solution is adjusted in pH to about 6.0 to about 8.0, the pH
adjustment step is followed by the steps utilized to process the
protein solution prepared from neutral dry powder pulse protein
product or the neutralized solution of low pH dry powder pulse
protein product described above, namely treatment with proteolytic
enzyme, heat treatment to inactivate the enzyme (alternatively heat
treatment may be effected after pH adjustment), pH adjustment to an
acid value, such as about pH 2 to about pH 4, centrifugation to
effect solids/liquid separation, concentration and optional
diafiltration of the centrate on a membrane filtration system and
drying the retentate or alternatively, similar processing without
the acidification step, to yield a pulse protein hydrolyzate having
a protein content of at least 60 wt % (N.times.6.25) d.b. and which
is acid soluble and provides little or no astringency when tasted
in acidic solution. When the acidification step is employed, the
resulting pulse protein hydrolyzate has an acidic natural pH in
solution, which facilitates the formulation of acidic beverage
products. As with the above-described procedures, the residual
solids, which have an improved Amino Acid Score compared to the
substrate protein may be further processed, such as by directly
drying or washing and then drying to provide a pulse protein
hydrolyzate having a protein content greater than about 60 wt %
(N.times.6.25) d.b. When the process with the acidification step is
employed, the solids may be adjusted in pH to about 6.0 to about
8.0 prior to the drying step or washed and then adjusted in pH to
about 6.0 to about 8.0 prior to the drying step. As a further
alternative for the process employing the acidification step, the
solids may be neutralized during the washing step by adjusting the
mixture of solids and wash water to a pH of about 6.0 to about 8.0
using food grade alkali solution, then collecting the solids by
centrifugation and drying the solids to provide a pulse protein
hydrolyzate having a protein content of at least about 60 wt %
(N.times.6.25) d.b.
[0031] When the optionally diluted protein solution derived from
the 810 process is processed without initial pH adjustment to the
range of about 6.0 to about 8.0, the acidic protein solution is
processed by the steps described above for the protein solution
prepared from the low pH dry powder pulse protein product without a
subsequent neutralization step, namely optional adjustment of the
solution pH within the range of about 1.5 to about 4.0, treatment
with proteolytic enzyme, heat treatment to inactivate the enzyme,
centrifugation to effect solids/liquid separation, concentration
and optional diafiltration of the centrate on a membrane filtration
system and drying the retentate to yield a pulse protein
hydrolyzate having a protein content of at least 60 wt % (N x 6.25)
d.b. and which is acid soluble and provides little or no
astringency when tasted in acidic solution. This pulse protein
hydrolyzate has an acidic natural pH in solution, which facilitates
the formulation of acidic beverage products. The residual solids,
which have an improved Amino Acid Score compared to the substrate
protein may be further processed, such as by directly drying or
washing and then drying to provide a pulse protein hydrolyzate
having a protein content greater than about 60 wt % (N.times.6.25)
d.b. Alternatively, the solids may be adjusted in pH to about 6.0
to about 8.0 prior to the drying step or may be washed then
adjusted in pH to about 6.0 to about 8.0 and prior to the drying
step. As a further alternative, the solids may be neutralized
during the washing step by adjusting the mixture of solids and wash
water to a pH of about 6.0 to about 8.0 using food grade alkali
solution, then collecting the solids by centrifugation and drying
the solids to provide a pulse protein hydrolyzate having a protein
content of at least about 60 wt % (N.times.6.25) d.b.
[0032] 4) Enzyme treatment on dryer feed for commercial
products.
[0033] In procedures 1) and 2), there is discussion of rehydrating
commercial pulse protein products and enzyme treating them. It
would be more practical to conduct the enzyme treatment on the
material before drying, and thus the invention includes treatment
of dryer feed according to the procedure described in 1) or 2)
above except that it would not be necessary to rehydrate the
protein product.
[0034] One class of pulse protein hydrolyzates arising from the
above procedures are substantially soluble over the pH range of
about 2 to about 7 and provide little or no astringent sensation
when tasted in acidic solution. Acidic solutions of the product of
the invention are preferably clear and heat stable. The second
class of pulse protein hydrolyzates arising from the above
procedures have an improved Amino Acid Score compared to the
substrate protein. Some degree of bitterness may be developed
during the protein hydrolysis step, but ideally the products of the
invention have little or no bitterness. Different proteolytic
enzymes have different activities at different pH values. Selection
of a proteolytic enzyme for use in the present invention may be
influenced by factors such as the pH of the hydrolysis and the
level of bitterness in the final product. The length of time for
the enzyme treatment may also influence the properties of the final
products. Generally, a relatively short treatment time, such as
about 30 minutes to about 60 minutes is preferred.
EXAMPLES
Example 1
[0035] This Example describes the preparation of pulse protein
hydrolyzates from neutral, dry powder pulse protein product
according to an embodiment of the method of the present
invention.
[0036] 36 kg of yellow pea protein concentrate was added to 600 L
of reverse osmosis purified water at ambient temperature and
agitated for 10 minutes to provide an aqueous protein solution. A
portion of the suspended solids were removed by centrifugation
using a decanter centrifuge and a protein solution having a protein
content of 2.34% by weight was collected. The pH of the protein
solution was lowered to 3.07 by the addition of HCl solution
(concentrated HCl diluted with an equal volume of water) and then
the solution was warmed to 50.degree. C., held for 10 minutes, then
centrifuged using a disc stack centrifuge. 519 L of acidified
protein solution and 77.44 kg of acid insoluble solid material were
collected.
[0037] The acidified protein solution, having a protein content of
0.82 wt %, was adjusted in pH to 1.92 and then reduced in volume
from 520 L to 70 L by concentration on a polyethersulfone membrane
having a molecular weight cut-off of 100,000 daltons, operated at a
temperature of about 51.degree. C. The protein solution, with a
protein content of 5.32 wt %, was then diafiltered on the same
membrane with 630 L of RO water adjusted to pH 2 with HCl solution,
followed by diafiltration with an additional 150 L of RO water. The
diafiltration operation was conducted at about 51.degree. C. The
diafiltered protein solution, having a protein content of 4.64 wt %
was then further concentrated to a protein content of 8.11 wt %.
34.5 kg of concentrated and diafiltered protein solution was
pasteurized at about 72.degree. C. for 16 seconds and then cooled.
28.38 kg of the pasteurized protein solution was adjusted in pH to
7.12 with a solution containing 12.5 wt % NaOH and 12.5 wt % KOH
(henceforth referred to as NaOH/KOH solution) and then spray dried
to yield a product found to have a protein content of 91.75%
(N.times.6.25) d.b. The product was termed YP29-E02-16A
YP810N-02.
[0038] 36 kg of yellow pea protein concentrate was added to 600 L
of reverse osmosis purified water at ambient temperature and
agitated for 10 minutes to provide an aqueous protein solution. A
portion of the suspended solids were removed by centrifugation
using a decanter centrifuge and a protein solution having a protein
content of 2.40% by weight was collected. The pH of the protein
solution was lowered to 3.00 by the addition of HCl solution
(concentrated HCl diluted with an equal volume of water) and then
the solution was warmed to 50.degree. C., held for 10 minutes then
centrifuged using a disc stack centrifuge. 508 L of acidified
protein solution and 69.39 kg of acid insoluble solid material were
collected.
[0039] The acidified protein solution, having a protein content of
1.04 wt %, was adjusted in pH to 2.07 and then reduced in volume
from 510 L to 70 L by concentration on a polyethersulfone membrane
having a molecular weight cut-off of 100,000 daltons, operated at a
temperature of about 50.degree. C. The protein solution, with a
protein content of 6.21 wt %, was then diafiltered on the same
membrane with 630 L of RO water adjusted to pH 2 with HCl solution,
followed by diafiltration with an additional 145 L of RO water. The
diafiltration operation was conducted at about 50.degree. C. The
diafiltered protein solution, having a protein content of 5.73 wt %
was then further concentrated to a protein content of 9.50 wt %.
31.7 kg of concentrated and diafiltered protein solution was
pasteurized at about 72.degree. C. for 16 seconds and then cooled.
The pasteurized solution was diluted to 38.44 kg. 18 kg of this
solution was adjusted in pH to 6.93 with NaOH/KOH solution and
diluted with 3 L of RO water then spray dried to yield a product
found to have a protein content of 91.42% (N.times.6.25) d.b. The
product was termed YP29-E04-16A YP810N-02.
[0040] 1.625 kg of YP29-E02-16A YP810N-02 and 0.667 kg of
YP29-E04-16A YP810N-02 were mixed with 20 L of RO water to form a
protein solution containing 2 kg of substrate protein. This
solution had a pH of 6.59. NaOH/KOH solution was added to raise the
pH of the sample to 6.95. The sample was then warmed to about
50.degree. C. and 20 ml of the proteolytic enzyme Flavourzyme
(Novozymes) was added. The sample was held at about 50.degree. C.
and mixed for 1 hour. The enzyme was inactivated by heat treating
the solution at about 90.degree. C. for 10 minutes and then the
treated protein solution was cooled to room temperature. This
sample had a pH of 6.89 and a protein content of 9.87 wt %. The pH
of the sample was then lowered to 3.02 using a solution of
concentrated HCl mixed with an equal volume of RO water. The
acidified sample was centrifuged batchwise at 6,000 rpm for 9 or 10
minutes in the HG-4L rotor of a Sorvall RC-3 centrifuge to provide
15.94 kg of centrate (soluble fraction) and 5.05 kg of residual
solids.
[0041] 15.9 L of centrate was combined with one volume of pH 3 RO
water and the sample concentrated to the original volume on a Dow
Filmtec NF-2540 nanofiltration membrane operated at ambient
temperature. This batchwise diafiltration was repeated nine
additional times. The sample was then reduced in volume to 5.4 kg
by concentration on the same membrane. The diafiltered and
concentrated protein hydrolyzate solution had a protein content of
9.62 wt %. This protein hydrolyzate solution was pasteurized at
about 72.degree. C. for less than 5 minutes. The pasteurized
solution was then spray dried to yield a pea protein hydrolyzate
having a protein content of 94.31 (N.times.6.25) d.b. The product
was termed YP29-H11-16A YP820A.
[0042] 3 kg of residual solids were mixed with 4 volumes of RO
water and then the pH of the sample raised to about 7 with NaOH/KOH
solution. The sample was then centrifuged to provide 2.955 kg of
washed solids. The 2.955 kg of washed solids were combined with
2.955 kg of RO water (to facilitate spray drying) and then spray
dried to yield a pea protein hydrolyzate having a protein content
of 92.31% (N.times.6.25) d.b. The product was termed YP29-H11-16A
YP820PN.
Example 2
[0043] This Example describes another example of the preparation of
pulse protein hydrolyzates from neutral, dry powder pulse protein
product according to an embodiment of the method of the present
invention.
[0044] 36 kg of yellow pea protein concentrate was added to 600 L
of reverse osmosis purified water at ambient temperature and
agitated for 10 minutes to provide an aqueous protein solution. A
portion of the suspended solids were removed by centrifugation
using a decanter centrifuge and a protein solution having a protein
content of 2.72% by weight was collected. The pH of the protein
solution was lowered to a pH of about 3 by the addition of HCl
solution (concentrated HCl diluted with an equal volume of water),
the solution was warmed to 50.degree. C., held for 10 minutes and
then centrifuged using a disc stack centrifuge. 474.8 L of
acidified protein solution and 85.85 kg of acid insoluble solid
material were collected.
[0045] The acidified protein solution, having a protein content of
1.66 wt %, was diluted with 25 L of water and then reduced in
volume to 145 L by concentration on a microfiltration membrane
having a molecular weight cut-off of 0.80 .mu.m and operated at
about 52.degree. C. The protein solution was then further
concentrated while concurrently diafiltering it with RO water
adjusted to pH 2 with HCl solution, the concentration and
diafiltration being conducted at a temperature of about 52.degree.
C. A total of 580 L of microfiltration permeate (clarified,
acidified protein solution) having a protein content of 1.28 wt %
was collected. This solution was reduced in volume to 125 L by
concentration on a polyethersulfone membrane having a molecular
weight cut-off of 100,000 daltons, operated at a temperature of
about 47.degree. C. The protein solution, with a protein content of
5.17 wt %, was then diafiltered on the same membrane with 1125 L of
RO water adjusted to pH 2 with HCl solution, followed by
diafiltration with an additional 125 L of RO water. The
diafiltration operation was conducted at about 50.degree. C. The
diafiltered protein solution, having a protein content of 4.50 wt %
was then further concentrated to a protein content of 10.01 wt %.
47 L of concentrated and diafiltered protein solution was
pasteurized at about 74.degree. C. for 16 seconds and then cooled.
56.56 kg of the pasteurized protein solution was combined with
13.25 kg of RO water and sufficient NaOH/KOH solution to adjust the
pH to 6.94 and then the mixture was spray dried to yield a product
found to have a protein content of 90.37% (N.times.6.25) d.b. The
product was termed YP35-G19-16A YP810N.
[0046] 2.322 kg of YP35-G19-16A YP810N was mixed with 20 L of RO
water to form a protein solution containing 2 kg of substrate
protein. This solution had a pH of 6.87. The sample was warmed to
about 50.degree. C. and 5 g of proteolytic enzyme (Enzeco Bromelain
Concentrate, Enzyme Development Corporation) was added. The sample
was held at about 50.degree. C. and mixed for 30 minutes. The
enzyme was inactivated by heat treating the solution about
80.degree. C. for 10 minutes and then the treated protein solution
cooled to room temperature. This sample had a pH of 6.47 and a
protein content of 9.79%. The pH of the sample was then lowered to
1.93 using a solution of concentrated HCl mixed with an equal
volume of RO water. The acidified sample was centrifuged batchwise
at 6,000 rpm for 10 minutes in the HG-4L rotor of a Sorvall RC-3
centrifuge to provide 16.46 kg of centrate (soluble fraction) and
4.58 kg of residual solids.
[0047] 16.46 kg of centrate was combined with two volumes of pH 2
RO water and the sample concentrated to the original volume on a
Dow Filmtec NF-2540 nanofiltration membrane operated at ambient
temperature. This batchwise diafiltration was repeated four
additional times. The sample was then reduced in volume to 8.90 kg
by concentration on the same membrane. The diafiltered and
concentrated protein hydrolyzate solution had a protein content of
8.01 wt %. This protein hydrolyzate solution was pasteurized at
about 73.degree. C. for less than 2 minutes. The pasteurized
solution was then spray dried to yield a pea protein hydrolyzate
having a protein content of 96.33% (N.times.6.25) d.b. The product
was termed YP35-I19-16A YP820A.
[0048] 126.5 g of residual solids were mixed with 506 g of RO water
and then the pH of the sample raised to about 7 with NaOH/KOH
solution. The sample was centrifuged and 100.70 g of washed solids
were freeze dried to yield a pea protein hydrolyzate having a
protein content 84.69% (N.times.6.25) w.b. The product was termed
YP35-I19-16A YP820PN.
Example 3
[0049] This Example describes another example of the preparation of
pulse protein hydrolyzates from neutral, dry powder pulse protein
product according to an embodiment of the method of the present
invention.
[0050] 96 kg of yellow pea protein flour was added to 600 L of
reverse osmosis purified water at ambient temperature and agitated
for 10 minutes to provide an aqueous protein solution. A portion of
the suspended solids were removed by centrifugation using a
decanter centrifuge and a protein solution having a protein content
of 3.94% by weight was collected. The pH of the protein solution
was lowered to 2.05 by the addition of HCl solution (concentrated
HCl diluted with an equal volume of water), the solution was warmed
to 50.degree. C., held for 10 minutes and then centrifuged using a
disc stack centrifuge. 524 L of acidified protein solution and
83.98 kg of acid insoluble solid material were collected.
[0051] The acidified protein solution, having a protein content of
3.46 wt %, was reduced in volume to 190 L by concentration on a
microfiltration membrane having a molecular weight cut-off of 0.80
.mu.m operated at about 54.degree. C. The protein solution was then
further concentrated while concurrently diafiltering it with pH 2
RO water at about 54.degree. C. A total of 520 L of microfiltration
permeate (clarified, acidified protein solution) having a protein
content of 2.05 wt % was collected. This solution was reduced in
volume to 150 L by concentration on a polyethersulfone membrane
having a molecular weight cut-off of 100,000 daltons, operated at a
temperature of about 50.degree. C. The protein solution, with a
protein content of 5.20 wt %, was then diafiltered on the same
membrane with 1350 L of RO water adjusted to pH 2 with HCl
solution, followed by diafiltration with an additional 180 L of RO
water. The diafiltration operation was conducted at about
51.degree. C. The diafiltered protein solution, having a protein
content of 5.30 wt % was then further concentrated to a protein
content of 9.69 wt %. 80 L of concentrated and diafiltered protein
solution was diluted with 20 L of water and then pasteurized at
about 77.degree. C. for 16 seconds and then cooled. The pasteurized
protein solution was adjusted to pH 6.98 with NaOH/KOH solution and
then spray dried to yield a product found to have a protein content
of 89.38% (N.times.6.25) d.b. The product was termed YP34-G27-16A
YP810N.
[0052] 2.32 kg of YP34-G27-16A YP810N was mixed with 20 L of RO
water to form a protein solution containing 2 kg of substrate
protein. This solution had a pH of 6.77. NaOH/KOH solution was
added to raise the pH of the sample to 7.01. The sample was then
warmed to about 50.degree. C., diluted with 10 L RO water and then
10 ml of proteolytic enzyme (Liquipanol T-200, Enzyme Development
Corporation) was added. The sample was held at about 50.degree. C.
and mixed for 30 minutes. The enzyme was inactivated by heat
treating the solution about 80.degree. C. for 10 minutes and then
the treated protein solution cooled to room temperature. This
sample had a pH of 6.57 and a protein content of 6.61%. The pH of
the sample was then lowered to 1.96 using a solution of
concentrated HCl mixed with an equal volume of RO water. The
acidified sample was centrifuged batchwise at 6,000 rpm for 10
minutes in the HG-4L rotor of a Sorvall RC-3 centrifuge to provide
26.94 kg of centrate (soluble fraction) and 4.84 kg of residual
solids.
[0053] 26.94 kg of centrate was combined with 2 volumes of pH 2 RO
water and the sample concentrated to the original volume on a Dow
Filmtec NF2540 nanofiltration membrane operated at ambient
temperature. This batchwise diafiltration was repeated four
additional times. The sample was then reduced in volume by
concentration on the same membrane. The diafiltered and
concentrated protein hydrolyzate solution had a protein content of
7.31 wt %. This protein hydrolyzate solution was pasteurized at
about 72.degree. C. for 16 seconds. The pasteurized solution was
then spray dried to yield a pea protein hydrolyzate having a
protein content of 95.75% (N.times.6.25) d.b. The product was
termed YP34-I26-16A YP820A.
[0054] 139.7 g of residual solids were mixed with 559 g of RO water
and then the pH of the sample raised to about 7 with NaOH/KOH
solution. The sample was centrifuged at 6,000 rpm for 10 minutes in
the HG-4L rotor of a Sorvall RC-3 centrifuge to provide 117 g of
washed solids, 100 g of which were freeze dried to yield a pea
protein hydrolyzate having a protein content 80.32% (N.times.6.25)
w.b. The product was termed YP34-I26-16A YP820PN.
Example 4
[0055] This Example describes an example of the preparation of
pulse protein hydrolyzates from pH adjusted, concentrated protein
solution prepared according to the process of U.S. Ser. No.
14/811,052, according to an embodiment of the method of the present
invention.
[0056] 18 kg of yellow pea protein concentrate was added to 300 L
of reverse osmosis purified water at ambient temperature and
agitated for 10 minutes to provide an aqueous protein solution. A
portion of the suspended solids were removed by centrifugation
using a decanter centrifuge and a protein solution having a protein
content of 2.80% by weight was collected. The pH of the protein
solution was lowered to 2.03 by the addition of HCl solution
(concentrated HCl diluted with an equal volume of water), the
solution was warmed to 50.degree. C., held for 10 minutes and then
centrifuged using a disc stack centrifuge. 245 L of acidified
protein solution and an unrecorded weight of acid insoluble solid
material were collected.
[0057] The acidified protein solution was reduced in volume to 93 L
by concentration on a polyethersulfone membrane having a molecular
weight cut-off of 100,000 daltons, operated at a temperature of
about 44.degree. C. The protein solution, with a protein content of
5.41 wt %, was then diafiltered on the same membrane with 855 L of
RO water adjusted to pH 2 with HCl solution, followed by
diafiltration with additional RO water (volume not recorded). The
diafiltration operation was conducted at about 51.degree. C. The
diafiltered protein solution, having a protein content of 4.48 wt %
was then further concentrated to a protein content of 10.52 wt
%.
[0058] 23.86 kg of concentrated and diafiltered protein solution
was adjusted to pH 7.23 with NaOH/KOH solution. The sample was then
warmed to about 50.degree. C. and 25 g of proteolytic enzyme
(Flavourzyme, Novozymes) was added. The sample was held at about
50.degree. C. and mixed for 1 hour. The enzyme was inactivated by
heat treating the solution at about 90.degree. C. for 10 minutes
and then the treated protein solution cooled to room temperature.
This sample had a pH of 7.10 and a protein content of 10.03%. The
pH of the sample was then lowered to 3.12 using a solution of
concentrated HCl mixed with an equal volume of RO water. The
acidified sample was centrifuged to provide 13.38 kg of centrate
(soluble fraction) and 9.36 kg of residual solids.
[0059] 13.38 kg of centrate, having a protein content of 2.49 wt %
was combined with 26.76 kg of RO water adjusted to pH 3 with HCl
solution and the sample concentrated to 13.38 kg on a Dow Filmtec
NF-2540 nanofiltration membrane operated at ambient temperature.
This batchwise diafiltration was repeated four additional times.
The sample was then reduced in volume by concentration on the same
membrane. The diafiltered and concentrated protein hydrolyzate
solution had a protein content of 2.88 wt %. This protein
hydrolyzate solution was pasteurized at about 72.degree. C. for 16
seconds. The pasteurized solution was then spray dried to yield a
pea protein hydrolyzate having a protein content of 91.13%
(N.times.6.25) d.b. The product was termed YP35-J06-16A YP822A.
[0060] 200 g of residual solids were mixed with 800 g of RO water
and then the pH of the sample raised to about 7 with NaOH/KOH
solution. The sample was centrifuged to provide 216.08 g of washed
solids, a portion of which were freeze dried to yield a pea protein
hydrolyzate having a protein content 77.72% (N.times.6.25) w.b. The
product was termed YP35-J06-16A YP822PN.
Example 5
[0061] This Example describes preparation of the pulse protein
hydrolyzates of the present invention from pH adjusted,
concentrated protein solution prepared according to the process of
U.S. Ser. No. 14/811,052, according to an embodiment of the method
of the present invention.
[0062] 18 kg of yellow pea protein concentrate was added to 300 L
of reverse osmosis purified water at ambient temperature and
agitated for 10 minutes to provide an aqueous protein solution. A
portion of the suspended solids were removed by centrifugation
using a decanter centrifuge and a protein solution having a protein
content of 2.85% by weight was collected. The pH of the protein
solution was lowered to 2.07 by the addition of HCl solution
(concentrated HCl diluted with an equal volume of water), the
solution was warmed to 50.degree. C., held for 10 minutes and then
centrifuged using a disc stack centrifuge. 240 L of acidified
protein solution and an unrecorded weight of acid insoluble solid
material were collected.
[0063] The acidified protein solution, having a protein content of
2.37 wt %, was reduced in volume to 110 L by concentration on a
polyethersulfone membrane having a molecular weight cut-off of
100,000 daltons, operated at a temperature of about 47.degree. C.
The protein solution, with a protein content of 4.96 wt %, was then
diafiltered on the same membrane with 990 L of RO water adjusted to
pH 2 with HCl solution, followed by diafiltration with an
additional 110 L of RO water. The diafiltration operation was
conducted at about 51.degree. C. The diafiltered protein solution,
having a protein content of 4.64 wt % was then further concentrated
to provide 53.36 kg of concentrated and diafiltered protein
solution having a protein content of 7.76 wt %.
[0064] 40.18 kg of concentrated and diafiltered protein solution
was adjusted to pH 7.07 with NaOH/KOH solution. The sample was then
warmed to about 50.degree. C. and 3.35 g of proteolytic enzyme
(Liquipanol T-200, Enzyme Development Corporation) was added. The
sample was held at about 50.degree. C. and mixed for 30 minutes.
The enzyme was inactivated by heat treating the solution about
90.degree. C. for 10 minutes and then the treated protein solution
cooled to room temperature. This sample had a pH of 6.77 and a
protein content of 8.02 wt %. The pH of the sample was then lowered
to 2.91 using a solution of concentrated HCl mixed with an equal
volume of RO water. The acidified sample was centrifuged to provide
28.26 kg of centrate (soluble fraction) and 11.26 kg of residual
solids.
[0065] 28.26 kg of centrate, having a protein content of 3.26 wt %
was combined with 60 L of pH 3 RO water and the sample concentrated
to the original volume on a Dow Filmtec NF-2540 nanofiltration
membrane operated at ambient temperature. This batchwise
diafiltration was repeated three additional times. Then an
additional batchwise diafiltration step was conducted where 50 L of
pH 3 RO water was added and the sample concentrated to the original
volume. The sample was then reduced in volume to 10.48 kg by
concentration on the same membrane. The diafiltered and
concentrated protein hydrolyzate solution had a protein content of
8.26 wt %. This protein hydrolyzate solution was pasteurized at
about 72.degree. C. for 16 seconds. The pasteurized solution was
then spray dried to yield a pea protein hydrolyzate having a
protein content of 100.14% (N.times.6.25) d.b. The product was
termed YP35-J11-16A YP822A.
[0066] 11.26 kg of residual solids were mixed with 45.02 kg of RO
water and then the pH of the sample raised to about 7 with NaOH/KOH
solution. The sample was centrifuged with a disc stack centrifuge
to provide 17.75 kg of washed solids. These washed solids were
diluted with 5 L of RO water and pasteurized at about 72.degree. C.
for 16 seconds. The pasteurized sample was diluted with 5 L of RO
water to facilitate drying, then spray dried to yield a pea protein
hydrolyzate having a protein content of 80.33% (N.times.6.25) d.b.
The product was termed YP35-J11-16A YP822PN.
Example 6
[0067] This Example describes preparation of pulse protein
hydrolyzates from acidic, concentrated protein solution prepared
according to the process of U.S. Ser. No. 14/811,052, according to
an embodiment of the method of the present invention.
[0068] 36 kg of yellow pea protein concentrate was added to 600 L
of reverse osmosis purified water at ambient temperature and
agitated for 10 minutes to provide an aqueous protein solution. A
portion of the suspended solids were removed by centrifugation
using a decanter centrifuge and a protein solution having a protein
content of 2.54% by weight was collected. The pH of the protein
solution was lowered to 2.02 by the addition of HCl solution
(concentrated HCl diluted with an equal volume of water) and then
the solution was warmed to about 50.degree. C., held for 10 minutes
and then centrifuged using a disc stack centrifuge. 535 L of
acidified protein solution was collected having a pH of 2.10 and a
protein content of 2.30 wt %.
[0069] The acidified protein solution was reduced in volume to 170
L by concentration on a polyethersulfone membrane having a
molecular weight cut-off of 100,000 daltons, operated at a
temperature of about 48.degree. C. The protein solution, with a
protein content of 5.98 wt %, was then diafiltered on the same
membrane with 1530 L of RO water adjusted to pH 2 with HCl
solution, followed by diafiltration with an additional 270 L of RO
water. The diafiltration operation was conducted at about
50.degree. C. The diafiltered protein solution, having a protein
content of 5.67 wt % was then further concentrated to a protein
content of 9.68 wt %. 85 L of concentrated and diafiltered protein
solution was pasteurized at about 72.degree. C. for 16 seconds and
then cooled.
[0070] 55.5 kg of the pasteurized, concentrated and diafiltered
protein solution was mixed with 34.5 L of RO water to form a
protein solution. This solution had a pH of 2.98 and a protein
content of 4.73 wt %. The sample was warmed to about 50.degree. C.
and 22.5 g of proteolytic enzyme (Enzeco Fungal Acid Protease
Concentrate, Enzyme Development Corporation) was added. The sample
was held at about 50.degree. C. and mixed for 1 hour. The enzyme
was inactivated by heat treating the solution at about 70.degree.
C. for 10 minutes and then cooling it to room temperature. The heat
treated sample had a pH of 3.70 and a protein content of 4.46 wt %.
This sample was then centrifuged with a disc stack centrifuge to
provide 84 L of centrate (soluble fraction) and 18.54 kg of
residual solids.
[0071] The 84 L of centrate was concentrated to 10.04 kg on a Dow
Filmtec NF-2540 nanofiltration membrane operated at about
26.degree. C. The concentrated protein hydrolyzate solution had a
protein content of 13.64%. The concentrated protein hydrolyzate
solution was pasteurized at about 72.degree. C. for 16 seconds. The
pasteurized solution was spray dried to yield a pea protein
hydrolyzate having a protein content of 101.08 (N.times.6.25) d.b.
The product was termed YP35-L07-16A YP823A.
[0072] The residual solids were washed with 40 L of RO water
adjusted to pH 3 with HCl solution and then the slurry centrifuged
with a disc stack centrifuge to collect 27.3 kg of washed solids.
The washed solids were then combined with 60 L of RO water and
sufficient NaOH/KOH solution to adjust the pH to about 7. This
slurry was centrifuged with a disc stack centrifuge to provide
19.24 kg of neutralized, washed solids. These solids were
pasteurized at about 72.degree. C. for 30 seconds and then spray
dried to yield a pea protein hydrolyzate having a protein content
81.16% (N.times.6.25) d.b. The product was termed YP35-L07-16A
YP823PN.
Example 7
[0073] This Example contains an evaluation of the solubility in
water of the pulse protein hydrolyzates produced by the methods of
Examples 1 to 6. Solubility was tested based on protein solubility
(termed protein method, a modified version of the procedure of Morr
et al., J. Food Sci. 50:1715-1718).
[0074] Sufficient protein hydrolyzate powder to supply 0.5 g of
protein was weighed into a beaker and then a small amount of
reverse osmosis (RO) purified water was added and the mixture
stirred until a smooth paste formed. Additional water was then
added to bring the volume to approximately 45 ml. The contents of
the beaker were then slowly stirred for 60 minutes using a magnetic
stirrer. The pH was determined immediately after dispersing the
protein and was adjusted to the appropriate level (2, 3, 4, 5, 6 or
7) with diluted NaOH or HCI. A sample was also prepared at natural
pH. For the pH adjusted samples, the pH was measured and corrected
periodically during the 60 minutes stirring. After the 60 minutes
of stirring, the samples were made up to 50 ml total volume with RO
water, yielding a 1% w/v protein dispersion. The protein content of
the dispersions was measured by combustion analysis using a Leco
Nitrogen Determinator (N.times.6.25). Aliquots of the dispersions
were then centrifuged at 7,800 g for 10 minutes, which sedimented
insoluble material and yielded a supernatant. The protein content
of the supematant was measured by combustion analysis
(N.times.6.25) and the protein solubility of the product calculated
as follows:
Protein Solubility (%)=(% protein in supernatant/% protein in
initial dispersion).times.100
[0075] Values calculated as greater than 100% were reported as
100%.
[0076] The natural pH values of the hydrolyzed protein products of
Examples 1 to 6 in water (1% protein) are shown in Table 1:
TABLE-US-00001 TABLE 1 Natural pH of pulse protein hydrolyzates in
water at 1% protein Product Natural pH YP29-H11-16A YP820A 3.69
YP35-I19-16A YP820A 2.89 YP34-I26-16A YP820A 3.07 YP35-J06-16A
YP822A 3.65 YP29-J11-16A YP822A 3.69 YP35-L07-16A YP823A 3.76
[0077] The protein solubility results obtained are set forth in the
following Table 2:
TABLE-US-00002 TABLE 2 Protein solubility of products at different
pH values Protein Solubility (%) Product pH 2 pH 3 pH 4 pH 5 pH 6
pH 7 Nat. pH YP29-H11-16A 98.1 100 100 100 100 100 100 YP820A
YP35-I19-16A 100 100 100 100 100 95.2 95.0 YP820A YP34-I26-16A 100
100 93.2 97.2 98.0 99.0 100 YP820A YP35-J06-16A 92.1 97.3 100 100
100 100 100 YP822A YP29-J11-16A 100 98.0 95.0 91.2 98.0 99.0 99.0
YP822A YP35-L07-16A 100 100 100 100 98.9 100 92.9 YP823A
[0078] As can be seen from the results presented in Table 2, the
pulse protein hydrolyzates had high protein solubility across the
pH range tested.
Example 8
[0079] This Example illustrates the production of pulse protein
isolate according to the procedure of U.S. patent applications Ser.
No. 13/103,528 filed May 9, 2011 (US Patent Publication No.
2011/0274797 published Nov. 10, 2011), Ser. No. 13/556,357 filed
Jul. 24, 2012 (US Patent Publication No. 2013/00189408 published
Jul. 25, 2013), Ser. No. 13/642,003 filed Jan. 7, 2013 (US Patent
Publication No. 2013/0129901 published May 23, 2013) and Ser. No.
15/041,193 filed Feb. 11, 2016 (US Patent Publication No.
2016/0227833 published Aug. 11, 2016 ("YP701")). As previously
mentioned, pulse protein isolate prepared by this method provides
an astringent sensation in the mouth when consumed in acidic
solution. The product prepared according to this Example was used
in sensory evaluations of astringency, as described in Examples
9-14, 18 and 19.
[0080] 30 kg of yellow pea protein concentrate was combined with
300 L of 0.15 M CaCl.sub.2 solution at ambient temperature and
agitated for 30 minutes to provide an aqueous protein solution. The
residual solids were removed by centrifugation to produce a
centrate having a protein content of 2.68% by weight. 262 L of
centrate was added to 274 L of RO water and the pH of the sample
lowered to 2.85 with HCl solution (concentrated HCl diluted with an
equal volume of water). The diluted and acidified centrate was
further clarified by filtration to provide a clear protein solution
with a protein content of 1.15% by weight and having a pH of
3.23.
[0081] The filtered protein solution was warmed then reduced in
volume from 662 L to 57 L by concentration on a polyethersulfone
membrane, having a molecular weight cutoff of 100,000 Daltons,
operated at a temperature of about 49.degree. C. The concentrated
acidified protein solution, with a protein content of 9.33% by
weight, was diafiltered with 285 L of RO water, with the
diafiltration operation conducted at about 52.degree. C. 50.20 kg
of acidified, diafiltered, concentrated protein solution was
obtained having a protein content of 7.91 wt %. The acidified,
diafiltered, concentrated protein solution was then heated at about
76.degree. C. for 16 seconds and then dried to yield a product
found to have a protein content of 101.50 wt % (N.times.6.25) d.b.
The product was termed YP35-K28-16A YP701.
Example 9
[0082] This Example illustrates a comparison of the astringency
level of the YP29-H11-16A YP820A prepared as described in Example 1
with that of the YP35-K28-16A YP701 prepared as described in
Example 8.
[0083] Samples were prepared for sensory evaluation by dissolving
sufficient protein powder to supply 2 g protein in 100 ml of
purified drinking water. The pH of the YP820A solution was 3.62.
The pH of the YP701 solution was 3.56. An informal panel of 7
panellists was asked to blindly taste the samples and indicate
which was less astringent.
[0084] Four out of seven panellists indicated that the YP29-H11-16A
YP820A was less astringent and three panellists indicated that the
YP35-K28-16A YP701 was less astringent.
Example 10
[0085] This Example illustrates a comparison of the astringency
level of the YP35-I19-16A YP820A prepared as described in Example 2
with that of the YP35-K28-16A YP701 prepared as described in
Example 8.
[0086] Samples were prepared for sensory evaluation by dissolving
sufficient protein powder to supply 2 g protein in 100 ml of
purified drinking water. The pH of the YP820A solution was 2.83.
The pH of the YP701 solution was lowered from 3.45 to 2.87 by the
addition of food grade HCl solution. An informal panel of 7
panellists was asked to blindly taste the samples and indicate
which was less astringent.
[0087] Five out of seven panellists indicated that the YP35-I19-16A
YP820A was less astringent and two panellists indicated that the
YP35-K28-16A YP701 was less astringent.
Example 11
[0088] This Example illustrates a comparison of the astringency
level of the YP34-I26-16A YP820A prepared as described in Example 3
with that of the YP35-K28-16A YP701 prepared as described in
Example 8.
[0089] Samples were prepared for sensory evaluation by dissolving
sufficient protein powder to supply 2 g protein in 100 ml of
purified drinking water. The pH of the YP820A solution was 3.04.
The pH of the YP701 solution was lowered from 3.69 to 3.08 by the
addition of food grade HCl solution. An informal panel of 6
panellists was asked to blindly taste the samples and indicate
which was less astringent.
[0090] Five out of six panellists indicated that the YP34-I26-16A
YP820A was less astringent and one panellist indicated that the
YP35-K28-16A YP701 was less astringent.
Example 12
[0091] This Example illustrates a comparison of the astringency
level of the YP35-J06-16A YP822A prepared as described in Example 4
with that of the YP35-K28-16A YP701 prepared as described in
Example 8.
[0092] Samples were prepared for sensory evaluation by dissolving
sufficient protein powder to supply 2 g protein in 100 ml of
purified drinking water. The pH of the YP822A solution was 3.63.
The pH of the YP701 solution was 3.60. An informal panel of 6
panellists was asked to blindly taste the samples and indicate
which was less astringent.
[0093] All six panellists indicated that the YP35-J06-16A YP822A
was less astringent.
Example 13
[0094] This Example illustrates a comparison of the astringency
level of the YP29-J11-16A YP822A prepared as described in Example 5
with that of the YP35-K28-16A YP701 prepared as described in
Example 8.
[0095] Samples were prepared for sensory evaluation by dissolving
sufficient protein powder to supply 3 g protein in 150 ml of
purified drinking water. The pH of the YP822A solution was 3.68.
The pH of the YP701 solution was 3.63. An informal panel of 10
panellists was asked to blindly taste the samples and indicate
which was less astringent.
[0096] Seven out of ten panellists indicated that the YP29-J11-16A
YP822A was less astringent, two panellists indicated that the
YP35-K28-16A YP701 was less astringent and one panellist could not
detect a difference in astringency level.
Example 14
[0097] This Example illustrates a comparison of the astringency
level of the YP35-L07-16A YP823A prepared as described in Example 6
with that of the YP35-K28-16A YP701 prepared as described in
Example 8.
[0098] Samples were prepared for sensory evaluation by dissolving
sufficient protein powder to supply 3 g protein in 150 ml of
purified drinking water. The pH of the YP823A solution was 3.75.
The pH of the YP701 solution was 3.63. An informal panel of 10
panellists was asked to blindly taste the samples and indicate
which was less astringent.
[0099] Eight out of ten panellists indicated that the YP35-L07-16A
YP823A was less astringent, while two panellists indicated that the
YP35-K28-16A YP701 was less astringent.
Example 15
[0100] This Example describes another example of the preparation of
pulse protein hydrolyzates from neutral, dry powder pulse protein
product according to an embodiment of the method of the present
invention.
[0101] 96 kg of yellow pea flour was added to 600 L of reverse
osmosis purified water at ambient temperature and agitated for 10
minutes to provide an aqueous protein solution. A portion of the
suspended solids were removed by centrifugation using a decanter
centrifuge and a protein solution having a protein content of 2.86%
by weight was collected. The pH of the protein solution was lowered
to 2.04 by the addition of HCl solution (concentrated HCl diluted
with an equal volume of water), the solution was warmed to
50.degree. C., held for 10 minutes and then centrifuged using a
disc stack centrifuge. 477.3 L of acidified protein solution and an
unrecorded weight of acid insoluble solid material were
collected.
[0102] The acidified protein solution, having a protein content of
2.46 wt %, was reduced in volume to 135 L by concentration on a
microfiltration membrane having a molecular weight cut-off of 0.80
.mu.m and operated at about 51.degree. C. The protein solution was
then further concentrated while concurrently diafiltering it with
135 L of RO water adjusted to pH 2 with HCl solution, the
concentration and diafiltration being conducted at a temperature of
about 52.degree. C. A total of 576 L of microfiltration permeate
(clarified, acidified protein solution) having a protein content of
1.85 wt % was collected. This solution was reduced in volume to 180
L by concentration on a polyethersulfone membrane having a
molecular weight cut-off of 100,000 daltons, operated at a
temperature of about 48.degree. C. The protein solution, with a
protein content of 5.31 wt %, was then diafiltered on the same
membrane with 1620 L of RO water adjusted to pH 2 with HCl
solution, followed by diafiltration with an additional 75 L of RO
water. The diafiltration operation was conducted at about
53.degree. C. The diafiltered protein solution, having a protein
content of 5.23 wt % was then further concentrated to a protein
content of 9.01 wt %. 90 L of concentrated and diafiltered protein
solution was pasteurized at about 73.degree. C. for 16 seconds and
then cooled. 90 kg of the pasteurized protein solution was combined
with 20 kg of RO water, filtered through a 2 .mu.m sock filter and
then sufficient NaOH/KOH solution to adjust the pH to 6.96 was
added and the mixture spray dried to yield a product found to have
a protein content of 87.74% (N.times.6.25) d.b. The product was
termed YP36-B28-17A YP810N.
[0103] 6.565 kg of YP36-B28-17A YP810N was mixed with 70 L of RO
water at 50.degree. C. to form a protein solution containing 5.42
kg of substrate protein. This solution had a pH of 6.84. To the
sample was added 25 g of proteolytic enzyme (Protease P, Amano).
The sample was held at about 50.degree. C. and mixed for 60
minutes. The enzyme was inactivated by heat treating the solution
for 20 minutes between 62.degree. and 72.degree. C. The treated
protein solution was then cooled to room temperature. 76.18 kg of
sample was obtained having a pH of 6.29 and a protein content of
7.49%.
[0104] A 19.56 kg portion of the enzyme treated material was
centrifuged in the HG-4L rotor of a Sorvall RC-3 centrifuge to
provide 14.56 kg of centrate (soluble fraction) and 5 kg of
residual solids. The centrate had a protein content of 4.75 wt %.
The centrate was pasteurized by heating to about 73.degree. C. for
16 seconds and then cooled. The pasteurized solution was spray
dried to yield a pulse protein hydrolyzate having a protein content
of 90.81 wt % (N.times.6.25) d.b. This product was termed
YP36-D20-17A YP840N. The residual solids were freeze dried to
provide a pulse protein hydrolyzate having a protein content of
82.59 wt % (N.times.6.25) d.b. This product was termed YP36-D20-17A
YP840PN.
[0105] A 46.44 kg portion of the enzyme treated material was
lowered in pH to 2.94 using a solution of concentrated HCl mixed
with an equal volume of RO water. The acidified sample was
centrifuged using a desludger centrifuge to provide 45.24 kg of
centrate (soluble fraction) and 10.90 kg of residual solids. 45.24
kg of centrate was concentrated to about 15 L on a Dow Filmtec
nanofiltration membrane operated at about 26.degree. C. 30 L of RO
water adjusted to pH 3 with HCl solution was added and then the
sample reconcentrated on the same membrane to about 15 L. This
batchwise diafiltration was repeated four additional times. The
temperature of the diafiltration increased from about 24.degree. C.
for the first diafiltration step to about 34.degree. C. for the
last diafiltration step. 16.96 kg of diafiltered and concentrated
protein hydrolyzate solution was obtained having a protein content
of 6.26 wt %. This protein solution was pasteurized at about
72.degree. C. for 16 seconds. The pasteurized solution was then
spray dried to yield a pea protein hydrolyzate having a protein
content of 100.01% (N.times.6.25) d.b. The product was termed
YP36-D20-17A YP820A. 634 g of residual solids were freeze dried to
yield a pulse protein hydrolyzate having a protein content of
78.33% (N.times.6.25) d.b. The product was termed YP36-D20-17A
YP820PA.
Example 16
[0106] This Example describes the preparation of pulse protein
hydrolyzate from acidic, dry powder pulse protein product according
to an embodiment of the method of the present invention.
[0107] 96 kg of yellow pea flour was added to 600 L of reverse
osmosis purified water at ambient temperature and agitated for 10
minutes to provide an aqueous protein solution. A portion of the
suspended solids were removed by centrifugation using a decanter
centrifuge and a protein solution having a protein content of 2.87%
by weight was collected. The pH of the protein solution was lowered
to 1.89 by the addition of HCl solution (concentrated HCl diluted
with an equal volume of water), the solution was warmed to
50.degree. C., held for 10 minutes and then centrifuged using a
disc stack centrifuge. 482 L of acidified protein solution and an
unrecorded weight of acid insoluble solid material were
collected.
[0108] 462 L of the acidified protein solution, having a protein
content of 2.45 wt %, was reduced in volume to 180 L by
concentration on a polyethersulfone membrane having a molecular
weight cut-off of 100,000 daltons, operated at a temperature of
about 49.degree. C. The protein solution, with a protein content of
5.68 wt %, was then diafiltered on the same membrane with 1620 L of
RO water adjusted to pH 2 with HCl solution, followed by
diafiltration with an additional 180 L of RO water. The
diafiltration operation was conducted at about 52.degree. C. The
diafiltered protein solution, having a protein content of 5.24 wt %
was then further concentrated to a protein content of 10.20 wt %.
90 L of concentrated and diafiltered protein solution was
pasteurized at about 73.degree. C. for 16 seconds and then cooled.
The pasteurized protein solution (95L) was further diluted with
about 20 L of RO water then spray dried to yield a product found to
have a protein content of 89.53% (N.times.6.25) d.b. The product
was termed YP36-I11-17A YP810A.
[0109] 55.8 g of YP36-I11-17A YP810A was mixed with RO water to
prepare 1000 ml of protein solution containing 48.1 g of substrate
protein. The pH of the protein solution was raised from 2.83 to 3
by the addition of 2M NaOH and then the temperature of the solution
raised to about 50.degree. C. 0.25 g of Protease M (Amano) was
added to the sample, which was mixed at 50.degree. C. for 60
minutes. The enzyme was then inactivated by heating the sample to
90.degree. C. for 10 minutes. The sample was then cooled to room
temperature and centrifuged for 10 minutes at 7,000 g using a
laboratory centrifuge. The centrate (soluble fraction) was
discarded. The residual solids were collected and resuspended in 4
volumes of RO water adjusted to pH 3 with HCl solution. The sample
was then centrifuged for 10 minutes at 7,000 g using a laboratory
centrifuge, the centrate discarded and the washed residual solids
collected and freeze dried. 23.40 g of freeze dried material was
collected, containing a protein content of 80.79% (N.times.6.25)
w.b. The product was termed benchscale YP821PA.
Example 17
[0110] This Example contains an evaluation of the solubility in
water of the pulse protein hydrolyzates derived from the soluble
fractions in the method of Example 15. Solubility was tested based
on protein solubility (termed protein method, a modified version of
the procedure of Morr et al., J. Food Sci. 50:1715-1718).
[0111] Sufficient protein hydrolyzate powder to supply 0.5 g of
protein was weighed into a beaker and then a small amount of
reverse osmosis (RO) purified water was added and the mixture
stirred until a smooth paste formed. Additional water was then
added to bring the volume to approximately 45 ml. The contents of
the beaker were then slowly stirred for 60 minutes using a magnetic
stirrer. The pH was determined immediately after dispersing the
protein and was adjusted to the appropriate level (2, 3, 4, 5, 6 or
7) with diluted NaOH or HCI. A sample was also prepared at natural
pH. For the pH adjusted samples, the pH was measured and corrected
periodically during the 60 minutes stirring. After the 60 minutes
of stirring, the samples were made up to 50 ml total volume with RO
water, yielding a 1% w/v protein dispersion. The protein content of
the dispersions was measured by combustion analysis using a Leco
Nitrogen Determinator (N.times.6.25). Aliquots of the dispersions
were then centrifuged at 7,800 g for 10 minutes, which sedimented
insoluble material and yielded a supernatant. The protein content
of the supematant was measured by combustion analysis
(N.times.6.25) and the protein solubility of the product calculated
as follows:
Protein Solubility (%)=(% protein in supernatant/% protein in
initial dispersion).times.100
[0112] Values calculated as greater than 100% were reported as
100%.
[0113] The natural pH values of the pulse protein hydrolyzates of
Example 15 in water (1% protein) are shown in Table 3:
TABLE-US-00003 TABLE 3 Natural pH of pulse protein hydrolyzates in
water at 1% protein Product Natural pH YP36-D20-17A YP840N 6.21
YP36-D20-17A YP820A 3.36
[0114] The protein solubility results obtained are set forth in the
following Table 4:
TABLE-US-00004 TABLE 4 Protein solubility of products at different
pH values Protein Solubility (%) Product pH 2 pH 3 pH 4 pH 5 pH 6
pH 7 Nat. pH YP36-D20-17A 100 100 97.1 96.2 100 100 100 YP840N
YP36-D20-17A 95.1 93.9 92.6 98.0 100 100 97.9 YP820A
[0115] As can be seen from the results presented in Table 4, the
enzyme hydrolyzed protein products were very soluble across the pH
range tested.
Example 18
[0116] This Example illustrates a comparison of the astringency
level of the YP36-D20-17A YP840N prepared as described in Example
15 with that of the YP35-K28-16A YP701 prepared as described in
Example 8.
[0117] Samples were prepared for sensory evaluation by dissolving
sufficient protein powder to supply 2 g protein in 100 ml of
purified drinking water. The pH of the YP701 solution was 3.39. The
pH of the YP840N solution was lowered from 6.25 to 3.41 by the
addition of food grade HCl solution. An informal panel of
panellists was asked to blindly taste the samples and indicate
which was less astringent.
[0118] Five out of seven panelists indicated that the YP36-D20-17A
YP840N was less astringent, while two panelists indicated that the
YP35-K28-16A YP701 was less astringent.
Example 19
[0119] This Example illustrates a comparison of the astringency
level of the YP36-D20-17A YP820A prepared as described in Example
15 with that of the YP35-K28-16A YP701 prepared as described in
Example 8.
[0120] Samples were prepared for sensory evaluation by dissolving
sufficient protein powder to supply 2 g protein in 100 ml of
purified drinking water. The pH of the YP820A solution was 3.23.
The pH of the YP701 solution was 3.34. An informal panel of 7
panellists was asked to blindly taste the samples and indicate
which was less astringent.
[0121] Five out of seven panelists indicated that the YP36-D20-17A
YP840N was less astringent, while two panelists indicated that the
YP35-K28-16A YP701 was less astringent.
Example 20
[0122] This Example describes the Amino Acid Score of products
derived from the residual solids after enzyme treatment compared to
the substrate material.
[0123] The reference amino acid pattern used to determine the Amino
Acid Scores was the FAO/WHO/UNU 1985 (Report of Joint FAO/WHO/UNU
Expert Consultation (1985) Energy and Protein Requirements, WHO
Technical Report Series 724) pattern for children 2-5 (Report of
Joint FAO/WHO Expert Consultation (1991) Protein Quality
Evaluation, FAO Food and Nutrition Paper 51). This pattern is shown
in Table 5.
TABLE-US-00005 TABLE 5 Reference essential amino acid pattern used
to calculate Amino Acid Scores Essential amino acid(s)
Concentration (mg/g protein) Histidine 19 Isoleucine 28 Leucine 66
Lysine 58 Methionine + Cystine 25 Phenylalanine + Tyrosine 63
Threonine 34 Tryptophan 11 Valine 35
[0124] Amino Acid Score is calculated by dividing the content of
each essential amino acid (mg/g protein) in the test protein by the
content of the same essential amino acid in the reference pattern
(mg/g protein). The lowest resulting value, obtained for the most
limiting essential amino acid, is considered the Amino Acid Score
(AAS) (Report of Joint FAO/WHO Expert Consultation (1991) Protein
Quality Evaluation, FAO Food and Nutrition Paper 51; Schaarfsma, G.
2000. J. Nutr., 130: 1865S).
[0125] Amino acid profiles of the pulse protein hydrolyzates
derived from the residual, enzyme treated solids in Examples 2, 5,
6, 15 and 16 as well as the dry pulse protein substrates used in
Examples 2, 15 and 16 were assessed experimentally. A complete
amino acid profile analysis was done, to quantify tryptophan,
cysteine/methionine and the remaining amino acids.
[0126] Essential amino acid profiles and calculated Amino Acid
Scores for the substrates and the pulse protein hydrolyzates
derived from the residual solids are shown in Tables 6 to 10
below.
TABLE-US-00006 TABLE 6 Essential amino acid profiles and Amino Acid
Scores for substrate and residual solids derived pulse protein
hydrolyzate of Example 2 YP35-G19-16A YP810N YP29-I19-16A YP820PN
Concentra- Conc./ Conc./ Essential amino tion (mg/g reference
Concentration reference acid(s) protein) conc. (mg/g protein) conc.
Histidine 25.80 1.36 24.79 1.30 Isoleucine 46.64 1.67 63.80 2.28
Leucine 76.56 1.16 100.86 1.53 Lysine 85.51 1.47 74.72 1.29
Methionine + 19.35 0.77 26.42 1.06 Cystine Phenylalanine + 91.17
1.45 127.73 2.03 Tyrosine Threonine 45.82 1.35 48.59 1.43
Tryptophan 8.96 0.81 15.46 1.41 Valine 49.59 1.42 64.05 1.83 AAS
0.77 AAS 1.06
TABLE-US-00007 TABLE 7 Essential amino acid profile and Amino Acid
Score for residual solids derived pulse protein hydrolyzate of
Example 5 YP35-J11-16A YP822PN Essential amino Concentration
Conc./reference acid(s) (mg/g protein) value Histidine 26.45 1.39
Isoleucine 60.66 2.17 Leucine 102.76 1.56 Lysine 82.24 1.42
Methionine + 24.89 1.00 Cystine Phenylalanine + 122.63 1.95
Tyrosine Threonine 45.00 1.32 Tryptophan 12.51 1.14 Valine 59.21
1.69 AAS 1.00
TABLE-US-00008 TABLE 8 Essential amino acid profile and Amino Acid
Score for residual solids derived pulse protein hydrolyzate of
Example 6 YP35-L07-16A YP823PN Essential amino Concentration
Conc./reference acid(s) (mg/g protein) value Histidine 23.55 1.24
Isoleucine 56.71 2.03 Leucine 92.50 1.40 Lysine 75.00 1.29
Methionine + 24.62 0.98 Cystine Phenylalanine + 108.42 1.72
Tyrosine Threonine 41.97 1.23 Tryptophan 11.08 1.01 Valine 57.63
1.65 AAS 0.98
TABLE-US-00009 TABLE 9 Essential amino acid profiles and Amino Acid
Scores for substrate and residual solids derived pulse protein
hydrolyzates of Example 15 YP36-B28-17A YP36-D20-17A YP36-D20-17A
YP810N YP820PA YP840PN Conc. Conc./ Conc. Conc./ Conc. Conc./
Essential (mg/g ref. (mg/g ref. (mg/g ref. amino acid(s) protein)
value protein) value protein) value Histidine 26.00 1.37 25.39 1.34
25.03 1.32 Isoleucine 46.67 1.67 53.26 1.90 54.33 1.94 Leucine
79.44 1.20 85.94 1.30 89.13 1.35 Lysine 81.86 1.41 75.65 1.30 75.09
1.29 Methionine + 20.47 0.82 25.13 1.01 24.93 1.00 Cystine Phen-
92.63 1.47 107.30 1.70 110.01 1.75 ylalanine + Tyrosine Threonine
43.29 1.27 42.71 1.26 44.20 1.30 Tryptophan 8.75 0.80 12.28 1.12
12.21 1.11 Valine 49.46 1.41 55.47 1.58 56.41 1.61 AAS 0.80 AAS
1.01 AAS 1.00
TABLE-US-00010 TABLE 10 Essential amino acid profiles and Amino
Acid Scores for substrate and residual solids derived pulse protein
hydrolyzate of Example 16 YP36-I11-17A YP810A benchscale YP821PA
Conc./ Concentra- Conc./ Essential amino Concentration reference
tion (mg/g reference acid(s) (mg/g protein) value protein) value
Histidine 25.75 1.36 23.82 1.25 Isoleucine 45.36 1.62 57.23 2.04
Leucine 77.84 1.18 93.02 1.41 Lysine 80.28 1.38 70.45 1.21
Methionine + 21.99 0.88 24.71 0.99 Cystine Phenylalanine + 91.54
1.45 108.85 1.73 Tyrosine Threonine 42.92 1.26 43.02 1.27
Tryptophan 9.15 0.83 10.65 0.97 Valine 49.07 1.40 57.11 1.63 AAS
0.83 AAS 0.97
[0127] As may be seen from the results in Tables 6 to 10, the pulse
protein hydrolyzates prepared from the residual solids after enzyme
treatment had Amino Acid Scores in the range of 0.97-1.06. This was
an improvement on the Amino Acid Scores of the substrate proteins,
which were in the range of 0.77-0.83.
Example 21
[0128] This Example illustrates the molecular weight profile of the
pulse protein hydrolyzates derived from the soluble fraction in
Examples 1-6 and 15.
[0129] Molecular weight profiles were determined by size exclusion
chromatography using a Varian ProStar HPLC system equipped with a
300.times.7.8 mm Phenomenex Yarra SEC-2000 series column. The
column contained hydrophilic bonded silica rigid support media, 3
micron diameter, with 145 Angstrom pore size.
[0130] A standard curve was prepared using a Biorad gel filtration
standard (Biorad product #151-1901) containing proteins with known
molecular weights between 17,000 Daltons (myoglobulin) and 670,000
Daltons (thyroglobulin) with Vitamin B12 added as a low molecular
weight marker at 1,350 Daltons. A 0.9% w/v solution of the gel
filtration standard was prepared in running buffer (0.05M
phosphate/0.15M NaCl, pH 6 containing 0.02% sodium azide), filtered
with a 0.45 .mu.m pore size filter disc then a 25 .mu.L aliquot run
on the column using a mobile phase of 0.05M phosphate/0.15M NaCl,
pH 6 containing 0.02% sodium azide. The mobile phase flow rate was
1 mL/min and components were detected based on absorbance at 214
nm. Based on the retention times of these molecules of known
molecular weight, a regression formula was developed relating the
natural log of the molecular weight to the retention time in
minutes.
[0131] For the analysis of the pulse protein hydrolyzate samples,
0.05M phosphate/0.15M NaCl, pH 6 containing 0.02% sodium azide was
used as the mobile phase and also to dissolve dry samples. Protein
hydrolyzate samples were mixed with mobile phase solution to a
concentration of 1% w/v, placed on a shaker for at least 1 hour
then filtered using 0.45 .mu.m pore size filter discs. Sample
injection size was 100 .mu.L. The mobile phase flow rate was 1
mL/minute and components were detected based on absorbance at 214
nm.
[0132] The regression formula relating molecular weight and
retention time was used to calculate retention times that
corresponded to molecular weights of 100,000 Da, 15,000 Da, 5,000
Da and 1,000 Da. The HPLC ProStar system was used to calculate the
peak areas within these retention time ranges and the percentage of
material ((range peak area/sum of all range peak areas).times.100)
falling in a given molecular weight range was calculated.
[0133] The molecular weight profiles of the pulse protein
hydrolyzates are shown in the Table below.
TABLE-US-00011 TABLE 11 Molecular weight profiles of soluble
fraction derived pulse protein hydrolyzates Product % > 100,000
Da % 15,000-100,000 Da % 5,000-15,000 Da % 1,000-5,000 Da % <
1,000 Da YP29-H11-16A 8 27 20 32 14 YP820A YP35-I19-16A 0 4 27 54
15 YP820A YP34-I26-16A 0 8 25 50 17 YP820A YP35-J06-16A 14 30 21 27
8 YP822A YP35-J11-16A 2 22 29 37 11 YP822A YP35-L07-16A 2 4 21 51
23 YP823A YP36-D20-17A 1 8 24 48 20 YP840N YP36-D20-17A 1 9 25 47
17 YP820A
[0134] As may be seen from the results of Table 11, the profiles of
all samples indicated the presence of some larger molecular weight
material.
SUMMARY OF THE DISCLOSURE
[0135] In summary of this disclosure, the present invention is
concerned with the preparation of pulse protein hydrolyzates
involving hydrolysis of the starting pulse protein product,
optional pH adjustment and separation of the resulting material.
The soluble portion after enzyme hydrolysis and separation is
further processed to provide a low astringent, acid soluble pulse
protein hydrolyzate. The residual solids after the enzyme
hydrolysis and separation are further processed to provide a pulse
protein hydrolyzate with an improved Amino Acid Score compared to
the substrate pulse protein. Modifications are possible within the
scope of this invention.
* * * * *