Note: Descriptions are shown in the official language in which they were submitted.
CA 02362948 2001-08-23
Title
METHOD FOR PREPARING AN ALBUMIN ISOLATE FROM A SUBSTANCE
CONTAINING ALBUMIN
Field of the Invention
The invention relates to a method for preparing an albumin isolate from a
substance containing
albumin, the substance being ground to a flour, the flour being suspended in
an aqueous solution,
the albumin being extracted from the flour into the solution using at least
one countercurrent
protease, and the albumin being precipitated from the solution using mineral
acid and being
neutralized.
Background of the Invention
Several methods are known to extract albumin from vegetable raw materials, an
alkaline
extraction of the albumin at a pH of between 7 and 9 and at a temperature of
40 to 50 °C being
the basis of these methods. The extracted albumin is then purified by
centrifugation and acidified
with a mineral acid until the isoelectric point of the albumin is reached. The
albumin precipitated
at the isoelectric point is again concentrated and purified by means of
centrifugation. In this way
yields of 70 % as related to the albumin contained in the raw materials are
possible. However, it
is a serious drawback that only such raw materials may be used in which the
albumin has a low
level of denaturation. Especially in exploring the most important vegetable
albumin isolate, i. e.
soya albumin isolate, the limitation to so called "white flakes" as a raw
material is an economic
drawback. White flakes are only obtained by means of a special drying method
for the
remainders of Soya oil production. Normally, the remainders are dried less
carefully, and they are
then present in a form of so called "toasted flakes" in which the albumin is
strongly denatured.
From DE I 203 588 it is known to enhance the alkaline extraction process in
exploring albumins
from an albumin containing substance by means of a pre-treatment of an aqueous
suspension of
the substance with hydrogen peroxide within the alkaline range and with
proteolytic enzymes. To
this end, the pH of the suspension of the albumin containing substance is at
first raised, then
hydrogen peroxide is added and the temperature is raised to initiate
peroxidation. Then, the pH is
CA 02362948 2001-08-23
adjusted to 4 to 9, and enzymes are added to the suspension, the activity
minimum of which is
within this pH-range. Afterwards, the suspension is stirred for two hours.
Preferably, vegetable
enzymes like bromelaine, ficine and papaine are used. After the enzymatic
hydrolysis, the
albumin is dissolved in that the pH is raised to 9 to 12. At the same time,
high temperatures are
applied from which it is known that they result in albumin damages
particularly in combination
with the high pH. The multiple change of the pH in this known method requires
great efforts in
large scale applications and results in a high consumption of alkali and acid
followed by a strong
formation of salt.
It is also known from DE 44 29 787 C2 to use proteolytic enzymes for enhancing
the solubility of
albumins. Here, the starting material is at first extracted with alcohol, only
afterwards it is
mechanically broken up and then further enzymatically broken up in the acid to
neutral range.
The extraction of attendant material to the albumin is effected by means of a
countercurrent
process. A treatment with hydrogen peroxide can take place after the
extraction of the albumin in
this known method, too. Due to the use of alcohol for the extraction, the
method requires high
efforts which are disadvantageous. The explosion danger of the suspension and
the inactivation
of the used enzymes by the presence of alcohol have to be permanently
considered. In the known
method, the proteolyses is per definitionem limited to a pH-range of < 9,5,
only the pH-range of
< 7,5 being concretely described in DE 44 29 787 C2. On the other hand it is
known to those
skilled in the art that the solubility of albumin is particularly great, if
the pH is over 9. All at all,
the efficiency of the known method as compared to the cost to be spend for its
application is only
low so that the prepared albumin isolates are not competitive.
A method according the preamble of claim 1 is known from DE 43 39 743 C 1.
This method does
without the use of alcohol. For dissolving the protein, however, a pH of over
11,5, particularly of
about 12,5, is needed which is associated with a high consumption of alkali
and of acid for a later
neutralization. Prior to the proteolysis, the albumin containing substance is
treated with a
protease at a pH > 10.5. The pre-treatment requires a comparatively large
amount of enzyme,
which results in a drawback with regard to the production cost. The
comparatively high salt
content at the end of the albumin extraction of this known method, which in
praxis requires an
additional washing step, is a further drawback.
2 CA 02362948 2001-08-23
A method for extraction of albumin from an albumin containing substance which
operates with
high amounts of enzyme is also know from JP 2 076 597 A. An alkaline protease
is added to an
alkaline suspension of the albumin containing substance, the pH of which is
between 10 and 13,
and incubated at a temperature of 30 to 50 °C for 1 to 20 hours.
Afterwards, a neutralization and
a filtration of the suspension take place. These indeed dramatic conditions of
the albumin
extraction result in extended albumin damages. As a result, it is only
possible to obtain
hydrolysates for cosmetic applications by the known method, it is unsuitable,
however, for the
preparation of albumin isolates as food.
The application of albumin cleaving enzymes prior to the alkaline extraction
which enhances the
extractability of the albumins in the following alkaline extraction may be
regarded as the
common step of the methods which have been described in detail above.
The present invention is based on the problem to disclose a method for
preparing an albumin
isolate from a substance containing albumin which results in a better albumin
yield with at the
same time limited use of the agents water, alkali, acid and enzyme, without
reducing the quality
of the prepared albumin isolate both with albumin containing substances with
high and with low
albumin solubility.
CA 02362948 2001-08-23
Summary of the Invention
The invention discloses a method for preparing an albumin isolate from a
substance containing
albumin, the method comprising the steps of grinding the substance to a flour;
suspending the
flour being suspended in an aqueous solution; extracting the albumin from the
flour into the
solution using at least one countercurrent protease; precipitating the albumin
from the solution
using mineral acid; and neutralizing the precipitated albumin; wherein the
step of extracting the
albumin from the flour into the solution comprises the further step of
subjecting the flour
suspended in the aqueous solution to an at least two stage treatment with the
at least one protease,
with a pH greater than 8 and with heat of temperatures between 30 and 60
°C; the flour being
treated in the first stage with a lower concentration of the at least one
protease in relation to the
albumin weight, with a lower pH and with a higher temperature than in the
second stage; at the
end of the first stage, a first over flow being separated from a fraction
containing the flour, from
which over flow the albumin is precipitated; and at the end of the second
stage, a second over
flow being separated from a fraction containing the flour, which over flow is
fed back to the fist
stage.
In a preferred embodiment of the method, the albumin containing substance is
ground to a flour
with an average particle size of 30 to 100 Vim.
Further, it is preferred that the flour suspended in the aqueous solution is
homogenized by energy
input at the beginning of the second stage.
Further, it is preferred that the flour suspended in the aqueous solution is
subjected to a three
stage treatment with the at least one protease, with pH-values of greater than
8 and with heat of
temperatures between 30 and 60 °C; the flour being treated in the third
stage with a higher
concentration of the at least one protease in relation to the albumin weight
and with a lower
temperature than in the second stage; and a third over flow being separated
from a fraction
containing the flour after the third stage, which over flow is fed back into
the second stage.
Further, it is preferred that the protease is added to the solution in the
third stage.
Further, it is preferred that the at least one protease is selected from the
group comprising serine,
CA 02362948 2001-08-23
cysteine, asparagine and metal proteases.
Further, it is preferred that the at least one protease is inactivated in the
albumin by means of a
heat treatment.
Further, it is preferred that the albumin is extracted from the flour into the
solution using at least
one alcohol and/or hydrogen peroxide.
Further, it is preferred that, before the step of extracting the albumin into
the solution, the flour is
subjected to a pre-treatment using at least one substance selected from the
group comprising
lyes, acids and enzymes.
Further, it is preferred that at least one of the over flows is separated from
the fraction containing
the flour in that the flour suspended in the solution is centrifugated on a
decanter under vacuum.
CA 02362948 2001-08-23
In the new method, the dissolution of the albumins is effected out with the
aid of an alkaline
countercurrent extraction by means of a temperature gradient, different pH-
values and a
simultaneous application of albumin cleaving enzymes during the extraction
cascade. Within an
individual case, this extraction can be supported by the application of
hydrogen peroxide and/or
alcohols.
At first, the starting material, if it does not already have such a small
particle size, is milled into a
flour with a particle size of 50 to 100 Vim, and mixed with water at a ratio
of 1:5 to 1:8, the
suspension being raised to a temperature of 30 to 60 °C, particularly
of about 50 °C. The pH of
the suspension is adjusted to 8 to 10, particularly to about 9, by means of
alkaline or alkaline
earth hydroxides. To enhance the albumin solubility in this stage of
extraction, a protease may be
added already here. Preferably, however, only the cleared over flow or the
filtrate of the
following stage of extraction is added. The enzyme/albumin-ratio obtained
should be between 1
to 2000 and 1 to 6000. After an incubation time of 10 to 60 min., particularly
of about 20 min, a
first separation takes place.
In general, all common preparation techniques are useable. The use of a
Sedikanter obtained from
Flottweg GmbH, Germany providing a force field of 6000 x g for the separation
is preferred.
During the separation, a formation of foam may be essentially avoided by means
of a vacuum
surrounding. A suitable vacuum is in the range of an absolute pressure of 300
to 500 mbar. In this
way, processing of albumin suspensions without an antifoam agent is
successful. If it is not
possible to carry out the separation under vacuum it has to be cared for a
sufficient degassing of
the suspension prior to the separation, if no antifoam agents shall be used.
After the separation has been completed, the non-dissolved solid matter is
dispersed in water,
then homogenized and then again subjected to an alkaline extraction. To this
end, the temperature
is adjusted to between 30 to 45 °C, particularly to about 40 °C,
and the pH is adjusted to between
10,5 and 11,5, particularly to about 11,2. Generally, homogenization may take
place at each
extraction stage. Particularly effective, however, is the homogenization prior
to the stage of
extraction described here, which may thus be regarded as sufficient for
carrying out the new
method. It is important to add the cleared over flow or the filtrate of the
following stage of
extraction to the stage of extraction described here so that an enzyme/albumin-
ratio of between 1:
6 CA 02362948 2001-08-23
500 and 1:2000, particularly of about 1:1250, is present. After an extraction
time of 10 to 60
min., a second separation takes place.
Afterwards, the solid matter is suspended with water for a third time. The pH
self adjusts to
between 10,2 and 10,5 depending of the water quality. The temperature is
adjusted to between 25
and 40 °C, particularly to about 35 °C. To this suspension a
protease is originally added. The
enzyme/albumin-ratio here is between 1:100 to and 1:500, particularly about
1:400. After adding
the protease which also may be a protease mixture, an enzymatic hydrolysis
takes place for 3 to
30 min. The hydrolysis times depends of the used amount of enzyme, the
respective enzyme
activity and the kind of protease. Suitable proteases may by selected from the
groups of the serine
proteases, the cysteine proteases, the asparagine proteases and/or the metal
proteases.
Some albumin containing substances as raw material for the new method show
sensorics which
make the resulting albumin isolate inedible, if no further measures are taken.
Thus, it has been
proved advantageous to add hydrogen peroxide to the second alkaline stage of
extraction. The
amount of hydrogen peroxide as related to a 35 % hydrogen peroxide solution
may be between 5
and 50 ml per kg albumin, particularly 20 ml/kg albumin. The concentration has
to be adjusted in
such a way that damages to the used proteases do not occur.
If the albumin containing starting material contains organic substances which
may not be
removed by means of an aqueous pre-treatment, it is advantageous to carry out
the alkaline
extraction in the presence of 5 to 20 % alcohol, particularly of 10 % alcohol.
Herein, the
solubility of the organic substances is raised so that they remain in the
aqueous solution when the
albumins are precipitated at the isoelectric point. It is also sufficient to
add the alcohol within the
second stage of extraction, if it is cared for that the desired alcohol
concentration after feeding
back the clear over flow of the second separation into the first stage of
extraction is not below 5
%.
In the new method, the clear over flow of the first stage of extraction is, in
a way which is known
as such, adjusted to the product depending isoelectric point of the albumin
which is generally in
the pH-range of 4,2 to 4,6 by means of mineral acids or organic acids. By
means of this, the
albumin is precipitated, and a separation can be carried out with known
separation techniques,
7 CA 02362948 2001-08-23
especially with decanters. The quark-like albumin isolate obtained in this way
can be washed and
neutralized before drying, to adjust a desired pH for the end product.
Surprisingly, the step by step alkaline extraction using proteases with
feeding back the extraction
agents results in an albumin quality which is clearly above of the products of
known methods.
This is the case both with regard to the yield and the sensorics of the method
products. At the
same time, the process course in the new method can be tuned in such a way,
that a water
consumption of about 81 fresh water per kg raw material used is sufficient for
all steps of the
procedure. Only 25 % of this have to be warm water. The arising sewage water
is only about 5 1
per kg raw material used.
Because of the comparatively high solid matter contents of the processed
suspensions, despite the
higher pH no greater amounts of acid and/or alkali are used in the new method
as compared to
classic extraction methods. Instead, a reduction of the amounts of acid and
alkali and thus of the
resulting salt of up to 20 % may be obtained as compared to commercial methods
presently used
to prepare albumin isolates.
The albumin isolates obtained by the method according to the invention have a
typical high
albumin content of 90 to 94 % as related to the dry matter content. Depending
of the kind of
drying applied, the remaining water may be between 4 and 8 %. The yield as
related to the
albumin content of the starting material is typically between 80 and 87 %.
With a tuned process
course, the taste of the method products is neutral. A special taste resulting
from the starting
material can be removed without problems. The protein damages are low. This is
documented,
for example, by a very low lysinoalanine content of only about 50 to 150 ppm
as related to the
albumin in the obtained albumin isolate.
Short description of the Drawing
Fig. l shows a flow diagram of an embodiment of the method for preparing an
albumin isolate
from a substance containing albumin according to the invention.
CA 02362948 2001-08-23
Description of the preferred Embodiment of the Invention
The flow diagram of the method according to the invention which is shown in
the accompanying
Fig. 1 indicates the preferred ranges of pH and temperature, and starts with a
mash which is
prepared by mixing the albumin containing starting material having a small
particle size with the
clear over flow of a separation step. In the right hand part of Fig. 1, the
three alkaline/enzymatic
stages of extraction of the preferred embodiment of the invention are
indicated. In the left hand
part, the processing of the over flow 1 of the separation l, i. e. the
separation after the first stage
of extraction, is shown. At the beginning, this over flow contains the albumin
in a dissolved form.
Non dissolved substances within the over flow are removed by clearing.
Afterwards, a
precipitation of the albumins at the isoelectric point takes place. The
precipitate of this
precipitation is removed by separation and again suspended in water. By means
of a following
heat deactivation the enzymes from the extraction of the albumins are
inactivated. In the
following separation, the over flow is obtained with which the mash is
prepared. This over flow
does not contain relevant enzyme activity. The downstream neutralization and
drying of the solid
matter results into the desired albumin isolate.
CA 02362948 2001-08-23
Examples
In the following, the invention is further explained and described by means of
examples.
1. Soya Albumin Isolate
Toasted soya meal is at first processed into a albumin concentrate. The
albumin concentrate
having a particle size of about 50 ~m is suspended in 50 °C warm water
to a solid matter
concentration of the resulting suspension of 12,5 %. This corresponds to the
mash in Fig. 1.
Then, this suspension is added with the fed back flow of the downstream second
stage of
extraction. Afterwards, the pH is adjusted to 9 with 25 % sodium hydroxide.
The
enzyme/albumin-ratio is 1:5500. The suspension is stirred for 15 min at 45
°C. Then, the
suspension is separated over a Sedikanter at 6000 x g, a vacuum of 300 mbar
absolute pressure
being present in the Sedikanter. The over flow of the Sedikanter contains the
already dissolved
albumin, the under flow contains the solid matter with the still bonded
albumin.
The under flow is again suspended in water so that a solid matter content of 8
% is adjusted. The
water may be cold. The suspension obtained in this way is homogenized by means
of a pressure
drop of 80 bar and added with the cleared over flow of the third stage of
extraction. The self
adjusted temperature is 38 °C, the enzyme/albumin ratio is 1:1250.
Then, the pH is adjusted to
11,2 again with 25 % sodium hydroxide, and it is stirred for 15 min.
Afterwards, a second
separation via a Sedikanter under vacuum takes place. The over flow of this
Sedikanter is fed
back into the first stage of extraction. The solid matter output, i. e. the
under flow is dispersed
with cold water and adjusted to a solid matter content of 6 %. The self
adjusting temperature
should be below 40 °C. Now, proteases having a total activity of 6
Anson Units/kg albumin are
added to this suspension, which corresponds to an enzyme/albumin-ratio of
1:400. This
suspension reacts for 5 min., and is then centrifugated in a Sedikanter in the
presence of vacuum
in a third separation. The over flow is fed back to the second stage of
extraction. The solid matter
output or under flow may be neutralized and used as a fibre containing
swelling agent, for
example. Within the method described here, however, it is not further used.
The over flow of the first stage of extraction is adjusted to pH 4,3 by means
of 15 % hydrogen
chloride and then centrifugated in a Sedikanter at 300 mbar. The temperature
of the input to the
I0 CA 02362948 2001-08-23
Sedikanter is 45 °C. The solid matter output has a dry matter content
of 28 %, i. e. the under flow
of the Sedikanter contains the albumin, the over flow contains the attendant
material and soluble
albumin, which is directed to the sewage water. The solid matter is then mixed
with water of best
quality to 15 % and for a short time heated up to 110 °C. By means of
this, the solid matter is
washed, and the enzymes contained in it are inactivated. After cooling down to
50 °C a
separation of the solid matter from the added water takes place in a standard
decanter at about
4000 x g. The over flow of this separation is used for preparing the mash for
the first stage of
extraction. The under flow is again diluted with water to 15 % dry matter
content, neutralized
with sodium hydroxide and then spray dried.
2. Potato Albumin Isolate
Albumin containing byproducts occurring in the production of starch are at
first milled down to a
particle size of 50~m and then suspended in 50 °C warm water in such a
way that a suspension
having a solid matter content of 15 % is produced. A protease having an
activity of 5 Anson
Units/kg protein is added to this suspension. After 5 min. of stirring, the pH
is adjusted to 8,5,
and then it is stirred for further 10 min. Afterwards, the pH of the
suspension is adjusted to 4,2 by
means of hydrogen chloride, and the suspension is centrifugated on a decanter
at 4000 x g. The
over flow is removed; the solid matter is again suspended in 50 °C warm
water and then process
according to the steps of the new method shown in Fig. 1. To this end, the
over flow from the
second stage of extraction is added to the solid matter suspended in water.
The
enzyme/albumin-ratio is 1:6500, then. The solid matter content is 10 %. The pH
is adjusted to 9,
and afterwards it is stirred for 15 min. Here, the temperature should be 45
°C. Afterwards a
centrifugation in a Sedikanter at 6000 x g takes place. Here, the over flow
contains the dissolved
protein. The under flow, i. e. the solid matter output having a solid matter
content of 26 %, is
suspended in cold water and added with the over flow of the following third
stage of extraction.
The temperature self adjusts to 38 °C. The pH is raised to 11,2 by a 25
% sodium hydroxide
solution. The enzyme/albumin-ratio is 1:1600. This suspension is stirred for
l5min, 20 pl of 25
hydrogen peroxide per kg of solid matter being added after 10 min. After the
stirring time of 15
min, the suspension is homogenised by via a pressure drop of 80 bars, and then
separated in a
second Sedikanter under vacuum of 300 mbar. The over flow is added to the
first stage of
extraction; the under flow containing the solid matter is again suspended in
cold water. The
11 CA 02362948 2001-08-23
temperature of the dispersed solid matter self adjusts to 32 °C. The pH
reaches 10,5. Now,
proteases having an activity of 4 Anson Units/kg proteins are added, resulting
in an
enzyme/albumin-ratio of 1:500. After 5 min., this dispersion is centrifugated
in a Sedikanter for a
third time, again under vacuum of 300 mbar absolute pressure. The over flow of
this separation is
fed back to the second stage of extraction. The solid matter output, i. e. the
under flow, is
disposed.
The over flow of the first stage of extraction is adjusted to pH 4,3 by means
of hydrogen chloride.
Afterwards, it is centrifuged in a Sedikanter at an under pressure of 300 mbar
absolute. The over
flow of the Sedikanter is disposed. The quark-like under flow is adjusted to a
solid matter content
of 15 % with demineralised water. This dispersion is heated up to 110
°C for a short time, then
cooled down to 50 °C and centrifuged in a decanter at 4000 x g. The
cleared flow or over flow
obtained here is used for preparing the mash from the flour of the starting
materials for the first
stage of extraction. The solid matter output, i. e. the under flow, is
neutralized and dried.
Instead of hydrogen peroxide also ethanol can be used in the preparation of
potato albumin
isolate for extracting undesired organic attendant materials.
