Note: Descriptions are shown in the official language in which they were submitted.
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Background of the invention
, .
This invention relates to a process for the preparation of highly
pure proteins from rapeseed.
In view of the world-wide shortage in proteins, oilseeds such as
soybean, peanut and cottonseed are becoming of increasing importance as
sources for the production of edible proteins. Rapeseed, Brassica napus,
which is the major oilseed crop of the temperate zones, had so far found
little application in the production of protein, mainly due to the presence
of undesirable constituents, such as erucic acid and glucosinolates, in the
seeds. The breeding of new varieties of rapeseed for better quality of oil
and meal have led to improved nutritional properties of rapeseed products.
The seed oils of these new varieties are free or almost free of erucic acid
and the meals contain much smaller proportions of glucosinolates than conven-
' tional rapeseed.
According to estimates of the United Nations Food and Agriculture
Organization (FAO), by 1980, the world production of rapeseed can be expected
` to reach at least 12 mill. tons, annually. The new varieties of rapeseed
will contribute substantially to filling the world's need of edible protein,
provided suitable processes for the isolation of rapeseed protein can be
found. Moreover, rapeseed proteins may be used in preparing technical pro-
ducts, such as plastics, coatings and adhesives.
In recent years, considerable efforts has been made to find con-
ditions for the removal of toxic substances from rapeseed. It has been
possible to obtain 'protein concentrates' which contain 50-60 % of protein
; from some oilseeds, such as soybean and cottonseed, simply by dehulling the
seeds and extracting the oils. Similarly, protein concentrates have also
been prepared from conventional rapeseed by dehulling, extraction of
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glucosinolates and defatting. The yields of rapeseed protein concentrates
have ranged from 23 to 28 % of the seeds.
Protein concentrates from soybeanJ cottonseed and, to a very small
extend, from rapeseed, are finding use as animal feed. For human consumption,
however, and in most technical applications, proteins of much higher purity,
'protein isolates', are required.
In the isolation of proteins from oilseeds on an industrial scale,
; two major steps are usually involved: Firstly, the proteins are extracted
from defatted meal with water or an aqueous electrolyte solution. Secondly,
the proteins are recovered from the extracts either by lypholization, drying
or precipitation.
The extraction of the proteins is carried out either by single
step or multiple stage processes using one or more than one solvents. All of
these procedures require rather large volumes of solvent to attain a satis-
factory degree of extraction. The recovery of proteins by lypholization or
drying leads to products containing practically all the protein present in
the extract. These products, however, are contaminated with non-protein
material derived from the meal and the solvent. Alternatively, the proteins
are recovered from the extract at the mean isoelectric point. The proteins
~; 20 isolated this way might be of higher purity than those obtained by lypholiza-
tion or drying of the extracts, but the yields are generally lower.
Summary of the invention
The present invention is directed toward a process for the pro-
duction of protein isolates which are free of insoluble and, especially for
children, indigestable carbohydrates, free of factors which are detrimental
to health and free of undesirable flavor constituents or bitter principles
or other components that might be harmful in human nutrition. These protein
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isolates are suitable for human consumption as well as animal feed, they can
be used to enrich other foodstuff with valuable proteins.
We have found that a maximum of protein present in defatted rape-
seed meals can be extracted by a countercurrent process using water or aqueous
solutions of electrolytes. In addition, we have discovered, that almost all
of the protein present in the extract can be recovered by stepwise precipita-
tion with aqueous mineral acid at different pH values.
Description of preferred embodiments
The overall process for the preparation of protein isolates from
rapeseed consists of two procedures: First, the proteins are extracted by
a countercurrent process, continuously or discontinuously, using water, at
pH 7.0, or aqueous solutions of sodium hydroxide or potassium hydroxide~ at
pH values between 7.5 and 10.0, preferably at pH 9Ø Second, the proteins
in the extract are precipitated in two consecutive steps by the addition of
a mineral acid, such as hydrochloric acid, to attain a pH value between 5.0
and 6.5, preferably between pH 5.7 and 6.0 (Protein Isolate I), and then a
pH value between 3.0 and 4.0, preferably pH 3.6 (Protein Isolate II). The
precipitated proteins are washed with water and/or an organic solvent and,
after filtration, they are dried in the usual manner.
The major advantages of the countercurrent extraction are,
- almost quantitative dissolution (>95%) of protein at
- least requirement of sdlvents and chemicals due to optimal utilization
of the dissolving capacity of the solvent,
- easy adaptibility to either a continuous or discontinuous process,
- low space requirement of the plant due to the
- small volume of equipment, and also,
- low cost of plant, equipment, utilities and operation.
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The major advantages of the stepwise precipitation of proteins at
different pH values are,
- the almost quantitative recovery of proteins from the extract at a
minimum of chemicals and energy,
- the feasibility of continuous or discontinuous processing,
- the availability of two or more distinct types of proteins,
- the very high protein content (>95%) of the products isolated~
- the negligible content of glucosinolates in the protein isolates and
- the favorable amino acid composition of the protein isolates.
The protein isolates have a light color, bland taste and no odor;
they are suitable for human consumption as well as for various technical
applications.
The entire process described in this invention is feasible on a
large scale because each of the two procedures involved, - extraction of the
proteins from defatted meal as well as precipitation of the proteins from
the extract, - can be carried out in a continuous fashion. The overall
yield of protein isolates is very high because each of these two procedures
results in high recovery. Both the countercurrent extraction and the step-
wise precipitation of proteins, as unit processes, contribute to the low
cost of the isolation of proteins from rapeseed. The solid residue remaining
after extraction of the proteins has a high fiber content and may serve as
a filler, whereas the liquid remaining after precipitation, due to its high
; carbohydrate content, may be used in fermentation industry.
In conclusion, the process described constitutes a significant
advance in the isolation of proteins from rapeseed. Both the countercurrent
extraction of proteins and their stepwise precipitation and certainly, the
combination of these processes are entirely new.
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The following specific examples illustrate the present invention
: more fully. These two examples are for illustrative purposes only and are not
~- intended to limit the scope of the invention.
Example I
Figure 1 is a schematic representation of the countercurrent ex-
traction of proteins from defatted oilseeds.
Fresh solvent
meal t meal t meal 1 meal
Stage A I l ~ J I ~ IV Extract A
Fresh solvent
from ~ from t from 1 from
Stage B Stage A ~ Stage A ~ Stage A ~ Stage A Extract B
Fresh solvent
from ~ from ~ from ~ from
Stage C Stage B ~ Stage B ~ Stage B ~ Stage B
Extract C
Fresh solvent
from l from ~ from ~ from
Stage D Stage C ~ Stage C J Stage C ~ Stage C Extract D
Figure 1: Sche~e for the countercurrent extraction of proteins.
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In a typical example, four 250 g samples of hexane-defatted
rapeseed meal ~Brassica napus, Erglu), designated I-IV, were extracted with
0.02 M sodium hydroxide. In each of the four stages, A-D, the first sample
was extracted with 6250 ml of fresh solvent at a meal to solvent ratio of
1:25. Extraction was carried out at ambient temperature for 10 minutes using
a high-speed homogenizer. Subsequently, the samples were filtered under
vacuum through cotton cloth. At each stage the filtrate from one sample was
used to extract the next sample after measuring its volume and taking aliquots
for nitrogen determination. At the beginning of each stage, the first sample
in the previous stage was moved to the end of the series. The final extracts
from the four stages were combined (pH 9.2), centrifuged at 5000 x g for 15
m mutes, and stored at 5C. Out of the 25 liters of 0.02 M sodium hydroxide
used a total of 23 liters of extract was recovered. The results of the
countercurrent extraction are presented in Table 1. The percentage of meal
nitrogen extracted from the sample was calculated from the total amount of
nitrogen of protein extracted up to the stage involved, and the amount of
nitrogen originally present in the sample. The percentage of total meal
nitrogen extracted at the various stages was calculated from the nitrogen
content of protein extracts obtained-from-the four samples, at a given stage,
and the amount of nitrogen originally-present in the four samples. The
results show that about 94 % of the nitrogen present in the meal is ex-
tracted.
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Table ]: Countercurrent extraction of rapeseed (Brassica napus,
Erglu) protein
Extraction Sample Meal Nitrogen Total Meal
Extracted from Nitrogen Extracted
Stage No. the sample at each Stage
(%~ (%)
.
A I 67.1
II 12.0
III 31.1
rv 26.0 34.1
B II 90.6
III 49.1
IV 48.8
I 71.0 30.8
C III 83.4
IU 81.9
I 82.1
II 93.1 20.3
D IV 97.2
I 92.0
II 94.2
III 91.2 8.6
It is obvious that the countercurrent extraction of proteins as
described in the present example offers great advantages over single step
extractions-becaus-e of the high degree of protein recovery at a minimum re-
quirement of solvent. Anothe~ major advantage of working with more concen-
trated suspensions in a countercurrent process is that less material has to
be treated for a very high output. The air-dried residue resulting from the
countercurrent extraction contained 4.Q % protein and 21.1 % fiber.
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The combined protein extract (23 liters) was transferred to a pre-
cipitation vat. This solution, which contained soluble proteins, carbo-
hydrates, minerals and glucosinolates had a pH value of 9.2; it was acidified
to a pH value of 6.0 by the addition of 93 ml of 6 N hydrochloric acid in
order to precipitate "Protein Isolate I". The protein curd was allowed to
settle for about two hours. The supernatent liquid was siphoned off and
transferred to another precipitation vat. The precipitated curd was centri-
fuged at 5000 x g for 10 minutes and the supernatent was added to the solution
in the second vat. The curd was washed with 400 ml of water and the washings
were combined with the solution in the vat. The water-washed curd was then
leached twice with 400 ml, each, of acetone. The protein isolate was air-
dried at room temperature, Protein Isolate I (220 g) is light grey and has a
bland taste.
The combined supernatent and the water washings from Protein
Isolate I (pH ~.21 were acidified to a pH value of 3.6 by the addition of 103
ml of 6 N hydrochloric acid in order to precipitate "Protein Isolate II".
The protein curd was allowed to settle for 4 hours. The supernatent liquid
was siphoned off and discarded. The precipitated curd of Protein Isolate II
was washed once with 200 ml of water and twice with 200 ml of acetone, each.
Protein Isolate II was air-dried at room temperature. Protein Isolate II (77
is white and has a bland taste.
The yields of Protein Isolates I and II and the total yield are as
follows:
Protein Isolate I Protein Isolate II Total
~ % % %
Yield65.2 22.9 88.1
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Analyses of the two rapeseed protein isolates are given in Table 2.
Table 2: Analyses of rapeseed ~Brassica napus, Erglu) protein isolates
Protein Isolate I Protein Isolate II
Moisture % 10.5 8.1
Protein (dry basis) %
~% protein-N x 6.25) 92.9 98.6
Lipids % o.oo o.oo
Ash % 0.70 0.31
Crude fiber % - 0.06 0.02
Glycosinolates % traces traces
Static foam (~) 310 395
.
These analytical data show that the protein isolates are of high
purity. These isolates conta~n little ash and fiber, and they are free of
glucosinolates and other toxic ingredients. The sigma values indicate that
the two isolates from rapeseed yield foams more than twice as stable as foams
produced from commercial partially degraded soy protein.
Analyses of the amino acid composition of the two isolates show a
fairly hîgh lysine content. Isoleucine, which is considered the limiting
amino acid in rapeseed meal, occurs at a higher level in the isolates than in
the meal used as starting material.
It is cl~early demonstrated by this example that pure, light colored,
bland protein isolates having a desirable amino acid composition can be obtain-
ed from rapeseed (Erglu) at a high yield through a combination of counter-
current extraction and stepwise precipitation.
Example II
The countercurrent extraction (Figure l) described was carried out
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on a large lot of hexane-defatted rapeseed meal (Brassica napus, Lesira).
Four 1250 g samples were extracted with 0.02 M sodium hydroxide
solution using a ~eal to solvent ratio of 1:20. The mixture was filtered
under a pressure of three atmospheres. The results of the countercurrent ex-
traction are given in Table 3. The percentage of total meal nitrogen extract-
ed at each stage was calculated from the amount of nitrogen in the protein
extract at the stage involved and the amount of nitrogen origi~ally present
in the four samples.
Table 3: Countercurrent extraction of rapeseed (Brassica napus, Lesira)
.
1 0 protein
Extaction Total Meal Nitrogen
Stage Extracted at each Stage
~%)
A 49.2
B 27.2
C 14.4
D 1.9
.
These data demonstr~te that even after scaling up the process, a
high efficiency of the countercurrent extraction for the dissolution of the
protein is obtained. As much as 93 % of the protein-nitrogen present in the
meal is thus extracted at a minimum solvent requirement.
The combined extracts from the four stages ~91 liters) were centri-
fuged at 5000 x g and transferred to a precipitation vat. The stepwise
precipitation process as well as the washing and drying of the precipitated
curds were carried out as in Example I. The extract having a pM value of 9.2
was acidified to pH 5.7 by the addition of 391 ml of 6 N hydrochloric acid in
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order to precipitate Protein Isolate I. Subsequently, Protein Isolate II was
precipitated from the supernatent and washings at a pH value of 3.6 by the
addition of 378 ml of 6 N hydrochloric acid. The air-dried Protein Isolate I
~1168 g) is light grey, and the air-dried Protein Isolate II ~287 g) is white.
Both isolates have bland taste.
The yields of Protein Isolates I and II and the total yield are as
follows:
Protein Isolate I Protein Isolate II Total
~%) ~%) ~%)
- Yield 73.2 18.0 91.2
Analyses of the two rapeseed protein isolates are given in Table 4
Table 4: Analysis of rapeseed (Brassica napus, Lesira) protein isolates
. _ _ _ _
Protein Isolate I Protein Isolate II
~oisture % 7 3 7.2
Protein ~dry basis) %
C% protein-N x 6.25) 99.6 99.3
Lipids % traces traces
Ash % 0 04
Crude fiber % traces traces
Glucosinolates % traces traces
Static foam ~) 411 450
These analytical data show that the protein isolates are of high
purity. These isolates contain little ash and fiber, and they are free of
glucosinolates and other toxic ingredients. The sigma values indicate that
the two isolates from rapeseed yield foams more than thrice as stable as foams
produced from commercial partially degraded soy protein.
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Analyses of the amino acid composition of the two isolates show a
fairly high lysine content. Isoleucine, which is considered the limiting
amino acid in rapeseed meal, occurs at a higher level in the isolates than in
the meal used as starting material.
It is clearly demonstrated by this example, that pure, light colored,
bland protein isolates having a desirable amino acid composition can be
ob*ained from rapeseed (Lesiral at a high yield through a combination of
countercurrent extraction and stepwise precipitation.
Ramification of the invention
The process described in this invention can be subjected to several
modifications in order to suit special requirements.
Thus, more than two protein fractions can be produced by the step-
wise addition of a mineral acid to the protein extract whereby the pH value
~i is adjusted to values between 6.5 and 3.0; the various protein isolates pre- -
cipitated are filtered off or centrifuged, washed and dried.
In some cases it is possible to modify the process of countercurrent
extraction in such a way, that the total proteins are dissolved in a single
step or in a multiple stage extraction, either with water, at pH 7.0, or with
aqueous solutions of sodium hydroxide or potassium hydroxide, at a pH value
between 7.5 and 10Ø
The principle of the process described in this invention can also
be applied to the production of proteins from seeds other than rapeseed.
Nutritive value of protein isolates
Experiments in chicks ~A.S. El Nockrashy, M. Kiewitt, H.K. Mangold
and K.D. Mukherjee, Nutr. Metab. 19, 145-152 ~1975~) revealed better per-
formance of the protein isolates as compared to the corresponding meals. Both
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the feed consumption and weight gain of chicks fed protein isolates were
higher than those of chicks that received the corresponding meals. The
protein efficiency ratios of diets containing 20 % rapeseed protein varied
from 2.2 to 3.4, which indicates that protein isolates from rapeseed are
eminently suitable for feeding chicks. In comparison, the meals of soybean,
sunflower seed and sesame seed have protein efficiency ratios in chicks of
2.1, 2.1 and 0.85, respectively.
Similar results were obtained in nutritional experiments with rats.
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