Field of the Invention
The present invention relates to a process for producing
a soybean protein hydrolysate. More specifically, it relates to a process for producing
a soybean protein hydrolyzed with an enzyme in high yield with minimal formation
of dregs upon dissolution.
Products obtained by hydrolyzing proteins with a proteolytic
enzyme have better absorbability upon digestion than those of proteins without proteolysis,
and are utilized in various fields such as health food. In particular, their use
is postulated in sports drinks and drinks for nutrition.
Up to now, JP 61-254153 A, JP 1-269499 A, JP 2-23885 A,
4-190797 A, JP 8-322471 A, JP 10-271958 A, etc., disclose processes for producing
enzymatic decomposition products obtained by hydrolyzing animal and vegetable proteins
with enzymes. In general, after hydrolysis of proteins with an enzyme, heat treatment
is carried out in these known processes so as to inactivate the enzyme, sterilizing
the products, and so on. In particular, anaerobic thermophilic bacteria often cause
problems in these kinds of products and, normally, thorough heat sterilization is
EP-A-408,063 discloses a process for producing hydrolyzed
vegetable proteins such as soy protein. In this process, soy protein is enzymatically
hydrolyzed and the hydrolysate is heated under acidic conditions to deamidate the
hydrolysate, followed by neutralising the deamidate hydrolysate.
US-A-5,716,801, discloses a method for producing a vegetable
protein such as soy protein by enzymatic hydrolysis. In this process, the enzyme
is inactivated by heating just after hydrolysis and the sterilisation is carried
out by ultrafiltration.
EP-A-797,928 discloses a method for producing a soy protein
hydrolysate with a low content glycinin by allowing a proteolytic enzyme to act
on soy protein at a pH of 1.0 to 2.8 to selectively decompose glycinin in the soybean
EP-A-797,927 discloses a method for producing a soy protein
hydrolysate with a low content of &bgr;-conglycinin by allowing a proteolytic
enzyme to act on soybean protein at a temperature of higher than 50°C to less
than 90°C to selectively decompose &bgr;-conglycinin in the soybean protein.
EP-A-963,704 relates to the treatment of proteinaceous
material with a transglutaminase and an oxido-reductase.
US-A-3,830,942 discloses a method for producing protein
products which comprises the steps of forming an aqueous slurry containing defatted
oleaginous seed materials, adjusting the pH of the aqueous slurry to approximately
the isoelectric point of the oleaginous seed materials, heating said aqueous slurry
to elevated temperatures, adding a predetermined amount of enzyme to the aqueous
slurry, agitating the mixture during digestion of the protein material and thereafter
separating the undigested protein from the digested protein.
In addition, although a water-soluble fraction and a water-insoluble
fraction are separated after hydrolysis, conventional drinks containing protein
hydrolysates are liable to form a small amount of a precipitate (dregs) during storage
and this is a problem. In general, improvement of quality causes a decrease in yield,
whereas increase in yield is liable to form more dregs during storage. This is also
Objects of the Invention
One object of the present invention is to provide a process
for producing a soybean protein hydrolyzed with an enzyme in a high yield with minimal
formation of dregs upon dissolution.
Summary of the Invention
The present inventors have studied intensively to solve
the above problems. As a result, it has been found that the above problems can be
solved by a two-step heat treatment, wherein, after hydrolysis of a protein with
an enzyme, the hydrolyzation mixture is subjected to a step of heating lightly,
followed by cooling to separate insolubles before subjecting the mixture to heat
sterilization. Thus, the present invention has been completed.
That is, the present invention is a process for producing
a soybean protein hydrolysate which comprises hydrolyzing a soybean protein solution
with a proteolytic enzyme to a degree of hydrolysis of 20% to 98% in terms of a
soybean protein decomposition rate expressed by a solubilization degree of a protein
component in 15% trichloroacetic acid, heating (a) the hydrolyzation mixture at
a temperature of 75°C or higher which does not inactivate the enzyme for 10(5.25-0.05
×T) minutes (wherein T is heating temperature (°C)) or shorter,
cooling the hydrolyzation mixture until the temperature drops to 30°C or lower
separating and removing insolubles from the mixture to obtain a supernatant, and
heat-sterilizing (b) the supernatant for longer than 10 (5.25-0.05 ×T)
minutes (wherein T is heating temperature (°C)) to such a degree that the enzyme
remaining is substantially inactivated and a remaining viable count is 10 or less.
Preferably, insolubles are separated and removed at a pH
of the soybean protein solution of 4.0 to 6.2, or the soybean protein solution contains
an alkaline earth metal compound or a protein flocculating agent.
Detailed Description of the Preferred Embodiments
As a raw material for preparing the soybean protein solution
of the present invention, which is derived from soybeans and is available inexpensively,
there can be used soybean milk, concentrated soybean protein, isolated soybean protein,
defatted-soybeans and, soybean whey protein. Among these, soybean milk or isolated
soybean protein is preferred. When concentrated soybean protein or defatted-soybeans
are used, separation of "okara (insoluble residue)" after enzymatic decomposition
tends to be difficult. Also, it takes much time to collect whey protein, and whey
protein has an inferior flavor. As an alkali to be used for preparing the soybean
protein solution, or adjusting the pH of the hydrolyzation mixture, sodium hydroxide
can be used. Potassium hydroxide can also be used with a view to nutrition. As an
acid, preferably, an organic acid such as citric acid is used with a view to flavor.
The concentration of the soybean protein solution to be
subjected to the enzymatic treatment is 1 to 30% by weight, preferably 5 to 15%
by weight, more preferably 8 to 12% by weight. Even if the concentration is low,
it will not be an obstacle to the process itself. However, productivity is reduced,
which causes an increase in the production cost of a soybean protein hydrolysate.
When the concentration of the soybean protein solution is too high, a large amount
of an enzyme is required for decomposing the protein sufficiently. This may be caused
by polymerization of protein hydrolysates once formed by hydrolysis with one another,
and is undesirable.
As the proteolytic enzyme to be used in the present invention
(protease), an exoprotease or endoprotease can be used alone or in combination.
The enzyme may be derived from animals, vegetables or microorganisms. Specifically,
serine proteases (trypsin, chymotrypsin, etc. derived from animals; subtilisin,
carboxypeptidase, etc. derived from microorganisms; etc.), thiol proteases (papain
ficin, bromelain, etc. derived from vegetables) and carboxy proteases (pepsin derived
from animals) can be used. Further, specific examples include Protin FN (trade name
of protease manufactured by Daiwakasei K.K.) derived from Aspergillus oryzae,
Actinase (trade name of protease manufactured by Kaken Seiyaku K.K.) derived from
Streptomyces griseus, Alkalase (trade name of protease manufactured by Novo)
derived from Bacillus licheniformis, Protein A (trade name of protease manufactured
by Daiwakasei K.K.) derived from Bacillus subtilis, and the like. In addition,
examples of enzyme preparations containing endoproteases include Protease S manufactured
by Amano Seiyaku K.K. and Protin AC-10 manufactured by Daiwakasei K.K. Examples
of proteolytic enzymes containing exo- and endoproteases include Protease M manufactured
by Amano Seiyaku K.K.
Conditions for hydrolysis of the present invention vary
to some extent according to the particular kind of proteolytic enzyme to be used.
However, in general, it is preferred to use the enzyme in an amount sufficient for
hydrolyzing soybean protein in a pH range at a temperature effective for the enzyme
activity. When the pH is 5 to 10, preferably 6 to 9, formation of a salt by neutralization
can be reduced and this is desired with a view to using the hydrolysate for a salt-restriction
diet (e.g., alimental infusion, etc.).
The degree of hydrolysis is 20 to 98%, more preferably
50 to 90% in terms of the soybean protein decomposition rate expressed by the degree
of solubilization of the protein component in 15% trichloroacetic acid. Although
the time for action of the proteolytic enzyme varies depending upon the activity
of the particular proteolytic enzyme to be used and its amount, normally, it may
be 5 minutes to 24 hours, preferably about 30 minutes to 9 hours. When the enzymatic
decomposition time is too long, putrefaction is liable to take place.
The hydrolyzed soybean protein solution is subjected to
heating (a) and cooling prior to the step of separating and removing insolubles
therefrom. This heating (a) is effected lightly in comparison with that for heat
sterilization. When this heating is effected excessively so that the heating time
is in excess of 10(5.25-0.05 × T) minutes, a material causing dregs
upon dissolution of the decomposed product is formed, presumably due to formation
of a fraction eluted from a precipitated fraction of the soybean protein hydrolysate
formed by hydrolysis. This is undesirable. On the contrary, when this heating is
not effected, flocculation capability of insolubles is poor, which results in difficulty
in separation between a supernatant and insolubles with a practical continuous separation
means. Flocculation capability can be readily judged by, for example, collecting
a 10 cc sample of an enzyme hydrolyzation mixture at 25°C, which has resulted
from enzymatic decomposition of a soybean protein solution, followed by heat treatment,
in a graduated centrifuge tube, centrifuging at 1,500 G for 20 minutes to precipitate
a sludge (precipitate) and then comparing the volume of the sludge to that of a
sample obtained in the same manner except that the heat treatment is not effected.
Specifically, it is preferred to effect this heating to such a degree that the volume
of the former is 2/3 or less of the latter. The required heating time can be readily
determined within the range of 75 to 160°C, preferably 80 to 140°C. A
shorter heating time can be employed if the heating temperature is higher. Normally,
a heating time (minutes) of 10(3.5-0.05 × T) or longer is sufficient.
For example, it is sufficient to raise the temperature to about 110°C, followed
by maintaining this temperature for about 0.01 minute and then cooling.
It is suitable to effect the cooling of the next step so
that the temperature is reduced to
30°C or less, preferably 15°C or less. When this
cooling is omitted, it makes the separation of insolubles difficult. Then in the
case of centrifugation, a high centrifugal force or longer holding time is required
for separation of insolubles. Specifically, when heating and cooling were not carried
out prior to separation, a longer holding time such as 20 minutes at 1,500 g was
required for separation of insolubles by centrifugation. However, according to the
present invention, insolubles can be separated by centrifugation within a shorter
holding time such as for several seconds to 5 minutes at 1,500 g. Therefore, according
to the present invention, continuous centrifugation can be employed, whereas continuous
centrifugation is hardly employed in a conventional process. In addition, as to
the degree of insolubilization, a larger amount of dregs tend to form as the difference
between the heating temperature and that of the cooling medium becomes larger. Then,
as to cooling conditions, it is preferred that the difference between the heating
temperature and that of a cooling medium is larger.
Further, although cooling can be carried out by allowing
to stand (natural cooling), artificial cooling with a cooling medium is preferred
because cooling can be carried out quickly to insolubilize components of the dregs
quickly, thereby facilitating prevention of formation of dregs upon dissolution
of the product.
Separation of insolubles can be carried out by a filtration
means such as a filter press, membrane or filter. However, normally, centrifugation
is employed and, in particular, a continuous centrifugal separator, a liquid cyclone,
etc. can be used.
Normally, the pH of the hydrolyzation mixture is within
the range of 3 to 8. In order to accelerate or improve the separation/flocculation
capability of the above insolubles, it is suitable that the pH is preferably 4 to
6.2, more preferably 4.5 to 5.5 because insolubles containing undecomposed materials
tend to flocculate at about the isoelectric point of soybean protein. Alternatively,
separation/flocculation capability can also be accelerated or improved by addition
of an alkaline earth metal compound such as a salt, for example, a chloride or sulfate,
or a hydroxide of calcium, magnesium, etc. or a flocculating agent such as sodium
polyacrylate, aliginic acid, chitin, chitosan, etc. to the hydrolyzation mixture.
After separation and removal of insolubles, heat sterilization
(b) is carried out. This treatment can be carried out according to a known method.
However, when the above heating (a) is effective for heat sterilization and inactivation
of the enzyme though it is weak, the conditions of this heating can be milder taking
into consideration the effect of the heating (a). Suitably, this heating is carried
out to such a degree that the enzyme remaining in the soybean protein hydrolysate
is substantially inactivated and the remaining viable count is 10 or less. Normally,
it is preferred to carry out heating in excess of the above-described heating time,
i.e., for longer than 10(5.25-0.05 × T) minutes (wherein T is heating
The pH of the hydrolyzation mixture to be subjected to
this heat sterilization (b) is preferably determined by the particular use of the
end product and, normally, it is within the range pH 3 to 8. When the hydrolysate
of the present invention is used for neutral drinks, preferably, the end product
is within pH 6 to 7. When the hydrolysate is used for acidic drinks, pH 3.5 to 4.5
is suitable. The degree of sterilization varies to some extent according to this
pH value. In the case of weakly acidic to neutral, sterilization of thermophilic
anearobes such as Clostridium, etc. is required and, preferably, the heating
is carried out in excess of, i.e., for longer than 10(6.25-0.95 × 7)
minutes. On the other hand, when the final pH is about 4.5 or less, a heating time
of 10(6.25-0.05 × T) minutes or shorter is sufficient because growth
of almost all pathogenic bacteria, putrefactive bacteria and sporangia hardly takes
The product resulting from the heat sterilization (b) can
be stored as it is, or after concentration, by sealing in a container. Alternatively,
the product can be dried and pulverized or atomized for storage.
The embodiments of the present invention are illustrated
by the following Examples.
An aqueous 0.9% solution (pH 7.0) of isolated soybean protein
("New Fuji Pro-R" manufactured by Fuji Oil, Co., Ltd.) (30 kg) was prepared and
subjected to an enzymatic reaction with a proteolytic enzyme ("Protease S" manufactured
by Amano Seiyaku K.K.) (1.2 kg) to hydrolyze the protein at 60°C for 5 hours
(15% TCA solubilisation degree: 85%). Then the hydrolyzation mixture was adjusted
to pH 5.5 by addition of citric acid. Steam at 8 kg/cm2 was blown into
the mixture to raise its temperature to 95°C and the mixture was held at this
temperature for 1 minute (heating (a)). The mixture was cooled at 12°C with
a heat exchanger plate through which cooling water was passed, followed by centrifugation
with a high-speed continuous centrifugal separator (SB-7 manufactured by WESTFALLIA
SEPARATOR) by adjusting the feed rate to 100 L/hour to separate and remove the precipitate
fraction formed. The precipitate fraction was sufficiently firm to retain it for
20 minutes until it was discharged. The resultant supernatant (yield of solids:
72.4%) was adjusted to pH 6.5 and sterilized at 150°C for 1 minute (heat-sterilization
(b)). Immediately after sterilization, the supernatant was spray-dried to obtain
a dry powder. The resultant dried powder was dissolved in water at a concentration
of 5%. When this solution was stored at 5°C for 24 hours, dregs were not observed
The above hydrolyzation mixture (10 cc) was collected in
a graded centrifuge tube before heating (a) and its temperature adjusted to 25°C
and was centrifuged at 1,500 G for 20 minutes. The volume of the sludge was 32%
of that of the hydrolyzation mixture. By heating (a), the volume of the sludge was
condensed to 10% of that of the hydrolyzation mixture.
Comparative Example 1
(The first heating step was omitted).
In the same manner as described in Example 1, a dried powder
was obtained except that the soybean protein adjusted to pH 5.5 was not subjected
to the heat treatment, but was directly cooled to 12°C with a heat exchanger
plate, followed by centrifugation. In this case, the precipitate fraction formed
in the centrifugal separate was insufficiently firm. Then, the fraction could be
retained only for 5 minutes until it was discharged. The resultant dried powder
was dissolved in water at a concentration of 5% and the solution was stored at 5°C
for 24 hours. Although dregs were not formed at all, the yield of solids of the
supernatant was only 53.2%.
Comparative Examples 2 and 3
(Heat sterilization was effected at the first heating step).
In the same manner as described in Example 1, a dried powder
was obtained except that the hydrolyzation mixture was raised to 95°C and maintained
at that temperature for 20 minutes, or raised to 80°C and maintained at that
temperature for 60 minutes instead of raising to 95°C and maintaining at that
temperature for 1 minute, and heat sterilization was omitted. The volume of sludge
determined by adjusting the temperature to 25°C and centrifuging at 1,500 G
for 20 minutes was almost the same as that of Example 1. However, when the resultant
dried powder was dissolved in water at a concentration of 5% and the solution was
stored at 5°C for 24 hours, dregs were clearly recognized with the naked eye
in both cases.
Example 2 and Comparative Example 4
(Cooling was slow cooling or omitted).
In the same manner as described in Example 1, a dried powder
was obtained except that the hydrolyzation mixture was allowed to cool for 4 hours
to room temperature and then centrifuged (Example 2), or was centrifuged directly
without cooling (Comparative Example 4) instead of subjecting to heat treatment
at 95°C for 1 minute, cooling to 12°C and then centrifugation.
The dried powder was dissolved in water at a concentration
of 5% and stored at 5°C for 24 hours. As a result, there was slight formation
of dregs in Example 1, whereas dregs were clearly formed in Comparative Example
Example 3 and Comparative Examples 5 and 6
In the same manner as described in Example 1, a dried powder
was prepared except that the hydrolyzation mixture was adjusted to pH 4.5 by addition
of citric acid, the heating (a) was carried out by holding at 100°C for 6 seconds,
the mixture was cooled to 15°C, the centrifugation was carried out in a continuous
centrifugal separate (MD-10 manufactured by Ishikawajima-Harima Heavy Industries
Co., Ltd.) by adjusting the feed rate to 30 L/hour, and the heat sterilization (b)
was carried out at 125°C for 10 seconds (Example 3). When the dried powder
was dissolved in water at a concentration of 5% and stored at 5°C for 24 hours,
dregs were not formed at all.
The volume of sludge determined before heating (a) by raising
the temperature to 25°C and centrifuging at 1,500 G for 20 minutes was 30%
based on the volume of the hydrolyzation mixture, whereas the volume of sludge determined
after heating (b) was 10% based on the volume of the hydrolyzation mixture. The
yield of the supernatant by continuous centrifugation was 65%.
The soybean protein was treated in the same manner as described
in Example 3 except that the heating (a) was omitted (Comparative Example 5). However,
separation with the continuous centrifugal separator was bad. In addition, in the
same manner as described in Example 3, a dried powder was produced except that the
heating (a) was carried out by holding at 104°C for 5 minutes (Comparative
Example 6). When the dried powder was dissolved in water at a concentration of 5%
and stored at 5°C for 24 hours, dregs were clearly formed.
Example 4 and Comparative Example 7
An aqueous 9% soybean protein solution (pH 7.0) was prepared
using the same isolated soybean protein as that in Example 1 (30 kg) and subjected
to an enzymatic reaction with a proteolytic enzyme ("Protease M" manufactured by
Amano Seiyaku K.K.) (E/S ratio = 2%) in a continuous enzymatic reaction vessel for
2 hours. After addition of CaSO4 in an amount of 0.5% by weight based
on the weight of the substrate, steam was blown into the hydrolyzation mixture so
that the temperature was raised to 130°C and heating (a) was stopped. The mixture
was cooled to 15°C with a heat exchanger plate through which cooling water
was passed, followed by treatment with a continuous separator, a liquid cyclone
(NHS-10 manufactured by Nippon Kagaku Kikai Seizo) adjusting the feed rate to 400
L/hour to separate and remove insoluble components. The resultant supernatant was
adjusted to pH 6.5 and sterilized at 150°C for 1 minute. Immediately after
sterilization, the supernatant was spray-dried to obtain a dried powder. The resultant
dried powder was dissolved in water at a concentration of 5%. When this solution
was stored at 5°C for 24 hours, dregs were not formed at all.
As Comparative Example 7, the soybean protein was treated
in the same manner as in Example 4 except that heating to 130°C by blowing
steam into the hydrolyzation mixture was omitted and the mixture was directly cooled
to 15°C. However, the insoluble components could not be separated by using
the liquid cyclone.
Effect of the Invention
According to the present invention, a precipitate formed
after enzymatic decomposition of soybean protein is readily separated, thereby improving
a yield. Also, when the resultant enzymatic decomposition product is used for drinks,
formation of dregs can be minimized.