Title: Process for producing protein hydrolyzate
Abstract: The present invention relates to a method which can prevent browning of hydrolyzed protein obtained by enzymatic hydrolysis of vegetable protein material.A vegetable protein material containing saccharides is mixed with the fungal culture and is subjected to enzymatic hydrolysis in a liquid reaction system. The reaction is conducted first at a temperature ranging from 15.degree. C. to 39.degree. C. with aeration and agitation, and then, after stopping the aeration, the reaction is conducted and completed at a temperature ranging from 40.degree. C. to 60.degree. C.
Patent Number: 6,858,405 Issued on 02/22/2005 to Nakamura, et al.
| Inventors:
|
Nakamura; Michinobu (Kawasaki, JP);
Seki; Mitsuyoshi (Kawasaki, JP);
Nawata; Miyoko (Kawasaki, JP);
Nakazawa; Hidetsugu (Kawasaki, JP);
Okamura; Hideki (Kawasaki, JP)
|
| Assignee:
|
Ajinomoto Co., Inc. (Tokyo, JP)
|
| Appl. No.:
|
674280 |
| Filed:
|
December 21, 2000 |
| PCT Filed:
|
April 23, 1999
|
| PCT NO:
|
PCT/JP99/02171
|
| 371 Date:
|
December 21, 2000
|
| 102(e) Date:
|
December 21, 2000
|
| PCT PUB.NO.:
|
WO99/57302 |
| PCT PUB. Date:
|
November 11, 1999 |
Foreign Application Priority Data
| Apr 30, 1998[JP] | 10/121029 |
| Apr 30, 1998[JP] | 10/121030 |
| Current U.S. Class: |
435/68.1; 435/171; 435/911; 435/913; 435/918 |
| Intern'l Class: |
C12P 021//06; C12P 001//02; C12N 001//14 |
| Field of Search: |
435/68.1,171,911,913,918
424/115
|
References Cited [Referenced By]
U.S. Patent Documents
| 3655396 | Apr., 1972 | Goto et al. | 99/9.
|
| 4808419 | Feb., 1989 | Hsu | 426/13.
|
| 5888561 | Mar., 1999 | Niederberger et al. | 426/20.
|
| 6045819 | Apr., 2000 | Takebe | 424/442.
|
| Foreign Patent Documents |
| 50019996 | Mar., 1975 | JP.
| |
| 52-079084 | Jul., 1977 | JP.
| |
| 6-245790 | Sep., 1994 | JP.
| |
| WO 95/28853 | Nov., 1995 | WO.
| |
Other References
Muramatsu et al. Nippon Jozo Kyokaishi. (1992), 87(3), pp. 219-223.
|
Primary Examiner: Afremova; Vera
Attorney, Agent or Firm: Oblon, Spivak, McClelland, Maier & Neustadt, P.C.
Claims
What is claimed is:
1. A method for producing hydrolyzed protein by subjecting a vegetable
protein material containing saccharides to enzymatic hydrolysis,
comprising:
(1) conducting cultivation of a koji mold in a submerged culture
fermenter-type reaction vessel to obtain a fungal culture;
(2) mixing a dispersion of said vegetable protein material with said fungal
culture to obtain a mixture; and
(3) subjecting said mixture to enzymatic hydrolysis first at a temperature
ranging from 15.degree. C. to 39.degree. C. with aeration and agitation
and then at a temperature ranging from 41.degree. C. to 50.degree. C.,
to obtain said hydrolyzed protein,
wherein a ratio of reducing sugars present in said hydrolyzed protein
obtained is 5% by weight or less based on the total solid content in said
hydrolyzed protein, and
wherein the temperature is shifted from a temperature ranging from
15.degree. C. to 39.degree. C. to a temperature ranging from 41.degree. C.
to 50.degree. C. when from 10% to 60% of the total period of time required
for completion of the enzymatic hydrolysis has passed;
wherein each of (1) to (3) are in a liquid state,
wherein said vegetable protein material is prepared for said enzymatic
hydrolysis by a process comprising:
(a) pulverizing a vegetable protein material which exists at least
partially in a solid state to a size of 300 .mu.m or less to obtain
pulverized vegetable protein material;
(b) dispersing said pulverized vegetable protein material in hot water at a
temperature higher than 80.degree. C., to obtain a vegetable protein
material dispersion;
(c) removing air bubbles from said vegetable protein material dispersion;
and
(d) subjecting said vegetable protein material dispersion to sterilization
immediately after said air bubbles have been substantially removed
and wherein said method is in the absence of an added bacteriostatic
substance.
2. The method of claim 1, wherein said vegetable protein material is
selected from the group consisting of wheat gluten, corn gluten, de-fatted
soybean, and treated products thereof.
3. The method of claim 1, wherein said subjecting said mixture to enzymatic
hydrolysis is first at a temperature ranging from 25.degree. C. to
38.degree. C. with aeration and agitation.
4. The method of claim 1, wherein said enzymatic hydrolysis is completed at
a temperature ranging from 41.degree. C. to 50.degree. C.
5. The method of claim 1, wherein said subjecting said mixture to enzymatic
hydrolysis is first at a temperature ranging from 25.degree. C. to
38.degree. C. with aeration and agitation, and wherein said enzymatic
hydrolysis is completed at a temperature ranging from 41.degree. C. to
50.degree. C.
6. The method of claim 1, wherein said enzymatic hydrolysis is first at a
temperature ranging from 15.degree. C. to 39.degree. C. and is shifted to
a temperature ranging from 41.degree. C. to 50.degree. C. so that the
ratio of reducing sugars present in said hydrolyzed protein obtained at
the completion of said enzymatic hydrolysis is 3% by weight or less based
on the total solid content in said hydrolyzed protein.
7. The method of claim 1, wherein said enzymatic hydrolysis is first at a
temperature ranging from 15.degree. C. to 39.degree. C. and is shifted to
a temperature ranging from 41.degree. C. to 50.degree. C. so that the
ratio of reducing sugars present in said hydrolyzed protein obtained at
the completion of said enzymatic hydrolysis is 1.5% by weight or less
based on the total solid content in said hydrolyzed protein.
Description
TECHNICAL FIELD
The present invention relates to a method for producing hydrolyzed protein.
More specifically, it relates to a method for producing hydrolyzed
protein, wherein in a step of enzymatically hydrolyzing a vegetable
protein material containing a protein in a solid state, the hydrolysis is
conducted in a specific manner, whereby the resulting hydrolyzate is not
browned or a period of time that lapses until the resulting hydrolyzate is
browned can markedly be prolonged.
BACKGROUND ART
With respect to a process for obtaining amino acids by enzymatically
hydrolyzing a protein starting material containing a vegetable protein in
a solid state, a large number of processes have been already known.
For example, JP-A-51-35461 describes a process for producing a liquid
seasoning comprising, in combination, a first step of reacting modified
de-fatted soybeans having a soluble nitrogen index of 50 or less with an
alkaline protease having a pH of from 9 to 12 for 2 hours, thereby
solubilizing and extracting 70% or more of a protein-derived nitrogen
component to conduct solid-liquid separation, and a second step of
subjecting the extracted to hydrolysis with a peptidase in a sealed
container at from 40.degree. C. to 60.degree. C.
Further, JP-A-6-125734 describes an enzyme preparation which is obtained
from an organic solvent-dipped product of koji formed through solid
culture of a microorganism and which contains an exopeptidase obtained
through autolysis of the koji, and a process for producing a proteinous
liquid seasoning, which comprises reacting an animal or vegetable protein
material with protein solubilizing enzymes, and then reacting the reaction
product with a exopeptidase-containing enzyme preparation.
Still further, JP-A-9-75032 describes a process for producing a seasoning,
wherein when koji for soy sauce is charged in the presence of alcohol for
enzymatic hydrolysis at from 35.degree. C. to 45.degree. C., evaporation
is forcibly conducted such that the alcohol concentration in the
completion of the hydrolysis is 2% or less, and this hydrolyzate is
fermented and aged.
Furthermore, JP-A-9-121807 describes a multi-purpose seasoning having a
high glutaminate content and having a flavor peculiar to an
acid-hydrolysis seasoning without a soy sauce smell and a brewing smell.
This seasoning is prepared by simultaneously conducting the cultivation of
a koji mold (Aspergillus) and the hydrolysis of proteins in the medium by
the enzymes contained in the culture of the koji mold in the absence of
salt or in the presence of a small amount of salt.
However, the problem has remained that when the hydrolyzate produced by
these conventional processes is stored, coloring occurs within a
relatively short period of time, and it is browned rapidly, with the
result that the commercial value thereof is notably reduced.
Any known processes for producing amino acids by enzymatically hydrolyzing
a protein material containing a solid protein have involved problems that
microorganisms other than those as the enzyme source in the hydrolysis
step, so-called contaminants are grown, reducing the quality of
hydrolyzates and decreasing the yield of amino acids. In order to solve
the problems, the presence of bacteriostatic materials such as alcohol,
sodium chloride and ethyl acetate has been employed in the hydrolysis step
in the conventional processes. In these processes, an additional step of
separating and removing the bacteriostatic materials after the completion
of the hydrolysis step is required. Especially when the presence of sodium
chloride is employed as a bacteriostatic means, it has been quite
difficult to remove sodium chloride to less than an appropriate
concentration without reducing the quality of the resulting hydrolyzate.
Further, it has been almost impossible to avoid occurrence of a so-called
brewing smell or soy sauce smell in the product obtained through the
hydrolysis step in the presence of the bacteriostatic materials, which
results in extremely limiting the use range of the hydrolyzed protein
obtained.
In addition, in the conventional processes as well, an approach has
naturally been attempted in which contaminants incorporated or contained
in a protein material containing a solid protein or a microbial culture as
the enzyme source are removed or killed, and the hydrolysis step is then
conducted. The process in which the starting material is subjected to the
proteolysis reaction after the sterilization is said to be relatively easy
on a laboratory scale. However, in the industrial mass-production, it
involves quite serious problems such as the control of contaminants in the
sterilization step and the proteolysis step.
DISCLOSURE OF THE INVENTION
One object in the present invention is to establish a method in which the
browning of a hydrolyzed protein obtained by enzymatically hydrolyzing a
protein material containing a vegetable protein in a solid state is
prevented to stably maintain the commercial value thereof over a long
period of time.
Another object of the present invention is to provide a process for
producing hydrolyzed protein which is useful as a multi-purpose seasoning
material or a multi-purpose food material without contamination with germs
even in the absence of a bacteriostatic substance, which process can be
practiced in the industrial mass-production.
In order to solve the problem, the present inventors have conducted a large
number of experiments and assiduous studies regarding a method for
enzymatic hydrolysis of protein materials containing various vegetable
proteins and a relationship of the conditions thereof and the browning of
the resulting hydrolyzate. Consequently, they have obtained the following
new findings (1) to (3).
(1) Occurrence and progression of the hydrolyzate browning is closely
related with a concentration of reducing sugars contained in the reaction
product immediately after the hydrolysis reaction. That is, when the
concentration of reducing sugars is high, the browning occurs immediately
after the reaction, and it rapidly proceeds even under an ordinary storage
environment.
(2) Once the browning occurs, it is quite difficult to find an effective
method for inhibiting the progression of the browning.
(3) The concentration of reducing sugars contained in the reaction product
immediately after the hydrolysis can be controlled to less than a
predetermined concentration by specifying the hydrolysis method and the
conditions thereof. Besides, in the thus-specified hydrolysis method and
conditions, the hydrolysis rate of the protein itself and the composition
ratio of the resulting amino acids is substantially unchanged.
The present inventors have also obtained the following new findings (4) to
(6) with respect to the sterilization of the protein material and the
culture medium.
(4) Contaminants grown in the hydrolysis step are those present in the
protein material in a solid state and in the microbial culture as the
enzyme source.
(5) In case the protein material and the culture medium can completely be
sterilized, the hydrolysis step can be conducted substantially in the
absence of contaminants.
(6) The sterilization of the protein material and the culture medium is
extremely inhibited by the presence of air and bubbles contained therein.
In other words, when the heat sterilization is conducted after air and
bubbles contained therein are completely removed, the protein material
which is substantially in a sterile state and the culture of
microorganisms as the enzyme source which is substantially free from
contamination with germs can be obtained.
The present inventors have completed the inventions on the basis of these
new findings.
That is, the first invention is a method for producing hydrolyzed protein
by subjecting a vegetable protein material containing saccharides to
enzymatic hydrolysis using a fungal culture in a liquid reaction system,
comprising mixing the vegetable protein material with the fungal culture,
conducting a reaction first at a temperature ranging from 15.degree. C. to
39.degree. C. with aeration and agitation, and then, after stopping the
aeration, conducting and completing the reaction at a temperature ranging
from 40.degree. C. to 60.degree. C.
The second invention is the method for producing hydrolyzed protein
according to the first invention, wherein the vegetable protein material
is selected from the group consisting of wheat gluten, corn gluten,
de-fatted soybean and treated products thereof.
The third invention is the method for producing hydrolyzed protein
according to the first invention, wherein the reaction which is conducted
at a temperature ranging from 15.degree. C. to 39.degree. C. is shifted to
the reaction which is conducted at a temperature ranging from 40.degree.
C. to 60.degree. C. when from 10% to 60% of the total period of time
required from the start-up to the completion of the reaction passes after
the start-up of the reaction.
The fourth invention is the method for producing hydrolyzed protein
according to the first invention, wherein a ratio of reducing sugars
present in the reaction product obtained at the completion of the reaction
is adjusted to 5% by weight or less based on the total solid content in
the reaction product.
The fifth invention is the method for producing hydrolyzed protein
according to the first invention, wherein the preparation of the fungal
culture and the enzymatic hydrolysis of the vegetable protein material are
conducted in a submerged culture tank-type reaction vessel.
The sixth invention is the method for producing hydrolyzed protein
according to the first invention, wherein the vegetable protein material
is at least partially in a solid state, and is pulverized to 300 .mu.m or
less prior to the enzymatic hydrolysis, dispersed in hot water at higher
than 80.degree. C., subjected to the sterilization immediately after air
bubbles contained in the pulverized product are substantially removed.
The present inventions are described in detail below.
The starting material used in the present invention is a vegetable protein
material containing saccharides. That is, it is a vegetable protein
starring material having a high content of an edible vegetable protein
which is at least partially in a solid state, and containing saccharides
including starch and various saccharides other than starch, such as
glucose, fructose, sucrose and galactose.
The form of these vegetable protein materials used is not particularly
limited. Starting materials having the various forms such as powder,
granules, pellets, a dispersion in an aqueous solvent and paste, are used.
Further, so long as it is the vegetable protein material, its origin is
not limited.
Specific examples of the vegetable protein material include starting
materials such as wheat gluten, corn gluten, de-fatted soybean, separated
soybean protein, separated potato protein and treated products of these
vegetable protein materials. Of these vegetable protein materials, wheat
gluten and de-fatted soybean are especially important protein materials in
the invention.
The treatment of enzymatically hydrolyzing the vegetable protein material
is a step of dispersing in an aqueous solvent the sterilized protein
material or the protein material kept in a bacteriostatic state, and
contacting the dispersion with a fungal culture having a high proteolytic
activity in the aqueous solvent to hydrolyze the protein material.
It is important to employ an embodiment of conducting aeration and
agitation at the initial stage of the hydrolysis reaction, confirming,
after a certain period of time, that the reaction system is in a
predetermined state, and then clearly shifting the reaction temperature to
a high temperature region and continuing the contact. In this respect, the
method is markedly different from an ordinary method for enzymatically
hydrolyzing a protein material. Thus, it is a main characteristic feature
in the method of the invention.
For practicing this embodiment specifically, it is required that a
hydrolysis reaction vessel or tank is provided with at least a temperature
control equipment, an aeration equipment and a agitation equipment. In the
reaction, a rate of aeration of 1/1 vvm or less is sufficient. The
agitation equipment is not particularly limited so long as it corresponds
to the size of the reaction vessel and fully withstands a load such as a
viscosity of the starting dispersion or the reaction system. Various
agitation devices are available. For example, a submerged culture device
used in fermentation for amino acid production is an especially preferable
reaction device.
The starting material used is pulverized or finely divided so as not to
inhibit the agitation procedure, at the time of sterilization of the
starting material or just before the hydrolysis reaction. The
sterilization treatment is conducted by a method and a device which are
ordinarily employed in the fermentation industry. In order to conduct the
hydrolysis reaction without germ contamination, cultivation of fungi as
the enzyme source is carried out under the non-contamination conditions.
Further, as a matter of course, a measure to cope with germ contamination
and a management of the process during the reaction step should be
perfectly conducted.
It is preferable that the protein material is, before subjected to the
proteolysis step, pulverized to a size of 300 .mu.m or less, and dispersed
in hot water at 80.degree. C. or higher. The pulverization of the protein
material may be conducted with respect to a dry protein material. However,
when it is conducted simultaneously with the treatment of dispersing in
hot water the protein material which has been roughly pulverized, the step
can continuously be shifted to the sterilization step conveniently.
The conditions of the pulverization and the temperature condition of hot
water for dispersion have inductively been determined as a result of many
tests on various protein materials. When the pulverization is conducted as
much as possible under these conditions and the treatment is conducted at
a high temperature of approximately a boiling point, preferable
sterilization effects can be expected in the subsequent sterilization
step.
That is, when the dispersion containing particles having particle size of
300 .mu.m or more is treated with a heat exchanger, the particles of the
protein material in the dispersion are precipitated, and there is a risk
that clogging might occur in the flow path of the heat exchanger.
Accordingly, the sterilization treatment becomes actually impossible.
Meanwhile, a phenomenon has been found that the viscosity of the dispersion
of fine particles of the protein material is abruptly decreased at
80.degree. C. or higher.
FIG. 1 is a line graph showing a relationship of a viscosity and a
temperature of a dispersion of wheat gluten in hot water having a
concentration of 32% at temperatures ranging from 60.degree. C. to
90.degree. C. In FIG. 1, the ordinate axis indicates a viscosity at
regular intervals with a unit of 10.sup.4 cps (centipoises), and the
abscissa axis indicates a temperature at regular intervals with a unit of
.degree. C. In this example, it can be observed that the viscosity is
remarkably decreased at a temperature of from 80.degree. C. to 85.degree.
C.
One of the technical progresses in the invention exists in this point. That
is, the marked improvement of the handleability with the abrupt decrease
in the viscosity of the protein material dispersion within the specific
temperature range is linked with the effective heat sterilization.
When the protein material is subjected to the pulverization and the hot
water dispersion, the dispersion of the protein material shows an
emulsified state in many cases. However, the viscosity of the dispersion
is not increased, and the dispersion becomes a non-tacky solution having a
low viscosity. Consequently, air and bubbles are not included into the
dispersion treated.
When the protein material is pulverized and dispersed in hot water, a
method and an apparatus that meet the purpose can be employed. For
example, a method can be employed in which the powdery protein material is
fed to a tank containing an aqueous solution which has been maintained at
a predetermined temperature, and fed to an emulsifier while being stirred
for emulsification and dispersion.
What is important in the dispersion treatment is to confirm that air and
bubbles are not adhered to, nor contained in, the fine particles of the
protein material present in the dispersion after the dispersion treatment.
At this time, the dispersion after the disppersion treatment is observed
in a weak enlarged visual field of a microscope, and it is identified that
bubbles are substantially not adhered to the fine particles dispersed and
that the fine particles dispersed are brought into direct contact with a
liquid phase portion.
In case bubbles are present in the dispersion after the dispersion
treatment, no predetermined sterilization effect can be expected even when
the high-temperature treatment is conducted in the subsequent
sterilization step. Further, in the operation of a sterilizer, there is a
fear that serious troubles such as clogging might occur.
When bubbles are present in the dispersion, the sterilization cannot
completely be conducted presumably because the heat is not uniformly
distributed in the sterilizer and cannot act on germ cells or spores
surrounded by the bubbles.
The protein material dispersed in hot water is, after the dispersion
treatment, successively subjected to the sterilization step.
Amethodandadevice for the sterilization step are not particularly limited.
A continuous sterilization method or a batchwise sterilization method in a
device for hydrolysis is useful for smoothly practicing the whole step.
The dispersion of the protein material becomes substantially sterile by
this sterilization treatment. Further, it is also possible that the sample
is collected and identified to be sterile as required.
As a device used for continuous sterilization, a plate-type heat exchanger
or a nozzle-type heater is especially appropriate. When the hot water
dispersion of the protein material which is produced by the
above-mentioned method and is identified to be free from bubbles is
treated with these heat sterilization devices under usual operation
conditions, accidents such as clogging and scorching in the devices do not
occur. Further, cleaning and maintenance procedures of the devices which
are conducted after the completion of the treatment are quite easy.
As the fungal culture used in the hydrolysis reaction, a culture newly
prepared by growing a fungal strain with a high protease productivity,
from which an activity of a protease produced can be anticipated, is
appropriate.
As the fungus having the high protease productivity, various fungi can be
used regardless of the taxonomical classification. In consideration of the
fact that a product is used for food, it is advisable to select fungi
which have been used so far in the field of food or brewing industry,
especially a koji mold (Aspergillus). In practicing the proteolysis
reaction, the koji mold is convenient in the aspect of the control of the
hydrolysis reaction or the purification and the post-treatment of the
reaction product.
As the koji mold, strains newly separated from commercial koji rice and
commercial koji for soy sauce brewing and having fixed strain properties
may be used. Needless to say, the deposited strains of these
microorganisms may be used.
The fungal culture having the high protease activity which is used in the
hydrolysis reaction is added to, and mixed with, the protein material
which has been sterilized in the form of liquid koji. The starting
material constituting the liquid koji may be the same as, or different
from, the protein material to be hydrolyzed. However, when the hydrolysis
is conducted in a state free from germ contamination, germs should not be
present in the liquid koji prepared. Accordingly, special care must be
taken in the sterilization of the protein material for preparing liquid
koji.
By the way, when there is a fear that the sterilization treatment might not
be conducted effectively in the hydrolysis reaction system or when the
sterilization treatment cannot satisfactorily be conducted for some
reasons, it is also possible to conduct the hydrolysis reaction in the
presence of a bacteriostatic substance that inhibits the growth of germs
to co-exist in the same system.
Examples of the bacteriostatic substance which is addied to the hydrolysis
reaction system include sodium chloride, ethanol and ethyl acetate.
Further, with respect to the mode of the addition, appropriate amounts of
these bacteriostatic substances are added to the system, and moreover,
regarding ethanol, yeast having an ability to form ethanol effectively may
be caused to co-exist in the system.
In case any of these bacteriostatic substances is used, there is a need to
remove the bacteriostatic substances from the reaction mixture by
separation after the completion of the hydrolysis reaction. It is
considerably difficult to effectively conduct the removal by separation
without decreasing the qualities of the resulting hydrolyzate, and this is
not said to be economically advantageous. Especially for completely
removing sodium chloride by separation, a new equipment is required.
Accordingly, there is no alternative but to obtain a hydrolyzate
containing a considerable amount of sodium chloride. The use of such a
product is limited as a matter of course.
FIG. 2 is a line graph showing a concentration (unit: mg/dl) of glutamic
acid (GH) formed and accumulated in a hydrolysis system at each
temperature after each reaction time. Further, FIG. 3 is a line graph
showing a concentration (unit: mg/dl) of glucose (Glc) in a hydrolysis
system at each temperature after each reaction time.
As is clearly seen upon comparing FIG. 2 with FIG. 3, it can be understood
that, by shifting the reaction temperature on purpose during the
hydrolysis reaction, the amount of saccharides typified by the
concentration of glucose formed and accumulated, especially, the amount of
reducing sugars can selectively be reduced without substantially
influencing the rate of the proteolysis reaction, namely, the rate of
amino acid formation typified by the concentration of glutamic acid formed
and accumulated. Further, it can also be understood that the amount and
the concentration of sugar present in the reaction product finally
obtained can be adjusted to less than predetermined levels.
FIG. 2 reveals that the concentration of glutamic acid formed and
accumulated is increased with the increase in the reaction temperature and
with the lapse of the reaction time. Meanwhile, FIG. 3 reveals that the
concentration of glucose formed and accumulated is abruptly decreased at
the reaction temperature of from 36.degree. C. to 39.degree. C. with the
lapse of the reaction time (after from 5 to 10 hours). From this fact, it
can be anticipated that under the reaction temperature condition of from
36.degree. C. to 39.degree. C., glucose once formed is rapidly decomposed
and consumed by the fungi over the course of the reaction time.
That is, the sterile vegetable protein material and the fungal culture, the
liquid koji, are mixed in a hydrolysis reaction vessel. Thereafter, the
mixture is reacted first at a temperature ranging from 15.degree. C. to
39.degree. C., preferably from 25.degree. C. to 38.degree. C. with
aeration and agitation, and the aeration is then stopped to complete the
reaction at a temperature ranging from 40.degree. C. to 60.degree. C.,
preferably from 41.degree. C. to 50.degree. C. Consequently, the amount of
sugar, especially reducing sugars, which is formed, accumulated and
present in the hydrolysis reaction system, can selectively be decreased
without substantially influencing the proteolysis rate, namely the rate of
amino acid formation, and the ratio of reducing sugars present in the
reaction product finally obtained can be adjusted to 5% or less based on
the total solid content in the reaction product.
Moreover, the time when the reaction which is conducted at a temperature
ranging from 15.degree. C. to 39.degree. C. is shifted to the reaction
which is conducted at a temperature ranging from 40.degree. C. to
60.degree. C. is set at a time when from 10% to 60% of the total period of
time required from the start-up to the completion of the reaction passes
after the start-up of the reaction. Consequently, the amount of sugar,
especially reducing sugars, which is formed, accumulated and present in
the hydrolysis reaction system, can selectively be decreased without
substantially influencing the proteolysis rate, namely the rate of amino
acid formation and the ratio of reducing sugars present in the reaction
product finally obtained can be adjusted to 5% or less based on the total
solid content in the reaction product.
The hydrolysis reaction product obtained by adjusting the ratio of reducing
sugars present in the resulting reaction product to 5% or less based on
the total solid content in the reaction product can maintain its quality
stably over a long period of time without getting brown.
FIG. 4 is a line graph showing the results of a cruel heating test which
was conducted using the above-mentioned product and a control product
obtained by conducting the whole procedure at 45.degree. C. constantly
without changing the reaction temperature during the hydrolysis reaction.
In FIG. 4, the ordinate axis indicates a rate of increase in an absorbance
of light of a wavelength of 545 nm, and the abscissa axis indicates a time
for which the temperature is maintained at 105.degree. C. Further, the
blank arrow in FIG. 4 shows that the browning of the product could
markedly be inhibited as shown by a downward length of the arrow.
This cruel heating test was conducted by maintaining the liquid product and
the control liquid product which were adjusted to a 20% Brix concentration
in a sealed state at 105.degree. C. for 6 hours. The test conditions
correspond to such conditions that the product is maintained at room
temperature for 12 months. It suggests that although the product is stored
for 12 months, it can keep stable qualities without being browned.
In the hydrolysis reaction, the setting of the two types of the temperature
ranges, the setting of the time when the temperature is shifted and the
ratio of reducing sugars present in the final reaction product are
inductively specified from the results obtained in a large number of trial
experiments conducted using various vegetable protein materials and a
plurality of liquid kojis of different seed strains under various
conditions.
Further, from the results of these many trial experiments, it is advisable
to distinguish the above-mentioned two types of the temperature ranges as
clearly as possible. That is, it is advisable that the hydrolysis reaction
is first started at a relatively low temperature and after the temperature
is shifted, the reaction is conducted at a relatively high temperature.
Further, in consideration of the fact that the overall period of time
required for the hydrolysis reaction is approximately 24 hours in many
cases, it has been found that the time when the temperature is shifted is
set at a time when approximately 8 hours passes from the start-up of the
reaction, that is, a time when approximately 30% of the overall period of
time anticipated passes, whereby good results are provided. Still further,
the ratio of reducing sugars present in the final reaction product should
be 5% or less, preferably 3% or less, more preferably 1.5% or less based
on the total solid content. That is, the ratio of 5% is the upper limit
thereof.
Accordingly, with respect to the two temperature ranges used when the
hydrolysis reaction is conducted with a specific vegetable protein
material and a specific liquid koji, and the specific point of time to be
set when the temperature is shifted, it is necessary to determine the
optimum ranges and the values within the above-mentioned ranges upon
conducting preliminary trial experiments on the specific protein
materials.
The hydrolyzate obtained under the above-mentioned hydrolysis reaction
conditions is a light yellow, semi-transparent liquid having koji mold
cells dispersed therein. A light yellow, clear liquid obtained by the
solid-liquid separation after the decoloring and deodorizing treatments
with the addition of activated carbon is an amino acid solution having a
dense, palatable taste, and it is free from a specific unpleasant taste or
offensive smell.
The enzymatically hydrolyzed protein solution obtained through the
hydrolysis reaction mentioned above is directly used as a seasoning
material. However, in many cases, it is subjected to decoloring treatment,
deodorizing treatment, for example, activated carbon treatment, or
purification treatment such as concentration treatment to provide a
product. Alternatively, according to the use Purpose, it is formed into a
concentrated paste, a flaky powder, a spray-dried powder, granules or a
cubic block. Incidentally, the product obtained without using the
bacteriostatic substance such as sodium chloride in the hydrolysis
reaction step has multi-purpose characteristics for which it can find wide
acceptance, in addition to the property that it is not browned easily.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 is a line graph showing the relationship of viscosity and
temperature of a dispersion of wheat protein in hot water.
FIG. 2 is a line graph showing a relationship of a concentration of
glutamic acid formed and accumulated in a hydrolysis reaction system when
employing various reaction temperatures and a reaction time.
FIG. 3 is a line graph showing a relationship of a concentration of glucose
formed and accumulated in a hydrolysis reaction system when employing
various reaction temperatures and a reaction time.
FIG. 4 is a line graph showing that there is a marked difference in the
increase of an absorbance in a cruel heating test conducted with respect
to a product obtained by shifting hydrolysis reaction temperature
conditions during the reaction and a product obtained by not shifting the
temperature conditions.
BEST MODE FOR CARRYING OUT THE INVENTION
The invention is illustrated by referring to specific Examples of the
invention. By the way, the following Examples do not limit the technical
scope of the invention.
EXAMPLE 1
Production of a Wheat Gluten Hydrolyzate Resistant to Browning
(Emulsification Pretreatment of Wheat Gluten)
Four-hundred liters of city water were introduced into a 1,000-liter tank
connected with an emulsifier for emulsification by impact shearing,
Homomicline Mill (supplied by Tokushu Kikako K.K.). Water in the tank was
heated. When the temperature of water reached 95.degree. C., the operation
of the emulsifier started. Twenty kilograms of a powder of active wheat
gluten were charged into the tank. The wheat gluten became a completely
emulsified dispersion in 30 minutes after the operation started, and at
the same time, the viscoelasticity peculiar to wheat gluten disappeared.
In the dispersion, neither incorporation of a coagulum (so-called clump)
of wheat gluten nor inclusion of bubbles was observed at all in a slightly
enlarged visual field of a microscope.
The particle diameter of the wheat gluten particles in the emulsified
dispersion was on the average 150 .mu.m, at least 10 .mu.m and at most 900
.mu.m. Further, the concentration of the wheat gluten particles was 50
g/liter.
(Pretreatment of De-fatted Soybeans for Liquid Koji) A de-fatted soybean
powder obtained by roughly pulverizing unmodified de-fatted soybeans
(supplied by Toyo Seiyu K.K.) was heated while being mixed with a mixer
capable of heating procedure to conduct hot heat treatment at 98.degree.
C. for 20 minutes. (Sterilization Treatment of De-fatted Soybeans for
Liquid Koji)
Three kilograms of the de-fatted soybean powder heat-treated were charged
into 200 liters of water having a temperature of 25.degree. C. which had
been introduced into a submerged culture fermenter-type reaction vessel
for amino acid production while being stirred to obtain a uniform
de-fatted soybean powder dispersion free from inclusion of bubbles.
Subsequently, the dispersion was subjected to batchwise heat sterilization
through heating with a superheated steam at 120.degree. C. for 20 minutes.
(Production of Liquid Koji)
One percent by volume of a seed culture of a koji mold, Aspergillus oryzae
ATCC 15240, which had been incubated after inoculating spores at a
concentration of 10.sup.4 spores/ml in a medium containing 1.5% de-fatted
soybean powder in a 5-liter jar fermenter, was inoculated in this
heat-sterilized dispersion of de-fatted soybean powder. After the
inoculation, the cultivation was conducted at 30.degree. C. for 24 hours
with aeration at a rate of 1/4 vvm and agitation at 520 rpm to obtain
liquid koji.
(Evaluation of Liquid Koji)
The protease activity of the resulting liquid koji was 304 units/ml, and
neither contamination of germs other than the koji mold nor growth thereof
was observed.
(Hydrolysis of Wheat Gluten)
The total amount of the wheat gluten emulsified dispersion obtained by the
above-mentioned method was transferred to a 1-kiloliter fermenter used in
fermentation for amino acid production. Subsequently, the wheat gluten
dispersion was subjected to batchwise heat sterilization through heating
with a superheated steam at 120.degree. C. for 20 minutes. When the
temperature of the solution was lowered to 50.degree. C. after the
completion of the heat sterilization, the half amount of the liquid koji
was added thereto. The hydrolysis reaction was conducted until hour 8 from
the start-up of the reaction with aeration at a rate of 1/4 vvm and
agitation at 200 rpm while controlling the temperature of the dispersion
to 35.degree. C., and from hour 8 to hour 24 when the reaction was
completed, without conducting the aeration while controlling the
temperature of the dispersion to 45.degree. C.
During the reaction, the concentration of amino acids formed in terms of
the glutamic acid concentration in the reaction system was progressively
increased from the start-up of the reaction. Meanwhile, the concentration
of reducing sugars in terms of the glucose concentration in the reaction
system was rapidly increased until hour 3 from the start-up of the
reaction and was maintained approximately at the maximum value from then
to hour 8. However, while the reaction was continued upon raising the
temperature of the dispersion in the reaction system from hour 8 and
maintaining and controlling it to 45.degree. C., the concentration of
reducing sugars was abruptly decreased. On hour 24 when the reaction was
completed, the glucose concentration was decreased to 1.0% by weight or
less based on the total solid content of the reaction product.
(Hydrolysis of Wheat Gluten Conducted as a Control)
Two-hundred liters of a wheat gluten emulsified dispersion which had been
obtained in the above-mentioned manner were charged into a 1-kiloliter
fermenter used in fermentation of an amino acid. Then, the wheat gluten
dispersion was subjected to batchwise heat sterilization through heating
with a superheated steam at 120.degree. C. for 20 minutes. When the
temperature of the dispersion was lowered to 50.degree. C. after the heat
sterilization, the half amount of the liquid koji was added thereto. The
hydrolysis reaction was conducted with aeration and agitation while
maintaining the temperature of the dispersion at 45.degree. C. constantly
and without shifting the temperature during the whole reaction period from
the start-up of the reaction to hour 24 when the reaction was completed.
During the reaction, the concentration of amino acids in terms of the
glutamic acid concentration in the reaction system was progressively
increased from the start-up of the reaction. Meanwhile, the concentration
of reducing sugars in terms of the glucose concentration in the reaction
system was rapidly increased until hour 3 from the start-up of the
reaction and was maintained at approximately the maximum value from hour 8
to hour 24 when the reaction was completed. On hour 24 when the reaction
was completed, the glucose concentration reached 6.6%. It was identified
that during the overall reaction period, the glutamic acid concentration
tended to be always increased somewhat slowly in comparison with the
above-mentioned case of raising and controlling the temperature of the
solution during the reaction. (Test for Storage of the Hydrolyzate
Obtained)
The test hydrolyzate of wheat gluten (hereinafter referred to as a "test
product") obtained by the above-mentioned method and the control
hydrolyzate of wheat gluten (hereinafter referred to as a "control
product") for comparison were subjected to centrifugation to separate and
remove the koji mold cells. The residues were then passed through a
granular activated carbon layer for brewing to obtain hydrolyzates
purified. Each of these hydrolyzates purified was charged into a
500-milliliter colorless transparent glass bottle having a stopper such
that no space was given on the upper portion.
With respect to the samples in the bottles stored under scattered light in
a room in which the temperature was not particularly controlled, the state
of occurrence and progression of browning was visually observed
immediately after the charging and after 1 week, 2 weeks, 1 month, 3
months, 6 months and 12 months. The results are shown in Table 1 together
with the state of the change of a smell immediately after releasing the
stopper after these storage periods. In Table 1, "+" in the column
"browning" indicates evaluation with 5 grades. That is, a state in which
browning proceeds most is defined as 5, and a state in which browning is
slightly observed is defined as 1. Further, "+" in the column "burnt
smell" indicates evaluation with 5 grades. That is a state in which an
irritating smell accompanied by browning, a so-called "burnt smell"
notably occurs is defined as 5, and a state in which the burnt smell is
slightly found is defined as 1. By the way, the evaluation was conducted
by five panelists. The evaluation points given by the panelists were
averaged, and rounded. The resulting points were indicated in terms of the
number of "+".
TABLE 1
Browning of a wheat gluten hydrolyzate
Test product Control product
Storage period Browning Burnt smell Browning Burnt smell
Immediately after No No No No
charging
After 1 week No No No +
After 2 weeks No No + +
After 1 month No No ++ +
After 3 months No No +++ ++
After 6 months + No ++++ +++
After 12 months + + +++++ +++++
As shown in Table 1, in the test product, even after 12 months, the degree
of browning was week, and the occurrence of the "burnt smell" was little
found. Thus, the test product was judged to maintain a satisfactory
commercial value. Meanwhile, in the control product, immediately after the
charging into the bottle, the sign of the browning was already observed,
and the presence of the "burnt smell" was found though weak. After 1
month, the "browning" and the "burnt smell" were clearly found. After 3
months, the "browning" and the "burnt smell" were both notably found.
Accordingly, the control product was judged to extremely impair a
commercial value.
EXAMPLE 2
Production of Hydrolyzed Protein Resistant to Browning from the Other
Vegetable Protein Materials
Hydrolyzed protein resistant to browning were produced from corn gluten and
de-fatted soybeans in the same manner as in Example 1.
(Pretreatment of Vegetable Protein Materials)
Powdery corn gluten from Minnesota, U.S.A. was emulsified as in Example 1.
Further, unmodified de-fatted soybeans (supplied by Toyo Seiyu K.K.) were
pulverized, and then emulsified as in Example 1. Neither formation of a
coagulum (so-called a clump) nor inclusion and presence of bubbles was
observed at all in any of the emulsified products.
(Production of Liquid Koji)
Liquid koji was produced from the de-fatted soybean powder in the same
manner as in Example 1.
(Hydrolysis of the Vegetable Protein Materials)
Each of the corn gluten emulsified dispersion and the de-fatted soybean
emulsified dispersion was transferred to a 30-kiloliter fermenter, and
sterilized. When the temperature of the dispersion was lowered to
50.degree. C., the liquid koji was added to each of the fermenters as in
Example 1. The conditions of the hydrolysis reaction were the same as
those in Example 1. That is, the hydrolysis reaction was conducted until
hour 8 from the start-up of the reaction with aeration and agitation while
controlling the temperature of the dispersion to 35.degree. C., and from
hour 8 to hour 24 when the reaction was completed without aeration while
controlling the temperature of the dispersion to 45.degree. C. In the
completion of the hydrolysis reaction, the glucose concentration of the
reaction product was 0.9% by weight based on the total solid content of
the reaction product.
(Hydrolysis of the Vegetable Starting Materials Conducted as a Control)
Each of a corn gluten emulsified dispersion and a defatted soybean
emulsified dispersion obtained according to the above-mentioned method was
transferred to a 30-kiloliter fermenter, and sterilized. When the
temperature of the dispersion was lowered to 50.degree. C., the liquid
koji was added to each fermenter. The hydrolysis reaction was conducted
with agitation while controlling the temperature of the dispersion to
45.degree. C. without shifting the temperature during the reaction from
the start-up of the reaction to hour 24 when the reaction was completed.
When the hydrolysis reaction was completed, the glucose concentration in
the reaction product was 6.4% by weight based on the total solid content.
(Test for Storage of the Resulting Hydrolyzates)
The resulting hydrolyzates were subjected to the storage test in the same
manner as in Example 1. The results thereof for comparison are shown in
Tables 2 and 3.
TABLE 2
Browning of a corn gluten hydrolyzate
Test product Control product
Storage period Browning Burnt smell Browning Burnt smell
Immediately after No No No No
charging
After 1 week No No No No
After 2 weeks No No + +
After 1 month No No ++ +
After 3 months No No +++ ++
After 6 months + No ++++ +++
After 12 months + + +++++ +++++
TABLE 3
Browning of a de-fatted soybea hydrolyzate
Test product Control product
Storage period Browning Burnt smell Browning Burnt smell
Immediately after No No No No
charging
After 1 week No No > >
After 2 weeks No No + +
After 1 month No No ++ ++
After 3 months No No +++ +++
After 6 months + No ++++ ++++
After 12 months + + +++++ +++++
As shown in Tables 2 and 3, in the corn gluten test product and the
de-fatted soybean test product, even after 12 months, the degree of
browning was week and the occurrence of the "burnt smell" was little
found. Thus, the test products were judged to maintain a satisfactory
commercial value. Meanwhile, in the gluten control product and the
de-fatted soybean control product, immediately after the charging into the
bottle, the sign of the browning was already observed though there was a
slight difference therebetween, and the presence of the "burnt smell" was
found though weak. After 1 month, the "browning" and the "burnt smell"
were clearly found. After 3 months, the "browning" and the "burnt smell"
were both notably found. Accordingly, the control products were judged to
extremely impair a commercial value.
INDUSTRIAL APPLICABILITY
Hydrolyzed protein produced from a vegetable protein material in a liquid
reaction system using a fungal culture according to the method of the
present invention can maintain a stable commercial value without being
browned over a long period of time.
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