Method of making a soybean protein food product

A soybean protein food product made by use of a newly discovered microorganism which releases a soybean protein clotting enzyme, and a method of making such soybean protein food product is disclosed. The new enzyme functions to clot a soybean protein (soymilk), thus permitting an effective production of new fermented soybean products, such as a soymilk cheese or a soymilk cream, as well as any other wide varieties of soybean protein food products.

BACKGROUND OF THE INVENTION 
1. Field of the Invention 
The present invention relates to a soybean protein product and method of 
producing the same, and in particular, to a soybean protein clotting 
enzyme which is utilized in the process of producing a soybean protein 
product, a method of producing the soybean protein clotting enzyme 
effeciently, and a method of making a soybean protein food product by use 
of a soybean protein clotting enzyme. 
2. Description of the Prior Art 
In recent years, a vegetable food has received a great attention from many 
people having an increased concern about their health in view of the 
tendency for most of people to eat an excessive amount of animal foods or 
fats being the cause of such fatal diseases as heart diseases, or 
arteriosclerosis. 
Generally, most of commonly available vegetable food products are 
artificially made from vegetable oils and fats, as quasi-vegetable foods, 
and as such, a pure vegetable food product is not available easily from 
local shops, nowadays. 
Among other vegetables, a soybean is, not to mention its high quality of 
vegetable protein, a major food that has long been utilized in a variety 
of food products, and is said to contain almost all essential nutrition 
for human health. 
Study and development have been made in an attempt to use a soybean protein 
for production of a pure vegetable food. However, a difficulty has still 
remained in producing a fermented soybean protein food similar to a cheese 
which is made from milk. The reason is that a cheese is produced via a 
rennin enzyme which clots milk proteins, but nobody has hitherto 
discovered an enzyme capable of clotting the soybean proteins in a manner 
analogous to such rennin enzyme. 
SUMMARY OF THE INVENTION 
It is a primary purpose of the present invention to provide a novel soybean 
protein food product. 
In accomplishment of this purpose, in accordance with the present 
invention, a bacteria is discovered by the inventor, which is deemed to 
belong to the genus Bacillus and releases a soybean protein clotting 
enzyme. The bacteria is name 26D7. It is therefore found that the addition 
of such enzyme released by the 26D7 into a soybean protein permits an 
effective production of a new soybean protein food, such as for example, a 
soymilk cheese or a soymilk cream. 
It is another purpose of the present invention to provide a method of 
producing a soybean protein clotting enzyme efficiently. 
In achieving this purpose, in accordance with the present invention, the 
above-mentioned bacteria (26D7) is cultured at a medium comprising 0.1% 
yeast extract, 0.02% casamino acid, 0.1% ammonium sulfate, 0.05% sodium 
citrate, 0.01% magnesium sulfate, 1.0% phosphate, and 5.0% soymilk 
(adjusted to pH6.0 by addition of KOH). 
Accordingly, in the presence of 5.0% soymilk, the strain 26D7 is found to 
produce a sufficient activity of a soybean protein clotting enzyme so as 
to clot the soymilk in a satisfactory manner. 
It is still another purpose of the present invention to provide a soybean 
protein clotting enzyme per se which is effective for clotting a soybean 
protein. 
To this end, in accordance with the present invention, there is obtained a 
soybean protein clotting enzyme of the following characteristics. 
Molecular weight: approx. 30000 
Optimal temperature: approx. 80.degree. C. 
Thermal stability: stable at 35.degree. C.-40.degree. C. for 30 min. 
Optimal pH: below pH 6.0 
pH stability: stable at pH4-pH9 for 12 h. 
Influence of metal ions: null 
Influence of inhibitors: enzymatic activity is halted by phenyl methyl 
sulfonyl fluoride and tosyl fluoride. 
Proteolytic activity and enzyme: a sort of serine protease

DETAILED DESCRIPTION OF PREFERRED EMBODIMETNS OF THE INVENTION 
Firstly, we, the inventors of the present invention, effected a number of 
screening procedures in order to find a microorganism, or a bacteria 
capable of releasing a soybean protein clotting enzyme. Soils and plants 
were mainly collected and subjected to screening procedures in a known 
conventional way. 
As a result, we discovered and succeeded in isolating a new bacteria with 
the property of releasing a soybean protein clotting enzyme in the 
presence of a soybean protein, and hereby let it be known that the 
bacteria strain is named 26D7 and has been deposited under the deposit 
number FERM BP-1778 at the authorized Japanese depositary, Agency of 
Industrial Science and Technology, with its address at 1-3, Higashi 
1-chome, Tsukuba-shi, Ibaraki-ken, Japan, in conformity with the 
provisions of Butapest Treaty on the international recognition of the 
deposit of microorganisms for the purposes of patent procedure. 
Now, description will be given of the bacterial properties and culture of 
the above-mentioned strain 26D7. 
CULTURE MEDIUM 
The microorganism identified as 26D7 was cultured in a medium prepared from 
the ingredients shown in the Table 1 below. 
TABLE 1 
______________________________________ 
Beef extract 10 g 
Polypeptone 10 g 
NaCl 5 g 
Distilled water 1000 ml 
______________________________________ 
The foregoing medium may be formed on a plate as a plate medium by addition 
of 1.5% agar thereinto. 
BACTERIAL PROPERTIES 
Thus-cultured strain 26D7 was determined its bacterial properties. The 
results are shown in the Table 2 below. 
TABLE 2 
______________________________________ 
Shape rod 
Sporulation + 
Gram staining + 
Growth at 45.degree. C. 
+ 
65.degree. C. - 
Growth in 7% NaCl + 
Voges-Proskaver reaction 
+ 
Catalase + 
Growth in anaerobic state 
+ 
Motility +++ 
Utilization of carbohydrate 
glucose.+-. + 
______________________________________ 
The above results suggest that the strain 26D7 in question might belong to 
the genus Bacillus. 
Next, description will be made in regard to a a soybean protein clotting 
enzyme release by the 26D7, and procedures for isolating and purifying 
such enzyme, hereinafter. 
The first stage for encouraging the aforementioned strain 26D7 to produce 
soybean protein clotting enzymes is such that the strain 26D7 is cultured 
in a best preferred medium prepared in accordance with the Table 3 below. 
Or alternatively, any other kinds of media may be used, which contains a 
required nutrition sources, including nitrogen and carbon sources, insofar 
as the media per se contain nitrogen source (soybean protein) and 
phosphate. It is important that nitrogen source (soybean protein) and 
phosphate must be added in the medium in order to insure the release of 
soybean protein clotting enzymes from the bacteria 26D7, which function to 
cloth the soymilk in this instance. 
TABLE 3 
______________________________________ 
Yeast extract 0.1% 
Casamino acid 0.02% 
Ammonium sulfate 0.1% 
Phosphate 1.0% 
Sodium citrate 0.05% 
Magnesium sulfate 
0.01% 
Soymilk 5.0% 
______________________________________ 
Note: 
Adjusted to pH 6.0 by adding KOH. 
Regarding the above-listed composition of medium in Table 3, it is 
preferable that the concentration of the soymilk should srictly be set at 
5.0% as above, since experiments show that the soymilk concentration below 
or above 5.0% results in the reduced production of the soybean protein 
clotting enzyme as understandable from FIG. 1. 
The strain 26D7 was inoculated into the above-mentioned medium, and 
cultured therein in an aerobic condition by means of a conventional 
shaking-type aeration and agitation device at the temperature of 
45.degree. C. for 48-72 hours. In terms of the aeration, care must be 
taken to control the air supply amount in the medium at a moderate degree, 
avoiding an excessive larger or lower amount of air supplied, because it 
adversely affects the productivity of the soybean protein clotting enzyme. 
Preferably, the aeration and agitation should be carried out by a suitable 
rotary shaker at 200-400 rpm, keeping the air supply at 0.5-0.8 VVm. 
Under those conditions, the 26D7 was grown and soybean protein clotting 
enzymes were produced, as shown in FIG. 2. In this connection, we also 
determined the proteolytic activity of the same soybean protein clotting 
enzymes, for the sake of comparison between the soybean protein clotting 
activity and proteolytic activity of the soybean protein clotting enzymes. 
After the above-described culture of 26D7, the purification and isolation 
of the soybean protein clotting enzyme were conducted, which will be set 
forth below. 
The cultured medium (in liquid) was filtered through a kieselguhr, and a 
residual layer on the kieselguhr, which contains crude soybean protein 
clotting enzymes, was injected into a 55% saturated ammonium sulfate 
solution for precipitation purpose. Thereafter, the precipitate of crude 
soybean protein clotting enzymes was desalted by a gel filtration 
chromatography using a Sephadex G25 gel column (available from Pharmacia 
Fine Chemicals) to thereby obtain the fractions of the crude soybean 
protein clotting enzymes. 
The desalted enzyme fractions were purified chromatographically using a 
CM-cellulose column (Pharmacia Fine Chemicals). Specifically, after the 
desalted fractions were chromatographed on such cellulose powder column, 
an appropriate volume (3-5 ml) of different NaCl solutions with gradually 
different NaCl concentrations ranged from 0M to 2M were prepared so that a 
linear NaCl gradient was provided for obtaining multiple fractions in 
accordance with the NaCl linear gradient. Then, by dispensing each of such 
different NaCl solutions into the column, each eluate dropped from the 
column was received in a cell, so that plural cells of eluate were 
prepared and labelled their respective fraction numbers. Then, the eluate 
cells were measured by a spectrophotometer at 280 nm. The resultant 
absorbance of each eluate is shown by the dotted line in FIG. 3, which 
reveals the peak absorbace values at the fraction Nos. 75-100. On the 
other hand, the activity of the soybean protein clotting enzymes was 
determined at each of those eluate cells, with the result that the 
enzymatic activity ( ) was observed substantially at the fraction Nos. 
75-100 as a first peak fraction group (I), thus coinciding with the 
foregoing peak absorbance values of the same eluate, and further observed 
substantially at fraction Nos. 120-130 as a second peak fraction group 
(II), as shown in FIG. 3. 
Accordingly, it is seen that the soybean protein clotting enzymes exist at 
the fraction Nos. 75-100 as well as 120-130. 
Next, the eluate in the cells with those fraction Nos. 75-100 and 120-130 
were furthermore purified by a gel filtration chromatography using 
Sephadex G-100 gel column (Pharmacia Fine Chemicals) in order to obtain an 
isolated soybean protein clotting enzyme. Each eluate therefrom was 
renumbered its fraction number and measured its absorbance at 280 nm and 
determined the activity of the soybean protein clotting enzyme. The 
results are shown in FIG. 4, and the chromatogram of the first peak 
fraction group (I) on this Sephadex G-100 gel column represents a single 
band pattern in a sense similar to that developed by SDS electrophoresis. 
The measurement of such fraction group (I) indictates that the molecular 
weight of the isolated soybean protein clotting enzyme is approximately 
30000. 
The optimal temperature range and thermal stability for the peak acitivity 
of the above-discussed soybean protein clotting enzymes were examined, and 
the results are shown in FIGS. 5(A) and 5(B), respectively. It is observed 
that the optimal acitvity temperature for the enzymes at the first peak 
fraction group (I) is approx. 80.degree. C., whereas as for the crude 
enzymes at the second peak fraction group (II), the optimal activity 
temperature thereof is approx. 65.degree. C., as in FIG. 5(A), and that, 
in terms of the thermal stability, the finally isolated enzyme obtained 
by the Sephadex G-100 stands stable at 35.degree.-40.degree. C. for 30 
min., as in FIG. 5(B). 
The pH effect and pH stability of the soybean protein clotting enzymes at 
the first and second peak fraction groups (I) and (II) were determined, 
and the results are shown in FIGS. 6(A) and 6(B). From FIG. 6(A), it is 
noted that the optimal pH range for the enzyme activity at both peak 
fractions (I) and (II) lies below pH 6.5, while on the contrary, at the pH 
level above pH 6.5, most of the enzyme activity is terminated. The pH 
stability of the enzymes at those peak fraction groups, as in FIG. 6(B) is 
maintained at pH 4 to pH 9; in other words, at the pH ranges from pH 4 to 
pH 9, about 70% of both enzymes remains alive and active for 17 hours at 
the temperature of 4.degree. C. 
Further, the influence of metal ions and inhibitors upon the soybean 
protein clotting enzymes was examined, and as a result thereof, there was 
almost no metal ion influence on both of the soybean protein clotting 
enzymes at the first and second peak fraction groups (I) and (II), but, 
according to the inhibitor experiments, the clotting activity of the 
enzyme at the first fraction group (I) was completely inhibited by phenyl 
methyl sulfonyl fluoride (PMSF) and tosyl fluoride (TSF), whereas only by 
ethylene diamine tetra-acetic acid (EDTA), was completely inhibited the 
clotting activity of the enzyme at the second peak fraction group (II). 
This result of inhibitor experiments indicates that the first peak 
fraction group (I) contains a kind of serine protease and the second peak 
fraction group (II) contains a kind of metal protease. The details of 
those metal and inhibitor experiments are shown in the Table 4 below. 
TABLE 4 
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Relative activity (%) 
Chemicals Conc.(M) (I) (II) 
______________________________________ 
ZnSO.sub.4 7H.sub.2 O 
1 .times. 10.sup.-3 
100 100 
CuSO.sub.4 5H.sub.2 O 
" 100 93.3 
MgCl.sub.2 6H.sub.2 O 
" 94.3 96.4 
MnSO.sub.4 4H.sub.2 O 
" 100 100 
CaCl.sub.2 2H.sub.2 O 
" 91.6 100 
BaCl.sub.2 2H.sub.2 O 
" 100 100 
FeSO.sub.4 7H.sub.2 O 
" 100 96.2 
LiOH H.sub.2 O 
" 100 100 
HgCl.sub.2 " 100 90.2 
TSF 1 .times. 10.sup.-2 
0 100 
EDTA " 100 0 
PMSF " 0 100 
None 100 100 
______________________________________ 
FIGS. 7 and 8 are intended to show the optimal pH range and temperature for 
the proteolytic activity of the isolated soybean protein clotting eznyme, 
respectively. 
METHOD OF PRODUCING A SOYBEAN PROTEIN FOOD 
Now, we will describe the method of producing a cheese-like soybean protein 
food, as one of the preferred embodiments, by use of the above-stated 
soybean protein clotting enzyme. 
In accordance with the present invention, a best preferred method for 
making such chees-like food involves, at first, preparing a base material 
by admixing the undermentioned items, and sterilizing the base material by 
a heating treatment at 80.degree. C. for 30 min. 
(a) Soybean protein solution prepared by liquifying a soybean protein 
powder so that the soybean protein concentration amounts to 7.5%. 
(b) Lactose: 2% 
(c) Fat: 25% (as a solid fat) 
(d) Emulsifier: 2% against the fat 
While in the present embodiment, the soybean protein powder is utilized, it 
should be understood that a soymilk commonly on the market, or a soymilk 
for industrial use in other soybean food products, may also be utilized. 
During the heat sterilizing treatment, the soybean protein is denatured. 
Thereafter, the aforementioned base material is homogenized by means of a 
suitable homogenizer at 150 kg/cm.sup.2 at the temperature of 70.degree. 
C., and then, the hogenized base material is placed in a suitable 
thermostat or thermostatic vessel (such as a cheese fermentation vessel) 
and maintained therein at the temperature of 40.degree. C. (preferably 
under the conditions of pH 6.5-6.6 and approx. 0.2 acidity). At this step, 
2-3% lactic acid bacteria starter (pH 4.5-pH4.7 and 0.2-0.75 acidity) is 
added into the the thermostatic vessel. The base material is let stand 
under this condition for about 30 min. 
Meanwhile, a soybean protein clotting enzyme solution is prepared by adding 
0.03-0.06% enzyme powder of the previously described soybean protein 
clotting enzyme into 0.2M NaCl solution. 
When the pH and acidity conditions in the thermostatic vessel becomes at pH 
6.4-4.7 and about 0.25, respectively, the soybean protein clotting enzyme 
solution is injected into the vessel, whereupon the base material becomes 
clotted into a curd by virtue of the soymilk in the base material being 
clotted by the soybean protein clotting enzyme. 
The base material is transformed into a cheese-like curd for about 2 hr. 
and 30 min, and when it is acertained that the curd state of the base 
material reached at a proper degree, the curd is cut into a predetermined 
pieces, and each pieces of curd is left as it stands for about 30 min., 
allowing separation of a whey therefrom. 
Thereafter, the curd is mounted onto a mold device (a hoop) comprising a 
flat upper die and a lower die of a substantially top-opened cubic shape 
having plual holes perforated in its lateral walls and bottom wall, and 
then, after placing the curd in the lower die, the upper die is lowered 
down into the lower die, to thereby press the curd, forcing out water from 
the curd through the plural holes of the lower die. 
At this stage, it is important to note that variations of the pressure 
against the curd provide a number of different natures of fermented 
soybean protein food products. For example, to keep applying a pressure of 
4-5 kg/cm.sup.2 to the curd for about 10 hours at the ambient temperature 
of 12.degree.-15.degree. C. results in producing a soft cheese-like 
soybean protein food product. A hard cheese-like food product, or a 
yogurt-like food product may be made by adjustment of pressure against the 
curd, as desired. In addition, a soybean protein cream may be produced if 
the lactic acid bacteria is not added. Moreover, addition of a flavor or 
other seasoning may avoid a bitter taste inherent in this fermented food 
product, and give a more smooth taste thereto. 
While having described the present invention as above, it should be 
understood that the invention is not limited to the illustrated 
embodiments, but other replacements, modifications, or additions may be 
possible without departing from the scope and spirit of the appended 
claims for the invention. 
Accordingly, from the above description, it is to be appreciated that the 
soybean protein clotting enzymes in accordance with the present invention 
is quite effective in clotting a soymilk or other soybean proteins, and 
may find use in a great wide variety of applications for producing many 
new soybean protein food products.