JPH03112483A - Bacillus licheniformis - Google Patents

Abstract

PURPOSE:To obtain Bacillus licheniformis mutant low in sore forming ability with high productivity for soybean protein coagulation enzyme useful for producing soybean protein-processed foods by pitting Bacillus licheniformis to mutation. CONSTITUTION:Bacillus licheniformis B-6-4J is put to mutation to obtain the objective mutant low in spore forming ability with high productivity for soybean protein-coagnlation enzyme. An example of the present mutant is Bacillus licheniformis 26D-7 (FERM P1778). The present mutant is incubated in a medium, and using a soybean protein-cagulation enzyme collected from the cultured product, soybean milk is coagulated to obtain a soybean processed food. For example, lactic bacteria and soybean protein coagulation enzyme are added to a mixture of soybean milk, lactose, fatty oil and emulsifier to effect fermentation, and whey is removed from the fermented product followed by pressing, thus obtaining a food similar to cheese.

Description

[Detailed description of the invention] [Industrial application field]

The present invention relates to Bacillus licheniformis which produces soybean protein coagulase.

[Technical background]

Foods are made by coagulating animal proteins with coagulating enzymes, just as cheese is made from milk. It has been well known for a long time. On the other hand, in recent years, for health and economic reasons, there has been a desire to produce processed soybean foods by coagulating soybean protein using inexpensive and abundant soybeans.

[Prior art]

In view of this background, the present inventor discovered and isolated Bacillus licheniformis, which produces soybean protein coagulase extracellularly, among bacteria existing in the natural world. The strain was named rB-6-4JJ and submitted to the Institute of Microbial Technology, Agency of Industrial Science and Technology, as part of the 8th Microbiological Research Institute.
No. 626 (FERMP-8626), and a patent application has been filed for a soybean protein coagulase produced using a Mycobacterium strain and a method for producing the same (Japanese Patent Laid-Open No. 62
(Refer to Publication No.-179386). The obtained soybean protein coagulase has no bitter taste and is suitable as a food material.

[Problem to be solved by the invention]

However, the above Bacillus licheniformis B-6-4J
(Feikoken Bibori No. 8626) produces soybean protein coagulase, but the amount is not very large, and it is necessary to industrially produce large quantities of soybean processed foods by coagulating soybean protein. It was difficult to obtain a sufficient amount of soybean protein coagulase. In addition, this Bacillus licheniformis B-6-4J has a high spore formation frequency, and when the produced soybean protein coagulase is separated from the bacterial cells and extracted, spores are likely to be mixed in. There is a problem in that 6-45 acts on soybean protein together with the enzyme, imparting an unintended taste to the finished soybean protein food. The present invention was made in order to solve the above problems, and its purpose is to produce a large amount of soybean protein coagulase and to develop a new method that allows only the enzyme to be easily separated and extracted from bacterial cells. To provide Bacillus licheniformis.

[Means to solve the problem]

The present invention relates to Bacillus licheniformis, and its first invention resides in Bacillus licheniformis, which has a low spore-forming ability and a high ability to produce soybean protein coagulase. In addition, the second invention of the present invention is Bacillus licheniformis, which has a high production ability of soybean protein coagulase.
It's in the issue. Therefore, Bacillus licheniformis according to the present invention is a mutant strain whose parent strain is Bacillus licheniformis B-6-4J. This means that the formation frequency is 11,500 times or less compared to the parent strain Bacillus licheniformis, and the production ability of soybean protein coagulase is high. 5 times or more.

[Actions and effects of the invention]

Since the Bacillus licheniformis according to the present invention has extremely low spore-forming ability and high production ability of soybean protein coagulase, the enzyme is separated from the bacterial cells from the soybean protein coagulase production system and spores are mixed in. The enzyme can be extracted in large quantities without any bitterness, and the resulting enzyme forms a soybean protein coagulate with no bitterness and a smooth texture. Therefore, according to Bacillus licheniformis according to the present invention, it is possible to obtain a large amount of soybean protein coagulating enzyme suitable for producing processed soybean protein foods.

【Example】

Bacillus licheniformis according to the present invention is a mutant strain obtained through intermediate mutants of the above-mentioned Bacillus licheniformis B-6-4J (wild strain), and these strains are isolated, produced, cultured, and mutated. , conducted experiments on their usefulness, etc., and measured their characteristics. a. Screening for soybean protein coagulase producing bacteria (al)
Isolation Source: Samples were widely collected from plants, soil, and excrement from livestock such as pigs and cows at the National Institute of Genetics in Mishima City, Shizuoka Prefecture, and at the foot of the mountains in Hakone. (al) Separation method (soybean protein coagulase) Each collected material (about the size of an earpick made by crushing plants, soil, excrement, etc.) was placed into test tubes each containing 3 m7 of sterilized soy milk. Put it in for 12-16 hours, 3
Culture the soybean milk with shaking at 7°C (primary screening), measure the p II of the soymilk in the test tube that has formed a curd (including the coagulated product), and find the soybean milk with a pH of 5.5 or higher. The same screening as above was repeated for in vitro materials. In addition, p from the solidified sample
The reason for excluding those with less than H5.5 is to exclude materials formed into cards by acid. In order to purify the target microorganism, screening using soy milk was performed up to the fifth stage. Through such screening up to the fifth stage, it was confirmed that microorganisms capable of coagulating soymilk could be transferred from curd-formed soymilk to sterilized soymilk. As a result, ■ Among the materials screened above,
It was confirmed that there is a microorganism capable of coagulating soybean protein, and that this microorganism is breeding in soymilk. Thereafter, the microorganisms obtained through the 5th screening were grown in a growth medium adjusted to pH 6.0 with the composition shown in Table 1 below.
The cells were subcultured by shaking culture at 37° C. for 12 to 16 hours. 1 Ammonium sulfate 0.1% phosphate
1.0% magnesium sulfate
0.01% Sodium Citrate 0
．． 05% Casamino Acid 0.02% Yeast Extract 0.1% Glucose
Purification of 0.5%b soybean protein coagulase-producing bacteria A strain with the highest soybean protein coagulation ability was obtained as a single strain by plate dilution from samples subjected to the fifth screening. This strain was named rB-6-4JJ and was deposited at the Institute of Microbial Technology, Agency of Industrial Science and Technology. The deposit number is Tetsukoken Bacillus No. 8626 (FERM P-8626)
It is. This bacterial strain was concentrated in bamboo leaves and roots. Next, in the obtained bacterial strain (B-6-4J), it was confirmed whether a substance having soybean protein coagulation ability was present inside or outside the bacterial cell. First, this strain was incubated for 24 hours in a production medium adjusted to pH 6.0 with the composition shown in Table 2 above.
Culture was carried out with shaking at 37°C. Ammonium sulfate Potassium dihydrogen phosphate Magnesium sulfate Sodium citrate Casamino acid Yeast extract Glucose 11.1% Calcium chloride solution 0.1% 1.0% 0.01% 0.05% 0.02% 0.1% 0 .5% 0.01% Soy milk (soy protein concentration 10%) 5% The thus cultured product was incubated at 0°C and 6000 rp.
The cells were centrifuged for 10 minutes at m, and the supernatant and bacterial cells were separated and their respective activities were examined. Regarding the supernatant liquid, the supernatant liquid was passed through a filter with an average pore diameter of 0.22 μm to remove bacteria, and the soybean milk coagulation activity of the supernatant liquid was examined. Regarding the bacterial cells, the bacterial cells were thoroughly ground with quartz sand in a milk pestle, a phosphate buffer solution of pH 7.0 was added thereto, and the bacterial cells obtained were filtered through a 0.22 μm filter in the same manner as above. The soy milk coagulation activity of substances contained in the body extract was investigated. In this case, the purpose of adding a phosphate buffer is to restore the pH that has changed due to the crushing of the bacterial cells and eliminate the effects of acid on soymilk coagulation. The purpose of passing it through a filter is to remove unnecessary components. As a result, it was confirmed that the supernatant liquid had an activity to coagulate soymilk, and the bacterial cell extract had no activity to coagulate soymilk. Furthermore, as a result of heating the supernatant at 80° C. for 30 minutes and examining whether the heated supernatant had soymilk coagulation activity, the activity was found to be absent. was confirmed to disappear. Through these experiments, it was confirmed that the substance having soymilk coagulation activity was not an inorganic substance but an extracellular enzyme produced by the microorganism. Study of culture conditions to increase C0 enzyme productivity (cl) This strain (B-6-4J) was cultured under various conditions to study culture conditions to increase soybean protein coagulating enzyme productivity. In such cases, the basic medium having the composition shown in Table 2 above was used. This medium composition contains carbon sources, nitrogen sources, nutrient sources, etc. that can be assimilated (the bacterial strain can inoculate the medium as nutrients and grow in the medium), and secretes soybean protein coagulase. Any medium may be used as long as it can be used. As a result of the above experiment, we learned the following regarding the culture conditions for the strain that increases the production of soybean protein coagulase. For convenience of explanation, a medium having the composition shown in Table 2 above will be used as an example. ■Inorganic salts ((Nl14hsOa, KI12PO4,
Mg5Oa.7+120), sodium citrate, glucose, yeast extract, and casamino acids are used to proliferate bacterial cells, and are not very relevant to increasing the productivity of soybean protein coagulase. In other words, these compounds have little to do with bringing out the soybean protein coagulation activity. ■Whether or not to add soy milk (protein concentration = 10%) has a large effect on the ability to produce soy protein coagulase. Figure 1 shows the relationship between the amount of soymilk added to the production medium shown in Table 2 and soybean protein coagulation activity.
These are the results obtained when shaking culture was carried out for 4 hours, and the coagulation activity was calculated based on the definition described below. According to Figure 1, adding about 2.5 to 7.5% of soy milk (10% soy protein) to the medium can effectively bring out the soybean protein coagulation activity of bacterial cells, and that adding soy milk (10% soy protein) to the medium at 5% It can be seen that the soybean protein coagulation activity of the bacterial cells can be most effectively brought out by adding the soybean protein at a certain level. Furthermore, in this case, it is thought that soymilk acts as an inducer for this strain to increase its ability to produce soybean protein coagulase. ■Figure 2 shows the number of bacteria (colony formiB
The relationship between unit/+j) and soybean protein coagulation activity is shown. According to this result, the culture time is required to be 20 hours or more, and it can be seen that the coagulation activity remains until 50 hours. In addition, as a result of performing shaking culture under the above culture conditions by changing the temperature at the optimum time and changing the pH at the optimum time and temperature, it was found that the appropriate culture temperature is around 45°C, and the culture medium The pH was stable at 6-7. (C2) Definition of soybean protein coagulation activity SCA The reference time for adding a culture solution to soybean milk (protein concentration: 10%) and forming a curd at 65°C is set to 1 hour, and it is calculated using the following formula. S CA = Soymilk Clotting
Activity (unit) = (S/E) x (
3600/l) S: ffi of substrate (soy milk) (m)) E: Volume of supernatant after centrifuging the culture solution and removing bacterial cells (m)
) 2 Time for card formation (seconds) Improvement of bacterial cells to increase the amount of d1-secreting enzyme (obtaining mutant strains) Increasing the amount of extracellular enzyme secreted by the strain (B-6-4J) For this purpose, mutant strains were obtained using the following method. After culturing this strain with shaking at 45°C and pH 6.0 using a commercially available nutrient growth medium shown in Table 3, only the bacterial cells were collected by centrifugation, and in order to keep the collected bacterial cells alive for a long time, they were placed in a bottle. The same bacterial cells were suspended in phosphate buffer and used in the following experiment. 3゜Antibiotic Meat extract Yeast extract Peptone dextrose Sodium chloride Dipotassium hydrogen phosphate Potassium dihydrogen phosphate 1 2 (Product name manufactured by Difco) 0615% 0.15% 0.5% 0.1% 0.35% 0.388% 0.132% Mediuen (dl) Ultraviolet (UV) treatment The suspended bacterial cells obtained by the above treatment were diluted 100 times, and vegetative growth was carried out using the composition shown in Table 3 as a medium. It was smeared onto a flat plate and irradiated with ultraviolet light under various conditions. After such irradiation, the cells were cultured on the plate at 37° C. for 1 day and night. After the culture, mutant strains with low spore-forming ability were extracted, and nine mutant strains were obtained. The obtained mutant strain was examined for soybean protein coagulation activity, but no strain was found that had soybean protein coagulation activity. (d2) γ-ray irradiation treatment The same strain diluted 100 times above was irradiated with γ-rays to 20%, then the bacterial cells were plated on a vegetative growth plate using a medium with the composition shown in Table 3, and kept at 45°C for 3 days. Cultured. We searched for strains with lower spore-forming ability than the strains grown on the plates, but none were found. Furthermore, no strain was found that had soybean protein coagulation activity. (d3) Nitrosoguanidine (NTG) treatment NTG5m
z is phosphoric acid II! It was dissolved in 4.5 d of IWJ solution, 0.5 mt of the 100-fold diluted bacterial cell solution was added to this solution, and the mixture was stirred at 37°C for 60 minutes. Next, the cells were washed three times with phosphate buffer, then spread on the above-mentioned vegetative growth plate, and cultured at 45° C. for 1 day. As a result, 22 strains that seemed to have low spore-forming ability were obtained. Among these 22 strains, 3 strains having soybean protein coagulation activity were found. Among these, the strain with the highest coagulation activity was designated intermediate mutant strain 26D, and was used for subsequent experiments. The properties of this intermediate mutant strain 26D relative to the wild strain B-6-4J are described below. ■ Spore formation frequency 1/1000 to 1/10000 times that of wild strain B-6-4J ■ Comparison of soybean protein coagulation activity Approximately twice that of wild strain These properties are higher than when culturing wild strain B-4-6J. , observed under culture conditions with low aeration. Note that other culture conditions are the same as those for strains B-6 and -4J described in (cl) above. In addition, the spore formation frequency was calculated by treating and measuring the bacterial strain using the method described below. a: Transfer the colonies of the bacterial strain one by one to the plate. Make two of these same plates. b: A petri dish with a full drop of chloroform at the bottom.
Place one of the plates above on the lid, and place the other plate on the lid of the petri dish to which chloroform has not been added (
control), the strains on these plates at 45°C,
Incubate for 16 hours. Strains with weak spore-forming ability or strains that form few spores will not grow due to the influence of chloroform. C: The coagulation activity of the control strain corresponding to the non-growing strain (chloroform side) was examined, and the strain with coagulation activity was grown in the nutrient growth medium shown in Table 3. d: Heat-treat the bacterial cells at 80°C for 15 minutes, measure the number of bacteria before and after the heat treatment, and calculate the spore formation frequency using the following formula. Spore formation frequency = (Number of bacteria after heat treatment) / (Number of bacteria before heat treatment) When 100% of the proliferated bacteria form spores, the spore formation frequency is 1. (d4) NTG treatment of intermediate mutant strain 26D intermediate mutant strain 26
In order to further obtain mutant strains from D., the following treatments were performed. (2) Intermediate mutant strain 26D was cultured overnight at 45°C in 3 m of the nutrient growth medium shown in Table 3. ■ Add 0.1 mJ2 of the culture solution from ■ above to 3 m of the above nutrient growth medium.
g and cultured with shaking at 45°C until log phase. The reason for culturing until the logarithmic growth phase is that strains in this growth phase have the strongest tendency to mutate. ■ Dispense the culture solution from ■ above into 5 test tubes at 0.5n+/each, and add 11g/af of NTG to each tube at 0.0.
2.5. 5.0°10.0. Add 25.0 μl each. As a result, the NTGm degree in each test tube is 0. 5. 10. It becomes 20.50μg/mノ. (2) Incubate each test tube in (1) above at 45°C for 30 minutes. (2) After the treatment in (2) above, the bacterial cells in each test tube are washed three times with 1+ne of the above nutrient growth medium. ■After the treatment in (■) above, 0.5 ml of the above nutrient growth medium was added to each bacterial cell and Jl! TI becomes cloudy. (2) Add 30μ of each of the suspensions in (2) to 3111 of the above nutrient growth medium, and culture with shaking at 45°C overnight. After the treatment in (2) above, each culture solution was stored at -80°C and used for subsequent experiments. Next, although bacteria are normally sensitive to kanamycin (antibiotic 'Ii') (the intermediate mutant strain 26D obtained this time is also sensitive to kanamycin), the bacteria obtained by mutation are sensitive to kanamycin. In view of the fact that the strain may become resistant to
NoIII/! The cells were plated on vegetative growth plates containing kanamycin, and the NTG 1 group with a high frequency of mutations was selected. As a result, when the NTG21 degree was 20 Pg/m, the mute frequency was the highest, and the frequency was 3.52 x 10-3. NTG concentration 20
μg/ml group was added to the medium shown in Table 1 above with 0.5 μg/ml of protease activity measuring reagent (Hyde Powder brand name manufactured by Sigma).
% and agar to form a plate medium, and collect the strains that form a halo. The number of bacteria was 1.3xlO5, and one of the target bacterial strains (the strain with the highest protein coagulation activity) was obtained. This strain was mutated into strain 26.
It was named D=7 and internationally deposited with the Institute of Microbial Technology, Agency of Industrial Science and Technology. The deposit number is FE-KENJO deposit No. 1778 (FE
RM BP-1778). Table 4 below shows a comparison of the sporulation frequency and soybean protein coagulation activity of the wild strain (B-6-4J), intermediate mutant strain (26D), and mutant strain 26D-7. (Left below) Table 4 Mycological properties of soybean e, B-6-4J, and 26D-7 Both strains B-6-4 were tested using the B-2 medium shown in Table 5 above.
J, 26D-7 was cultured and rThe Genu
sBacillus J and rBergey's Man
ual of Determinat. Their mycological properties were investigated based on ive Bact, eriologyJ. (Leaving space below) Table 5゜゜゜Two-fork゜L-fun゜゜In addition, when using a medium with the above composition as a plate, 1.5%
Add agar powder. Table 5 below shows the mycological properties of B-6-4J and 26D-7. (Leaving space below) Table 6゜Seedling 1 Grape 1 Quality Based on the mycological properties shown in Table 6, both wild strain B-6-4J and mutant strain 26D-7 are considered to belong to the genus Bacillus, and furthermore, Within the genus, both images are related to Bacillus liceniformis because it forms colonies that adhere tightly to agar, has a high growth temperature, and grows under anaerobic conditions.
heniformis) species. Table 4 shows the soybean protein coagulase production ability and sporulation frequency of Kokura's wild strain B = 6-4 J, intermediate mutant 26D, and mutant 26D-7. Furthermore, in the process of the NTG treatment, wild strain B-6-
It was also confirmed that 4J and intermediate mutant strain 26D are sensitive to kanamycin as an antibiotic, but mutant strain 26D-7 is resistant to kanamycin. f. Isolation and Purification of Soybean Protein Coagulase 2 was sterilized by putting 1.51 volume of medium (medium of formula 2 above) into a jar fermenter with a capacity of 2, and then the mutant strain 26D was sterilized.
-7 was planted and cultured with aeration at 45°C for 48 hours. The culture solution was filtered through diatom ± (SSC) to remove bacterial cells, and then subjected to ammonium sulfate fractionation using 55% saturated ammonium sulfate.
adexG-100 (trade name) was used to desalt. The culture solution after 1'l Ii; The elution pattern of the culture solution in this case (absorbance at a wavelength of 280I measured by a spectrophotometric needle) is shown in broken lines in Figure 3, and
The solid line indicates the coagulation activity of soybean protein coagulase. Among these fractions, soybean protein coagulation activity was observed in the enzyme corresponding to beak ■ and the enzyme corresponding to beak H shown by the solid line in FIG. Furthermore, Beak 2, in which a large amount of enzyme was eluted, was purified into a single substance using the column of the gel filtration applicable packing material. FIG. 4 shows the elution pattern of this purified product by the above-mentioned sodium chloride linear concentration gradient method, but in this case as well, in the elution pattern like the broken line, the enzyme shown by the solid line has soybean protein. Coagulation activation ability was observed. As a result, the desired soybean protein coagulase is isolated and purified. g. Properties of soybean protein 111-binding enzyme The physicochemical properties of the enzymes corresponding to peaks I and II (hereinafter simply referred to as enzyme peaks I and II) were confirmed as follows as a result of various experiments. ■Both Enzyme Beak I and II coagulate soy protein when they act on soy milk. ■ Enzyme peaks I and ■ both have no bitterness and are not toxic and are suitable for food processing. ■Enzyme peak ■ is a single band in 5DSt aerophoresis, and the molecular weight is around 30,000. Since the production amount of enzyme beak (■) is small, the above measurement is not possible. ■The graph in Figure 5 shows the relationship between soybean protein coagulation activity of the enzyme and temperature measured under the following conditions (1), and the graph in Figure 6
F is the thermostability of the enzyme measured under the following conditions (II),
The graph in FIG. 7 shows the relationship between coagulation activity of the enzyme and pH measured under the following condition (III), and the graph in FIG. 8 shows the pH stability of the enzyme measured under the following condition (rV). From the results of these graphs, the optimal temperature that shows coagulation activity is the enzyme peak! The temperature is about 80℃ for the enzyme peak, and about 65℃ for the enzyme peak
It is stable for 1 hour at ℃. Furthermore, both of these enzymes show high coagulation activity on the acidic side, and almost lose their coagulation activity at pH > 7, but at 4°C, both enzymes are almost stable at pH 4 to 9, with at least 70% activity remains. Conditions (■): 200 μm each of enzyme peaks ■ and ■ at 180 μm
Add each to 0 μl of soy milk, adjust the pH to 6.5, and measure coagulation activity at each temperature. Condition (■): After holding the enzyme peaks r and rr at each temperature for 1 hour, the enzyme peak ■ is at 80°C and the enzyme peak ■ is at 65°C, and other conditions are the same as condition (1). Measures coagulation activity. Condition (■): The coagulation activity was measured in the same manner as in condition (1) except that the soymilk was adjusted to each pH and the coagulation activity measurement temperature was 80°C to 65°C. Condition (■): Enzyme peak! , ■ were adjusted to each pH and refrigerated at 4°C for 161 I4J, and coagulation activity was measured in the same manner as in condition (1) except that the coagulation activity measurement temperature was 80°C and 65°C. ■Table 7 shows the influence of each compound on the soybean protein coagulation activity of the enzyme measured under the following conditions.As shown in the table, the enzyme peak! In both cases, no influence by metal ions was observed. Enzyme peak ■ is PMSF (phenylmethylsulfonyl fluoride) and TSF ()
The enzyme peak (■) was 100% inhibited by EDTA (ethylenediaminetetra-acid). With these,
The enzyme peak ■ is considered to be a type of serine protease.
The enzyme peak ■ is considered to be a type of metalloenzyme. Table 7: Influence of each compound on soybean protein coagulase and usefulness of the enzyme By coagulating soymilk using the enzyme corresponding to the above peak I, processed soybean food can be produced. For example, soy milk, lactose,
A solid substance is obtained by adding lactic acid bacteria and the enzyme to a mixture of fats and oils and an emulsifier, fermenting the mixture, removing whey from the fermented product, and releasing water by pressing. In such cases, lactic acid bacteria are added to eliminate the odor and taste peculiar to soybeans. When this solid material is sterilized by adding water, a stabilizer, a taste for cheese, a flavor for cheese, etc., a dairy product similar to cheese is produced. In this case, hard type cheese, cream type cheese, etc. can be produced by varying the amounts of water and stabilizer added. Moreover, by adding a taste and flavor suitable for other foods in place of the taste and flavor for cheese, processed soybean foods different from cheese can also be produced. Foods produced in this way are low in cholesterol and are excellent as health foods. In addition, soybeans are abundant as raw materials, and processed soybean foods similar to dairy products can be provided at low cost.

[Brief explanation of drawings]

Figure 1 is a graph showing the relationship between the soybean protein coagulation activity of the enzyme produced by the wild strain of Bacillus keniformis and the amount of soymilk added. Figure 2 is a graph showing the relationship between culture time, number of bacteria, and coagulation activity. The figure shows the elution pattern of the enzyme produced by Bacillus licheniformis according to the present invention, Figure 4 shows the re-elution pattern of the enzyme corresponding to peak I in Figure 3, and Figure 5 shows the peaks I and II in Figure 3. A graph showing the relationship between the soybean protein coagulation activity and temperature of the enzyme corresponding to , FIG. 6 is a graph showing the stability of the same enzyme with respect to temperature, and FIG. 7 is a graph showing the relationship between the coagulation activity of the same enzyme and PH. FIG. 8 is a graph showing the stability of the same enzyme against pH.

Claims (2)

[Claims] (1) Bacillus licheniformis has low spore-forming ability and high soybean protein coagulase production ability. (2) Bacillus licheniformis Microtechnical Research Institute No. 1778, which has a high production capacity for soybean protein coagulase.