JP3088886B2 - Method for manufacturing viscous substances - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a method for culturing Bacillus natto,
And a method for producing a viscous substance produced by Bacillus natto used as food or cosmetics.

[0002]

2. Description of the Related Art In general, edible natto is prepared by immersing soybeans in water, absorbing water and steaming or boiling the soybeans, wrapping them in rice straw, and leaving them at a temperature of 40 to 43 ° C. for 12 to 16 hours. This is obtained in a state where the surface of the bean grain is covered with an off-white pellicle.

[0003] Bacillus natto, a pure culture of Bacillus natto, is also used.
There is also a product made by packaging in Kyogi (wood). It is said that the viscous substance produced from the natto thus produced is a very nutritious and healthy food.

[0004] However, no cultivation method for efficiently obtaining the viscous substance produced from natto has been studied at all, and the viscous substance produced from natto has a problem of stringiness and odor, so that it is widely used. Has not been reached.

In recent years, means for obtaining only viscous substances have been considered in order to improve such problems. For example, Japanese Patent Publication No. Sho 61-30541 discloses that natto bacteria are propagated on steamed soybeans and matured. A method for selectively extracting a viscous substance from edible natto obtained by the above method is disclosed.

[0006]

However, according to the above-mentioned prior art, edible natto obtained by cultivating natto on a steamed soybean and ripening it is used as a starting material. There is a problem that only viscous substances having no problem cannot be selectively and efficiently cultured.

Further, the above-mentioned prior art does not sufficiently disclose a technique for efficiently extracting a viscous substance having a high nutritive value in a culture product of Bacillus natto and particularly having a high protease activity.

Accordingly, the present invention solves the above-mentioned problems and provides a method for culturing Bacillus natto, which can selectively and efficiently culture only viscous substances. It is an object of the present invention to provide a method for efficiently extracting a viscous substance having high protease activity.

[0009]

[0010]

Means for Solving the Problems To solve the above problems,
In the method for producing a viscous substance produced by Bacillus natto according to the present invention, a liquid medium containing sucrose as a carbon source, soybean peptone or ground soybean as a nitrogen source, and a buffer having a pH value of 6 to 8 is added. , Bacillus Natt
o), inoculated and cultured at a temperature of 30 to 40 ° C., and then centrifuged to separate a liquid phase portion. This liquid was mixed with acetone to separate and precipitate a viscous substance. The viscous substance aqueous solution to which the liquid and water were added was mixed with ethyl alcohol, and the mixture was pulverized and allowed to stand in a cool and dark place, and then a means for separating the precipitated viscous substance was adopted.

[0011]

DESCRIPTION OF THE PREFERRED EMBODIMENTS As a carbon source of a liquid medium in the present invention, sucrose, which is a disaccharide, is used. In order to investigate the optimality of sucrose as a carbon source in a liquid medium and its suitable blending amount, a `` comparison test of carbohydrates as a carbon source '' and a `` comparison test of sucrose usage amount '' were performed. The results are shown in Tables 1 and 2.

The culture conditions and the method for measuring the amount of viscous substance employed in the comparative test examples described below are as follows:
(B). (A) Culture conditions The culture solution was placed in an Erlenmeyer flask, inoculated with Bacillus natto, and then incubated at 30 ° C.
For 14 days.

(B) Method for Measuring Viscous Substance Production After the completion of the culture under the above culture conditions, the medium containing the viscous substance was centrifuged. 16 ml of the supernatant obtained by centrifugation is placed in a centrifuge tube, mixed with 32 ml of cold acetone, and then centrifuged at 3000 rpm for 10 minutes to remove the supernatant, and the precipitate remaining in the centrifuge tube is dried and weighed. It was measured.

[0014]

[Table 1]

As is apparent from the results in Table 1, sucrose is suitable as a carbon source in the liquid medium.
In a medium using the monosaccharides glucose and fructose as carbon sources (Test Examples 2 and 3), it was not possible to produce a viscous substance as efficiently as expected.

[0016]

[Table 2]

From the results in Table 2, it can be seen that the amount of sucrose in the liquid medium was 1000
It is understood that the amount is preferably 20 to 40 parts by weight, more preferably about 30 parts by weight (Test Example 6) based on parts by weight. That is, in the liquid medium to which sucrose was added at a ratio of less than 20 parts by weight (Test Example 4), it was not possible to efficiently produce a viscous substance to the expected degree, and a large amount exceeded 40 parts by weight. This is because the added liquid medium is considered to be impractical from the viewpoint of economic efficiency.

The soybean peptone used in the present invention is a partially decomposed product of soybean, that is, a seed of Glycine max Merrill, by an alkali, an acid and an enzyme, and has a higher degree of decomposition than proteose. The soybean pulverized material used in the present invention is obtained by mechanically pulverizing soybeans into fine particles so that the soybean protein is easily leached into water.

In order to examine the optimality of soybean peptone as a nitrogen source in a liquid medium and its suitable blending amount, a "nitrogen source comparison test" and a "soybean peptone usage comparison test" were performed. 3 and Table 4.

[0020]

[Table 3]

As is clear from the results in Table 3, the medium composition using a pure amino acid or a salt thereof as a nitrogen source has a low production rate of viscous substances, and the medium composition using soy peptone should be used. Is preferable for efficiently maintaining the viscous substance production.

[0022]

[Table 4]

From the results in Table 4, it can be seen that in Experimental Example 15 in which the blending amount of soy peptone was 40 parts by weight, the production amount of the viscous substance was the highest, and the preferred blending amount of soy peptone was determined by the medium. 30 to 100 parts by weight of water to be added
It is understood that the content is preferably set to 50 parts by weight.

Next, in order to examine a suitable culture temperature of the liquid medium, a "comparison test of the amount of viscous substance produced according to the culture temperature" was performed, and the results are shown in Table 5. The composition of the liquid medium used was as follows.

Medium composition: sucrose 3
0 g Soy peptone 40 g Monopotassium phosphate 2.5 g Sodium chloride 5.0 g Buffer pH 6.0 100 ml Water 900 ml

[0026]

[Table 5]

As is clear from the results in Table 5, the culture temperature is optimally 35 ° C., and it is preferable to set the temperature to 30 ° C. to 40 ° C. in order to produce a viscous substance at a high rate.

To summarize the results of the comparative tests so far,
Saccharose is used as a saccharide as a carbon source, soybean peptone is used as a nitrogen source, and a sterilized medium is inoculated with Bacillus natto and allowed to stand at 30 to 40 ° C. It can be said that culturing is preferable.

[Examples 1 to 3] A culture medium having a composition shown in Table 6 was added to a Erlenmeyer flask containing a culture medium having a predetermined composition to which a McCluben buffer having a PH value shown in the table was added. (Bacillus Natto) and inoculated at 30 ° C. for 14 days.

Then, the production amount of the viscous substance was measured in exactly the same manner as the method for measuring the production amount of the viscous substance, and the results are also shown in Table 6.

[0031]

[Table 6]

Comparative Example 1 As shown in Table 6, PH5.
Culture was carried out in exactly the same manner as in Examples 1 to 3, except that a liquid medium to which McCluben buffer of 0 was added was used. Then, the production amount of the viscous substance was measured in exactly the same manner as the method for measuring the production amount of the viscous substance, and the results are also shown in Table 6.

As is clear from the results in Table 6, in Examples 1 to 3 in which the pH of the buffer is 6 to 8 and all other predetermined conditions are satisfied, the viscosity is 1.63 to 1.81 w.
Production rate was as high as t%. However, in Comparative Example 1 using a buffer solution that did not satisfy the predetermined PH value, the production amount of the viscous substance was as low as 1.38 wt%, which was not a preferable culture method.

Next, a method for producing a viscous substance for culturing natto bacteria efficiently and separating and extracting it more efficiently will be described.

Example 4 A medium having the following composition was 2,000 m
After sterilization in a l-volume flask, inoculated with a commercially available natto, 35
Static culture was carried out for 20 days in a thermostat at ℃.

Bacillus natto culture medium composition: sucrose 30.0 g soy peptone 40.0 g monopotassium phosphate 2.5 g sodium chloride 5.0 g biotin 100 μg PH 7.0 McCluben buffer 100 ml water 900 ml The liquid medium thus obtained was added at 3500 rpm for 20 minutes.
The solids were separated under centrifugation conditions for 1 minute, and the following operation I was performed on 1000 ml of the culture solution from which the solids had been removed.

Operation I: This was slowly added to 1.5 volumes of cold acetone with stirring to precipitate a viscous substance. This viscous substance was added to 50 m of pH 7 McCluben buffer.
and 350 ml of water were added, and the mixture was allowed to stand in a cool and dark place for 12 hours while stirring appropriately to obtain about 400 ml of an aqueous solution of a viscous substance.

Operation I was performed again on the viscous substance aqueous solution, and the resulting viscous substance aqueous solution was slowly added to a three-fold amount of cold ethyl alcohol with stirring to precipitate the viscous substance. After adding cold ethyl alcohol to the precipitated viscous substance, it was ground with a mixer. This cold ethyl alcohol mixed viscous substance solution is allowed to stand in a cool and dark place for about 4 hours, separated into a viscous substance-containing precipitate layer (layer A) and a supernatant layer (layer B), and the layer B is removed to take out the precipitate. Was. The precipitate is kept at 60 ° C.
After drying for an hour, the mixture was further dried at room temperature for 24 hours to obtain 22.1 g (yield 2.21%) of a fine viscous substance. Then, this viscous substance was pulverized in a mortar to obtain a powder of viscous substance produced by Bacillus natto.

The protease titer of the thus obtained viscous substance produced by Bacillus natto was examined by the following measuring method, and the results are shown in Table 7.

(Protein titer measurement method) (a) 1 ml of a 2% casein solution adjusted to a predetermined pH was taken as a substrate in a test tube and heated in a constant temperature bath at 40 ° C. (B) Add 1 ml of a viscous substance solution dissolved at 5% (20 times) to each pH (3, 6, 9) buffer, mix well, and add
Was reacted. (C) TCA 10 minutes after adding the viscous substance solution
2 ml of (0.4 M trichloracetic acid) was added, mixed well, and left for a while. (D) The reaction solution was subjected to gravity filtration to remove a precipitate. (E) Take 0.5 ml of the filtrate into a test tube, and add 0.4 M-Na
2.5 ml of ₂ CO ₃ solution was added and mixed well. (F) Add 0.5 ml of Folin reagent and mix well.
Heated at 0 ° C. for 10 minutes. (G) The absorbance at 660 nm was measured with a spectrophotometer.

(H) As a blank test, 1 ml of the substrate solution and 2 ml of the viscous substance solution were added to the test tube in this order, mixed well, and the operations of (d) to (g) were performed. (I) The value obtained by subtracting the value of the absorbance of the blank test in (h) from the value of the absorbance obtained in (g) was used as the measured value. (J) In order to convert the value of the absorbance into the tyrosine concentration, the operations of (e) to (g) were performed while changing the tyrosine concentration to prepare a standard curve in advance.

Here, the unit of the enzyme was 1 unit of the amount of the enzyme that gave a product equivalent to 1 μg of tyrosine per minute.
In addition, this protease measurement method conforms to the enzyme activity measurement method of "Biotechnology Experiment Book" edited by Japan Society for Biotechnology.

1 unit [PU] Acid protease: pH 3.0 Neutral protease: pH 6.0 Alkaline protease: pH 9.0

[0044]

[Table 7]

From the results, it was found that the viscous substance extracted by the culture / separation / extraction method of the present invention contained a large amount of active protease.

Therefore, when this product is used as food, it is also useful as a beauty and health food, and the obtained viscous substance has a very high water retention property, and is excellent in moisture retention and feeling when used in cosmetics. It is also possible to make it into a powder form, and it is also expected to be effective in removing protein stains on the skin by blending it in packs, detergents, bath additives and the like. Further, the obtained viscous substance is expected to promote secretion of a thrombolytic enzyme based on natto kinase, prevent thrombus, and have anti-inflammatory properties.

The following is a reference formulation example. In the formulation, the viscous substance obtained by the production method of the present invention is abbreviated as viscous substance A, and the dried and powdered substance is abbreviated as viscous substance B, and the mixing ratio is all wt%. .

[0048] (Preparation method) Add (2) to (1), stir and dissolve. Next, (3),
(4) was added and dissolved, and the mixture was stirred until it became uniform to obtain natto jelly.

The obtained natto jelly was very easy to eat without feeling of stringiness and smell, and had high nutritional value.

[0050] (Preparation method) (1) to (11) were sequentially added to (12), and the mixture was stirred until it became uniform to obtain a soft drink. The obtained soft drink had a mild (rich) feeling and was easy to drink.

Formulation Example 3 Functional Food The viscous substance A was gelatinized into gelatin capsules. Alternatively, the powdery substance was formed into tablets or granules with a granulator together with vitamin C. This is protein nutrition and
It was useful as a functional food to aid digestion.

[Formulation Example 4] Lotion [Formulation Example 4] [Comparative Example] (1) Purified water 86.75 87.75 (2) Viscous substance A 1.00-(3) 1,3-butylene glycol 6 0.000 6.00 (4) Methyl paraoxybenzoate 0.10 0.10 (5) Brucine-denatured alcohol 4.00 4.00 (6) Glycerin 2.00 2.00 (7) Sodium citrate 0.100 .10 (8) Citric acid 0.05 0.05 Total 100.00 Total 100.00 (Preparation method) Add (2) to (1) and dissolve. (3) ~
Add the solution of (5), stir and make uniform. Further (6)
To (8) was added and dissolved by stirring to obtain a lotion.

The obtained lotion was examined by a sensory test conducted by 20 adult women to find out the three points of moisturizing property, protective effect and excellence in feeling of use. The total score of all the members was calculated by setting “effective” to 1 and “no effect” to 0, and the effectiveness (%) was calculated from the following formula Effectiveness (%) = (Total score / 40) × 100 The values are enclosed in parentheses and are shown in Table 8 together with the total points.

[0054]

[Table 8]

From the results shown in Table 8, it was found that the lotion of Formulation Example 2 containing the viscous substance A was far superior to the lotion of Comparative Example in all the evaluation items.

[Formulation Example 5] Skin cream Oil phase: (1) Stearic acid 3.00 (2) Salami beeswax 1.00 (3) Cetyl palmitate 3.00 (4) Glycerin trioctanoate 2.00 (5) Behenyl alcohol 1.50 (6) methylpolysiloxane 0.50 (7) isotridecyl isononanoate 5.00 (8) glyceryl monostearate 0.50 (9) polyoxyethylene tetraoleate 1.00 sorbit (60EO) (10) Polyoxyethylene cetyl ether (7EO) 1.00 (11) Propyl parahydroxybenzoate 0.05 Aqueous phase: (12) Purified water 67.20 (13) Glycerin 10.00 (14) 1,3 -Butylene glycol 2.00 (15) xanthan gum 0.10 (16) Methyl benzoate 0.15 (17) Viscous substance A 2.00 Total 100.00 (Preparation method) (1) to (11) were heated and dissolved to adjust to 75 ° C. [Oil phase] (12) (16) was heated and dissolved in the same manner and adjusted to 75 ° C. [Aqueous phase] [Aqueous phase] was added to [Oil phase] to emulsify. Thereafter, the mixture was cooled, (17) was added at 45 ° C, and cooled to 35 ° C to obtain a skin cream.

The skin cream was subjected to a sensory test by 20 women in the same manner as in the case of the lotion. As a result, no stickiness was obtained when the skin cream was applied in the same manner, and a moist and smooth feeling was obtained.

Formulation Example 6 Emulsion Aqueous phase: (1) Purified water 82.85 (2) Isopropylene glycol 5.00 (3) Glycerin 1.00 (4) Carboxyvinyl monomer 0.15 (5) Xanthan gum 0.10 (6) Methyl parahydroxybenzoate 0.15 (7) Potassium hydroxide 0.20 Oil phase: (8) Jojoba oil 0.50 (9) Squalane 1.50 (10) Isodecyl isononanoate 2.50 ( 11) Cetanol 1.50 (12) Stearic acid 0.50 (13) Glycerin monostearate 0.50 (14) Polyoxyethylene tetraoleate 1.00 Sorbit (40EO) (15) Polyoxy monostearate Ethylene 0.50 Sorbitan (20EO) (16) Propyl parahydroxybenzoate 0.05 (17) Sex material A 2.00 Total 100.00 (Preparation method) (1) to (7) warming dissolved and adjusted to 75 ° C. was ... [aqueous phase] (8) - (16) likewise dissolved by heating The temperature was adjusted to 75 ° C. [Oil phase] [Oil phase] was added to [Aqueous phase], and emulsification was performed. Thereafter, the mixture was cooled and (17) was attached thereto at 45 ° C, and cooled to 40 ° C to obtain an emulsion. When this was used, it had a similarly good feel.

[0059] (Preparation method) (1) to (7) were mixed with a powder mixer to obtain a powdery rinse-off type pack material. When used, this is mixed with an appropriate amount of water to form a paste, applied to the face, allowed to stand for 10 to 15 minutes, and then washed away with water. When this pack was used, dirt such as sebum and protein was also cleanly removed, and the skin was smooth and had a very good feel.

[Formulation Example 8] Powdered face wash (1) Viscous substance B 1.50 (2) Maltitol 40.00 (3) Sodium N-lauroyl-L-glutamate 35.00 (4) N-coconut fatty acid Hardened tallow fatty acid acyl-L-sodium glutamate 23.50 Total 100.00 (Preparation method) (1) to (4) were mixed with a powder mixer to obtain a powdery face wash.

When this product was used, dirt such as sebum and protein was removed cleanly and well, and there was little irritation to the skin. After use, the product was moist and smooth and good.

[Formulation Example 9] Bath agent (1) Sodium carbonate 30.30 (2) Sodium bicarbonate 10.00 (3) Sodium sulfate 55.00 (4) Borax 1.50 (5) Uranine 0.20 (6) Perfume 1.00 (7) Viscous substance B 2.00 Total 100.00 (Preparation method) (1) to (7) were mixed with a powder mixer to obtain a powdered bath agent.

When this product was used, the feel of the skin after bathing was moist and smooth and very good.

[0064]

As described above, the present invention inoculates Bacillus natto in a liquid medium employing a predetermined carbon source and nitrogen source,
Since the culture is performed under predetermined conditions, there is an advantage that the method can be used to selectively and efficiently culture only viscous substances. In addition, those cultured in this way have a moisturizing effect, mucosal protection effect, protein degradation effect, thickening effect,
It is an odorless viscous substance having a high protease activity and having a thrombus prevention effect and a nutritional effect. The production method of the present invention has an advantage that such a viscous substance can be efficiently extracted by a predetermined operation.

Continued on the front page (51) Int.Cl. ⁷ Identification FI (C12N 9/50 C12R 1:07) (72) Inventor Takanori Hara 7-3-2 Honcho Higashi, Yono-shi, Saitama Pref.・ In the A Food Research Institute (72) Inventor Kozo Awatani 7-3-2 Honcho Higashi, Yono City, Saitama Prefecture Limited P & A Food Research Institute (72) Inventor Isao Yamauchi, Saitama Prefecture 7-3-2, Machihigashi Limited P & A Food Research Institute (56) References JP-A-4-79879 (JP, A) JP-A-51-142558 (JP, A) JP-A-61 −239897 (JP, A) JP-A-3-47087 (JP, A) (58) Fields investigated (Int. Cl. ⁷ , DB name) WPI (DIALOG) BIOSIS (DIALOG)

Claims (1)

(57) [Claims] 1. A liquid medium containing sucrose as a carbon source, soybean peptone or soybean pulverized material as a nitrogen source, and a buffer having a pH value of 6 to 8 is added to a liquid medium containing Bacillus N.
atto), incubate at a temperature of 30 to 40 ° C., centrifuge it to separate the liquid phase, mix this liquid in acetone to separate and precipitate viscous substances, and buffer it. A method for producing a viscous substance, comprising mixing an aqueous solution of a viscous substance to which a liquid and water are added, mixing the ethyl alcohol, leaving the mixture in a cool, dark place, and then separating the precipitated viscous substance.