JP3358957B2 - Method for producing acetic acid bacteria powder - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a method for producing acetic acid bacteria powder which can be stored for a long period of time, and more particularly to a method for producing acetic acid bacteria powder which can effectively suppress a decrease in the activity of aldehyde oxidase during storage. It relates to a manufacturing method.

[0002]

2. Description of the Related Art Various techniques for applying acetic acid bacteria to foods have been developed, and the present inventors have developed some techniques and applied for patents. For example, a method for reducing the unpleasant odor of alcoholic beverages by heating the acetic acid bacteria at 60 ° C. for 15 minutes to deactivate the membrane-bound alcohol dehydrogenase, and then contacting the acetic acid bacteria with alcoholic beverages containing a medium-chain aldehyde ( Japanese Patent Publication No. 8-22220), a method for preventing the deterioration of the quality of oil-containing food by contacting the oil-containing food with acetic acid bacteria (Patent No. 206)
No. 5994), in a method for producing a retort food in which cooked rice or noodles are filled and sealed in a heat-resistant container, and then subjected to pressurization and heat sterilization, the rice or noodles are contacted with acetic acid bacteria prior to the pressurization and heat sterilization. (Japanese Patent No. 2521519).

[0003] In order to utilize acetic acid bacteria in such various foods, it is desirable to powder the acetic acid bacteria in terms of simplicity of handling and weighing operation.

[0004]

Accordingly, the present inventors have focused on freeze-drying, which is commonly used, as a method for pulverizing acetic acid bacteria. When the acetic acid bacteria powder was prepared by freeze-drying according to the method and stored, a phenomenon in which the activity of the aldehyde oxidase was reduced in a short time was encountered.

In order to prevent such a phenomenon, as a result of research, it has been found that acetic acid bacteria are dispersed in a buffer having a pH of 5 to 7, a saccharide is added and dissolved in the buffer, and then lyophilized. It was found that an acetic acid bacterium powder retaining the activity of aldehyde oxidase for a long time can be obtained.

[0006] The present invention has been developed based on the above findings, and it is an object of the present invention to provide a method for producing acetic acid bacterium powder capable of effectively suppressing a decrease in the activity of aldehyde oxidase during storage.

[0007]

According to a first aspect of the present invention, there is provided:
A wet acetic acid bacterium is dispersed in a buffer solution containing saccharides at a pH of 5 to 7 and then freeze-dried. In a second aspect of the present invention, the saccharide is sucrose,
The method for producing acetic acid bacteria powder according to the first aspect, which is selected from disaccharides such as lactose and maltose, and monosaccharides such as glucose, galactose and fructose. The aspect of the present invention is the method for producing acetic acid bacteria powder according to the first aspect, wherein the sugar concentration in the buffer solution is a concentration corresponding to 10 to 30% by weight based on the wet cells.

[0008]

DETAILED DESCRIPTION OF THE INVENTION In the present invention, acetic acid bacteria are first prepared. The acetic acid bacteria may be dry cells or wet cells, but it is preferable to use wet cells from the viewpoint of the stability of the cells. The following method can be exemplified as a method for obtaining wet cells.

First, acetic acid bacteria are cultured and grown. Usually, the main culture is performed after the preculture. To give specific examples of pre-culture, as a medium, yeast extract, polypeptone, glycerol, potato extract,
Glucose can be exemplified, and the culture conditions can be exemplified by 25 to 35 ° C. for 24 to 32 hours. As a specific example of the main culture, the medium can be exemplified by yeast extract, polypeptone, glucose and ethanol, and the culture condition can be exemplified by 25 to 35 ° C. for 24 to 32 hours. .

[0010] If the temperature is lower than the above conditions, the growth is slow and the culture takes too much time, which is not preferable. On the contrary, if the temperature is higher than the above conditions, the bacteria are easily killed. After the culture, the culture is centrifuged, and the collected bacterial cells are dispersed in water and stirred to remove impurities such as medium components. Thereafter, the cells are collected by centrifugation again, dispersed in water again, stirred, and centrifuged. These steps are repeated several times to collect the washed cells. Centrifugation conditions are 7,000-10,000 rpm at 10-20.
Minutes can be exemplified.

The cells thus obtained are dispersed in a buffer. The cells are collected by centrifugation again. This step is repeated several times to obtain wet cells substituted with the buffer. Examples of the buffer include McClubine buffer using citric acid and Na ₂ HPO ₄ as food additives. In this case, it is preferable to use a buffer having a pH of 5 to 7 in order to achieve the object of the present invention.

Next, in the present invention, the cells are dispersed in a buffer solution. In this case, the buffer to be used may be the buffer exemplified above, but it is necessary to use a buffer having a pH of 5 to 7 and to dissolve saccharides in the buffer to achieve the object of the present invention. Important from. When a buffer solution outside the above pH conditions is used, it is difficult to effectively suppress the decrease in the activity of the aldehyde oxidase during storage of the obtained acetic acid bacteria powder.

The saccharides to be added and dissolved in the buffer include disaccharides such as sucrose, lactose and maltose, glucose, and the like.
There are monosaccharides such as galactose and fructose, and one or more of these can be used. Among these saccharides, it is most preferable to use sucrose and lactose in order to achieve the object of the present invention.

The amount of saccharide added is preferably such that the saccharide concentration of the buffer solution is equivalent to 10 to 30% by weight of the wet cells, and the saccharide concentration is 10% by weight or less. When it comes, it tends to be difficult to maintain the aldehyde oxidase activity. Conversely, when the sugar concentration becomes 30% by weight or more, there is a tendency that handling of freeze-dried acetic acid bacteria powder becomes slightly problematic.

The acetic acid bacteria dispersed in the buffer are then freeze-dried to obtain acetic acid bacteria powder. Freeze-drying may be performed according to a conventional method, and is not particularly limited.

[0016]

【Example】

Example 1 Tap water 1 to 20 g of sliced potato
After adding 00 ml, the mixture was autoclaved at 120 ° C. for 10 minutes and allowed to cool. Immediately, centrifugation is performed at 10,000 rpm for 20 minutes to remove solids and obtain a supernatant. 1 g of yeast extract, 1 g of polypeptone, 2 g of glycerol and 0.5 g of glucose were added to the total amount of the supernatant, and 10 g of tap water was added.
0 ml was placed in a 500 ml Sakaguchi flask,
Inoculate Acetobacter Aceti IFO 3284 and 3
Pre-culture was performed for 30 hours while shaking at 0 ° C.

Next, 2.5 g of yeast extract, 1 g of polypeptone, 15 g of glucose and 15 ml of 3% ethanol are added, the pH is adjusted to 6.5 with 1N NaOH, and the total volume is adjusted to 500 ml with tap water. It was placed in a 2 liter Erlenmeyer flask, and further cultured for 30 hours while shaking at 30 ° C. Therefore, the pH of the obtained culture solution was 3.1. Thereafter, the culture was centrifuged at 7,000 rpm for 10 minutes to collect the cells, washed well with cold distilled water, and centrifuged at 10,000 rpm for 10 minutes to obtain 4 g of washed cells.

Next, the cells were washed with a 1/2 concentration of McClubane's buffer (pH 6.0).
The operation of centrifugation at pm for 10 minutes is repeated 3 times to obtain 4 g
Was collected. Seven samples of wet cells are prepared by the same method as described above.

Then, seven 20 ml (pH 6.0) solutions of Mackulbain buffer having a concentration of 1/2 were prepared, and the sugar concentration of the buffer solution was adjusted to 10 with respect to the wet bacterial cells. The monosaccharides and disaccharides shown in FIG. Separately, McClubane's buffer without added saccharide was prepared. Thereafter, each of the McClubane's buffers is added to the above 7 samples of wet cells and uniformly suspended. Immediately thereafter, the mixture was immediately poured into a freeze-drying tray and frozen at -45 ° C.
The solution was freeze-dried at 16 ° C. for 16 hours to obtain 0.2 g each of 7 samples of acetic acid bacteria powder.

The acetic acid bacterium activity in the acetic acid bacteria powder was measured after storing the thus obtained seven samples of the acetic acid bacteria powder at 30 ° C. for one week. FIG. 1 shows the measurement results. As is clear from FIG. 1, the use of the buffer solution to which the monosaccharide and disaccharide sugars were added was superior in the aldehyde oxidase activity as compared with the case where the buffer solution to which the sugars were not added was used.

The aldehyde oxidase activity was determined by measuring the oxygen consumption rate at 25 ° C. using a dissolved oxygen meter (Horiba Seisakusho). The initial rate of oxygen consumption was determined and defined as one unit when the amount of oxygen consumed per minute corresponded to 1 μmol.

As a reaction system, 0.5 ml of a bacterial solution prepared by adding 5 ml of distilled water to 10 mg of acetic acid bacteria powder, and 4.3 ml of McClubane's buffer (pH 5.0) and 120 μmol of acetaldehyde were added to the total amount. 6.0
The enzyme reaction was performed in ml. The reaction was started by adding an acetaldehyde solution.

[0023]

Example 2 Six samples of wet cells were prepared by treating in the same manner as in Example 1. Next, six 20 ml (1/2) concentrations of McClubane's buffer (6 ml) were prepared, and the sugar concentration of the buffer was added to the 6 McClubane's buffers.
Sucrose was added to each of 0 wt%, 1 wt%, 5 wt%, 10 wt%, 20 wt%, and 30 wt% based on the wet cells. Thereafter, each of the McClubane's buffers is added to the above six samples of wet cells and uniformly suspended. Thereafter, freeze-drying was performed in the same manner as in Example 1 to obtain six samples of acetic acid bacteria powder.

The thus obtained six samples of acetic acid bacteria powder were stored at 5 ° C. for 9 weeks, and the time-dependent changes in aldehyde oxidase activity in the acetic acid bacteria powder were measured. FIG. 2 shows the measurement results. As is clear from FIG. 2, the sugar concentration of the buffer was
By using a buffer to which sucrose was added so as to be 10 to 30% by weight with respect to the wet cells, the aldehyde oxidase activity could be maintained for a long time.

[0025]

Example 4 A culture solution obtained by pre-culture and main culture in the same manner as in Example 1 was centrifuged to collect cells. Thereafter, the culture was centrifuged at 7,000 rpm for 10 minutes to collect the cells, washed well with cold distilled water, and washed at 10,000 rpm.
centrifugation at 10 m for 10 minutes to obtain 4 g of washed cells.

Next, the cells were washed with a 1/2 concentration of McClubane's buffer (pH 6.0), and then washed at 15,000 r.
The cells were collected by centrifugation at pm for 10 minutes. After this operation was repeated three times, 20 ml of a 1/2 concentration of McClubane's buffer (pH 6.0) containing lactose so as to be 30% by weight based on the wet cells was added to the wet cells and homogenized. Suspended in Immediately, the mixture was poured into a freeze-drying tray and frozen at -45 ° C, and then freeze-dried at a shelf temperature of 20 ° C for 16 hours to obtain about 0.2 g of acetic acid bacteria powder.

The acetic acid bacterium powder thus obtained was stored at 5 ° C., and the aldehyde oxidase activity was measured.
Even after storage for a week, the enzyme activity was sufficiently maintained.

[0028]

According to the present invention, even when the acetic acid bacteria powder obtained by freeze-drying is stored for a long period of time, it is possible to effectively suppress the decrease in the activity of aldehyde oxidase in the acetic acid bacteria powder. Can be. As a result, the use of acetic acid bacteria can be greatly expanded.

[0029]

[Brief description of the drawings]

FIG. 1 shows the aldehyde oxidase activity in acetic acid bacteria powder after storing the acetic acid bacteria powder obtained by the method of Example 1 at 30 ° C. for one week.

FIG. 2 shows the time course of the aldehyde oxidase activity in the acetic acid bacteria powder when the acetic acid bacteria powder obtained by the method of Example 2 was stored in a refrigerator at 5 ° C. for 9 weeks.

────────────────────────────────────────────────── (5) Continuation of the front page (51) Int.Cl. ⁷ Identification symbol FI (C12N 1/38 C12R 1:02) (56) References JP-A-63-96107 (JP, A) JP-A-6-22746 JP-A-53-104787 (JP, A) JP-A-50-71891 (JP, A) JP-A-60-172280 (JP, A) JP-A-61-108385 (JP, A) JP 39-7383 (JP, B1) Nippon Nogeikagaku ku Kaishi (1987), Vol. 9, p. 1079-1085

Claims (3)

(57) [Claims] 1. The method of claim 1, wherein the wet cells of the acetic acid bacterium are subjected to
A method for producing acetic acid bacterium powder which retains the activity of aldehyde oxidase for a long time , comprising dispersing in a buffer solution of 5 to 7 and freeze-drying. 2. The acetic acid bacteria powder according to claim 1, wherein the saccharide is selected from disaccharides such as sucrose, lactose and maltose, and monosaccharides such as glucose, galactose and fructose. Production method. 3. The method for producing acetic acid bacteria powder according to claim 1, wherein the sugar concentration in the buffer solution is a concentration corresponding to 10 to 30% by weight with respect to the wet cells.