JP6680609B2 - Acetobacter culture solution and method for producing the same - Google Patents

Description

TECHNICAL FIELD The present invention relates to an acetic acid bacterium culture solution having high growth ability and efficiently enhancing aldehyde oxidation ability, and a method for producing the same.

Aldehydes having 1 to 10 carbon atoms are known to be rich in oxidizing power and harmful to the living body. In particular, it is well known that acetaldehyde causes sickness during drinking. Moreover, many aldehydes have a peculiar odor, and it has been clarified that octanal and nonenal are causative agents of "aging odor".

It has been desired to effectively remove the above-mentioned unsaturated aldehyde, and an enzyme preparation containing an aldehyde dehydrogenase and a complex of a cell membrane to which the aldehyde dehydrogenase is bound as an active ingredient has been reported (Patent Reference 1).

However, although the aldehyde dehydrogenase activity of the enzyme preparation was kept high, the proliferative ability of the acetic acid bacterium itself was not examined and was not sufficient.

PCT / JP2014 / 059593

  Therefore, an object of the present invention is to provide an acetic acid bacterium culture solution having a high proliferation ability and an efficiently enhanced aldehyde oxidation ability, and a method for producing the same.

As a result of intensive studies to achieve the above-mentioned object, the present inventors have started culturing acetic acid bacteria in a culture solution containing specific concentrations of ethanol and acetic acid, and have surprisingly high growth ability and aldehyde oxidation. The inventors have found that an acetic acid bacterium culture solution having an improved efficiency can be obtained, and completed the present invention.

That is, the present invention is
(1) Selected from Komagataeibacter hansenii, Gluconacetobacter liquefaciens, Komagataeibacter maltaceti, and Komagataeisis melinensis An acetic acid broth containing one or more acetic acid bacteria,
The turbidity (OD660 nm) at a wavelength of 660 nm of the acetic acid bacterium culture solution is 1 to 10,
The aldehyde dehydrogenase activity per 15 mL of the acetic acid bacterium culture solution is 4 to 60,
Acetic acid culture fluid,
(2) Start culture with a culture solution containing an ethanol concentration of 0% or more and less than 3% and an acetic acid concentration of 0% or more and less than 1.5%,
A method for producing an acetic acid culture solution,
(3) In the method for producing an acetic acid bacterium culture solution according to (2),
The total concentration of ethanol and acetic acid in the culture solution is 0% or more and less than 2.0%,
A method for producing an acetic acid culture solution,
(4) In the method for producing an acetic acid bacterium culture solution according to (2) or (3),
The culture solution contains one or two or more sugar alcohols selected from glycerol, sorbitol, or mannitol,
A method for producing an acetic acid culture solution,
(5) Including the acetic acid bacterium culture solution of (1) or a dried product thereof,
Aldehyde dehydrogenase composition,
(6) An acetic acid bacterium culture solution obtained by the method for producing an acetic acid bacterium culture solution of (2) to (4) or a dried product thereof,
Method for producing aldehyde dehydrogenase composition,
Is.

  According to the present invention, it is possible to provide an acetic acid bacterium culture solution having a high proliferation ability and an efficient aldehyde oxidation ability, and a method for producing the same.

  Hereinafter, the present invention will be described in detail. In the present invention, "%" means "mass%" and "part" means "part by mass".

<Characteristics of the present invention>
The present invention is selected from Komagataeibacter hansenii, Gluconacetobacter liquefaciens, Komagataeibacter maltaceti, and Komagataeisis medelensis. An acetic acid bacterium culture solution containing one or more acetic acid bacteria,
The turbidity (OD660 nm) at a wavelength of 660 nm of the acetic acid bacterium culture solution is 1 to 10,
The aldehyde dehydrogenase activity per 15 mL of the acetic acid bacterium culture solution is 4 to 60,
It is an acetic acid culture solution.

<Acetic acid bacteria>
Acetic acid bacterium is a general term for microorganisms that grow using sugar or sugar alcohol and oxidize ethanol to produce acetic acid. Typical types of acetic acid bacteria used for the production of brewed vinegar include genus Acetobacter, genus Gluconobacter, genus Gluconacetobacter, genus Komagataeibacter, etc. .
In the present invention, since an acetic acid bacterium culture solution having efficiently increased growth ability and aldehyde oxidation ability is obtained, Komagataeibacter hansenii, Gluconacetobacter liquefaciens , One or more selected from Komagataeibacter maltaceti and Komagataeibacter medellinensis.

<Acetic acid culture fluid>
The acetic acid bacterium culture solution of the present invention may be a culture solution containing an acetic acid bacterium, and may be dead or viable as long as it has aldehyde dehydrogenase activity.

In addition, the acetic acid bacterium culture solution of the present invention can be used as an aldehyde dehydrogenase composition by directly or after drying the culture solution. Here, the aldehyde dehydrogenase composition refers to a food, cosmetic, pharmaceutical composition or the like having aldehyde dehydrogenase activity.

<About proliferation ability>
<Turbidity>
The acetic acid bacterium culture solution of the present invention has a turbidity (OD660 nm) of 1 to 10 when measured with a spectrophotometer at a wavelength of 660 nm, and preferably 2 to 10.

Here, the turbidity (OD660 nm) is generally used as an index indicating the number of bacteria, and the higher the number of bacteria, the higher the value of turbidity.

<About aldehyde oxidizing ability>
<Aldehyde dehydrogenase activity>
In the present invention, the aldehyde dehydrogenase activity (hereinafter referred to as ALDH activity) is the absorbance of an aqueous solution of ferric ferrocyanide when ferric ferrocyanide is produced from potassium ferricyanide with the oxidation of acetaldehyde. (660 nm). The absorbance at this time is hereinafter referred to as absorbance _{(ALDH activity)} .
More specifically, this aldehyde hydrogenase activity can be measured as follows in accordance with the method of Wood et al. Using ferricyanide as an electron acceptor (Methods in Enzymology, Volume 5, 1962, Page 287-291). it can.

<Method of measuring ALDH activity>
That is, the culture broth after the culture is stopped is subjected to a centrifugal separation treatment to collect the acetic acid bacteria. Next, acetic acid bacterium dispersion liquid arbitrarily diluted with 50 mM potassium phosphate buffer (pH 6.5), 1 mL of an aqueous solution containing McClubein buffer solution (pH 5), acetaldehyde 0.02 mmol, and potassium ferricyanide at 25 ° C. By holding for 5 to 20 minutes, aldehyde dehydrogenase in the acetic acid bacterium dispersion is reacted with aldehyde to oxidize the aldehyde, and potassium ferricyanide is converted to ferric ferrocyanide (Prussian blue) as a side reaction of this oxidation. Give back. This reaction is stopped by adding 0.5 mL of Dupanol solution, 3.5 mL of distilled water is further added, and the absorbance of ferric ferrocyanide (Prussian blue) at 660 nm is measured. In this case, as a blank test, a sample containing no acetic acid bacterium dispersion liquid is measured.

<ALDH activity per 15 mL of culture solution>
The acetic acid bacterium culture medium of the present invention has an aldehyde dehydrogenase activity (ALDH activity) of 4 to 60, preferably 15 to 60, per 15 mL of the culture medium.
The ALDH activity per 15 mL of the culture solution was calculated by the following calculation formula (Equation 1) using the above-mentioned absorbance _{(ALDH activity)} .

(Formula 1) Aldehyde dehydrogenase activity per 15 mL of culture solution = absorbance _{(ALDH activity)} x dilution rate

In addition, the dilution rate is calculated by converting the dilution rate in the case of dissolving 15 mL of the culture liquid in 1 mL into 1 mL.

<Aldehyde dehydrogenase specific activity>
The aldehyde dehydrogenase specific activity of the acetic acid bacterium contained in the acetic acid bacterium culture solution of the present invention is preferably 1 to 10 U / mg, and more preferably 1 to 5 U / mg.

Here, the aldehyde dehydrogenase specific activity (hereinafter referred to as ALDH specific activity) refers to the ALDH activity per mg of the protein derived from acetic acid bacteria.
The ALDH specific activity is that the change in absorbance at 660 nm due to generation of Prussian blue is 4 when 1 μmol of acetaldehyde is oxidized, and the above-mentioned absorbance _{(ALDH activity)} is used to calculate the following formula (Equation 2). It was calculated by

(Formula 2) ALDH specific activity (U / mg) = absorbance _{(ALDH activity)} × 4 / reaction time (minutes) × total volume / sample amount / protein concentration in sample (mg / mL)

In addition, the absorbance _{(ALDH activity)} used here was obtained by collecting the acetic acid bacterial cells by centrifugation, diluting them with 50 mM potassium phosphate buffer (pH 6.5), and destroying the bacterial cells with a French press to obtain the acetic acid bacterial cell membrane. Absorbance is measured using the dispersion liquid.

<Ubiquinol oxidase specific activity>
The ubiquinol oxidase specific activity of the acetic acid bacterium contained in the acetic acid bacterium culture medium of the present invention is preferably 1 to 10 U / mg, and more preferably 1 to 5 U / mg.

Here, the ubiquinol oxidase specific activity (hereinafter referred to as Q1H2 specific activity) is the conversion of ubiquinol-1 (Q1H2) into ubiquinone-1 (Q1), and U (unit) per 1 mg of the protein derived from acetic acid bacterium. Is. When ALDH oxidizes acetaldehyde, Q1 changes to Q1H2. Ubiquinol oxidase restores Q1H2 to Q1, and the two enzymes work in tandem.

<Q1H2 specific activity measurement method>
As a method for measuring the Q1H2 specific activity, the absorption of Q1 when Q1H2 changes to Q1 is measured at a wavelength of 275 nm. Specifically, 990 μL of McIlvain buffer, 5 μL of 4% Tween 20 and 5 μL of cell membrane dispersion solution are mixed, and the absorbance of the blank is measured. Add 1 μL of 50 mM Q1H2 to this mixture and measure the absorbance. The Q1H2 specific activity was calculated by the following calculation formula (Formula 3).

(Equation 3) Q1H2 specific activity (U / mg) = Absorbance change amount per 1 min ÷ Q1 Absorption per 1 mM (12.25 mM-1) × total volume (1000 μL) ÷ sample amount (5 μL) × sample dilution ratio (50) ÷ Protein concentration (mg / mL)

<Aldehyde oxidase specific activity>
The aldehyde oxidase specific activity of the acetic acid bacterium contained in the acetic acid bacterium culture medium of the present invention is preferably 1 to 10 U / mg, and more preferably 1 to 5 U / mg.

Here, the aldehyde oxidase specific activity (hereinafter referred to as oxidase specific activity) is the amount of oxygen consumed when reoxidizing ubiquinone that is reduced as acetaldehyde is oxidized using oxygen.

<Method for measuring specific activity of aldehyde oxidase>
As a specific measuring method of the oxidase specific activity, when a substrate (aldehyde) is put into the enzyme solution, oxygen used for oxidation is measured.
Use a dedicated small glass container. Constant temperature water (25 ° C) can be put into the outside (jacket) and the reaction solution can be put into the inside. The bottom can be stirred with a stirrer. The mouth is sized to fit the oximetry electrode.
1400 μL of McKilvein buffer was put in a container, air bubbles in the container were removed, an electrode was inserted so as not to put bubbles, and oxygen consumption of the blank was measured.
To measure the total amount of oxygen in the buffer, dithionite (1 small spoonful) was added for measurement.
The sample measurement was carried out by adding 10 μL of the bacterial cell membrane dispersion solution and 90 μL of a 1M acetaldehyde solution to the buffer solution. The oxidase specific activity was calculated by the following calculation formula (formula 4).

(Equation 4) Specific activity of oxidase (U / mg) = dissolved oxygen amount at 25 ° C. (498 n atom / mL) × oxygen consumption per 1 min ÷ oxygen consumption in buffer × total volume (1500 μL) / sample Volume (10 μL) × sample dilution ratio (10) ÷ protein concentration (mg / mL

<Ratio of ALDH specific activity and Q1H2 specific activity>
The acetic acid bacterium contained in the acetic acid bacterium culture solution of the present invention preferably has an ALDH specific activity: Q1H2 specific activity of 5: 1 to 1: 5, and further preferably 3: 1 to 1: 3.

No matter how high the ALDH specific activity is, if the Q1H2 specific activity is low, the aldehyde oxidizing ability that actually functions will not be extended. Therefore, the above ratio means that by maintaining the ALDH specific activity and the Q1H2 specific activity at the specific ratios, the aldehyde oxidizing ability that actually functions can be efficiently increased.

<Screening method>
In addition, by measuring the above-mentioned ALDH specific activity and Q1H2 specific activity, it is possible to screen for acetic acid bacteria whose aldehyde oxidation ability is efficiently enhanced.
Specifically, it is a method for screening an acetic acid bacterium having an efficiently enhanced aldehyde oxidation ability, which is characterized by the following methods (A) and (B).
(A) A method of screening an acetic acid bacterium having an enhanced aldehyde dehydrogenase specific activity, using the conversion activity of potassium ferricyanide to ferric ferrocyanide as an index.
(B) A method of screening an acetic acid bacterium having an enhanced ubiquinone oxidase specific activity, using the conversion activity of ubiquinol-1 to ubiquinone-1 as an index.

<Culture conditions>
<Ethanol concentration>
In the production method of the present invention, the acetic acid bacterium converts ethanol into acetic acid, and assimilates acetic acid to obtain energy to enhance the growth ability. Therefore, the ethanol concentration in the culture medium at the start of culture is 0% or more 3 %, Preferably 0% or more and 2.0% or less, and 0% or more and 0.5% or less. On the other hand, if the ethanol concentration is 3% or more, the growth of acetic acid bacteria will not proceed.

<Acetic acid concentration>
In the production method of the present invention, since an acetic acid bacterium culture solution having high growth ability is obtained, the acetic acid concentration in the culture solution at the start of culture is 0% or more and less than 1.5%, and further 0% or more and 1% or less. Is good. When the acetic acid concentration is 1.5% or more, the pH of the culture solution is lowered and the growth of acetic acid bacteria is not promoted.

<Total ethanol concentration and acetic acid concentration>
In the production method of the present invention, it is preferable that the total concentration of ethanol and acetic acid in the culture solution is 0% or more and less than 2.0%.

<Sugar alcohol>
In the production method of the present invention, since it is easy to promote the growth of acetic acid bacteria by suppressing the decrease in pH in the culture solution, one or more kinds selected from glycerol, sorbitol, or mannitol can be added to the culture solution. It is good to contain sugar alcohol. The sugar alcohol concentration is not particularly limited, but may be, for example, 0.1 to 5.0%, and may be 0.5 to 2.0%.

<Cellulose membrane>
In the production method of the present invention, since it is easy to obtain an acetic acid bacterium culture solution having a high proliferation ability, it is preferable to perform culturing after the culturing so that a cellulose membrane is not formed on the surface of the culture solution.

<PH>
In the production method of the present invention, the pH of the acetic acid bacterium culture solution is preferably set to 3 to 7, since an acetic acid bacterium culture solution having a high growth ability can be easily obtained. If the pH of the culture solution is outside the above range, the growth of acetic acid bacteria and the ability to oxidize aldehydes may be suppressed.

  Next, the present invention will be further described based on Examples and Test Examples. The present invention is not limited to this.

<Example 1>
The culture solution 1 (15 mL) shown in the following composition table was added to a 300 mL Erlenmeyer flask with a baffle, and 0.01% of an acetic acid strain (Komagataeibacter hansenii NBRC14820) was inoculated. After culturing with stirring at a product temperature of 30 ° C. and a rotation speed of 230 rpm for 24 hours, the culture was collected to obtain the acetic acid bacterium culture solution of Example 1. At this time, no cellulose film was formed on the surface of the culture solution.

The turbidity (OD660 nm) of the acetic acid culture broth was 2.0, and the ALDH activity per 15 mL of the culture broth was 42.7. The pH of the culture solution at the end of the culture was 5.6.

<Composition table>

<Test Example 1>
In order to investigate the influence of the type of acetic acid bacteria on the growth ability and aldehyde oxidation ability, various acetic acid bacteria were cultured according to Example 1 using the culture solutions shown in Table 1 to obtain acetic acid culture solutions.
For each of the obtained acetic acid bacterium culture solutions, pH, turbidity of the culture solutions, and ALDH activity per 15 mL of the culture solution were measured and evaluated. The results are shown in Table 1.
KPJ Gluconacetobacter 2001 is a strain owned by Kewpie Brewing.

The evaluation is based on the following evaluation criteria.
<About turbidity (OD660nm)>
⊚: 5 or more and 10 or less was marked with ⊚, indicating that the growth was very good.
Good: Proliferation was shown to be good when it was 1 or more and less than 5.
Poor: A value of less than 1 was taken as x, indicating that the growth was poor.

<About ALDH activity per 15 mL of culture solution>
⊚: A value of 15 or more and 50 or less is marked with ⊚, and the ALDH activity is extremely high.
◯: A value of 4 or more and less than 15 is ◯, and the ALDH activity is high.
X: A value of less than 4 was evaluated as x, and the ALDH activity was low.

<Table 1>

From Table 1, Komagataeibacter hansenii, Gluconacetobacter liquefaciens, Komagataeibacter maltaceti, Komagataeisis medelensis selected from Komagataeisister medelensis It can be understood that the acetic acid bacterium culture liquid having high growth ability and high aldehyde oxidation ability can be obtained by using one or more kinds of acetic acid bacteria.

Specifically, the turbidity (OD660 nm) at a wavelength of 660 nm of the culture solution is 1 to 10,
The aldehyde dehydrogenase activity per 15 mL of the culture solution is 4 to 60,
An acetic acid bacterium culture solution is obtained.

<Test Example 2>
In order to investigate the influence of the ethanol concentration and the acetic acid concentration of the culture solution at the start of the culture, various acetic acid bacteria were cultured using the culture solution shown in Table 2 according to Example 1 to obtain an acetic acid culture solution. Specifically, ethanol and acetic acid were added to the culture solution 1 so as to have the concentrations shown in Table 2, and the rest was prepared with fresh water. The turbidity and ALDH activity per 15 mL of the culture solution were measured and evaluated for each of the obtained culture solutions of acetic acid bacteria. Table 2 shows the results.

<Table 2>

From Table 2, by starting the culture with a culture solution containing an ethanol concentration of 0% or more and less than 3% and an acetic acid concentration of 1% or more and less than 1.5%, acetic acid bacterium culture having high growth ability and enhanced aldehyde oxidation ability. It can be seen that a liquid is obtained.

Further, when the total concentration of ethanol and acetic acid in the culture solution is 0% or more and less than 2.0%, an acetic acid bacterium culture solution having a higher growth ability and an increased aldehyde oxidation ability is easily obtained.

<Test Example 3>
In order to investigate the influence of sugar alcohol in the culture medium, various acetic acid bacteria were cultured in the culture medium 1 using the carbon source (glycerol or glucose) shown in Table 2 according to Example 1 to obtain an acetic acid culture medium. It was The pH, turbidity, and ALDH activity per 15 mL of the culture solution were measured and evaluated for each of the obtained culture solutions of acetic acid bacteria. The results are shown in Table 3.

<Table 3>

From Table 3, in the production method of the present invention, when the culture solution contains one or two or more sugar alcohols selected from glycerol, sorbitol, and mannitol, the growth property is higher and the aldehyde oxidizing ability is increased. It can be understood that the acetic acid bacterium culture solution can be easily obtained.

<Test Example 4>
According to Example 1, various acetic acid bacteria were cultured using the culture solution shown in Table 4 to obtain an acetic acid culture solution. The ALDH specific activity, Q1H2 specific activity, and oxidase specific activity of each of the obtained acetic acid bacterium culture solutions were measured and evaluated. The results are shown in Table 4.
The specific method for measuring various activities is as described above.

The evaluation is based on the following evaluation criteria.
<About ALDH specific activity>
⊚: 5 U / mg or more and 10 U / mg or less is marked with ⊚, and the ALDH specific activity is extremely high.
◯: A value of 1 U / mg or more and less than 5 U / mg is ◯, and the ALDH specific activity is high.
Δ: Less than 1 U / mg is taken as Δ, and the ALDH specific activity is slightly low.

<About Q1H2 specific activity>
⊚: 5 U / mg or more and 10 U / mg or less is marked with ⊚, and the Q1H2 specific activity is extremely high.
◯: A value of 1 U / mg or more and less than 5 U / mg is ◯, and the Q1H2 specific activity is high.
Δ: Less than 1 U / mg is taken as Δ and the Q1H2 specific activity is slightly low.

<About oxidase specific activity>
⊚: 5 U / mg or more and 10 U / mg or less is marked with ⊚, and the oxidase specific activity is extremely high.
◯: A value of 1 U / mg or more and less than 5 U / mg is ◯, and the oxidase specific activity is high.
Δ: Less than 1 U / mg is taken as Δ and the oxidase specific activity is slightly low.

<ALDH specific activity: Q1H2 specific activity>
⊚: ALDH specific activity: Q1H2 specific activity is set to 3: 1 to 1: 3, and the aldehyde oxidation ability that actually functions is very efficiently enhanced.
◯: ALDH specific activity: Q1H2 specific activity is 5: 1 to 3: 1 or 1: 3 to 1: 5, and it is in a state in which the actually functioning aldehyde oxidizing ability is efficiently enhanced.
Δ: ALDH specific activity: Q1H2 specific activity is set to be Δ out of the range of 5: 1 to 1: 5, and the aldehyde oxidizing ability that actually functions is slightly low.

<Table 4>

From Table 4, the acetic acid bacterium contained in the acetic acid bacterium culture medium of the present invention has an ALDH specific activity of 1 to 10, a Q1H2 specific activity of 1 to 10 and an oxidase specific activity of 1 to 10.
Further, since the ALDH specific activity: Q1H2 specific activity is 5: 1 to 1: 5, it can be understood that not only the ALDH specific activity is high, but also the aldehyde oxidizing ability that actually functions is efficiently enhanced.

In addition, by measuring the ALDH specific activity and the Q1H2 specific activity, it is possible to screen acetic acid bacteria having an efficiently enhanced aldehyde oxidation ability.

Claims (3)

Komagataeibacter hansenii, Gluconacetobacter liquefaciens, Komagataeibacter maltaceti, Komagataeibacter medellinesis 1 or 2 species selected from Komagataeibacter maltaceti A method for producing an acetic acid bacterium culture solution containing at least one kind of acetic acid bacterium,
Start the culture with a culture medium containing an ethanol concentration of 0% or more and 1.0% or less (excluding 0%) and an acetic acid concentration of 0% or more and 0.2% or less (excluding 0%),
The total concentration of ethanol and acetic acid in the culture solution is 0% or more and 1.2% or less (excluding 0%),
The turbidity (OD660 nm) at a wavelength of 660 nm of the obtained acetic acid bacterium culture solution is 1 to 4 ,
The aldehyde dehydrogenase activity per 15 mL of the obtained acetic acid bacterium culture solution is 4 to 60,
Method for producing acetic acid culture broth. The method for producing an acetic acid bacterium culture solution according to claim 1,
The culture solution contains one or more sugar alcohols selected from glycerol, sorbitol, or mannitol,
Method for producing acetic acid culture broth. An acetic acid culture solution obtained by the method for producing an acetic acid culture solution according to claim 1 or 2, or a dried product thereof,
Method for producing aldehyde dehydrogenase composition.