JP5470807B2 - High temperature acetic acid fermentation acetic acid bacteria - Google Patents

Description

  The present invention relates to a natural mutant of Acetobacter.pasteurianus having acetic acid fermentation ability in the range of 39 to 41 ° C. by repeatedly increasing the culture temperature stepwise and a method for producing acetic acid using the mutant. .

  Acetic acid bacteria have the ability to oxidize various alcohols and sugar alcohols into acids. Using this acetic acid bacterium, acetic acid is produced industrially by oxidation from ethanol.

  Acetobacter acetic acid bacteria are widely used in industrial production of acetic acid. Acetobacter acetic acid bacteria are acetic acid bacteria isolated from fermented bacterial membranes on rice vinegar mash at the vinegar brewing site by the traditional stationary fermentation method in Japan. It has high acetic acid production capacity and acetic acid resistance, has many research achievements mainly in Japan, and is regarded as one of the standard strains for acetic acid bacteria research.

  Acetic acid fermentation using this Acetobacter genus acetic acid bacterium is widely carried out industrially, but accurate temperature control is necessary for efficient acetic acid fermentation. In the cultivation of Acetobacter acetic acid bacteria, when the temperature is higher than 30 ° C., the growth and acetic acid fermentation ability are rapidly reduced. However, in summer, the temperature becomes 30 ° C. or higher, and heat is generated by fermentation of microorganisms, so that the fermentation layer may become 40 to 45 ° C. or higher as acetic acid fermentation proceeds. Therefore, cooling equipment for keeping the fermenter at 25 ° C. to 30 ° C. and energy and water for cooling are required, but the cost burden is large. Although it depends on the production volume, it may cost several million yen per year for cooling water and energy to lower 1 ° C in a 1 ton fermenter. Therefore, research on high-temperature-resistant acetic acid bacteria capable of growing and acetic acid fermentation at a high temperature even at 1 ° C. is underway.

As a high-temperature-resistant acetic acid bacterium, Non-Patent Document 1 discloses that Acetobacter pasteurianus by the present inventors.
SKU1108 was isolated and this bacterium has been shown to have growth and acetic acid fermentability at 38 ° C. However, the growth and fermentation capacity are reduced at 39 ° C., and the growth and fermentation capacity are very weak at 40 ° C.

  Non-Patent Document 2 shows that Acetobacter aceti retains the same highest acetic acid producing ability as 35 ° C at 30 ° C, and has 68% of the activity when cultured at 30 ° C even at 37 ° C. It is shown. Since ordinary Acetobacter aceti loses the ability to produce acetic acid at 35 ° C, the high-temperature fermentation with this strain can reduce cooling costs somewhat. No acetic acid fermentation is observed at 0 ° C., and the cooling cost is still high.

  In Patent Document 1, a novel gene involved in temperature resistance is cloned from a practical acetic acid bacterium belonging to the genus Acetobacter, and the temperature resistance is remarkably improved in a transformed strain obtained by introducing the gene into the acetic acid bacterium. It is shown. In this document, it is shown that Acetobacter aceti transformants can be grown and acetic acid fermented even at 38 ° C. However, at 40 ° C., only the growth was observed, but no acetic acid fermentation was observed. In addition, it is a strain obtained by a genetic engineering technique, and high safety confirmation is required to produce acetic acid ingested as food using a strain obtained by a genetic recombination technique. In addition, some social emotions are unacceptable.

Furthermore, Non-Patent Document 3 discloses a cell fusion method of acetic acid bacteria belonging to the genus Acetobacter, that is, an acetic acid strain (Acetobacter aceti) that has temperature tolerance but does not have strong ability to ferment acetic acid, and acetic acid fermentation is strong but temperature resistance is not strong. Acetobacter
Although a strain having fermentability at high temperatures is shown by the method of fusing xylinus), 37 ° C. is the upper limit of growth and acetic acid fermentation, and growth and fermentation ability at 39 ° C. or higher are not shown.

Thus, the upper limit of efficient acetic acid fermentation was set to 38 ° C. in acetic acid bacteria known so far. However, in order to reduce the temperature to 38 ° C. or less, the cooling equipment and the cost burden are large. There has been a demand for acetic acid bacteria having acetic acid fermentation ability at high temperatures.
Republished WO2004-053122 A. Saeki., Etal., Biosci. Biotech. Biochem., 61, 138-145 (1997) S.OHMORI., Et al., Vol.44 No.12 Page.2901-2906 (1980.12) M.Fukaya., Etal., Agric.Biol.Chem., 53 (9), 2435-2440 (1989)

  As a result of intensive research aimed at obtaining a strain having higher temperature tolerance and acetic acid fermentation ability, the present inventor has increased the culture temperature step by step with respect to Acetobacter pasteurianus SKU1108. By repeatedly culturing in this way, a natural mutant strain having growth and acetic acid fermentation ability was successfully produced even at 39 ° C to 41 ° C, and the present invention was completed.

In the first aspect of the present invention, Acetobacter pasteurianus SKU1108 is repeatedly cultured in a medium having a growth limit temperature (38 ° C.), and a part of the obtained growth limit temperature-adapted strain is used as a preculture solution at 38.5 ° C. By repeatedly culturing in a new medium at 39.5 ° C. using a part of the adaptive strain in the obtained medium at 38.5 ° C. as a pre-culture solution, 39-41 ° C. A method for producing a mutant strain of acetic acid bacteria Acetobacter pasteurianus SKU1108 to obtain acetic acid bacteria having an acetic acid fermentation ability in the range of the above, wherein the cultivation of the cultured medium has a turbidity of Klett Unit of 60 to 120, acidity Is 1.0-1.8%, a part of the culture solution is inoculated into the next new medium as an inoculum, and the culture is repeated by culturing again at the same temperature. Acetobacter pasteurianus SKU1108 Of mutants It is a manufacturing method.

A second aspect of the present invention is a claim 1 Acetobacter acetic acid bacterium produced by the method for manufacturing a mutant strain of acetic acid bacteria Acetobacter pasteurianus SKU1108 according pasteurianu s T H-3 (consignment number NITE P-664).

A third aspect of the present invention, the acetic acid fermentation at 39 ° C. to 41 ° C. using a mutant strain of acetic acid bacteria Acetobacter pasteurianus SKU1108 made by a manufacturing method of a mutant strain of acetic acid bacteria Acetobacter pasteurianus SKU1108 of claim 1 Symbol placement This is a method for producing acetic acid.

  By utilizing the present invention, acetic acid fermentation can be efficiently performed even in the range of 39 to 41 ° C., so that the cost required for cooling equipment and cooling water in industrial acetic acid production can be greatly reduced. Furthermore, since it is a natural mutant, highly safe vinegar can be provided.

  In the present invention, the high temperature adaptability is defined as follows. That is, although growth and acetic acid fermentation can be performed at a normal temperature of 25 ° C. to 30 ° C., it means the ability to grow and acetic acid fermentation even in a high temperature medium of 30 ° C. to 41 ° C., particularly 38 ° C. to 41 ° C. Here, “growth” refers to a state in which the fungus is not alive, but is alive at that temperature, and exhibits efficient acetic acid fermentation (short induction period and high acidity).

  Acetobacter pasteurianus SKU1108 of the present invention was isolated from pineapple as Acetobacter lovaniensis by the present inventors and was deposited with the Patent Microorganism Depositary, National Institute of Technology and Evaluation on January 18, 2006. The deposit number is NBRC101655. It was later revealed by analysis of 16S rRNA gene that the present inventors belong to Acetobacter pasteurianus.

  A mutant strain of Acetobacter pasteurianus SKU1108 used in the present invention is a natural mutant strain having a high temperature adaptability obtained by repeatedly culturing Acetobacter pasteurianus SKU1108 while raising the temperature stepwise.

Here, “repeated culture” means a high temperature that causes a decrease in growth or fermentation, for example, Acetobacter
In the case of pasteurianus SKU1108, the culture is performed at around 38 ° C, and when the grown bacteria reach the target culture phase, a part of the culture is inoculated into the next new medium as an inoculum and cultured again at the same temperature. Is repeated. The target phase refers to the stage from the middle of the logarithmic growth phase to the beginning of the stationary phase and the increase in the reached acidity, and the turbidity of the medium is about 40 to 280, preferably in Klett Unit. Is about 60-120 in the middle of the logarithmic growth phase, and the achieved acidity is about 0.8-3.5, preferably about 1.0-1.8.

  By carrying out this repeated culture, a strain that has grown very slowly and has a low fermentation ability at the growth and fermentation limit temperature is mutated into a high-temperature adaptive strain having growth and acetic acid fermentation ability at that temperature. The number of “repetitions” in the repetitive culture is not particularly limited, but it is preferable to carry out until a strain having high temperature adaptability is obtained, that is, until the time required for starting is 24 hours, more preferably 15 hours.

  Next, a part of the strain obtained by the above repeated culture is used as a pre-culture solution, and the culture is repeated with a new medium having a higher temperature. The range of temperature increase is not particularly limited, but as described above, it must be below the limit temperature that causes the growth of bacteria and the decrease in fermentation. For example, the culture can be performed in a medium that has been increased in temperature by 0.5 ° C. It is essential. In this way, repeated cultivation is performed in a medium in which the temperature of the medium is gradually increased to obtain a high-temperature adapted strain at a higher temperature.

Using FIG. 1, 38 ° C. is the growth limit temperature (the temperature that causes the growth of bacteria and the decrease in fermentation).
An explanation will be given of the case where the culture is successively repeated at 38 ° C., 38.5 ° C. and 39.5 ° C. using pasteurianus SKU1108.

  Bacteria are cultured in a medium at 38 ° C., and Klett Unit and acidity are measured as they grow. When the target phase is reached according to the Klett Unit and acidity measurement, a portion of the culture is taken and inoculated into a new medium at the same temperature and cultured again. When the target phase is reached in this culture, a part of the culture solution is taken and inoculated into a new medium at the same temperature and further cultured. This is repeated, and when a high-temperature adaptable strain is obtained, a part of the culture solution is taken and inoculated into a medium at 38.5 ° C., and the culture is repeated as in the case of 38 ° C. When a high temperature adaptive strain is obtained at 38.5 ° C. by repeated culture, a portion of the culture solution is taken and inoculated into a 39.5 ° C. medium, and repeated culture is performed.

Among the strains obtained in this manner, the target phase is the middle of the logarithmic growth phase (about 60 to 120 in Klett Unit, acidity is 1 to 1.8%). TH-1 is a high-temperature adapted strain obtained by culturing at 38.5 ° C. Subsequently, repeated culture at 39.5 ° C. was performed, and the finally obtained bacterial group was spread on a plate, and the colonies of three types of sizes that could be confirmed were compared for growth. Was designated as a high temperature adaptable strain TH-3. The target phase is changed from the end of the logarithmic growth phase to the beginning of the stationary phase (Klett
When the culture is repeated at a unit of about 220 and an acidity of 2.7 to 3.5%), the high-temperature-adapted strain obtained by culturing at 38 ° C and 38.5 ° C is TH. -2. Acetobacter pasteurianus TH-1, TH-2, and TH-3 used in the present invention have been deposited with the Patent Microorganism Depositary Center for Product Evaluation Technology on October 22, 2008, and the deposit number is NITE AP-662, NITE AP-663 and NITE AP-664, respectively.

  In this way (1) Repeat culture at the growth limit temperature → (2) Obtain an adaptive strain at the medium temperature → (3) Inoculate a new medium with higher temperature → (4) Repeat culture → (5) A series of cycles of obtaining an adaptive strain at the medium temperature is performed by gradually increasing the temperature.

  The culture temperature may start from 38 ° C., which is the limit temperature for efficient acetate fermentation and growth of Acetobacter pasteurianus SKU1108, or the culture temperature may be gradually increased from below the growth limit temperature. The higher the final temperature reached, the better, but there is a limit to the ability of the bacteria to grow, and the upper limit is considered to be around 41 ° C.

  The pH conditions in acetic acid fermentation and bacterial culture using the fungus of the present invention can be adjusted by any known method, but the pH of the medium is preferably 7.5 or less. In order to prevent the pH of the medium from being lowered due to the acetic acid generated by the fermentation action of the acetic acid bacteria mutant of the present invention, for example, an appropriate buffer or alkali is added to the medium, or the medium is continuously or periodically added. Can be done by exchanging.

  When the strain is cultured to produce acetic acid, known medium composition (for example, YPG liquid medium containing 0.5% yeast extract, 0.5% polypeptone, 1% glycerin, etc.), aerobic culture such as shaking culture Can be carried out by liquid culture under typical conditions. It is also possible to use a yeast extract or add sugars such as fructose and sucrose. In the medium, alcohol, preferably ethanol, n-propanol, n-butanol, more preferably 3 to 5% ethanol is added. Furthermore, in order to efficiently perform acetic acid fermentation at a high temperature, aeration (aeration) can be increased. This aeration can be improved by blowing air into the culture medium. Acetic acid can be recovered by filtering the cells.

  In addition, after the growth of the bacteria is confirmed and fermentation is started, the alcohol is consumed and the alcohol concentration becomes 0.5 to 3% by volume, and the acidity is 0.1 to 10% and ethanol is 10 to 90% by volume. While controlling the additive solution having a composition of yeast extract 0.005-0.5 wt / vol% and glucose 0.005-1 wt / vol% so that the alcohol concentration is maintained at 0.4-3 vol%. Fermentation is continued by adding to the fermentation broth.

  As the ventilation method, a conventionally known method can be adopted and there is no limitation, and examples thereof include a method of supplying a gas containing oxygen such as air or oxygen gas through a ventilation pipe. The aeration amount may be appropriately set in consideration of the fermentation status and the like. For example, an aeration amount of 0.02 to 1 vvm (aeration capacity / amount of fermentation solution / min) is supplied to the lower part of the fermentation solution, and this is agitated. What is necessary is just to control by making it disperse | distribute and maintaining the dissolved oxygen in a fermentation liquid about 0.2-8 ppm.

(Pre-culture)
Acetobacter pasteurianus SKU1108 has a turbidity of 150 Klett
The cells were precultured at 37 ° C. and 200 rpm in a test tube containing 5 ml of potato medium (Table 1) until reaching Unit. 5 ml of this preculture was aseptically inoculated into a 500 ml flask containing 100 ml of YPGD (Table 2) medium, and 4% ethanol was added and cultured at 38 ° C. and 200 rpm.

(Measurement of growth and acetic acid fermentation)
At the stage where the culture progressed, the growth of the bacteria was measured by measuring the klett unit. For this measurement, a klett-summerson photoelectric colorimeter was used as the klett unit.

  Acetic acid fermentation was confirmed by measuring acidity. The acidity of the medium was determined by performing alkaline titration with 0.8N NaOH using phenolphthalein as an indicator for the fermentation broth, and multiplying the obtained titration amount by 0.12 to give the value converted to acetic acid concentration. Represented.

(Repeated culture)
When the growth of the fungus and acetic acid fermentation are observed and the target phase is reached, 5 ml of a part of the culture solution is taken as a seed fungus, inoculated into a new medium and cultured under the same conditions, that is, the “repeated culture” described above. Went.

  Here, when the target phase is the middle of the logarithmic growth phase (Klett Unit is about 60 to 120, acidity is 1 to 1.8%), and when the target phase is the end of the logarithmic growth phase. To the beginning of the stationary phase (about 220 in Klett Unit, acidity of 2.7-3.2%) and repeated culture.

  In the method in which the target phase is in the middle of the logarithmic growth phase, repeated culture at 38 ° C. is performed 7 times, and 5 ml of the 7th culture solution is used as an inoculum and aseptically placed in a 500 ml flask containing 100 ml of YPGD medium. Inoculated, 4% ethanol was added and cultured at 38.5 ° C. and 200 rpm. The culture was repeated about 10 times at 38.5 ° C., and the strain obtained by the 10th culture was designated as TH-1 (the upper part of FIG. 2). Furthermore, the TH-1 strain adapted to 38.5 ° C was repeatedly cultured 21 times at 39.5 ° C. Then, the bacteria group finally obtained from the 21st culture solution was spread on a plate, and the colonies of three types of sizes that could be confirmed were compared for growth. It was.

  The TH-1 strain was repeatedly cultured at 39.5 ° C. without passing through 39 ° C. This is because the TH-1 strain was able to perform acetic acid fermentation rapidly even at 39 ° C. .

  On the other hand, in the method of setting the target phase from the end of the logarithmic growth phase to the beginning of the stationary phase, repeated culture at 38 ° C. was performed 4 times, and 5 ml of the fourth culture was used as an inoculum, and 100 ml of YPGD medium was contained. A 500 ml flask was aseptically inoculated, 4% ethanol was added, and the mixture was cultured at 38.5 ° C. and 200 rpm. The culture was repeated 5 times at 38.5 ° C., and the strain obtained by the fifth culture was designated as TH-2 (lower part of FIG. 2).

(Acquisition of high temperature adaptability)
FIG. 3 shows the growth (A) and the acetic acid fermentation ability (B) when repeated culture at 39.5 ° C. is performed. In (A), the horizontal axis represents the culture time, the vertical axis represents the turbidity Klett Unit, and in (B), the horizontal axis represents the culture time, and the vertical axis represents the acidity (%). (A) shows the growth when the first and second repeat cultures are sequentially performed 21 times in order from the left, and the data indicating the acidity corresponding to each is (B). In the initial stage of culture, the growth was very slow and the increase in acidity was very slow, but repeated cultivation shortened the time to rise and the acidity also increased. Specifically, the bacteria that took about 160 hours to start acetic acid fermentation at the start of culture were finally improved to about 24 hours, and the acidity increased from about 2% to about 3.5%. . Thus, a strain capable of growing and acetic acid fermentation was obtained even by culturing at 39.5 ° C.

  In (A), the bacteria grow again after the stationary phase, and the acidity decreases as shown in (B). This is because energy is obtained by assimilating the acetic acid produced by the acetic acid bacteria. This is due to the properties commonly found in acetic acid bacteria.

(High temperature culture by jar fermenter)
Acetic acid bacteria Acetobacter pasteurianus obtained by the above
SKU1108 mutants TH-1, TH-2 and TH-3 and the pre-mutation Acetobacter pasteurianus
SKU1108 was pre-cultured in potato medium at 37 ° C. and 200 rpm in a 100 ml 500 ml flask until the turbidity reached 150 klett units. Place 2L of medium in a 5L jar fermenter (manufactured by Maruhishi Sangyo Co., Ltd.), aseptically inoculate all of the pre-culture solution (100ml), and each temperature of 37 ° C, 39 ° C, 40 ° C and 41 ° C And culturing at 500 rpm and aeration volume of 0.5 vvm. At this time, 4% ethanol was added. The results are shown in FIG. The horizontal axis represents the culture time, the right vertical axis represents acidity (%), and the left vertical axis represents turbidity (klett unit).

At 37 ° C., all have the same growth and fermentation ability. 15 hours after culturing, the turbidity exceeds 200 Klett Unit and the acidity exceeds 3%.
However, at 39 ° C, Acetobacter pasteurianus SKU1108 showed a sharp decline in both growth and acetic acid fermentation. About 15 minutes after cultivation, the turbidity was about 1/3 Klett Unit, and the acidity was about 5 minutes. 1%. On the other hand, in the TH-1, TH-2 and TH-3 strains, both the growth and fermentation ability are the same as the culture at 37 ° C., which exceeds 38 ° C., which has been regarded as the effective acetic acid fermentation and growth limit temperature so far. Successful growth of acetic acid bacteria and acetic acid fermentation at temperature.

  Furthermore, even at 40 ° C., it was revealed that TH-3 has growth and fermentation ability similar to that at 37 ° C. TH-1 also had a turbidity of about 2/3 Klett Unit and an acidity of about 2/3% compared to 37 ° C. In TH-2, compared to the case of 37 ° C., the turbidity was about 1/6 Klett Unit and the acidity was about 1/4%.

  Even at 41 ° C., the growth of bacteria and acetic acid fermentation decline in TH-3 compared to 37 ° C. However, by setting the culture time to 30 to 40 hours, acetic acid fermentation of about the same degree as 37 ° C. is achieved. Admitted.

  By using the naturally occurring Acetobacter pasteurianus mutant strain of the present invention, industrial acetic acid production can be efficiently performed while suppressing costs required for the cooling device and cooling water of the fermenter.

It is the figure which showed the repeated culture | cultivation at 38 degreeC, 38.5 degreeC, and 39.5 degreeC. It is a figure which shows culture | cultivation temperature in repetition culture | cultivation, the frequency | count of repetition, and obtained TH-1, TH-2, and TH-3. It shows (A) growth and (B) acetic acid fermentation ability when repeated culture is performed at 39.5 ° C. It is a figure which shows the culture | cultivation at 37 degreeC, 39 degreeC, 40 degreeC, and 41 degreeC by the mutant strains TH-1, TH-2, and TH-3 of the acetic acid bacterium Acetobacter pasteurianus SKU1108 and the unmutated strain Acetobacter pasteurianus SKU1108 .

Claims (3)

Acetobacter pasteurianus SKU1108 was repeatedly cultured in a medium with a growth limit temperature (38 ° C.), and a part of the obtained growth limit temperature-adapted strain was used as a pre-culture solution and repeatedly cultured in a new medium at 38.5 ° C., Acetic acid having an acetic acid fermentation ability in the range of 39 to 41 ° C. is obtained by repeatedly culturing in a new medium at 39.5 ° C. using a part of the obtained adaptive strain in the medium at 38.5 ° C. A method for producing a mutant strain of the acetic acid bacterium Acetobacter pasteurianus SKU1108 , which obtains a bacterium , wherein the turbidity of the cultured medium is 60 to 120 in terms of Klett Unit, and the acidity is 1.0 to 1.8%. The method for producing a mutant strain of the Acetobacter pasteurianus SKU1108 is a culture in which a part of the culture solution is inoculated as a seed fungus into the next new medium and cultured again at the same temperature . Claim 1, wherein the Acetobacter acetic acid bacteria produced by the method for manufacturing a mutant strain of acetic acid bacteria Acetobacter pasteurianus SKU1108 pasteurianu s T H- 3 ( consignment number NITE P-664). Production of acetic acid which is characterized in that the acetic acid fermentation at 39 ° C. to 41 ° C. using a mutant of a method for manufacturing acetic acid bacterium Acetobacter pasteurianus SKU1108 made by the mutant strain of claim 1 Symbol placement acetic acid bacteria Acetobacter pasteurianus SKU1108 Method.

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