JP5564719B2 - Storage method of acetic acid bacteria, manufacturing method of vinegar - Google Patents

Description

As the seed vinegar (fermented liquid containing “inoculum” that becomes the seed of fermentation), acetic acid bacteria collected from the vinegar fermented liquid being fermented by the deep fermentation method are subjected to acetic acid fermentation at a later date. for use, the method storage acetic acid bacteria to be stored while keeping the activity of the bacteria, those concerning the manufacturing process of vinegar.

  In the deep fermentation method of vinegar, it is necessary to add seed vinegar to the preparation liquid when starting fermentation. And conventionally, a method of collecting a part of the fermented vinegar fermented liquid during fermentation and leaving it as a seed vinegar is quickly and directly added to the next charged liquid. However, this method has a problem that it is necessary to always maintain fermentation of vinegar to be used as seed vinegar, and wasteful vinegar can be produced.

  In addition, a method in which a preculture solution pre-cultured in a medium for acetic acid bacteria for producing vinegar is prepared in advance and added to the preparation solution as seed vinegar when starting fermentation has been conventionally proposed (for example, patents). Reference 1).

  However, in the case of the conventional method described in Patent Document 1, a small-scale pre-culture fermentation apparatus must be operated separately from the fermentation apparatus for main fermentation, and there is a problem that work efficiency is poor twice. .

  Therefore, a technique for collecting and storing a vinegar fermented liquid and using it as a seed vine at a later date according to the production plan has been strongly desired. And as an example of such storage technology, the vinegar fermented liquor being fermented by the deep fermentation method is diluted so that the acetic acid concentration becomes 2 wt / vol% to 5 wt / vol% by adding a diluent solvent And the storage method of the vinegar fermented liquid which cools so that liquid temperature may be 0 degreeC or more and 15 degrees C or less, and stores for 24 hours or more is proposed (refer patent document 2).

JP 2004-33111 A JP 2007-166828 A

  However, since the storage method described in Patent Document 2 can be applied only to short-term storage of about one week or several months at the longest, it is not suitable for a case where it is desired to store for a longer period. Moreover, when the vinegar fermentation liquid stored by the storage method of patent document 2 is used as seed vinegar, dispersion | variation becomes large in the time (lag time) until vinegar fermentation starts, and a production plan cannot be made correctly. The malfunction is assumed. Furthermore, when it is stored for 3 months or more by the method described in Patent Document 2, for example, when the vinegar fermentation is performed using the stored vinegar fermented liquid as seed vinegar, it takes a long time to start fermentation (lag time). It is assumed that there is a problem that fermentation does not start.

  In the case of the storage method described in Patent Document 2, the fermented liquid to be stored includes acetic acid bacteria and other ingredients of the raw materials necessary for each vinegar type (for example, apple ingredients for apple vinegar and rice ingredients for rice vinegar). Component). Therefore, the range (kind of vinegar that can be used) in which the stored fermentation broth can be used is extremely limited.

  Here, as another conventional bacteria storage method, there is also the following method. That is, after collecting the fermentation broth during fermentation, it is centrifuged without dilution, and a microbe preservative such as glycerol or saccharified solution is added to the separated precipitate. And it is the method of freezing the microbe deposit to which the microbe preservative was added, and storing it at an extremely low temperature of about −80 ° C. The merit of this method is that it can be stored for a long time (for example, 3 months to 6 months).

  However, the above conventional method of storing at an extremely low temperature is unsuitable for storing a fermented liquid as a seed vinegar used in the actual production process of vinegar. This is because it takes a large amount of equipment and a lot of costs to store a fermentation broth containing the number of acetic acid bacteria that can quickly start fermentation in actual production at −80 ° C., which is not practical. . Moreover, it is because the acetic acid bacteria fermented liquid which added the microbe preservation | save agent is not preferable when using it as a seed vinegar of the vinegar fermentation process, since raw materials other than the original vinegar raw material may be mixed. Of course, there is no problem when the saccharified solution extracted from the vinegar production process is used as a microbe preservative, but at least the varieties that can be used are limited, which is also not preferable.

  The present invention has been made in view of the above problems, and its purpose is to withstand long-term storage of, for example, about six months to one year, and because the lag time is stable, it can be produced on an accurate schedule. It is another object of the present invention to provide a highly versatile storage method for acetic acid bacteria that can be used for vinegar production.

In order to solve the above-mentioned problems, the inventors of the present application have made extensive studies, and by diluting the fermentation liquid collected from the acetic acid fermentation liquid during the deep fermentation in advance and then performing centrifugation and dilution, the concentration is below a predetermined acetic acid concentration. And it was newly found that it can be stored for a long time by freezing it. Then, the inventor of the present application has made further studies based on this new knowledge, and completed the following problem solving means [1] to [ 5 ].

[1] By collecting a fermentation broth having an acetic acid bacteria concentration of 10 ⁷ cells / mL or more during acetic acid fermentation by a submerged culture method and adding a diluting solvent having a lower liquid temperature than that of the acetic acid bacteria fermentation solution , the acetic acid concentration A primary dilution step of diluting the acetic acid bacteria fermentation solution while reducing the temperature so that the concentration of the acetic acid bacteria fermentation solution is lower than 0.5 wt / vol% to 4 wt / vol%, and an acetic acid bacteria fermented solution reduced in temperature and diluted in the primary dilution step by adding centrifuged and diluted solvent, centrifugation acetate concentration is diluted to 0.001 wt / vol% 0.1 wt / vol% or less and acetic acid bacteria concentration of 10 ⁷ cells / mL or more And the secondary dilution step, the freezing step of freezing the acetic acid bacteria-containing solution containing the acetic acid bacteria having the acetic acid concentration and the acetic acid bacteria concentration through the centrifugation and secondary dilution steps, and the freezing through the freezing step The acetic acid bacteria-containing solution is -5 ° C or less And a storing storage step, storage method of the acetic acid bacterium and performing work within 72 hours before freezing the acetic acid bacterium-containing liquid fermentation liquor after collection in the acetic acid fermentation.

[ 2 ] The method for storing acetic acid bacteria according to the above means 1 , wherein in the centrifugation and secondary dilution step, centrifugation and dilution are simultaneously performed by pouring a dilution solvent during centrifugation.

[ 3 ] The method for storing acetic acid bacteria according to the above means 1 or 2 , wherein the bacteria cryopreservative is not added to the acetic acid bacteria-containing solution frozen in the freezing step.

[ 4 ] The method for storing acetic acid bacteria according to any one of the above means 1 to 3 , wherein in the storage step, the storage is performed for a period of 30 days or more.

[5] By collecting a fermentation broth having an acetic acid bacteria concentration of 10 ⁷ cells / mL or more during acetic acid fermentation by a submerged culture method and adding a diluting solvent having a lower liquid temperature than that of the acetic acid bacteria fermentation solution, the acetic acid concentration A primary dilution step of diluting the acetic acid bacteria fermentation solution while reducing the temperature so that the concentration of the acetic acid bacteria fermentation solution is lower than 0.5 wt / vol% to 4 wt / vol%, and an acetic acid bacteria fermented solution reduced in temperature and diluted in the primary dilution step by adding centrifuged and diluted solvent, centrifugation acetate concentration is diluted to 0.001 wt / vol% 0.1 wt / vol% or less and acetic acid bacteria concentration of 10 ⁷ cells / mL or more And the secondary dilution step, the freezing step of freezing the acetic acid bacteria-containing solution containing the acetic acid bacteria having the acetic acid concentration and the acetic acid bacteria concentration through the centrifugation and secondary dilution steps, and the freezing through the freezing step The acetic acid bacteria-containing solution is -5 ° C or less A storage step of storing, and an acetic acid fermentation step of performing acetic acid fermentation using the acetic acid bacteria stored through the storage step as an inoculum, and collecting the fermentation liquor during the acetic acid fermentation and then containing the acetic acid bacteria A method for producing vinegar, characterized in that the operation until the liquid is frozen is performed within 72 hours .

Therefore, according to the invention described in claims 1 to 4 , for example, it can withstand long-term storage of about six months to one year, and the lag time is stable, so that it can be produced on an accurate schedule, and any vinegar production A highly versatile storage method for acetic acid bacteria can be provided. Moreover, according to the manufacturing method of Claim 5 , efficient acetic acid fermentation is attained and the target vinegar can be manufactured reliably.

  Hereinafter, an embodiment of the present invention will be described in detail.

  Although it does not specifically limit as an acetic acid bacterium used by the deep culture method in this invention, For example, the acetic acid bacterium of the acetobacter genus is used. Specific examples of suitable Acetobacter acetic acid bacteria include Acetobacter aceti IFO 3281 (Acetobacter acetic IFO 3281) strain, Acetobacter aceti IFO 3283 (Acetobacter acetic IFO 3283) strain, and the like. These acetic acid bacteria are acetic acid bacteria used for fermenting production of vinegar at an acetic acid concentration of 10.0% by weight or less, but acetic acid bacteria for high acidity vinegar can also be used. Specific examples thereof include Acetobacter europaeus and Acetobacter altoacetigenes MH-24 (FERM BP-491).

  In the present invention, the acetic acid concentration means an acetic acid equivalent concentration (weight / volume%) obtained as a result of measurement and calculation as follows. That is, 5 mL of vinegar (fermented liquid) as a measurement sample was placed in a beaker, neutralized with 1N sodium hydroxide and neutralized with phenolphthalein as an indicator, and the titration (mL) obtained was multiplied by 1.2 to acetic acid. The concentration converted value was defined as acidity and expressed in%.

  In the present invention, the submerged culture method for obtaining a vinegar fermented liquid is to supply oxygen to the whole fermented liquid by actively aeration and agitation with respect to the fermented liquid in the culture apparatus. Refers to a culture method in which acetic acid bacteria are grown and fermentation is performed, and is usually compared with the surface fermentation method.

  The apparatus for performing the deep culture is not particularly limited, and a general aeration and stirring type deep fermentation apparatus can be used. A conventionally well-known means can be employ | adopted about stirring, for example, it is suitable to use stirring machines, such as a propeller and a rotor. Similarly, conventionally known means can be used for ventilation, and specific examples thereof include a method of supplying a gas such as air or oxygen through a ventilation pipe. About aeration, what is necessary is just to set suitably according to a fermentation condition. For example, the aeration rate of 0.02 to 1 vvm (aeration capacity / amount of fermentation broth / min) is supplied to the lower part of the fermentation broth, the gas is refined and diffused with a stirrer, and the dissolved oxygen in the fermentation broth is What is necessary is just to control so that it may be maintained at about 0.2-8 ppm.

  Moreover, about the fermentation format, the various methods conventionally implemented, such as a batch fermentation method, a semi-continuous fermentation method, and a two-stage fermentation method, are employable.

  And the vinegar fermented liquor during fermentation obtained by said deep culture method is diluted as follows (primary dilution).

  As the vinegar fermented liquor, it is necessary to collect a fermented liquid (that is, acetic acid produced), and more preferably a period excluding an initial stage and an end stage of fermentation (referred to as a medium stage for convenience). It is good to do. This is because the vinegar fermented liquid during this period contains a large amount of highly active acetic acid bacteria and is therefore suitable for storage. On the other hand, since the fermentation has not yet started sufficiently at the initial stage of fermentation, the number of cells is small, and at the final stage of fermentation, the activity of the cells is no longer high. Because it is not suitable to do.

The concentration of acetic acid bacteria at the time of collecting the vinegar fermented liquor during fermentation is not particularly limited and varies depending on the type of acetic acid bacteria to be used. For example, it is preferably 10 ⁶ / mL or more, particularly 10 ⁷ / It is preferable that it is mL or more. When stored for a long period of time, the number of acetic acid bacteria having activity is inevitably reduced, but if a high concentration of acetic acid bacteria is collected in advance, it becomes easier to secure the required number of acetic acid bacteria. .

  The liquid temperature of the vinegar fermented liquid during fermentation is so-called room temperature, and more specifically, 20 ° C. or higher and 40 ° C. or lower. Moreover, although the acetic acid concentration in the vinegar fermented liquor during fermentation changes with kinds of acetic acid bacteria to be used, it is generally 5 weight / volume% or more. In addition, alcohol (mainly ethyl alcohol) is usually contained in the vinegar fermented liquid during fermentation in an amount of about 0.1 to 3.0% by weight / volume.

  In measuring the alcohol concentration in the acetic acid fermentation process, it is preferable to use gas chromatography, a gas sensor, or the like, which is a highly accurate alcohol measuring device. For example, with a gas chromatography (GC-17A) manufactured by Shimadzu Corporation, a column made by GL Science (TC-WAX: 0.53 mm × 30 m) is used and kept at a detection temperature of 220 ° C. and a column temperature of 40 ° C. for 5 minutes, and 4 ° C. Examples include a method of using 1 μL of a sample under measurement conditions in which the temperature is raised to 220 ° C. under the conditions of / min and held at 220 ° C. for 10 minutes.

  The primary dilution in the present invention is carried out by mixing the collected vinegar fermented vinegar and a diluting solvent. The place where such mixing is performed may be in the fermentation apparatus for deep fermentation, or may be in a storage container prepared separately. Moreover, the form which is continuously mixed may be sufficient as the piping of the vinegar fermentation liquid branched from the fermenter and the piping to which the dilution solvent is supplied merge.

  Here, as the diluting solvent, any liquid can be used as long as it is a solution having a lower acetic acid concentration than the collected vinegar fermented liquid during fermentation. However, as a diluting solvent when it is scheduled to be used later as seed vinegar for vinegar fermentation, a liquid containing only components that are acceptable as vinegar components (for example, water, raw sugar solution, alcohol, etc.) is used. It is desirable. If a diluent solvent containing components other than water is used, the varieties of vinegar that can use the stored fungi are limited (for example, pure rice vinegar is produced using only rice and water as raw materials. In particular, it is desirable to use water. Of course, water (such as tap water) containing a slight amount of minerals that is limited to pure water may be used. Of course, the dilution solvent used in the centrifugation and secondary dilution steps is the same as described above, and water is preferable.

  And in a primary dilution process, the vinegar fermented liquor during fermentation is diluted so that an acetic acid concentration may be 4 weight / volume% or less by addition of such a dilution solvent. If dilution is not performed or the concentration of acetic acid is diluted to an extent exceeding 4% by weight / volume, acetic acid bacteria are damaged through the centrifugation step.

  At this stage, diluting more than necessary may not be preferable for the following reasons. That is, depending on the initial concentration of acetic acid and the amount to be diluted, if it is diluted more than necessary at this stage, the amount of acetic acid fermentation broth to be centrifuged increases, so the centrifugation step takes time. Resulting in. As a result, it is not preferable because it takes more time than necessary from collecting the fermentation broth during acetic acid fermentation until the acetic acid bacteria-containing liquid is frozen, and easily damages the bacteria. Therefore, it is preferable to keep the acetic acid concentration at 0.5 wt / vol% or more in the primary dilution step.

  In the primary dilution step, it is not essential to lower the liquid temperature, but a lower temperature is preferable because it is easy to suppress damage to bacteria. It is sufficient that the temperature is preferably 20 ° C. or lower, more preferably 10 ° C. or lower. The temperature at the time of dilution is preferably lower. As a method for lowering the liquid temperature, for example, primary dilution may be performed using a cooling device, or primary dilution may be performed using cold water or ice water as a dilution solvent without using a cooling device.

  The acetic acid bacteria fermentation liquor diluted in the primary dilution step is centrifuged by a centrifuge. The centrifuge used may be a continuous type or a batch type. A continuous centrifuge is a type that separates supernatant and sediment while continuously supplying a separation solution. For example, a centrifuge manufactured by Saito Centrifuge (ADS-1001CS) ) Etc. are available. In addition, the batch-type centrifuge is one in which the supply of the separation liquid and the operation of centrifugation are separated, and for example, KUBOTA 3700 from Kubota Corporation can be used. When the amount of treatment is large, a continuous type capable of collecting sediment in a short time is suitable. When the amount of processing is small, a batch system that does not require fine adjustment of processing conditions is simple and suitable.

  In the present invention, it is necessary to dilute the acetic acid concentration to 0.1 wt / vol% or less before freezing (secondary dilution). As the method, for example, the following can be employed.

  Centrifugation while allowing the primary diluted acetic acid fermentation broth to flow into a continuous centrifuge, after all the acetic acid fermentation broth has flowed in, the diluted solvent is then allowed to flow continuously into the centrifuge. The supernatant portion is mainly diluted by centrifuging for a predetermined time, and as a result, the acetic acid concentration in the centrifuge is lowered. And finally, the sediment layer accumulated in the centrifuge is taken out and used as a solution containing acetic acid bacteria before freezing.

  Alternatively, the primary diluted acetic acid bacteria fermentation broth is allowed to flow into a batch centrifuge and centrifuged for a predetermined time, and only the precipitate is collected and mixed with the diluting solvent. Thereafter, the mixture is further introduced into a batch-type centrifuge and centrifuged, and finally the sediment layer accumulated in the centrifuge is taken out to obtain a solution containing acetic acid bacteria before freezing.

  Use of a continuous centrifuge is preferable because workability is improved. In addition, the time from the primary dilution step to centrifugation can be shortened, and as a result, it is difficult to damage acetic acid bacteria in the presence of acetic acid (the number of active bacteria increases), which is preferable. .

  Note that the capacity, rotation speed, and power of the centrifuge may be appropriately selected according to the amount to be centrifuged.

  In the centrifugation and secondary dilution steps, the acetic acid bacteria-containing liquid is diluted until the acetic acid concentration is 0.1 wt / vol% or less. If the acetic acid concentration is diluted only to the extent that it exceeds 0.1% by weight / volume, the acetic acid bacteria are damaged during cryopreservation, and the number of active acetic acid bacteria decreases. As a result, when used as an inoculum for vinegar fermentation after storage for a predetermined period (for example, half a year), the lag time until the start of fermentation takes a long time (for example, 96 hours or more), which is not preferable.

  At this stage, diluting more than necessary may not be preferable for the following reasons. In other words, depending on the amount of the fermentation solution of acetic acid bacteria that undergoes centrifugation and secondary dilution, if you try to dilute more than necessary at this stage, the amount of centrifugation will increase, resulting in the amount of time until freezing. It will take a long time. Alternatively, if sufficient centrifugation is not performed so as not to spend time, the amount of the acetic acid bacteria-containing solution to be cryopreserved increases, which increases the storage facility costs and maintenance costs, which is not preferable. Therefore, it is preferable to keep the acetic acid concentration at 0.001 wt / vol% or more in the centrifugation and secondary dilution steps.

It is preferable that the acetic acid bacterium-containing liquid at the stage after the centrifugation and secondary dilution process (immediately before freezing) contains as many acetic acid bacteria as possible, specifically 10 ⁷ / mL or more, in particular It is preferable to contain 10 ⁸ pieces / mL or more. This is because if the concentration of acetic acid bacteria is high, it can be used as seed vinegar even when the storage period is long.

  In the present invention, any conventionally known method can be employed as a method for freezing the acetic acid bacteria-containing solution. When the suitable example is given, there exists a method etc. which put an acetic acid bacteria containing liquid in the screw-type cap tube for frozen storage, and leave it still in a freezer of -20-80 degreeC.

  Here, the speed when freezing is not particularly limited, but it is necessary to shorten the working time from collecting the fermentation broth during acetic acid fermentation to freezing the liquid containing acetic acid bacteria as much as possible (within 72 hours). Because of this, it is preferable to freeze as quickly as possible. The reason for setting the working time within 72 hours is to shift to a frozen and dormant state while maintaining the activity of the cells as much as possible. The time is preferably within 48 hours, more preferably within 24 hours, and the shorter the better.

  The acetic acid bacteria-containing liquid is stored at −5 ° C. or lower after freezing in order to maintain the dormant state of the acetic acid bacteria. In this case, the storage at a lower temperature can completely suppress the activity of the acetic acid bacteria being stored, and can be stored in a state where the number of active acetic acid bacteria is large when used as a seed fungus. It is preferable to store at −20 ° C. or lower, more preferably −30 ° C. or lower. However, even if stored at a lower temperature than this, the number of active acetic acid bacteria after storage does not change significantly, and the lag time when used as an inoculum does not change. Therefore, considering the equipment cost and the power cost, -20 to -30 ° C is preferable.

  Usually, in the case where microbial cells are cryopreserved in a laboratory or the like, about 15 to 50% of a microbe preservative such as glycerol or a saccharified solution is added. However, if the method of the present invention is used, long-term storage can be achieved in a state where the activity of acetic acid bacteria is high, without using such a microbe preservative. If glycerol or a saccharified solution is added to acetic acid bacteria to be stored, components other than the vinegar raw material may be mixed depending on the varieties of vinegar. That is, the varieties of vinegar that can be used as inoculum are limited.

  Examples of the microbe preservative include DMSO and skim milk, in addition to glycerol and saccharified solution.

  And the vinegar fermented liquid diluted and cooled as mentioned above is stored for a long time in the frozen state. In the case of short-term storage (for example, within 29 days), the method of Patent Document 2 can be used without applying the method of the present invention. Therefore, the method of the present invention is suitable for storing for 30 days or more, particularly for half a year to one year or more. For example, according to the storage method of the present invention, acetic acid bacteria having the optimum ability for the vinegar variety can be stored for small lot products that are produced only several times a year.

  The vinegar fermented liquid stored by the method described above maintains a suitable fungal activity even if stored for a long time. Therefore, if acetic acid fermentation is performed using this as another vinegar when performing another acetic acid fermentation, acetic acid fermentation can be started rapidly. Therefore, a desired vinegar can be efficiently manufactured by performing a deep culture method similarly to the last deep culture method. Moreover, according to the present invention, since the stored vinegar fermented liquid does not contain any fungicide cryopreservatives or other additives, the flavor, quality of vinegar, even if this is used as seed vinegar, It does not lead to a decrease in purity or the like.

  Examples that further embody the present invention will be described below, but the present invention is not limited to the examples.

[Example 1] Preparation of acetic acid bacteria for storage (Test Example 1)
(1) Preparation of acetic acid bacteria for storage by the method of the present invention

(A) 1 L of vinegar fermented liquor was prepared in advance using a fermentation tank capable of deep fermentation (5 L capacity: manufactured by Mitsuwa Richemical Co., Ltd.). This vinegar fermented broth was cultivated using Acetobacter aceti prepared from a feed solution made from rice, and the acetic acid concentration was 8.0 wt / vol%, the alcohol concentration was 0.3 vol / vol%, The liquid temperature is about 30 ° C. to 35 ° C., and the acetic acid bacteria concentration is about 10 ⁷ cells / mL.

  (B) 160 mL of such a vinegar fermented liquid is collected at the middle stage of fermentation, that is, at a stage where the activity of the fungus body is high and fermentation is actively taking place, and 20 mL of 15 ° C. water as a dilution solvent is preliminarily added thereto. 20 mL each was quickly added to the eight 50 mL centrifuge tubes (acid-resistant storage containers), and both solutions were mixed to make 40 mL. The acetic acid concentration after dilution was 4.0% w / v.

  (C) Next, the diluted vinegar fermented liquid was centrifuged at 8000 rpm for 5 minutes with a batch centrifuge (KUBOTA 3700, manufactured by Kubota Corporation). After centrifugation, all the supernatant was discarded, 40 mL of 27 ° C. water was added to suspend the precipitate, and centrifugation was again performed at 8000 rpm for 5 minutes. After centrifugation, all the supernatant was discarded, and the precipitate was suspended in 1 mL of water at 27 ° C. The acetic acid concentration after suspension was 0.1% by weight / volume.

(D) The suspended precipitation liquid was put into a 1.5 mL capacity polypropylene container having high airtightness, stored in a freezer, and cooled to −30 ° C. within 2 hours. In this way, the stored bacteria sample 1 was produced. The working time from collecting the fermentation broth during acetic acid fermentation to freezing the acetic acid bacteria-containing solution was 3 hours.
Furthermore, although it is basically the same as the above method, a sample in which the working time from collecting the fermentation broth during acetic acid fermentation to freezing the acetic acid bacteria-containing solution is 24 hours is prepared, and the stored bacteria sample 5 It was. Moreover, although it is the same as the said method fundamentally, what made the working time from collecting the fermented liquor during acetic acid fermentation until freezing the acetic acid bacteria containing liquid was produced, and the storage microbe sample 6 It was.

(2) Preparation of acetic acid bacteria for storage that was centrifuged and frozen without dilution

(A) 1 L of vinegar fermented liquor was prepared in advance using a fermentation tank capable of deep fermentation (5 L capacity: manufactured by Mitsuwa Richemical Co., Ltd.). This vinegar fermented broth was cultivated using Acetobacter aceti prepared from a feed solution made from rice, and the acetic acid concentration was 8.0 wt / vol%, the alcohol concentration was 0.3 vol / vol%, The liquid temperature is about 30 ° C. to 35 ° C., and the acetic acid bacteria concentration is about 10 ⁷ cells / mL.

  (B) 320 mL of such a vinegar fermented broth is collected at the middle stage of fermentation, that is, at a stage where the activity of the fungus body is high and fermentation is actively occurring, and eight 50 mL centrifuge tubes are used without performing primary dilution. Immediately 40 mL was put into (acid-resistant storage container).

  (C) Next, the charged vinegar fermented liquid was centrifuged at 8000 rpm for 5 minutes with a batch centrifuge (KUBOTA 3700, manufactured by Kubota Corporation). After centrifugation, all the supernatant was discarded. The acetic acid concentration of the precipitation liquid was 8.0% w / v.

  (D) And the container which collect | recovered precipitation was stored in a freezer as it was, without performing secondary dilution, and it cooled to -30 degreeC within 2 hours. In this way, the stored bacteria sample 2 was prepared. The working time from collecting the fermentation broth during acetic acid fermentation to freezing the acetic acid bacteria-containing solution was 3 hours.

(3) Centrifugation without primary dilution, and then storage acetic acid bacteria for storage as in the method of the present invention

(A) 1 L of vinegar fermented liquor was prepared in advance using a fermentation tank capable of deep fermentation (5 L capacity: manufactured by Mitsuwa Richemical Co., Ltd.). This vinegar fermented broth was cultivated using Acetobacter aceti prepared from a feed solution made from rice, and the acetic acid concentration was 8.0 wt / vol%, the alcohol concentration was 0.3 vol / vol%, The liquid temperature is about 30 ° C. to 35 ° C., and the acetic acid bacteria concentration is about 10 ⁷ cells / mL.

  (B) 320 mL of such vinegar fermented liquor is collected at the middle stage of fermentation, that is, at the stage where the activity of the bacterial cells is high and the fermentation is actively occurring, and this is collected into eight 50 mL centrifuge tubes (acid resistance is reduced). 40 mL each was quickly charged into the storage container).

  (C) Next, this vinegar fermented liquid was centrifuged at 8000 rpm for 5 minutes with a batch centrifuge (KUBOTA 3700, manufactured by Kubota Corporation). After centrifugation, all the supernatant was discarded, 40 mL of 27 ° C. water was added to suspend the precipitate, and centrifugation was again performed at about 8000 rpm for 5 minutes. After centrifugation, all the supernatant was discarded, and the precipitate was suspended in 1 mL of water at 27 ° C. The acetic acid concentration of the precipitation solution was 0.1% w / v.

  (D) The suspended precipitation liquid was put into a 1.5 mL capacity polypropylene container having high airtightness, stored in a freezer, and cooled to −30 ° C. within 2 hours. In this way, the stored bacteria sample 3 was prepared. The working time from collecting the fermentation broth during acetic acid fermentation to freezing the acetic acid bacteria-containing solution was 3 hours.

(4) Preparation of acetic acid bacteria for storage that undergoes centrifugation and secondary dilution steps after primary dilution but dilutes only to 0.2%

(A) 1 L of vinegar fermented liquor was prepared in advance using a fermentation tank capable of deep fermentation (5 L capacity: manufactured by Mitsuwa Richemical Co., Ltd.). This vinegar fermented broth was cultivated using a feed solution made from rice and Acetobacter aceti, with an acetic acid concentration of 7.0 wt / vol%, an alcohol concentration of 0.3 vol / vol%, The liquid temperature is about 30 ° C. to 35 ° C., and the acetic acid bacteria concentration is about 10 ⁷ cells / mL.

  (B) 160 mL of such vinegar fermented broth is collected at the middle stage of fermentation, that is, at the stage where the activity of the bacterial cells is high and the fermentation is actively occurring, and this is preliminarily charged with 15 ° C. water as a diluent solvent. 20 mL each was quickly added to the eight 50 mL centrifuge tubes (acid-resistant storage container), and both solutions were mixed to make 40 mL. The acetic acid concentration after dilution was 4.0% w / v.

  (C) Next, this vinegar fermented liquid was centrifuged at 8000 rpm for 5 minutes with a batch centrifuge (KUBOTA 3700, manufactured by Kubota Corporation). After centrifugation, 1 mL of the supernatant was left and discarded, and the precipitate was suspended in 16.5 mL of 27 ° C. water. The acetic acid concentration after suspension was 0.2% w / v.

  (D) The suspended precipitation liquid was put into a 1.5 mL capacity polypropylene container having high airtightness, stored in a freezer, and cooled to −30 ° C. within 2 hours. In this way, the stored bacteria sample 4 was produced. The working time from collecting the fermentation broth during acetic acid fermentation to freezing the acetic acid bacteria-containing solution was 3 hours.

[Example 2] Fermentation test using stored acetic acid bacteria (Test Example 2)
What stored the storage microbe samples 1-6 produced in Test Example 1 for half a year was used for vinegar fermentation and compared. Each stored bacteria sample was put into a fermentation tank for deep fermentation (5 L capacity: manufactured by Mitsuwa Riken Co., Ltd.) in which 3 L of a feed solution made from rice was put in advance. In addition, the amount of the stored bacteria sample input was unified with the initial number of bacteria as the amount of the stored bacteria sample prepared from the same amount of fermentation broth (15 mL of fermentation liquid) at the stage of preparing each stored bacteria sample. .

  Thereafter, the submerged fermentation of acetic acid bacteria was continuously performed for a predetermined time in accordance with a conventional method while aeration stirring was performed to produce brown rice vinegar. As the preparation liquid, a mixture of sake and seed vinegar (those from which the cells were removed) was used.

  Here, the time until the acetic acid concentration starts to rise by 0.1% after starting the culture (that is, the lag time until the start of fermentation) was measured for each of the stored bacteria samples 1-6. These results are shown in Table 1.

（試験結果）
表１によると、保管菌サンプル２〜４では、発酵開始までのラグタイムが７２時間以上となってしまうことがわかった。ここで、実用上好ましいラグタイムの程度が７２時間以内、好ましくは４８時間以内であると定義した場合、保管菌サンプル２〜４の方法では、長期にわたる保管ができないことが明らかとなった。

(Test results)
According to Table 1, it was found that in the stored bacteria samples 2 to 4, the lag time until the start of fermentation was 72 hours or more. Here, when it was defined that the practically preferred lag time was within 72 hours, preferably within 48 hours, it became clear that the method of the stored bacteria samples 2 to 4 could not be stored for a long time.

  On the other hand, the stored bacteria samples 1, 5, and 6 have a lag time of about 40 hours until the start of fermentation, and thus clearly fall within the above-mentioned preferred range and were within 48 hours. Therefore, it has been clarified that if the procedure of the stored bacteria samples 1, 5, 6 is followed, the storage is possible for a considerably long period. In addition, when the time until the acetic acid concentration started to increase by 1.5% was further investigated for the stored bacteria samples 1, 5 and 6, there was almost no difference in the stored bacteria samples 1 and 5, but in the stored bacteria sample 6 There was a tendency to increase the time.

[Example 3] Application experiment of stored bacteria obtained by the storage method of the present invention to multi-variety vinegar vinegar Sample 1 of storage bacteria of Test Example 1 was prepared using 3 L of feed liquid made from rice, brown rice, grapes and apples, respectively. It is put in a fermentation tank for deep fermentation (5 L capacity: manufactured by Mitsuwa Riken Kogyo Co., Ltd.) that has been put in advance, and the submerged fermentation of acetic acid bacteria is continuously carried out for a predetermined time according to a conventional method while stirring with aeration. , Black vinegar, grape vinegar, and apple cider vinegar. In the preparation liquid, rice vinegar is a mixture of sake made from rice and rice vinegar seed vinegar (those from which the cells have been removed), black vinegar is made from brown rice and black vinegar seed vinegar (from which the cells are removed) , Grape vinegar is a mixture of sake made from grapes and grape vinegar seed vinegar (with the cells removed), apple cider vinegar is made from apple and apple vinegar seed vinegar What mixed (thing which removed the microbial cell) was used. In addition, the amount of the stored bacteria sample input was unified with the initial number of bacteria as the amount of the stored bacteria sample prepared from the same amount of fermentation broth (15 mL of fermentation liquid) at the stage of preparing each stored bacteria sample. .

  The lag time from the storage of the stored bacteria sample to the start of fermentation is 60 hours for rice vinegar, 40 hours for black vinegar, 60 hours for grape vinegar and 40 hours for apple vinegar, all within 72 hours. It was a good result. Moreover, when the presence or absence of an excess component was analyzed, the components (amino acid, organic acid, sugar, etc.) derived from the vinegar fermented liquid (brown rice vinegar) which produced the storage microbe sample 1 were 1/100000 (1 / 100,000) or less. It is a concentration, and the content of brown rice is 0.0645 ppm or less, and it has been found that there is no problem even if the storage bacteria according to the present invention is used for producing varieties of vinegar.

[Example 4] Preparation of acetic acid bacteria for storage (a large amount of centrifugal separation treatment)
(1) A larger amount of fermentation broth than in Test Example 1 was subjected to a centrifugal separation treatment to produce stored bacteria. The procedure is described below.

(A) A vinegar fermented liquid deeply fermented in a large-scale fermentation tank of 5 kL was prepared. This vinegar fermented broth was cultivated using Acetobacter aceti prepared from a feed solution made from rice, and the acetic acid concentration was 8.0 wt / vol%, the alcohol concentration was 0.3 vol / vol%, The liquid temperature is about 30 ° C. to 35 ° C., and the acetic acid bacteria concentration is about 10 ⁷ cells / mL.

  (B) 20 L of such vinegar fermented liquor is collected at the middle stage of fermentation, that is, at the stage where the activity of the fungus body is high and the fermentation is actively occurring, and 180 L of water at 15 ° C. as a dilution solvent is collected in advance. The mixture was quickly added to the SUS tank (acid-resistant storage container), and both solutions were mixed. The acetic acid concentration of the mixed solution was 0.8% by weight / volume.

  (C) Next, the diluted vinegar fermented liquid was fed from the tank to a continuous centrifuge (ADS-1001CS, manufactured by Saito Centrifuge Co., Ltd.) at 200 L / h and centrifuged at 8000 rpm. At this time, the supernatant is automatically discharged out of the centrifuge, and the precipitate accumulates in the centrifuge. After all of the diluted vinegar fermented broth was centrifuged, 100 L of water at 15 ° C. was continuously fed to the centrifuge at 200 L / h to reduce the liquid acetic acid concentration in the centrifuge. After all 100 L of water had been put into the centrifuge, the precipitate accumulated in the centrifuge was discharged at once and recovered. The recovered liquid was about 3 L, and the acetic acid concentration was 0.1 wt / vol%. This precipitation solution was put into a 500 mL capacity polypropylene container (a container that can withstand freezing storage), stored in a freezer, and cooled to −30 ° C. within 4 hours. In this way, the stored bacteria sample 7 was prepared. The working time from collecting the fermentation broth during acetic acid fermentation to freezing the liquid containing acetic acid bacteria was 7 hours.

(2) The produced storage bacteria sample 7 which was stored for half a year was used for the actual production vinegar fermentation. That is, 2 kL of a stock solution using rice as a raw material was previously placed in a large-scale fermentation tank for deep fermentation, and the stored bacteria were put therein. The amount of the stored bacteria introduced was the amount of cells recovered from 20 L of the fermentation broth (total amount of 3 L of recovered solution).

  Thereafter, the submerged fermentation of acetic acid bacteria was continuously performed for a predetermined time in accordance with a conventional method while aeration stirring was performed to produce brown rice vinegar. As the preparation liquid, a mixture of sake made from rice and seed vinegar of brown rice vinegar (those from which cells were removed) was used.

  The lag time from the storage of the stored bacteria sample to the start of fermentation was 40 hours, which was a favorable result when used in actual production. Therefore, it has been clarified that if the procedure of the stored bacteria sample 7 is followed, it can be stored for a considerably long period of time and can be used as a starter in actual production fermentation.

(Conclusion as a whole)
Therefore, according to the present invention described above, the following effects can be obtained. That is, according to the method for fermenting acetic acid bacteria according to the present invention, a highly versatile inoculum of acetic acid bacteria that can be used for any vinegar production can be stored at a low cost for a long period (for example, about six months to one year). The acetic acid bacteria to be stored need not be prepared for each variety of vinegar and is extremely efficient. In addition, if vinegar fermentation is performed using the stored inoculum, the fermentation will start with a short lag time (for example, within 48 hours), so it is possible to respond flexibly to requests for production of various varieties and small lots of vinegar. it can. Moreover, the lag time itself is stable, and it is possible to carry out fermentation faithful to the production plan.

The technical ideas grasped by the above-described embodiment will be listed below.
(1) In any one of claims 1 to 7, the dilution solvent is a liquid containing only components allowed as vinegar components.
(2) In any one of claims 1 to 7, the dilution solvent is water.
(3) In any one of claims 1 to 7, the dilution solvent is cold water or ice water.
(4) In any one of claims 1 to 7, the vinegar fermentation liquid being fermented by the submerged culture method should be collected in a period excluding the initial stage and the final stage of fermentation.
(5) In any one of Claims 1 to 7, the vinegar fermentation liquid being fermented by the submerged culture method should be collected in a state where the growth rate of the acetic acid bacteria is stable.
(6) In any one of claims 1 to 7, a vinegar fermented liquid that is being fermented by a deep culture method is one having a concentration of acetic acid bacteria of 10 ⁷ cells / mL or more.
(7) In any one of claims 1 to 7, the alcohol concentration is diluted to 0.2% by weight or less by addition of a dilution solvent.
(8) In any one of claims 1 to 7, in the primary dilution step, the acetic acid concentration is diluted to 4 wt / vol% or less and 0.5 wt / vol% or more.
(9) In any one of claims 1 to 7, in the centrifugation and secondary dilution step, the acetic acid concentration is diluted to 0.1 wt / vol% or less and 0.001 wt / vol% or more. To do.
(10) In any one of claims 1 to 7, in the storage step, the acetic acid bacteria-containing liquid is stored at -30 ° C or higher and -20 ° C or lower.

Claims (5)

By collecting a fermentation broth having an acetic acid bacteria concentration of 10 ⁷ cells / mL or more during the acetic acid fermentation by the submerged culture method, and adding a diluting solvent having a lower temperature than that of the acetic acid bacteria fermentation broth , the acetic acid concentration becomes 0. A primary dilution step of diluting the fermentation solution of acetic acid bacteria at a low temperature so as to be 5% by weight or more and 4 % by weight or less;
By centrifuging the acetic acid bacterium fermentation solution that has been cooled and diluted in the primary dilution step and adding a diluting solvent, the acetic acid concentration is 0.001 wt / vol% or more and 0.1 wt / vol% or less and the acetic acid bacterium is added. Centrifuge and secondary dilution steps to dilute to a concentration of 10 ⁷ / mL or more ;
A freezing step of freezing the acetic acid bacteria-containing liquid containing the acetic acid bacteria having the acetic acid concentration and the acetic acid bacteria concentration through the centrifugation and secondary dilution steps;
A storage step of storing the acetic acid bacteria-containing solution frozen through the freezing step at −5 ° C. or lower;
A method for storing acetic acid bacteria, wherein the operation from collecting the fermentation broth during acetic acid fermentation to freezing the acetic acid bacteria-containing liquid is performed within 72 hours . The method for storing acetic acid bacteria according to claim 1 , wherein, in the centrifugation and secondary dilution step, centrifugation and dilution are simultaneously performed by pouring a diluent solvent at the time of centrifugation. The method for storing acetic acid bacteria according to claim 1 or 2 , wherein the acetic acid bacteria-containing solution to be frozen in the freezing step does not contain a bacteria cryopreservative. The method for storing acetic acid bacteria according to any one of claims 1 to 3 , wherein in the storage step, the product is stored for a period of 30 days or more. By collecting a fermentation broth having an acetic acid bacteria concentration of 10 ⁷ cells / mL or more during the acetic acid fermentation by the submerged culture method, and adding a diluting solvent having a lower temperature than that of the acetic acid bacteria fermentation broth, the acetic acid concentration becomes 0. A primary dilution step of diluting the fermentation solution of acetic acid bacteria at a low temperature so as to be 5% by weight or more and 4% by weight or less;
By centrifuging the acetic acid bacterium fermentation solution that has been cooled and diluted in the primary dilution step and adding a diluting solvent, the acetic acid concentration is 0.001 wt / vol% or more and 0.1 wt / vol% or less and the acetic acid bacterium is added. Centrifuge and secondary dilution steps to dilute to a concentration of 10 ⁷ / mL or more;
A freezing step of freezing the acetic acid bacteria-containing liquid containing the acetic acid bacteria having the acetic acid concentration and the acetic acid bacteria concentration through the centrifugation and secondary dilution steps;
A storage step of storing the acetic acid bacteria-containing solution frozen through the freezing step at −5 ° C. or lower;
An acetic acid fermentation process in which acetic acid fermentation is performed using the acetic acid bacteria stored through the storage process as an inoculum; and
A method for producing vinegar, characterized in that the operation from collecting the fermentation broth during acetic acid fermentation to freezing the liquid containing acetic acid bacteria is performed within 72 hours .