JP2019162080A - Method for decomposing lees of unrefined soy sauce and composition for decomposing lees of unrefined soy sauce - Google Patents

Abstract

To provide a novel method for decomposing lees of unrefined soy sauce and a new microbe and composition used therefor.SOLUTION: The method for decomposing lees of unrefined soy sauce comprises a step for bringing microbes deposited at an accession number NITE P-02649, microbes deposited at an accession number NITE P-02650, microbes deposited at an accession number NITE P-02651, or a mixture including two or more kinds selected from them, into contact with lees of unrefined soy sauce. There are also provided microbes and a composition for decomposing lees of unrefined soy sauce.SELECTED DRAWING: None

Description

  The present invention relates to a technique for treating a poorly soluble substance having a high salt concentration. More specifically, the present invention relates to a technique for decomposing soy sauce miso which is a by-product generated in the production process of soy sauce.

  Soy sauce moromi is a by-product generated in the process of producing soy sauce, and is produced in Japan about 100,000 tons per year. Soy sauce is generally manufactured by the following process: Mixing roasted and ground wheat and steamed soybeans, adding seed potatoes and fermenting them, then adding saline and fermenting for several months And get soy sauce moromi. The residue (squeezed residue) remaining after squeezing soy sauce moromi to obtain fresh soy sauce is soy moromi miso. Soy sauce moromi is a solid material that is typically plate-shaped. Soy sauce moromi has a water content of about 30% immediately after generation and contains about 7-8% of salt (sodium chloride). It is estimated that about 35% of insoluble solid content in soy sauce miso is cellulose and about 30% is protein (Non-patent Document 1).

  In order to dispose of soy sauce miso soup that is generated in large quantities in this way, some of them have been reused as livestock feed, mushroom culture medium, soil conditioner, boiler combustion aid, etc. Most of the waste is incinerated as industrial waste (Non-Patent Document 1).

  However, incinerating and discarding a large amount of soy sauce miso is a large cost for soy sauce manufacturers, and there is also a problem that the incinerator is easily damaged due to the high salt concentration as described above. Furthermore, incinerating a large amount of soy sauce miso increases the possibility of generating harmful substances including chlorine compounds such as dioxins. On the other hand, in order to reuse soy sauce miso, a desalting process, a drying process, etc. are required, and the cost is higher than the utility value. As described above, conventional methods for disposing soy sauce miso have problems from the viewpoint of cost and environmental conservation.

  Patent Document 1 describes that Aspergillus awamori (a type of mold mold) decomposed soy sauce cake. Patent Document 2 describes that symbiotic microorganisms separated from humus decomposed soy sauce cake. Non-Patent Document 1 describes that a specific filamentous fungus decomposed soy sauce cake and reduced its weight. In the experiments described in these documents, these bacteria are cultured under the condition that the soy sauce cake is diluted at least 10 times or more. Therefore, there remains doubt as to whether these bacteria can decompose soy sauce miso in the actual high salt concentration environment of soy sauce miso. In addition, in the experiments described in these documents, bacteria are cultured at 30 ° C., and it is not shown that these bacteria can degrade soy sauce miso even under low temperature conditions.

JP-A-10-99072 JP 2007-181436 A

National Food Research Institute No. 76, 33-38 (2012)

  As mentioned above, certain microorganisms are known to be able to degrade soy sauce moromi at least under conditions where the salt concentration is diluted, but the number of such known microorganisms is very limited. Is the current situation. It is meaningful to find more diverse soy sauce miso-degrading microorganisms, since different microorganisms can exhibit different degrading activities in different ambient environments (eg salt concentration, temperature and humidity).

  The present invention provides a novel method for degrading soy sauce miso and novel microorganisms and compositions that can be used for the method. A method for detecting such microorganisms is also provided.

The present invention includes at least the following embodiments.
[1]
Microorganism deposited with accession number NITE P-02649, microorganism deposited with accession number NITE P-02650, microorganism deposited with accession number NITE P-02651, or a mixture comprising two or more of them A method of decomposing soy sauce moromi miso, which comprises contacting the syrup with soy sauce miso miso.
[2]
The method according to [1], comprising performing the contact at a temperature of 1 to 10 ° C.
[3]
Microorganism deposited with accession number NITE P-02649.
[4]
Microorganism deposited with accession number NITE P-02650.
[5]
Microorganism deposited with accession number NITE P-02651.
[6]
[3] A composition for decomposing soy sauce miso soup comprising one or more microorganisms according to any one of [5] to [5].
[7]
Prepare a uniform aqueous suspension containing 1.6-2.4% (w / v) soy sauce miso, leave it for 20-40 minutes to settle the particles, and remove the supernatant. A mixture is prepared by combining 0.9 to 1.1 volumes of water, molten agar, and 3-20% (w / v) equivalent NaCl with the supernatant. Is poured into a culture container at a thickness of 1.2 to 2.8 mm to solidify the agar, soy sauce moromi miso agar medium,
A method of planting and culturing test microorganisms and detecting soy sauce miso-degrading microorganisms based on halo formation.

  Embodiments of the present invention include microorganisms capable of degrading soy sauce moromi in a high salt (NaCl) concentration environment of at least several percent, compositions comprising such microorganisms, methods for detecting such microorganisms, and A method of decomposing soy sauce moromi using such microorganisms is provided.

  According to the present invention, soy sauce moromi miso that contains salt and water can be decomposed and reduced at low cost.

FIG. 1 shows a soy sauce miso agar medium in which a halo is formed by soy sauce miso degrading bacteria. The arrow points near the outer edge of the halo. FIG. 2 shows the results of testing the soy sauce miso miso resolution in a liquid medium at 30 ° C. (a) and 4 ° C. (b). FIG. 3 shows the results of a salt tolerance test of soy sauce miso-degrading bacteria. The cells were cultured in a liquid medium of the displayed different NaCl concentrations, proliferation was measured degree based on the OD _660.

  Soy sauce moromi is a complex mixture of a wide variety of substances and is characterized by high salt concentrations. A method for efficiently detecting microorganisms capable of decomposing such soy sauce miso has not been established. Therefore, it has not been understood how much such microorganisms (especially bacteria) are present and how they can be identified and isolated. The inventor has developed a method for efficiently detecting microorganisms capable of degrading soy sauce moromi miso in a high salt concentration environment after trial and error. Using this technique, we screened a large number of microorganism specimens and succeeded in identifying and isolating microorganisms that can degrade soy sauce miso in a high salt concentration environment.

  In one aspect, the present disclosure provides a detection method and a screening method capable of efficiently detecting a microorganism capable of degrading soy sauce miso in a high salt concentration environment. First, this embodiment will be described.

  In addition, in this specification, "-" which shows a numerical range means that the numerical value described before and behind that is included as a lower limit and an upper limit, respectively. When possible numerical ranges such as “A to B” and “C to D” are separately described, a numerical range in which one lower limit or upper limit is combined with the other upper limit or lower limit (for example, “A to D "" C-B ") is also possible.

  In the present embodiment, the release of soy sauce miso-degrading activity by microorganisms is detected using a halo formed on an agar medium as an index, and soy sauce miso-degrading microorganisms are detected.

  First, soy sauce miso is combined with water and pulverized to obtain a suspension. Or you may pulverize soy sauce miso first and combine with water to obtain a suspension. The pulverization means that the solid content is crushed into small pieces, and does not necessarily mean that the powder is not suspended in the liquid. The soy sauce miso can be pulverized in a pulverizer such as a food processor or a mixer. If a fluid and homogeneous suspension is observed in the grinder, it is considered that sufficient grinding has been achieved. For example, sufficient grinding can be achieved by repeating the grinding process for 1 minute four times with a food processor. The soy sauce moromi miso concentration in this uniform suspension is preferably 1.6 to 2.4% (w / v), more preferably 1.8 to 2.2% (w / v). -2.1% (w / v) is most preferred. It was found that when the concentration of soy sauce moromi at this stage was outside the above range, it was difficult to observe a clear halo in the final agar medium.

  Once a uniform suspension is obtained, it is then allowed to settle to settle the particles. This eliminates excessive particles and enables highly sensitive detection. The standing time is preferably 20 to 40 minutes, more preferably 22 to 35 minutes, further preferably 25 to 32 minutes, and most preferably 28 to 30 minutes. If the standing time is outside these ranges, it will be difficult to observe a clear halo in the final agar medium. After the standing time, the supernatant (supernatant) is collected and used as a material for the agar medium. This supernatant contains soy sauce miso ingredients with an appropriate particle size. It is preferable to collect as a supernatant a liquid located above the middle line between the bottom of the liquid after standing and the liquid surface.

  The collected supernatant is combined with 0.9 to 1.1 times the volume of water, NaCl, and molten agar to obtain a mixture. By adding NaCl, it is possible to reproduce the high salt concentration that the actual soy sauce miso or its high concentration slurry will have. The amount of NaCl added here is preferably 3 to 20% (w / v) equivalent, more preferably 4 to 15% (w / v) equivalent, more preferably 5 to 12% (w / v). v) Equivalent amount, more preferably 6 to 10% (w / v) equivalent amount. As used herein, “equivalent amount” means that a slight amount of NaCl derived from diluted soy sauce moromi is ignored, and an amount of NaCl corresponding to the final concentration represented by these values is separately added to the agar medium. Means.

  The amount of agar added can be appropriately determined by those skilled in the art based on ordinary knowledge. The amount of agar added (final concentration) is, for example, 0.5 to 3% (w / v), preferably 1 to 2.5% (w / v), and particularly preferably 1.8 to 2.2. % (W / v). It is efficient and preferable to dissolve agar in water 0.9 to 1.1 times the volume of the supernatant in advance and combine it with the supernatant. Dissolving agar in water can be achieved, for example, by heating water to which agar has been added to 85 ° C. or higher. Although heating can be suitably achieved by an autoclave, possible heating means are not limited to this. It is preferable to mix the supernatant and the agar solution at a temperature of about 60 ° C. so that the agar coagulation does not start immediately after the combination with the supernatant.

  Depending on the particular application, embodiments are contemplated where additives other than those described above, such as additional nutrients, antibiotics, etc., are added to the agar medium.

  A mixed solution containing at least water, soy sauce miso, NaCl, and molten agar is poured into a culture vessel to solidify the agar, thereby obtaining a soy sauce miso agar medium. It has been found that the thickness of the agar medium is also important for observing the halo clearly. A thickness corresponding to an agar medium obtained by pouring 8 to 17 mL of the above mixed solution into a 90 mm diameter circular petri dish is preferred, a thickness corresponding to an agar medium obtained by pouring 9 to 16 ml is more preferred, and 10 to 15 ml A thickness corresponding to the agar medium obtained by pouring is particularly preferred. The thickness of the agar medium is, for example, 1.2 to 2.8 mm, preferably 1.4 to 2.6 mm, more preferably 1.5 to 2.5 mm, and particularly preferably 1.5 to 2.5 mm. 1.8 mm.

  A preferred embodiment of the agar medium is a uniform aqueous suspension containing 1.6-2.4% (w / v) soy sauce moromi miso, which is allowed to stand for 20-40 minutes to produce particles. The supernatant is removed, and a liquid mixture is prepared by combining 0.9 to 1.1 times the volume of water, molten agar, and 3-20% (w / v) NaCl equivalent to the supernatant. Is prepared by pouring into a culture vessel at a thickness of 1.2 to 2.8 mm and cooling. It is preferable that the culture vessel is transparent because halos can be more easily observed.

  By culturing and cultivating test microorganisms to determine the presence or absence of soy sauce mash miso decomposition ability in this agar medium, the soy sauce mash miso decomposition microorganisms can be detected. It is preferable to plant (inoculate) by puncture. The appropriate culture temperature may vary depending on the specific microorganism type and should be appropriately determined by those skilled in the art. For example, the culture can be performed at 4-38 ° C, 10-37 ° C, 20-32 ° C, or 25-30 ° C. Alternatively, by culturing at 1 to 10 ° C., 2 to 7 ° C., or 3 to 5 ° C., microorganisms having soy sauce miso-degrading activity at low temperatures can be detected. The culture time may also vary depending on the specific microorganism growth rate and degradation enzyme release rate, for example, 24 hours or more, 48 hours or more, 1 week or more, or 4 weeks or more. It may take more than 5 weeks for the halo to appear, but such examples are rare.

  By planting and culturing a plurality of candidate microorganisms whose soy sauce miso degrading ability is unknown in the soy sauce miso miso agar medium as described above, screening for identifying and isolating soy sauce miso miso decomposed microorganisms can be performed. Understood.

  Candidate microorganisms that have been confirmed to form halo in soy sauce moromi miso agar medium are further planted in a soy sauce moromi miso-containing liquid medium and cultured to observe the extent to which the turbidity of the liquid medium is reduced, which is particularly high in a liquid environment. It is also possible to identify microbial strains having soy sauce moromi miso resolution. In this case, from the viewpoint of making it easy to visually confirm the decrease in turbidity, the liquid medium in this case is the same volume of the supernatant collected after allowing the soy sauce moromi miso suspension to settle as described above. Those diluted with (0.9 to 1.1 times volume) water, that is, those obtained by removing agar from the composition of the soy sauce moromi miso agar medium are preferred. The salt concentration can also be adjusted in the same manner as in the soy sauce moromi miso agar medium. It is preferable to use only soy sauce miso as a carbon source.

  In this specification, the liquid environment is a fluid environment that is distinguished from a solid environment such as an agar medium, and means an aqueous suspension (including slurry) containing soy sauce miso.

  The inventor conducted a screening experiment using the above-mentioned soy sauce moromi miso agar medium, and identified and isolated a novel soy sauce moromi miso-degrading bacterium. Accordingly, in another aspect, the disclosure provides a microorganism deposited with accession number NITE P-02649, a microorganism deposited with accession number NITE P-02650, and a microorganism deposited with accession number NITE P-02651 I will provide a.

  These microorganisms were issued on March 16, 2018 at the National Institute for Product Evaluation and Technology (NITE) Patent Microorganism Depositary Center (NPMD) (2-5-8 Kazusa Kamashichi, Kisarazu City, Chiba Prefecture) under the accession number NITE. Deposited under P-02649, NITE P-02650, and NITE P-02651. These microorganisms are sometimes referred to herein as 1a, 1b, and 10a, respectively.

  The microorganism with the accession number NITE P-02649 (strain 1a) is a bacterium belonging to the genus Salinicola, based on the 16S rRNA gene sequence, and is most closely related to Salinicola salarius (also called Halomonas salaria) among known microorganisms. It can be seen. The microorganism having the accession number NITE P-02650 (strain 1b) and the microorganism having the accession number NITE P-02651 (strain 10a) are bacteria belonging to the genus Cobetia based on the 16S rRNA gene sequence. It seems to be most closely related to marina (also called Halomonas marina).

  There is also provided a composition for decomposing soy sauce miso, containing any one or more of these microorganisms. This composition may contain substances other than the microorganism, such as water. Further examples of substances that can be included in the composition for soy sauce miso decomposition include glycerol, salt, sugar, protein, protein hydrolysate, pH buffer, agar, yeast extract, and other microorganisms. It is not limited to. Since the microorganism disclosed in the present specification has the ability to decompose at least celluloses and insoluble proteins under high salt conditions, it is also useful in applications that decompose highly salt-insoluble substances other than soy sauce miso. obtain.

  In a further aspect, the disclosure provides a microorganism deposited with accession number NITE P-02649, a microorganism deposited with accession number NITE P-02650, a microorganism deposited with accession number NITE P-02651, or a Provided is a method for decomposing soy sauce miso, comprising contacting a mixture containing two or more of them with soy sauce miso.

  The temperature at which the microorganism and the soy sauce miso are brought into contact can be determined by those skilled in the art based on ordinary knowledge, and is, for example, 1 to 38 ° C.

  One embodiment preferably comprises contacting the microorganism with soy sauce miso at a temperature of 10-38 ° C. This contact temperature is more preferably 15 to 37 ° C, more preferably 20 to 35 ° C, still more preferably 25 to 33 ° C, and particularly preferably 28 to 32 ° C. These temperatures may be particularly suitable when strains 1a or 10a are used.

  Another embodiment preferably comprises contacting the microorganism with soy sauce miso at a temperature of 1-10 ° C. This contact temperature is more preferably 2 to 8 ° C, more preferably 3 to 5 ° C. These temperatures may be particularly suitable when using strains 1a or 1b. By using such a low temperature, for example, heating is unnecessary even in winter, which is preferable. In addition, by using such low temperature conditions, the soy sauce miso-degrading bacterium according to this embodiment becomes a dominant species and the state in which decomposition fermentation is promoted is obtained, and abnormal fermentation (so-called rot) due to contamination with various bacteria is prevented. This is preferable.

  It is not necessary to fix the temperature at which the microorganism and soy sauce moromi are brought into contact, and the contact may be performed while the temperature range varies. For example, when making contact outdoors, it is understood that the contact temperature may vary with the outside air temperature.

  The contact between the microorganism and the soy sauce moromi can be achieved by, for example, sprinkling a suspension of the microorganism on the soy moromi miso as a solid. In another embodiment, the contact between the microorganism and soy sauce moromi is performed in a liquid environment. In this case, soy sauce moromi is in a fluid state suspended in water, and microorganisms coexist in the suspension. The suspension may be agitated or shaken to promote degradation.

  The time for contacting the microorganism with the soy sauce moromi miso should be appropriately determined by a person skilled in the art according to the amount of soy sauce moromi miso to be decomposed, the required degree of degradation, and the like. It can be 4 weeks or more, or 2 months or more.

  As the soy sauce miso soup in the method of the present embodiment, a soy sauce miso paste that contains salt and water when it is generated as a by-product of soy sauce production can be suitably used. However, it is not excluded to use an embodiment that has undergone a certain degree of desalting or dehydration treatment, or conversely, that has undergone a treatment in which salt or water is further added.

  When the microorganism and soy sauce miso are brought into contact with each other, it is not necessary to change the pH naturally provided by the soy sauce miso miso. A pH of about 4.5 to 8.0 is considered appropriate, but is not necessarily limited thereto.

  The microorganism to be contacted with soy sauce miso may be provided in the form of the composition for decomposing soy sauce miso according to the above-described embodiment.

  EXAMPLES Hereinafter, although an Example is shown and this invention is demonstrated further in detail, this invention is not limited to the aspect of an Example.

[Development of soy sauce miso miso agar medium capable of detecting soy sauce miso-degrading bacteria]
The inventor said, “If soy sauce moromi paste-degrading bacteria exist, when cultured on an agar medium containing soy sauce moromi paste, the mash miso component is partially decomposed and the bacteria can be visually identified. I made the hypothesis. Therefore, first, preparation conditions of soy sauce miso agar medium that can be used for such an identification method were examined.

  Specifically, 200 mL of water (distilled water) is added to soy sauce moromi, pulverized with a food processor (1 minute × 4), uniformly suspended, and the suspension is allowed to stand to precipitate particles. Then, 100 mL of the supernatant was taken out with an electric pipettor, and the same volume of water was added, the NaCl concentration was adjusted, and molten agar was added so that the final agar concentration was 2% (w / v). More specifically, (A) 20 g of NaCl was dissolved in 100 mL of the supernatant to adjust the pH to 7, and (B) 4 g of agar was added to 100 mL of water and heated by autoclave (121 ° C., 20 Minutes), and then, both the A and B liquids, each adjusted to 60 ° C. using a constant temperature water bath, were mixed in a clean bench. This mixed solution in a state in which agar was dissolved was dispensed into a petri dish with an electric pipetter, and then cooled to room temperature to solidify the agar, thereby preparing a soy sauce moromi miso agar medium.

  It was considered that the detection sensitivity of the experiment could be improved by removing the excessive solid content by allowing the particles to settle. In addition, since salt derived from soy sauce miso miso itself is significantly diluted with water, in order to maintain a high salt concentration condition in this experiment, 10% (w / V) A considerable amount of NaCl was further added. This is the “adjustment of NaCl concentration” described above.

  In the above experiments, (1) the concentration of soy sauce moromi miso suspended in water, (2) the time for sedimentation of particles (stationary time), and (3) the amount dispensed to the petri dish (ie the thickness of the agar medium) )) And the conditions were examined.

  In this preliminary experiment, a salt-tolerant cellulase-producing filamentous fungus (fungus, No. 18-5 strain) previously identified and isolated by the inventors in another study was used as a control bacterium. That is, the control bacterium was inoculated and cultured on the agar medium prepared as described above.

  As a result, (1) the concentration of soy sauce moromi miso suspended in water was 2% (w / v), (2) the time for settling the particles (standing time) was 30 minutes, and (3) the diameter was 90 mm. Under conditions where 10 mL or 15 mL of medium is dispensed into a circular petri dish (medium thickness of about 1.6 mm or about 2.4 mm), it is clearly distinguishable from the background of the agar medium around the inoculated filamentous fungal colonies A halo was formed (FIG. 1). In contrast, (1) when the concentration of soy sauce miso suspended in water is 1% (w / v), (2) when the particle is allowed to settle (stationary time) is 60 minutes, or ( 3) When the amount of the medium dispensed into the petri dish was 20 mL, the visual contrast between the background of the agar medium and the halo was not sufficiently obtained. With regard to the item (2), the standing time was further divided into 20, 25, 30, 35, and 40 minutes, and a more detailed examination was performed. The halo was observed most clearly at the standing time of 30 minutes. I was able to. Overall, (1) the initial soy sauce moromi miso concentration 2% (w / v), (2) the sedimentation time 30 minutes, and (3) the dispensed volume 10 mL should be the optimum conditions for clearly detecting halo formation. Was found.

[Identification and isolation of soy sauce miso-degrading bacteria]
Using the soy sauce moromi miso agar medium under the optimum conditions described above, a number of microorganism sources were screened. Screened microbial sources are as follows.
(A) Six salt-tolerant cellulase-producing bacteria previously isolated in another study by the inventors. These are strains isolated using as an index the ability to degrade CMC (carboxymethylcellulose) under high salt concentration conditions. Since these are already isolated strains, they are hereinafter referred to as “isolates”.
(A) 130 source samples stored in the inventors' laboratory. These are a variety of specimens that are thought to potentially contain salt tolerant microorganisms, such as commercial salt, salted foods, marine products, and high salt soils.
(C) 668 commercially available salt samples stored elsewhere. These are commercial products such as salt collected all over the world, which are also considered to contain potentially salt-tolerant microorganisms.

  First, in the first experiment using the above-mentioned 6 isolates, inoculation was performed by dropping the bacterial suspension and inoculating by puncture with a toothpick for comparison. The culture was performed at 30 ° C. As a result, it was difficult to confirm halo in inoculation by dripping, but halo formation was clearly confirmed in all 6 strains by puncture culture with toothpick. Therefore, at least for these specific bacteria, puncture culture was considered suitable for confirming halo formation. In this regard, it was considered that puncture culture may cause the cells to be denser than dripping and increase the decomposition rate of moromi. In addition, it was considered that these isolates were facultative anaerobic bacteria, and the miso soup decomposition in the lower layer of the culture medium (the deeply punctured part) was promoted.

  Thus, 6 isolates were selected as soy sauce miso-degrading bacteria candidates based on the standard of halo formation on an agar medium. However, as described later, CMC-degrading bacteria tend to have a low ability to decompose soy sauce moromi, and these 6 isolates were not included in the subsequent analysis.

  Next, 130 samples of the separation source (A) were applied onto the agar medium and cultured at 30 ° C. As a result, 20 strains were isolated from 130 samples. When they were inoculated and cultured in a soy sauce moromi miso agar medium by puncture, a halo was formed around the colonies of a total of 11 strains (1a, 1b, 2, 5a, 5b, 6b, 7b, 8, 9, 10a, 15a) Was confirmed. The formation of halo in addition to colony formation suggests that the soy sauce miso degrading activity is released and diffused outside the cell, and thus shows an efficient soy sauce miso decomposing ability. .

  The soy sauce miso-degrading bacterium that has been found in the prior art exhibits a degrading activity at a relatively high temperature of about 30 ° C. However, in winter, for example, a heating facility is required to achieve a temperature of 30 ° C., and the cost for the soy sauce miso cracking process may increase. Further, at a relatively high temperature of about 30 ° C., competition with other bacteria is likely to occur. Therefore, a fungus exhibiting soy sauce miso-degrading activity even at low temperatures corresponding to non-heating conditions or non-heating conditions in winter can be more useful.

  Then, when the culture experiment by puncture inoculation was repeated at a low temperature of 4 ° C., halo formation was confirmed in 9 strains (1a, 1b, 2, 5a, 7b, 8, 9, 10a, 15a). These 9 strains were strains in which halo formation could be confirmed even at 30 ° C., and it was shown that they have the ability to decompose soy sauce miso even at low temperatures around 4 ° C.

  Six strains were isolated from the salt (668) sample of (c) above, and three of them were able to form colonies on the soy sauce miso agar medium, but halo formation could not be confirmed.

[Soy sauce miso cracking activity test using liquid medium]
In order to enhance the versatility of soy sauce miso decomposition treatment, it is desirable to obtain a bacterium that exhibits high soy sauce miso miso resolution not only in a solid environment such as an agar medium but also in a liquid environment. Therefore, in the next experiment, the candidate strain was inoculated into a liquid medium corresponding to the composition obtained by removing the agar from the soy sauce moromi miso agar medium under the optimum conditions described above, and cultured with shaking at 30 ° C. This liquid medium is prepared by mixing 100 mL of the supernatant obtained after leaving 200 mL of the 2% soy sauce moromi miso suspension for 30 minutes as described above with 100 mL of saline containing 20 g of NaCl. . The results in FIG. 2a showed that strains 1a and 10a caused a clear turbidity reduction of the liquid medium compared to the uninoculated control (blank). This indicates that the suspended solid particles of soy sauce moromi are reduced (possibly metabolized to low molecules such as carbon dioxide). That is, it was confirmed that these strains have the ability to decompose soy sauce moromi in a liquid environment relatively efficiently.

  Experiments using the same liquid medium were also performed at a low temperature of 4 ° C. The results in FIG. 2b showed that strains 1a and 1b caused a clear turbidity reduction of the liquid medium compared to the uninoculated control (blank). That is, it was confirmed that these strains have the ability to decompose soy sauce miso relatively efficiently even in a low temperature liquid environment of around 4 ° C.

[Enzyme activity test of candidate strains]
Soy sauce miso is a mixture of various substances, including cellulose, other polysaccharides, and proteins, and the candidate strain identified in the above experiment degrades one or more of these substances. It is considered that the enzyme activity is released. In order to analyze what kind of enzyme activity contributes to the soy sauce miso cracking ability of these bacteria, an experiment using an agar medium with different compositions was performed.

  The composition of the three types of agar medium used in this experiment is shown in Table 1 below. The balance of the composition is water.

  That is, the candidate strain was inoculated on a high salt concentration agar medium containing 1% CMC (carboxymethylcellulose), 10% acid-treated cellulose, or 1% skim milk. After static culture at 30 ° C. or 4 ° C., the presence or absence of halo formation was observed. For CMC agar and acid-treated cellulose agar, halos were visualized by Congo red staining. Halo formation on CMC medium suggests endoglucanase activity, halo formation on acid-treated cellulose medium indicates cellobiohydrolase activity, and halo formation on skim milk medium suggests release of protease activity.

  The results of halo formation on different agar media are summarized in Table 2 below. In the table, “+” means that halo formation was observed, and “−” means that halo formation was not observed.

  These results indicate that all candidate strains having soy sauce mash miso resolution provide activity for decomposing acid-treated cellulose and skim milk. Therefore, it is suggested that at least cellobiohydrolase and protease may contribute to soy sauce miso resolution.

  It has also been found that candidate strains with soy sauce moromi resolution lack CMC resolution in most cases. This suggests that the relevance between soy sauce miso resolution and endoglucanase is low. It also indicates that it would be difficult to find soy sauce miso-degrading bacteria using CMC degrading activity or endoglucanase activity as an index. That is, it is understood that just because a salt-tolerant cellulase is expressed (even if it is a salt-tolerant or halophilic bacterium), it cannot be used as a soy sauce miso-degrading bacterium.

  On the other hand, strains 15b and 15c showed both acid-treated cellulose-decomposing activity and skim milk-degrading activity, and also CMC-degrading activity, but none of these two strains was found to have soy sauce miso-degrading ability. It was. This suggests the complexity of the mechanism of soy sauce miso decomposition, such as the requirements for enzyme production and interaction with other enzymes, and it is difficult to identify useful strains using individual enzyme activity as an indicator. And the usefulness of screening using soy sauce moromi miso itself as shown in this application.

[Salt tolerance test]
In order to examine the salt tolerance of strains 1a, 1b and 10a in more detail, the culture was carried out in liquid medium with different salt (NaCl) concentrations (0%, 5%, 10%, 15% or 20%). It was. This liquid medium is a standard culture medium that does not contain soy sauce miso soup, which is obtained by removing agar from the CMC agar medium. More specifically, after pre-cultured in a 10% NaCl-1% CMC liquid medium, 1% (v / v) inoculated into a 24 mm diameter test tube containing 10 ml of the same medium having different salt concentrations, and cultured with shaking. Bacterial growth was measured based on the absorbance at 660 nm (OD ₆₆₀ ). The results are shown in FIG.

  The strain 1a showed growth at a salt concentration of 5 to 20% at 30 ° C. and 4 ° C., and the optimum salt concentration was 10%. The 10a strain showed growth at a salt concentration of 0 to 20% at 30 ° C., and the optimum salt concentration was 5%. Strain 1b showed growth at a salt concentration of 0-20% at 4 ° C., with an optimum salt concentration of 5%. Both strains showed not only halotolerant bacteria but also halophilic bacteria because the growth rate decreased at a salt concentration of 0%. All the strains were confirmed to be particularly suitable for growth in a soy sauce moromi miso environment with a salt concentration of 5 to 10% and a high growth rate.

[Species identification]
In order to systematically identify the bacterial strains of strains 1a, 1b, and 10a, the full-length 16S rRNA gene was sequenced according to a conventional method. When a homologous sequence in a known gene sequence database was searched by NCBI BLAST using the determined nucleic acid sequence as a query, strain 1a was most closely related to Salinicola salarius (Halomonas salaria) (ie, having the highest sequence homology). ) 1b and 10a strains were found to be most closely related to Cobetia marina (Halomonas marina), respectively. From the high homology of these 16S rRNA gene sequences, the strain 1a was determined to be a bacterium belonging to the genus Salinicola, and the strains 1b and 10a were determined to be a bacterium belonging to the genus Cobetia. For reference, information about Salinicola salarius (Halomonas salaria) and Cobetia marina (Halomonas marina) described in the public literature is shown in Table 3 below.

  Despite the high homology of the 16S rRNA gene, they are known based on at least the growth temperature of Salinicola salarius (above 10 ° C.) and the lack of casein hydrolytic resolution of Cobetia marina (casein is the main protein of skim milk). It is understood that the bacteria described in the literature are not identical to the strains isolated in this application.

  INDUSTRIAL APPLICABILITY The present invention can be useful at least in the industrial field related to soy sauce production and in the field of decomposition treatment of food production by-products and other high-salt concentration hardly soluble substances.

Claims (7)

  Microorganism deposited with accession number NITE P-02649, microorganism deposited with accession number NITE P-02650, microorganism deposited with accession number NITE P-02651, or a mixture comprising two or more of them A method of decomposing soy sauce moromi miso, which comprises contacting the bean paste with soy sauce moromi miso.   The method according to claim 1, comprising performing the contacting at a temperature of 1 to 10 ° C.   Microorganism deposited with accession number NITE P-02649.   Microorganism deposited with accession number NITE P-02650.   Microorganism deposited with accession number NITE P-02651.   A composition for decomposing soy sauce miso, comprising one or more of the microorganisms according to claim 3. Prepare a uniform aqueous suspension containing 1.6-2.4% (w / v) soy sauce miso, leave it for 20-40 minutes to settle the particles, and remove the supernatant. A mixture is prepared by combining 0.9 to 1.1 volumes of water, molten agar, and 3-20% (w / v) equivalent NaCl with the supernatant. Is poured into a culture container at a thickness of 1.2 to 2.8 mm to solidify the agar, soy sauce moromi miso agar medium,
A method of planting and culturing test microorganisms and detecting soy sauce miso-degrading microorganisms based on halo formation.