JP3027460B2 - Phage-resistant lactic acid bacteria and method for producing soy sauce using the same - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to an improved method for producing phage-resistant lactic acid bacteria and soy sauce in which phage-resistant soy sauce lactic acid bacteria are added to koji and / or moromi. The present invention relates to a method for producing soy sauce that stably obtains high-quality soy sauce.

[0002]

2. Description of the Related Art In fermentation of soy sauce moromi, soy sauce lactic acid bacteria [Pediococcus halophilus (Pediococcus halophilus)
us)] produces lactic acid from sugar to lower the pH of moromi to a desirable value, and also plays an important role in soy sauce quality such as decomposition conversion of amino acids, decomposition generation of organic acids, and reduction. Therefore, in order to produce high-quality soy sauce balanced in flavor, it is necessary to stably and surely perform lactic acid fermentation by soy sauce lactic acid bacteria having excellent properties.

[0003] In the traditional soy sauce production method, moromi fermentation is usually performed in an open system, and artificial addition of microorganisms such as lactic acid bacteria is hardly performed, and all lactic acid bacteria active in moromi are brewed. It was left to the natural contamination of bacteria (natural flora) that naturally settled in places, facilities, and equipment. This natural flora is very diverse, and naturally contained lactic acid bacteria may contain bacteria that are not always desirable in terms of the quality of soy sauce. In addition, since the contents change depending on the region or with the lapse of time, there are drawbacks such as a difference in product quality depending on the brewing place, and a failure to obtain soy sauce having a constant quality throughout the year.

[0004] From such a point of view, with the aim of producing brewed soy sauce of consistently constant quality regardless of regional or time restrictions, soy sauce of a pre-selected or especially breeding type having excellent properties. It has been widely used to artificially add lactic acid bacteria to koji and / or moromi to stably perform lactic acid fermentation in a preparation process.

[0005] However, in the usual method for producing soy sauce, since non-sterilized koji is used and moromi is managed in an open container microbiologically, lactic acid fermentation of soy sauce moromi cannot be specified. It is very difficult to maintain stable lactic acid fermentation by allowing stable growth of the added lactic acid bacteria over a long period of time.

[0006] The inventors of the present invention have conducted intensive studies for the purpose of always stably growing the added soy sauce lactic acid bacteria and ensuring lactic acid fermentation with a constant content.
From the moromi taste of soy sauce, various types of phage that infect and lyse soy sauce lactic acid bacteria are detected, and lactic acid bacteria added by these phages undergo lysis or growth inhibition, which is one of the major causes of lactic acid fermentation trouble. And found that resolving this factor would achieve the above objectives.
First, in a method for producing soy sauce in which soy sauce lactic acid bacteria are added to koji and / or moromi, a phage-resistant soy sauce lactic acid bacterium is used as the soy sauce lactic acid bacteria (see JP-A-63-216449). Was developed.

[0007]

In this method, phage-resistant soy sauce lactic acid bacteria are constructed by contacting soy sauce lactic acid bacteria with phage and isolating phage-resistant bacteria by spontaneous mutation, or subjecting the soy sauce lactic acid bacteria to mutation treatment. Then, the phage is contacted with the phage, and the other strains are the same as the parent strain and have only strong phage resistance. That is, the phage has a high concentration of phage-sensitive soy sauce lactic acid bacteria (parent strain) that cannot grow at all. It grows and forms a colony when agar plates are cultured even in the presence of agar.] A resistant strain is isolated.

However, the phage-resistant soy sauce lactic acid bacterium thus obtained may be a so-called lysogen which has a phage gene incorporated into the gene of the lactic acid bacterium. Is expressed and phage may be released into the culture solution. In such a case, it is difficult to continue the subsequent lactic acid fermentation. That is, the phage-resistant soy sauce lactic acid bacteria constructed by this method had a defect of lacking reliability.

[0009]

Accordingly, the present inventors have
A phage-resistant soy sauce lactic acid bacterium with high reliability was produced, and intensive studies were conducted to develop a method for producing a soy sauce that stably obtains high-quality soy sauce using the lactic acid bacterium. As a result, the present invention was finally completed.

That is, according to the present invention, a master plate on which a large number of lactic acid bacteria colonies have been formed is gently pressed against the surface of a sterilized carrier, the colonies on the master plate are printed, and then a phage suspension is applied to this surface in advance. The medium was gently pressed, the colonies printed on the surface of the carrier were copied to obtain replica plates, and after cultivation, the colonies formed on the master plates and the replica plates were compared, and the colonies formed on the replica plates were compared. And a phage-resistant lactic acid bacterium obtained by separating a colony on the master plate corresponding to the phage-resistant soy sauce lactic acid bacterium added to koji and / or moromi. A method for producing soy sauce, comprising using the phage-resistant lactic acid bacteria as soy sauce lactic acid bacteria.

Hereinafter, the present invention will be described in detail. The phage-resistant soy sauce lactic acid bacterium according to the present invention is a lactic acid bacterium which infects soy sauce lactic acid bacterium and which is resistant to phage which lyses the lactic acid bacterium, and can be obtained by the following method.

1. [Phage Separation] The phage of soy sauce lactic acid bacteria can be separated according to a medium and a method used for ordinary detection and separation of phage except that the phage contains 10 to 20% of sodium chloride (1970/1975).
Published by Asakura Shoten on October 10th, edited by Sadajiro Uemura and Arai Arai "Fermentation and Microorganisms 11", pp. 75-92, "Phage Experiment Method"). For example, to search for phage that infects any lactic acid bacteria of soy sauce, a sample in which the presence of phage is presumed or a naturally prepared soy sauce moromi that seems to be rich in wild lactic acid bacteria is filtered to obtain moromi sap. This is filtered through a membrane filter having a pore size of 0.2 to 0.4 μm to completely remove microorganisms, thereby obtaining a phage-containing sterile filtrate. This is diluted in multiple stages with 10 to 20% saline, and a part thereof, for example, 0.5 ml, is added to a culture solution of a specific soy sauce lactic acid bacterium.
5 ml, and a part of this mixture, for example, 100 μl
Was spread uniformly on a previously prepared agar plate medium, and anaerobic conditions [Gas Pak (manufactured by Becton-Dickson) or overlay culture] were applied according to the usual agar plate culture method for lactic acid bacteria. Incubate at 20-30 ° C for 2-6 days. If phage that infect soy sauce lactic acid bacteria (indicator bacteria) are present, plaque [Plaqu
e)] appears, and the phage infecting the lactic acid bacteria of soy sauce can be separated from the regenerated plaque by collecting the bacteria, mixing the bacteria again with the same indicator bacteria, and performing agar plate culture. Although phage cannot always be detected for all soy sauce lactic acid bacteria, phage that infects the strain can be obtained by selecting an appropriate sample as a separation source and repeating the experiment.

2. [Creation of Phage-Resistant Soy Sauce Lactic Acid Bacteria] Phage-resistant soy sauce lactic acid bacteria are masters that have formed a large number of soy sauce lactic acid bacteria colonies obtained by pre-selected or especially bred soy sauce lactic acid bacteria having excellent properties as they are or by artificial mutation. The plate is gently pressed against the surface of a carrier such as a sterilized velvet or colony transfer pad,
After printing the colonies on the master plate, the plate medium coated with the phage suspension in advance was gently pressed onto this surface, and the colonies printed on the carrier surface were copied to obtain a replica plate, which was then cultured. The colony formed on the master plate is compared with the colonies formed on the replica plate, and colonies on the master plate corresponding to the colonies formed on the replica plate are separated.

If it is desired to obtain a strain having particularly high phage resistance from these, the following operation may be further performed. MRS liquid medium 2 containing 1% phage solution and 15% salt was added to the strain obtained from the master plate.
After inoculating 00 μl with a platinum wire and culturing at 30 ° C. for 4 days,
Those cells in which the growth of the cells was clearly recognized by the naked eye were selected. Further, the culture solution of the strain selected here was converted into an MRS liquid medium containing 1% phage solution and 15% salt and an MRS liquid medium (comparative medium) containing no phage solution and containing 15% salt (contrast medium). 10 μl of each was inoculated, and cultured at 30 ° C. for 3 days.
The growth of the cells is better than that of the contrast medium, and a strain which is not affected by the addition of phage is selected.

Hereinafter, the present invention will be described more specifically with reference to examples.

[0016]

Example 1 Pediococcus halophilus D10 (parent strain) isolated from a naturally prepared soy sauce moromi was subjected to a mutation treatment with N-methyl-N'-nitro-N-nitrosoguanidine, and the diluted suspension (2 000
100 μl), 15% salt and 1.5% agar
Plate (Difco, 0881)
-01-3), and cultured at 30 ° C. for 6 days to obtain a master plate having a large number of soy sauce lactic acid bacteria formed colonies. Then, this was lightly pressed against the surface of a sterilized colony transfer pad (Repliplate, manufactured by FMC), and the colonies on the master plate were printed on the surface of the repliplate. Next, a plate medium coated with a phage suspension prepared by a method described later was gently pressed against the surface of the printed repliplate, and the colonies were copied to obtain replica plates. Then, after culturing the plate at 30 ° C. for 6 days, the colonies formed on the master plate and the replica plate are compared, and the colonies on the master plate corresponding to the colonies formed on the replica plate are separated. Was obtained.

Next, since the strains obtained here contained strains with low phage resistance, strains with high phage resistance were further selected as follows. The bacterial strain obtained from the master plate was inoculated into 200 μl of MRS liquid medium supplemented with 1% of phage solution and 15% of sodium chloride with a platinum wire.
After culturing at 0 ° C. for 4 days, those in which the growth of bacterial cells was clearly recognized with the naked eye were selected. Next, the culture solution of the selected strain was further added to an MRS liquid medium supplemented with 1% phage solution and 15% sodium chloride, and without a phage solution,
MRS liquid medium supplemented with 15% salt (contrast medium) 40
0 μl was inoculated in an amount of 10 μl each and cultured at 30 ° C. for 3 days. The growth of the cells was measured by the absorbance at 580 nm, and a strain having a better growth of the cells as compared with the contrast medium was selected. As described above, the soy sauce lactic acid bacterium Pediococcus has the same bacteriological properties as the parent strain but has very strong phage resistance.
Halofilus D10-No. One strain was isolated.

Next, the Pediococcus halophilus D10-No. 1 shows the results obtained by examining the presence or absence of phage release. That is, 50 μl of the suspension of the above strain was added to 1
After inoculating 5 ml of MRS liquid medium supplemented with 5% salt and culturing at 30 ° C. for 4 days, 50 μl of this culture medium was again inoculated into the above MRS liquid medium, and the same procedure was repeated 5 times in the same manner. The solution was filtered through a membrane filter having a pore size of 0.2 μm, and 2 μl of the permeate was dropped on an MRS plate medium containing 15% salt and 1.5% agar to which the parent strain was applied, and cultured at 30 ° C. for 4 days. Depending on the presence or absence of spots,
The presence of phage in the culture was examined. As a result, no lysis spot was detected at all.

Next, for comparison, the results of the same examination as to the presence or absence of phage release of lactic acid bacteria obtained by the conventional method of constructing phage-resistant soy sauce lactic acid bacteria are shown. That is, the parent strain Pediococcus halophilus D10 is mutated with N-methyl-N'-nitro-N-nitrosoguanidine. Then, 0.5 ml of the phage solution prepared by the method described later and 0.5 ml of the culture solution of the parent strain subjected to the above mutation treatment were mixed, and 100 μl of the mixture was added to 15 ml of salt solution
% And 1.5% agar on an MRS plate medium (Dif
Co., Ltd., 0881-01-3), 30 ° C
After 6 days of cultivation, the colonies that had formed had the same properties as the parent strain but had only a strong resistance to phage [that is, a high concentration of soy sauce lactic acid bacteria (parent strain) sensitive to phage could not grow at all. It grows on agar plate culture even in the presence of phage and forms colonies.] 100 resistant strains were isolated. Next, the presence or absence of phage release of the resistant strain isolated above will be described. That is, these strains were inoculated into 5 ml of an MRS liquid medium supplemented with 15% salt, cultured at 30 ° C. for 4 days, and 50 μl of the obtained culture was again inoculated into the MRS liquid medium. Planted 5 times in total.
Next, this culture solution was filtered through a membrane filter having a pore size of 0.2 μm, and 2 μl of the permeate was dropped onto an MRS plate medium to which 15% of salt coated with the parent strain and 1.5% of agar were added. For 4 days and examined for the presence of plaques. One hundred out of 100 resistant strains were obtained.
Bacterial lysis spots were observed in the 0 strain, and phages were detected. From this result, the phage-resistant strain constructed by the above method is contaminated with a strain incorporating the phage gene,
It turned out to be unreliable.

From the results of the present invention and the comparative examples, the phage-resistant soy sauce lactic acid bacterium obtained by the conventional method has low reliability, but the phage-resistant soy sauce lactic acid bacterium obtained by the present invention has no concern about phage release and is very safe. It turns out that it is high.

Preparation of phage solution The soy sauce moromi, which has been naturally prepared, is filtered to obtain a moromi juice, which is filtered through a membrane filter having a pore diameter of 0.2 μm to completely remove microorganisms, thereby obtaining a phage-containing sterile filtrate. This was diluted in multiple steps with 15% saline, and 0.5 ml was mixed with 0.5 ml of the culture of the parent strain. 100 μl of this mixture was added with 15% of sodium chloride and 1.5% of agar previously prepared. The lactic acid bacteria were uniformly spread on the plated MRS plate medium, and anaerobic conditions [Gas Pak (Becton-D
(manufactured by ickson)) at 30 ° C. for 4 days. Lytic plaques [Plaque] appeared depending on the dilution stage, so they were picked up, mixed again with the same parent strain and liquid-cultured, lysed, and the lysate was passed through a membrane filter with a pore size of 0.2 μm. Filtration was performed to completely remove the microorganisms to obtain a phage-containing sterile filtrate, which was used as a phage solution.

[0022]

Example 2 100 kg of defatted soybean and 100 kg of wheat were respectively denatured, inoculated with Aspergillus sojae, and soy sauce koji was made by a conventional method.
This was divided into three equal parts and charged into separate tanks A, B, and C together with 120 l of 25% saline solution. Next, in tank A, Pediococcus halophilus D10 (phage-sensitive bacterium) was placed, and in tank B, Pediococcus halophilus D10-No. 1
(Phage-resistant bacteria) each having a viable cell count of 1 × 10 ⁵ / g
Was added at the time of preparation so that No soy sauce lactic acid bacteria were added to tank C as a control. Next, since the spontaneous appearance of the phage in the moromi thus prepared cannot be predicted in advance, the tanks A, B, and C were infected with Pediococcus halophilus D10 in order to confirm the effects of the present invention. 20 ml of each phage solution to be inoculated was inoculated. Then, the mixture was carefully stirred so that no mutual infection occurred between the moromi in each tank, and the moromi was normally fermented and matured.

After the preparation, the change in the pH of the moromi over time until the 50th day was examined. In the tank B inoculated with the phage-resistant strain, lactic acid gradually increased in the moromi even after the phage inoculation. The phage-sensitive soy sauce lactic acid bacteria were inoculated with the tank A, but no lactic acid was produced at all, and the pH was almost the same as that of the control C containing no lactic acid bacteria. About one month after the preparation, an appropriate amount of main fermented yeast S. saccharomyces luxy was added, alcohol fermentation was carried out, and after aging by a conventional method, pressed, raw soy sauce was obtained.

The pH and lactic acid of the raw soy sauce thus obtained were analyzed, and a sensory test was conducted by 23 skilled panelists. The results shown in Table 1 were obtained. In the sensory test, the flavor was scored based on the following criteria, and the total score was shown. Excellent flavor: 1 point, poor flavor: 0 point. From the results in Table 1, it can be seen that when a phage-resistant bacterium is used as a soy sauce lactic acid bacterium, lactic acid fermentation is performed stably, and high-quality soy sauce is obtained.

[0025]

──────────────────────────────────────────────────続 き Continuation of the front page (56) References JP-A-63-216449 (JP, A) (58) Fields investigated (Int. Cl. ⁷ , DB name) C12N 1/00-1/38 A23L 1 / 238 101 A23L 1/238 103 BIOSIS (DIALOG) WPI (DIALOG)

Claims (2)

(57) [Claims] 1. A master plate having a large number of lactic acid bacteria colonies formed thereon is gently pressed against the surface of a sterilized carrier, the colonies on the master plate are printed, and a plate medium previously coated with a phage suspension is gently lightened. Pressing, copying the colonies printed on the surface of the carrier to obtain replica plates, culturing them, comparing the colonies formed on the master plates and the replica plates, and corresponding to the colonies formed on the replica plates Phage-resistant lactic acid bacteria obtained by separating colonies on the master plate. 2. A method for producing soy sauce in which phage-resistant soy sauce lactic acid bacteria are added to koji and / or moromi, a master plate on which a large number of soy sauce lactic acid bacteria colonies are formed as the phage-resistant soy sauce lactic acid bacteria is lightly pressed against the surface of a sterilized carrier. ,
After printing the colonies on the master plate, the plate medium coated with the phage suspension in advance was gently pressed onto this surface, and the colonies printed on the carrier surface were copied to obtain a replica plate, which was then cultured. Compare the colonies formed on the master plate and the replica plate,
A method for producing soy sauce, comprising using a phage-resistant soy sauce lactic acid bacterium obtained by separating a colony on the master plate corresponding to a colony formed on the replica plate.