JP2904879B2 - Low temperature sensitive lactic acid bacterium mutant, fermented plant produced using the same, and method for producing the same - Google Patents

Description

[Background of the Invention] <Industrial application field> The present invention relates to a technique for producing a fermented plant, specifically, a fermented plant represented by pickles. More particularly, the present invention relates to a microorganism which is a mutant strain of a bacterium belonging to the genus Lactobacillus exhibiting low temperature sensitivity, a method for producing a plant fermentation product using the microorganism, and a plant fermentation product.

<Conventional technology> Representative examples of fermented plant products include pickles in Japan and sauerkraut and pickles in foreign countries. Pickles have been produced for a long time and are edible, and typical examples include pickled vegetables such as Chinese cabbage, cucumber, radish and turnip. The usual method of manufacturing pickles is to first thoroughly wash vegetables and other raw materials with water, add a certain amount of salt, and pickle them.Several days later, natural fermentation by lactic acid bacteria such as Lactobacillus present in the raw materials is carried out. A pickle with an appropriate sourness is completed, and thereafter, it is stored at a low temperature in order to maintain a preferable acidity.

However, this cryopreservation does not completely suppress fermentation. The lactic acid bacteria proliferate even during the low temperature storage of the moderately pickled pickles, and as a result, the lactic acid in the pickles increases, whereby the acidity increases too much. That is, the pickles are in a so-called excessively pickled state. In addition, in the case of such natural fermentation, in addition to the lactic acid bacteria, various germs attached to the raw material also propagate at the same time, so that the flavor deteriorates.

As an improved method for suppressing the propagation of various bacteria, a method of fermenting by adding a large amount of microorganisms such as lactic acid bacteria has been studied.In this case, the microorganisms grow during low-temperature storage after fermentation, It becomes too pickled like natural fermentation.

For this reason, conventionally, a method of preventing the growth of lactic acid bacteria and other germs in the pickles and thus the increase of lactic acid by adding a natural or synthetic preservative has been taken.
That is, it has been difficult to maintain a desirable acidity of the pickles immediately after production without addition of a preservative during storage and distribution.

[Summary of the Invention]

<Summary> The present invention provides a fermented, for example, lactic acid fermented, plant fermented product that has been subjected to low-temperature preservation, so that desirable quality immediately after production without addition of a preservative, particularly acidity or flavor, is constant. It is an object of the present invention to provide a manufacturing technique that enables the maintenance to be performed.

The present inventors have conducted intensive studies, and as a result, vigorous lactic acid fermentation is carried out at around 30 ° C., but growth is weak during low-temperature storage, and lactic acid production is extremely low, and a low-temperature-sensitive Lactobacillus plantarum mutant strain is produced. And found that it was possible to achieve the above object by inoculating this mutant strain as a starter into a plant fermentation raw material and fermenting it, and storing it at low temperature. Thus, the present invention has been completed.

That is, the microorganism according to the present invention is a mutant strain of a bacterium belonging to the genus Lactobacillus, which exhibits low-temperature sensitivity when cells are inoculated in a nutrient medium and cultured at low temperature, and causes poor growth.

Further, the method for producing a plant fermented product according to the present invention comprises inoculating at least one of the above microorganisms as a starter during fermentation of a plant material for fermentation, fermenting at a temperature suitable for the growth of the bacterial species, It is characterized by suppressing microorganisms in a fermented product by storing at a low temperature.

Furthermore, the plant fermentation product according to the present invention is produced by the above-mentioned production method.

<Effects of the Invention> According to the present invention, by using a mutant strain of a bacterium belonging to the genus Lactobacillus according to the present invention exhibiting low-temperature sensitivity as a starter during fermentation of a plant fermentation raw material, the number of general bacteria even during low-temperature storage, Conventional increases in the number of lactic acid bacteria and the acidity are significantly suppressed. Therefore, without adding a preservative, it is possible to obtain a high-quality plant fermented product that is free from deterioration in flavor due to an increase in the number of general bacteria, the number of lactic acid bacteria and the acidity during storage at low temperatures.

[Specific description of the invention]

<Lactobacillus mutant strain> The mutant strain of the bacterium belonging to the genus Lactobacillus according to the present invention performs vigorous lactic fermentation at the growth temperature when the cells are inoculated in a nutrient medium, but grows weakly during low-temperature storage. Become
Lactic acid production shows extremely low temperature sensitivity,
The representative species is Lactobacillus plantarum.

Representative examples of the Lactobacillus mutant strains according to the present invention include Lactobacillus plantarum LTSM119 strain and Lactobacillus plantarum LTSM175 strain.

The present inventors have used, for example, Lactobacillus plantarum isolated from vegetables (such as Chinese cabbage), for example, Lactobacillus plantarum No. 14 strain as a parent strain. Mutant strains Lactobacillus plantarum LTSM119 and Lactobacillus plantarum LTSM175, which showed vigorous growth with almost no change but weak growth and low lactic acid production at a low temperature of about 10 ° C. or less, were isolated.

The mycological properties of Lactobacillus plantarum No. 14 strain (parent strain), Lactobacillus plantarum LTSM119 strain and Lactobacillus plantarum LTSM175 strain are as follows. These mycological properties are described in Bergey's Manual of Systematic Bacteriolo.
gy) Based on the method described in Volume 2 (1986) and the method described in "The World of Intestinal Bacteria" by Tomoko Mitsuoka and Sobunbunsha (1980).

Bacteriological properties of Lactobacillus plantarum No. 14 strain A. Morphological properties (1) Cell shape and size: Bacillus, 2-5μ × 1-1.5μ (2) Motility: None (3) Presence or absence of spores: None (4) Gram staining: Positive (5) Presence or absence of polyplasia of cells: None B. Growth on medium (1) MRS agar medium: white, round, about 3 mm in diameter at 30 ° C for 3 days To form colonies.

(2) MRS liquid medium: turbid at 30 ° C. for 3 days and precipitates at the bottom.

(3) MRS agar puncture culture: grows uniformly along the puncture and also grows on the surface.

C. Physiological properties (1) Does not reduce nitrate (2) Does not produce indole (3) Does not liquefy gelatin (4) Catalase reaction: negative (5) Does not hydrolyze starch (6) Glucose fermentation format: homofermentation (7) Lactic acid produced: DL-lactic acid (8) Salt tolerance: NaCl grows up to 10% (w / v) and 11% (w / v)
Cannot grow in v).

(9) Generation of acid from saccharide and generation of gas Generation of acid Generation of gas Generation of amygdalin +-arabinose--cellobiose +-esculin +-fructose +-galactose +-gluconic acid +-lactose +-maltose +-mannitol + -Mannose +-melezitose +-melibiose +-raffinose +-rhamnose--ribose +-salicin +-sorbitol +-sucrose +-trehalose +-xylose--(10) Growth temperature: optimal temperature 28 to 31 ° C 7 to 42 ° C (11) Growth pH: optimum pH 6.9 Growth range 3.0 to 7.8 (12) Litmus milk: acidification (13) Urease: negative (14) Oxidase: negative (15) Hydrogen sulfide generation: generated (16) VP reaction: negative (17) MR reaction: positive Lactobacillus plantara Bacteriological properties of LTSM119 strain The bacteriological properties of Lactobacillus plantarum LTSM119 strain are as described in the item C of “Bacterial properties of Lactobacillus plantarum No. 14”, “(9) Acid from sugars and Among the "presence / absence of gas generation", the properties of galactose, melibiose, raffinose, and gluconic acid for sugar differ as follows. And the growth temperature is about 13-40 ° C,
Except that the growth pH range is about 4.0-7.8, it is exactly the same as the mycological properties of Lactobacillus plantarum strain No. 14.

(9) Generation of acid from saccharide and generation of gas Generation of acid Generation of gas Generation of amygdalin +-arabinose--cellobiose +-esculin +-fructose +-galactose--gluconic acid--lactose +-maltose +-mannitol + -Mannose +-Merezitose +-Melibiose--Raffinose--Rhamnose--Ribose +-Salicin +-Sorbitol +-Sucrose +-Trehalose +-Xylose--Mycological properties of Lactobacillus plantarum LTSM175 strain Lactobacillus The bacteriological properties of the plantarum LTSM175 strain are described in “(9) Presence or absence of generation of acid and gas from saccharides” in item C of the bacteriological properties of Lactobacillus plantarum No. 14 strain described above. Galactose, melibiose, raffino Vinegar, nature to the sugar of gluconate is different as follows. And the growth temperature is about 10 ° C to 38 ° C,
Except that the growth pH range is about 4.0-7.8, it is exactly the same as the mycological properties of Lactobacillus plantarum strain No. 14.

(9) Generation of acid from saccharide and generation of gas Generation of acid Generation of gas Generation of amygdalin +-arabinose--cellobiose +-esculin +-fructose +-galactose--gluconic acid--lactose +-maltose +-mannitol + -Mannose +-melezitose +-melibiose--raffinose--rhamnose--ribose +-salicin +-sorbitol +-sucrose +-trehalose +-xylose-- A mutagen is added to a suspension of high cells to perform a mutagenesis treatment, and the mutagen is removed before cultivation. The culture shows vigorous growth at a growth temperature (for example, about 30 ° C.) and a low temperature (for example, (Less than about 15 ° C, preferably less than about 10 ° C) Such things can be done by picking out. This mutation treatment is described in general literature, for example, "Experiments in
Molecular Genetics "(Jeffery H. Miller), publisher C
This can be performed with reference to the old Spring Harbor Laboratory (1972).

Mutants used in the mutation treatment include N-methyl-N '
-Nitro-N-nitrosoguanidine (hereinafter abbreviated as NTG) is typical, but ethyl methanesulfonate,
Mutants such as acridine orange, ultraviolet irradiation, X-ray irradiation and the like are also possible. Furthermore, by repeating these mutation treatments several times, mutants can be more efficiently isolated.

Microorganisms related to the present invention, including the representative strains described above, in the field of plant fermentation, show vigorous growth at the optimal growth temperature, susceptible at low temperatures, Lactobacillus sp. It is all inclusive.

The medium used for culturing the mutant strain according to the present invention may be any medium as long as it is used for culturing lactic acid bacteria. For example, MRS medium, Rogosa
Medium or GPY medium. For example, these microorganisms are inoculated into these steam-sterilized culture media, and usually at 20 ° C to 38 ° C.
Cultivate at about ° C, preferably 28 ° C to 35 ° C. The culturing time is usually about 15 hours to 72 hours, preferably 15 hours to 24 hours.

Microorganism Deposit Lactobacillus plantarum No. 14 strain was sent to the Institute of Microbial Industry and Technology by the National Institute of Advanced Industrial Science and Technology
P-11550).

Lactobacillus plantarum LTSM119 strain and Lactobacillus plantarum LTSM175 strain have been sent to Microorganisms Institute of Technology, National Institute of Advanced Industrial Science and Technology by
1551) and No. 11552 (FERM P-11552).

<Method for Producing Plant Fermented Product> The method for producing a plant fermented product according to the present invention comprises inoculating as a starter at least one kind of a microorganism that is a mutant strain of the genus Lactobacillus exhibiting low temperature sensitivity as described above during fermentation of a plant material for fermentation. After fermentation at a temperature suitable for the growth of the bacterial species, the microorganism is stored at a low temperature to suppress the growth of microorganisms in the fermented product.

(1) Fermentation plant raw material The fermentation plant raw material used as the raw material of the plant fermentation product may be any plant-based material. For example, vegetables include cucumber, Chinese cabbage, radish, carrot, tomato, onion,
Bell pepper, cabbage, celery and the like. In fruits, apples, pears, bananas, strawberries, persimmons, peaches, etc.
Soybeans, wheat, red beans and the like can be mentioned.

(2) Fermentation and Preservation The above-mentioned plant material for fermentation is usually firstly washed with water or a bactericide, such as sodium hypochlorite, or sterilized by heating, and then preferably water and, if necessary, salt. Alternatively, after adding a seasoning such as an amino acid or a nucleic acid (a desired amount may be added), a culture solution of the microorganism of the present invention (usually at a concentration of 10 ⁶ to 10 ⁹ cells / ml, preferably 10 ⁸ cells / m 2).
(concentration of l) is inoculated from 0.1% to 10%, preferably about 1% (V / V each). Thereafter, fermentation is performed at about 25 ° C. to 38 ° C., preferably 28 ° C. to 35 ° C., usually for 8 hours to 24 hours, preferably 8 hours to 15 hours. Here, the inoculated microorganism of the present invention grows and performs lactic acid fermentation. When the fermentation state reaches a preferable state (for example, when the pH is lowered by about 0.5 to 1.0 from that at the time of starting fermentation), the temperature is immediately lowered to a low temperature, preferably about 10%. Store below ℃. Thereafter, the growth of the inoculated microorganism of the present invention becomes extremely weak, and the production of lactic acid stops.
In other words, this production method makes it possible to maintain a favorable fermentation state before storage at low temperatures for a long time.

In addition, the growth during fermentation of various bacteria derived from the plant material for fermentation is suppressed by the inoculated microorganism of the present invention predominantly growing. It is preferable to carry out a sterilization treatment with a disinfectant such as acid soda or a sterilization treatment by heating.

<Plant fermented product> The plant fermented product according to the present invention is produced by the above-described production method, and as shown in Examples described below, the product obtained by the conventional method can be stored at 10 ° C, For example, the number of general bacteria and lactic acid bacteria in Chinese cabbage increases with the number of days (see FIG. 1), and eventually the fermentation becomes excessive due to the propagation of various bacteria or lactic acid bacteria, resulting in a so-called overpickled state. On the other hand, preferred quality immediately after production, particularly acidity or flavor, is maintained without adding a preservative.

Typical examples of the plant fermented product include Japanese pickles, sour craft (such as Germany) and pickles (such as the United States) in foreign countries.

The fermented plant product according to the present invention may be added with an appropriate preservative, if necessary.

<Example> The following shows an example of the present invention, but the present invention is not limited thereto.

Example 1: Cold-sensitive mutant Lactobacillus plantarum LTSM119 strain and Lactobacillus plantarum LTSM1
Isolation of 75 strains Lactobacillus plantarum No. 14 strain isolated from commercially available vegetables was inoculated into 100 ml of GPY medium (see Table 1), and
The cells were cultured at ℃. 0.4 at 660 nm (mid-log phase)
The culture was terminated when The cells collected from this culture by centrifugation (10,000 rpm / 10 min / 4 ° C.) by a conventional method were mixed with a phosphate buffer (1/15 M Na ₂ HPO ₄
And 1 / 15M KH ₂ PO ₄ mixed at a ratio of 6: 4, pH 7.0) 2
After times washed, and suspended in the same buffer 10 ml (10 ⁹ cells / ml).

Next, 0.5 ml of an NTG solution (2.5 mg / ml) was added to 2 ml of the bacterial suspension and reacted at 30 ° C. for 60 minutes.

After completion of the reaction, a 10-fold amount of a phosphate buffer was added to dilute the mixture, and immediately, washing was performed twice by centrifugation to completely remove NTG. Then, 2 ml of a phosphate buffer was added to the cells after removing the NTG and suspended.

1 ml of the bacterial suspension was inoculated into 100 ml of GPY medium, and cultured sufficiently at 30 ° C. Thereafter, the same NTG treatment was repeated twice.

After the NTG treatment, 1 ml of the culture solution was used for penicillin G (Sigma) 10
100 ml of GPY medium supplemented with 0 units / ml was inoculated and cultured at 20 ° C. for 4 days. This culture solution is centrifuged,
After collecting and washing the cells, the cells were diluted with GPY agar medium.

After culturing at 30 ° C for 3 days, colonies that appeared were MR one by one.
S medium (OXOID) was inoculated and cultured at 30 ° C and 15 ° C.
Then, those that showed vigorous growth at 30 ° C and weakly grown at 15 ° C were selected.

Next, those whose growth was weak at 15 ° C were subcultured 5 times at 30 ° C. Lactobacillus plantarum LTSM119 is one of those that grow only slightly at 15 ° C after subculture.
Strain and Lactobacillus plantarum LTSM175 strain were isolated.

Table 1 Glucose 10 g Polypeptone 10 g Yeast extract 10 g Sodium acetate 3 g Distilled water 1000 ml pH 6.8 Example 2: Low temperature sensitive mutant strains Lactobacillus plantarum LTSM119 and Lactobacillus plantarum LTSM175 strain obtained in Example 1, and furthermore Growth of the parent strain Lactobacillus plantarum No. 14 at 30 ° C. and 10 ° C. using an MRS medium (OXOID) was compared as follows.

The test strain was subcultured several times in MRS medium to stabilize the growth, and then inoculated at 1% (v / v) into the same medium at 30 ° C and 1 ° C.
The cells were cultured at 0 ° C. Then, the growth of the bacteria was compared by measuring the OD value at 660 nm over time (FIG. 2,
(See FIGS. 3 and 4).

As is clear from FIGS. 2, 3 and 4, the growth of Lactobacillus plantarum LTSM119 strain and Lactobacillus plantarum LTSM175 strain at 30 ° C. is almost the same as that of Lactobacillus plantarum No. 14 strain. It did not change. At 10 ° C., Lactobacillus plantarum strain No. 14 grows more slowly than at 30 ° C. In contrast, Lactobacillus plantarum LT
Growth of SM119 strain and Lactobacillus plantarum LTSM175 strain is weak and hardly grows.

Example 3: Cold-sensitive mutant Lactobacillus plantarum LTSM
The 119 strains and their parent strain Lactobacillus plantarum No. 14 were each cultured in MRS medium for 1 day, washed twice with the same volume of saline by centrifugation, and suspended in the same volume of saline. A bacterial suspension (10 ⁸ cells / ml) was prepared.

300 g of commercially available cucumbers were immersed in a 200 ppm aqueous solution of hypochlorous acid for 1 minute, and washed with water, 300 g of water and 18 g of common salt
(Final salt concentration: 3% (w / w)) was placed in a 1 L glass container with a lid. This was inoculated with 1% of each of the above bacterial suspensions. A non-inoculated one was used as a control. These were fermented at 35 ° C for 15 hours and then stored at 10 ° C. At this time, the number of general bacteria over the storage days,
Changes over time in the number of lactic acid bacteria and the acidity are shown in FIGS. 5, 6, and 7. The number of general bacteria was the number of bacteria grown on a normal agar medium, and the number of lactic acid bacteria was the number of bacteria grown on an MRS medium.

After storage at 10 ° C. for 14 days, those using the Lactobacillus plantarum LTSM119 strain as a starter showed slight increases in the number of general bacteria, lactic acid bacteria and acidity. And good flavor immediately after fermentation was maintained. In contrast, those using Lactobacillus plantarum No. 14 as a starter showed an increase in the number of general bacteria, lactic acid bacteria and acidity during storage at 10 ° C.,
It was over fermented. In addition, 10 ° C even for uninoculated ones
During storage, the numbers of general bacteria and lactic acid bacteria increased. Furthermore, uninfected ones began to feel off-flavors, which are considered to be germs in flavor.

Example 4: In the same manner as in Example 3, the bacteria were cultured and a bacterial suspension (10 ⁸
Cells / ml). Further, a commercially available cucumber was immersed in a 200 ppm aqueous solution of hypochlorous acid containing salt for 5 minutes, and sufficiently washed with water. Next, a 5% (w / v) saline solution was prepared and adjusted to pH 4.5. A plastic container (113 mm x 113 mm x 30 mm) was prepared, into which two slices of the above cucumber were poured, and saline was poured. This was inoculated with a 1% (v / v) bacterial suspension and sealed with a sealing machine. After fermenting this at 35 ° C for 4 hours,
Stored at 10 ° C. for 7 days. In addition, the cells which were not inoculated with the bacteria and stored at 10 ° C. immediately after packing were used as a control. 10 in Table 2
The storage stability after 7 days at 7 ° C was shown.

After storage at 10 ° C. for 7 days, the uninoculated one became cloudy due to the propagation of various bacteria, and gas generation was observed. Inoculated with Lactobacillus plantarum No. 14 strain,
The bacteria grew considerably so that white precipitates of the cells were observed, and the fermentation was in an over-fermented state. On the other hand, inoculation with Lactobacillus plantarum LTSM119 strain and Lactobacillus plantarum LTSM175 strain, the growth of the bacteria stopped, and no gas generation was observed.

[Brief description of the drawings]

FIG. 1 is an explanatory diagram showing the change in the number of bacteria of pickled Chinese cabbage at 10 ° C. FIG. 2 is an explanatory diagram showing a growth curve of Lactobacillus plantarum LTSM119 strain. FIG. 3 is an explanatory diagram showing a growth curve of Lactobacillus plantarum strain LTSM175. FIG. 4 is an explanatory diagram showing a growth curve of Lactobacillus plantarum No. 14 strain. Fig. 5 shows the salting of cucumbers by starter inoculation.
FIG. 4 is an explanatory diagram showing a change in the number of general bacteria when stored at ℃. Fig. 6 shows salting of cucumbers by starter inoculation.
FIG. 3 is an explanatory diagram showing a change in the number of lactic acid bacteria when stored at ℃. Fig. 7 shows salting of cucumbers by starter inoculation.
FIG. 3 is an explanatory diagram showing a change in acidity when stored at ℃.

 ────────────────────────────────────────────────── ─── Continued on the front page (72) Koji Izumo, Inventor 410 Kasukabe City, Saitama Prefecture, Akanuma-Dodo 410 Inside the Momoya Research Laboratories (56) References JP-A-62-268 (JP, A) JP-A-62 -130680 (JP, A) JP-A-63-209542 (JP, A)

Claims (5)

(57) [Claims] 1. A microorganism belonging to a mutant strain of Lactobacillus plantarum, which is sensitive to low temperature, and inoculated with 1% by volume of a bacterial cell liquid obtained by subculturing cells in an MRS medium and stabilizing the growth. OD660nm when cultured at 10 ° C for 5 days
A microorganism belonging to a Lactobacillus plantarum mutant strain having a value of 0.1 or less or 0.4 or less. 2. A Lactobacillus plantarum LTSM119 strain having an OD660nm value of 0.1 or less, or an OD660nm value of 0.4.
The microorganism belonging to the Lactobacillus plantarum mutant strain according to claim 1, which is a Lactobacillus plantarum LTSM175 strain, which shows the following: 3. Inoculation of at least one of the microorganisms according to claim 1 or 2 as a starter during fermentation of a plant material for fermentation, and fermentation at a temperature suitable for propagation of the species.
A method for producing a fermented plant, comprising storing at a low temperature the growth of microorganisms in the fermented product. 4. The method according to claim 3, wherein the fermentation temperature is about 28 ° C. to 35 ° C. and the storage temperature is about 10 ° C. or less. 5. A fermented plant product obtained by the production method according to claim 3 or 4.