JPH0463587A - Low temperature-sensitive lactic acid bacterium mutant, fermented plant produced using thereof and production of same plant - Google Patents

Abstract

NEW MATERIAL:A microorganism as a mutant of bacterium belonging to genus Lactobacillus exhibiting low temperature sensitivity and inducing hypogenesis in inoculating strain in a nutritive medium and culturing at a low temperature. EXAMPLE:A Lactobacillus plantarum LTSM 119 strain (FERM P-11551). USE:Used for preparation of fermented plant such pickles. PREPARATION:For instance, Lactobacillus plantarum No.14 strain isolated from market-sold vegetables is cultured and cultured solution is centrifuged, then the strain is collected, thus suspended in a phosphoric acid buffer solution and a solution of N-methyl-N'-nitro-N-nitrosoguanidine(NTG) as a mutation agent is added to the suspended buffer solution, then reacted at 30 deg.C for 60min, thus strain is collected after finish of the reaction by centrifuge and resultant strain is cultured in a medium to afford the aimed mutant Lactobacillus plantarum LTSM 119 strain.

Description

DETAILED DESCRIPTION OF THE INVENTION [Background of the Invention] <Industrial Application Field> The present invention relates to a technology for producing fermented plant products, specifically fermented plant products typified by pickles. More specifically, the present invention relates to a microorganism that is a mutant strain of a Lactobacillus bacterium that is sensitive to low temperatures, a method for producing a fermented plant product using the microorganism, and a fermented plant product.

<Prior Art> Typical fermented plant products include pickles in Japan, and sour clouds and pickles in other countries. Pickles have been produced for a long time and are considered to be edible, with typical pickles such as Chinese cabbage, cucumbers, daikon radish, and turnips. The usual method for making pickles is to first thoroughly wash the raw materials, such as vegetables, with water, add a certain amount of salt, and pickle. Fermentation produces pickles with a moderate amount of sourness, which is then stored at low temperatures to maintain the desired acidity.

However, fermentation is not completely inhibited by this low temperature storage. In moderately pickled pickles, lactic acid bacteria proliferate even during low-temperature storage, resulting in an increase in lactic acid in the pickles, resulting in an excessive increase in acidity.

In other words, the pickles become so-called over-pickled. In addition to lactic acid bacteria, such natural fermentation produces
Various bacteria attached to the raw materials also grow at the same time, resulting in poor flavor.

As an improved method for suppressing the growth of bacteria, research has been conducted on fermentation by adding large amounts of microorganisms such as lactic acid bacteria, but even in this case, microorganisms multiply during low-temperature storage after completion of fermentation. As with natural fermentation, the rice will be overly pickled.

To this end, conventional methods have been taken to prevent the growth of lactic acid bacteria and other bacteria in pickles, and to prevent the increase in lactic acid, by adding natural or synthetic preservatives. That is, it has been considered difficult to maintain the desirable sourness of pickles immediately after production, even during storage and distribution, without adding preservatives.

[Summary of the invention]

Summary of the Invention The present invention allows fermented plant products that have been subjected to fermentation, such as lactic acid fermentation, to be distributed at low temperatures, thereby maintaining desirable quality immediately after production, especially acidity or flavor, without adding preservatives. The purpose is to provide a manufacturing technology that allows for constant maintenance.

As a result of intensive research, the present inventors found that a low-temperature-sensitive Lactobacillus plantarum mutant strain that performs vigorous lactic acid fermentation at around 30°C, but exhibits weak growth during low-temperature storage and produces very little lactic acid. We succeeded in creating and isolating this mutant strain, and discovered that the above purpose could be achieved by inoculating plant fermentation raw materials as a starter and fermenting them, and storing them at low temperatures.Based on this knowledge, we The present invention was finally completed.

That is, the microorganism according to the present invention is a mutant strain of a bacterium belonging to Lactobacillus that exhibits low temperature sensitivity and causes poor growth when the microorganisms are inoculated into a nutrient medium and cultured at low temperatures.

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

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

<Effects of the Invention> According to the present invention, by using the mutant strain of the bacteria belonging to the genus Lactobacillus according to the present invention that is sensitive to low temperatures as a starter during the fermentation of plant fermentation raw materials, general bacteria can be maintained even during low temperature storage. The conventional increases in the number of lactic acid bacteria, the number of lactic acid bacteria, and the acidity are significantly suppressed. Therefore, without adding preservatives, it is possible to obtain a fermented plant product of excellent quality that does not suffer from deterioration of flavor due to increases in the number of general bacteria, lactic acid bacteria, or acidity during low-temperature storage.

[Detailed description of the invention]

Lactobacillus mutant strain> The mutant strain of Lactobacillus bacteria according to the present invention performs vigorous lactic acid fermentation at the growth temperature when the bacterial cells are inoculated into a nutrient medium, but the growth is weak during low-temperature storage. It exhibits low-temperature sensitivity with extremely low lactic acid production, and its representative species is Lactobacillus plantarum.

As a representative example of the Lactobacillus mutant strain according to the present invention,
Examples include Lactobacillus plantarum LTSM119 strain and Lactobacillus plantarum LTSM175 strain.

The present inventors used Lactobacillus plantarum isolated from vegetables (Chinese cabbage, etc.) as a parent strain, such as Lactobacillus plantarum No. 14, as a parent strain. We isolated the mutant strains Lactobacillus plantarum LTSM119 and Lactobacillus plantarum LTSM175, which continue to grow vigorously, but grow weakly at low temperatures of about 10° C. or lower, and produce weak lactic acid.

Lactobacillus plantarum No. 14 strains (parent strain)
The mycological properties of Lactobacillus plantarum LTSM119 strain and Lactobacillus plantarum LTSM175 strain are as shown below.

These mycological properties are described in the Purge Aids Manual on Systematic Bacteriology (Berge
y's Manual or 5ystesatlc
Bacteriology) Volume 2 (1,986)
Methods described and “The World of Intestinal Bacteria” by Tomozoku Mitsuoka, Soubunsha (
(1980).

Mycological properties A and morphological properties of Lactobacillus plantarum Nα14 strain (1) Cell shape and size: Bacillus, 2-5μ x 1-1.
5 μ (2) Motility: None 3) Presence of spores: None 4) Durham staining: Positive (5) Presence or absence of cell pleoplasia: None B, Growth status on medium (1) MR3 agar medium at 30°C , about 3mm in diameter in 3 days
Forms two white, circular colonies.

(2) MR8 liquid medium becomes turbid at 30°C for 3 days and precipitates at the bottom.

(3) MR3 agar puncture culture: uniform growth along the puncture;
It also grows on surfaces.

C3 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 type: homofermentation ( 7) Lactic acid produced: DL-lactic acid (8) Salt tolerance: Grows up to NaC110% (W/V),
Unable to grow at 11% (w/v).

(9) Production of acid from sugars and gas production Gas production + amygdalin arabinose cellobiose esculin fructose galactose gluconate lactose maltose + mannitol + mannose + melezitose + melibiose raffinose + rhamnose ribose + salicin + sorbitol + Sucrose + Trehalose Xylose (lO) growth temperature: Optimum temperature 28-31℃ growth range
7-42℃ (11) Growth pH: Optimum pH 6.9 Growth range 3.0-7.8 (12) Lidomus milk: acidification (13) Urease: negative (14) Oxidase: negative (15) Production of hydrogen sulfide : Not produced (1B) Vp reaction: Negative (17) MR reaction: Positive Mycological properties of Lactobacillus plantarum LTSM119 The mycological properties of Lactobacillus plantarum LTSM119 are similar to the Lactobacillus plantarum described above. Talam Nα1
In item C of the mycological properties of the four strains, "(9) Presence or absence of acid and gas production from sugars", the properties towards sugars of galactose, melibiose, raffinose, and gluconic acid differ as follows.

The growth temperature is approximately 13°C to 40°C, and the growth temperature is approximately 13°C to 40°C.
The mycological properties are exactly the same as the Lactobacillus plantarum Nα14 strain, except that the H range is approximately 4.0 to 7.8.

(9) Production of acid from sugars and production of acids and gases Production of gas Amygdalin + Arabinose Cellobiose + Aesculin fructose Galactose Gluconate lactose Maltose Mannitol Mannose Merezitose Melibiose Raffinose Rhamnose Ribose Salicin Sorbitol Sucrose Strehalose Sylose Mycological properties of Lactobacillus plantarum LTSM175 strain The mycological properties of Lactobacillus plantarum LTSM175 strain are the above-mentioned Lactobacillus plantarum No.
In Item C of the mycological properties of the 14 strains, ``(9) Presence or absence of acid and gas production from sugars,'' the 14 strains differ in their properties toward sugars such as galactose, melbiose, raffinose, and gluconic acid, as shown below.

The growth temperature is approximately 10°C to 38°C, and the growth temperature is approximately 10°C to 38°C.
The mycological properties are exactly the same as those of Lactobacillus plantarum No. 14, except that the H range is approximately 460 to 7.8.

(9) Generation of acid from sugars and generation of gas (acids and non-acids) Gas generation + amygdalin arabinose cellobiose esculin fructose galactose + + lactose decagluconate + maltose + mannitol + mannose melezitose + melibiose raffinose rhamnose ribose + Salicin + Sorbitol + Sucrose + Trehalose + Xylose Cold-sensitive mutant strains are basically created by adding a mutagen to a suspension of bacterial cells with high growth activity, performing mutation treatment, and removing the mutagen. This can be carried out by culturing and selecting those cultured bacteria that show vigorous growth at a growth temperature (for example, about 30°C) and weak growth at low temperatures (for example, about 15°C or lower, preferably about 10°C or lower). can.

This mutation process is carried out by - in-shell literature, e.g. rlExpe
rimentsln Molecular Genet
ics J (Jef'rrey If, Mill
er), Publisher Cold Spring Harbor
Laboratory (1972).

The mutagen used for mutation treatment is N-methyl-N'-
Nitro N-nitrosoguanidine (hereinafter abbreviated as NTG) is typical, but ethyl methanesulfonate,
Mutant agents such as acridine orange, ultraviolet irradiation, X-ray irradiation, etc. are also possible. Furthermore, by repeating these mutation treatments several times, mutant strains can be isolated more efficiently.

The microorganisms involved in the present invention include the representative strains mentioned above, as well as Lactobacillus microorganisms that are used in the field of plant fermentation to grow vigorously at optimal growth temperatures, but are sensitive to low temperatures and cause poor growth. It encompasses all of the above.

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. Examples include MR5 medium, Rogos
Examples include a medium and GPY medium. For example, the microorganisms are inoculated onto these steam-sterilized media, and the temperature is usually 20°C.
to 38°C, preferably 28°C to 35°C. The culture time is usually about 15 to 72 hours, preferably 15 to 24 hours.

Deposit of microorganisms 14 strains of Lactobacillus plantarum were deposited with the Institute of Microbial Technology, Agency of Industrial Science and Technology, as part of the Microbial Research Institute No. 11550 (F
ERM P-11550).

Rustobacillus blankrum strain LTSM119 and Lactobacillus Φ plantarum strain LTSM175 were submitted to the National Institute of Microbiological Technology, Agency of Industrial Science and Technology, No. 1155.
No. 1 (FERM P-11551) and No. 1155
No. 2 (FERMP-11552).

Method for producing a fermented plant product> The method for producing a fermented plant product according to the present invention involves inoculating at least one microorganism that is a mutant strain of the genus Lactobacillus exhibiting low temperature sensitivity as described above as a starter during fermentation of a plant material for fermentation. The fermentation product is characterized in that, after fermentation is performed at a temperature suitable for the growth of the bacterial species, the growth of microorganisms in the fermented product is suppressed by storing it at a low temperature.

(1) Plant materials for fermentation Plant materials for fermentation that serve as raw materials for fermented products may be any plant material, but examples of vegetables include cucumbers, Chinese cabbage, radish, carrots, tomatoes, onions, Examples include green peppers, cabbage, and celery. Examples of fruits include apples, pears, bananas, strawberries, persimmons, peaches, etc., and examples of grains include soybeans, wheat, adzuki beans, etc.

(2) Fermentation and Preservation The above-mentioned plant materials for fermentation are usually first washed with water or sterilized with a disinfectant such as sodium hypochlorite, or by heating, preferably with water and, if necessary, with salt. Alternatively, after adding seasonings such as amino acids and nucleic acids (the desired amount may be added), add the culture solution of the microorganism of the present invention (usually at a concentration of 106 to 109 cells/ml, preferably 10 cells/ml).
0.08 cells/ml). Inoculate from 1% to 10%, preferably about 1% (V/V each). Thereafter, fermentation is carried out at about 25°C to 38°C, preferably 28°C to 35°C, for usually 8 to 24 hours, preferably 8 to 15 hours. The inoculated microorganisms of the present invention proliferate and carry out lactic acid fermentation, but as soon as a desirable fermentation condition (for example, the point at which the pH decreases by 0.5 to 160 degrees from that at the start of fermentation) is reached, the microorganisms of the present invention that have been inoculated grow and the lactic acid fermentation is carried out. is about 10℃
Save below. After that, the growth of the inoculated microorganism of the present invention becomes extremely weak and the production of lactic acid also stops. That is, this production method makes it possible to maintain a favorable fermentation state for a long period of time before storage at low temperature.

In addition, the growth of various bacteria originating from plant materials for fermentation during fermentation can be suppressed by the inoculated microorganisms of the present invention dominating and multiplying; It is preferable to perform sterilization treatment using a sterilizing agent such as acid soda or sterilization treatment by heating.

Fermented plant product> The fermented plant product according to the present invention is produced by the above-mentioned production method, and as shown in the examples below,
Even if the products obtained by the conventional method are stored at 10℃,
For example, the number of general bacteria and lactic acid bacteria in Chinese cabbage increases with the passage of time (see Figure 1), eventually resulting in excessive fermentation due to the proliferation of various bacteria or lactic acid bacteria, resulting in what is called an over-pickled state. On the other hand, it maintains its desirable quality immediately after production, especially its sour taste or flavor, without adding preservatives.

Typical examples of this fermented plant product include Japanese pickles,
or sour cloud in foreign countries (such as Germany),
Examples include pickles (American, etc.).

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

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

Example 1: Isolation of cold-sensitive mutant strains Lactobacillus plantarum LTSMI 19 and Lactobacillus plantarum LTSM 175 14 strains of Lactobacillus plantarum isolated from commercially available vegetables were cultured in GPY medium (
(See Table 1) was inoculated into 100 ml and cultured at 30°C. The culture was terminated when the O, D of 660 nl reached 0.4 (mid-logarithmic growth phase). This culture solution was centrifuged using a conventional method (10,00 rpm; /10 min/
The bacterial cells collected at 4℃) were added to the same volume of phosphate buffer (1
/15M Na2HPO4 and 1/15MKH2PO
After washing twice with a 6:4 mixture of 4 and 4, pH 7.0), the cells were suspended in 10 ml of the same buffer (109 cells/ml).
).

Next, add NTG solution (2.5s + g/m
1) Add 0.5 ml and react at 30°C for 60 minutes.

After the reaction was completed, NTG was completely removed by diluting by adding 10 times the amount of phosphate buffer and immediately washing twice by centrifugation.

Then, 2 ml of phosphate buffer was added to the bacterial cells after NTG removal to suspend them.

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

GPY medium 1 to which 1 ml of the culture solution after NTG treatment was added with 100 units/ml of penicillin G (Sigma)
00ml and cultured at 20°C for 4 days.

This culture solution was collected and washed by centrifugation, and then poured onto a GPY agar medium.

After culturing at 30°C for 3 days, one colony that appeared was M
The cells were inoculated into RS medium (OXOID) and cultured at 30°C and 15°C. It showed vigorous growth at 30℃, and
Plants with weak growth at 5°C were selected.

Next, those that showed weak growth at 15°C were subcultured five times at 30°C. Lactobacillus plantarum LT was selected from among those with weak growth at 15°C even after subculturing.
SM119 strain and Lactobacillus plantarum LT
Strain SM175 was isolated.

Table 1 Glucose 10g Polypeptone 10g Yeast extract 10g Sodium acetate 3g Distilled water 1000ml 00O,8 Example 2: Cold-sensitive mutant strain Lactobacillus obtained in Example 1
plantarum LTSM119 strain and Lactobacillus
Lactobacillus plantarum LTSM175 strain, the parent strain Lactobacillus plantarum positive, and 14 strains in MR8 medium (O
The growth at 30° C. and 10° C. when using XOID Co., Ltd.) was compared as follows.

The test strain was subcultured several times in MR3 medium to stabilize its growth, and then inoculated at 1% (v/v) into the same medium.
Culture was carried out at 0°C and 10°C. The growth of the bacteria was compared by measuring the O, D, and D values at 660 nil over time (see Figures 2, 3, and 4).

As is clear from Figures 2, 3, and 4, the growth of Lactobacillus plantarum strain LTSM119 and Lactobacillus plantarum strain LTSM175 at 30°C is almost different from that of Lactobacillus plantarum strain 14. There wasn't. At 10°C, Lactobacillus blankrum N [Li2 strain grows more slowly than at 30°C. On the other hand, Lactobacillus plantarum LTSM119 strain and Lactobacillus plantarum LTSM175 strain have weak growth and rarely grow.

Example 3: Cold-sensitive mutant strain Lactobacillus plantarum LTS
M119 strain and its parent strain Lactobacillus plantarum N
Each α14 strain was cultured in MR3 medium for 1 day, washed twice with the same amount of physiological saline by centrifugation, and then suspended in the same amount of physiological saline to obtain a bacterial suspension (108 cells/ml).
) was adjusted.

300g of commercially available cucumbers with hypochlorous acid 2001)I)l
Water 3 for those immersed in an aqueous solution for 1 minute and washed with water.
00g and 18g of common salt were added (final salt concentration 3% (w)
/v))) was placed in an IL capacity glass container with a lid. Each of the above bacterial suspensions was inoculated at 1%. In addition, non-inoculated animals were used as controls. 1 at 35℃
After fermentation for 5 hours, it was stored at 10°C. Figures 5, 6, and 7 show changes over time in the number of general bacteria, the number of lactic acid bacteria, and acidity as the storage days progressed. The number of general bacteria was defined as the number of bacteria grown on ordinary agar medium, and the number of lactic acid bacteria was defined as the number of bacteria grown on MR3 medium.

When stored at 10°C for 14 days, the number of general bacteria, lactic acid bacteria, and acidity increased only slightly in the case where Lactobacillus plantarum LTSM119 strain was used as a starter. And, the good flavor immediately after fermentation was maintained. On the other hand, Lactobacillus plantarum yang,
In the case where strain 14 was used as a starter, an increase in the number of general bacteria, lactic acid bacteria, and acidity was clearly observed during storage at 10°C, indicating an overfermented state. Furthermore, an increase in the number of general bacteria and lactic acid bacteria was observed during storage at 10°C even in uninoculated samples. In addition, uninoculated seeds began to have a strange odor that was thought to be caused by bacteria.

Example 4: Bacteria were cultured in the same manner as in Example 3 and cultured in Bacteria Suspension 7IA solution (1
08 cells/ml). In addition, commercially available cucumbers were rubbed with salt, immersed in a 200 ppm hypochlorous acid aqueous solution for 5 minutes, and thoroughly washed with water. Next, a 5% (v/v) saline solution was prepared and adjusted to pH 4.5. A plastic container (113 mm + iX 113 mm x 30 mm) was prepared, and the two cucumber slices described above were placed in it and the saline solution was poured. This was inoculated with 1% (V/V) bacterial suspension and sealed using a sealing machine.

After fermenting this at 35°C for 4 hours, it was stored at 10°C for 7 days. In addition, without inoculating the bacteria, immediately after packing at 10°C.
The sample stored in Table 2 shows the evaluation of the storage stability after 7 days at 10°C.

After storage at 10° C. for 7 days, the pickling solution of uninoculated samples became cloudy due to proliferation of bacteria, and gas generation was observed. In those inoculated with Lactobacillus plantarum strain 14, the bacteria grew so much that a white precipitate of bacterial cells was observed.
It was in a state of excess and fermentation. In contrast, when Lactobacillus plantarum LTSM119 strain and Lactobacillus plantarum LTSM175 strain were inoculated, bacterial growth stopped and no gas generation was observed.

Figure 5 shows how to pickle cucumbers by inoculating starter bacteria.
It is an explanatory diagram showing changes in the number of general bacteria when stored at 0°C.

Figure 6 shows how to pickle cucumbers by inoculating starter bacteria.
It is an explanatory diagram showing changes in the number of lactic acid bacteria when stored at 0°C.

Figure 7 shows how to pickle cucumbers by inoculating starter bacteria.
It is an explanatory diagram showing changes in acidity when stored at 0°C.

[Brief explanation of drawings]

FIG. 1 is an explanatory diagram showing changes in the number of bacteria in pickled Chinese cabbage at 10°C. Figure 2 is Lactobacillus plantarum LTSM119
It is an explanatory diagram showing a growth curve of a strain. Figure 3 is Lactobacillus plantarum LTSM175
It is an explanatory diagram showing a growth curve of a strain. FIG. 4 is an explanatory diagram showing the growth curves of 14 strains of Lactobacillus plantarum.

Claims (1)

[Scope of Claims] 1. A microorganism that is a mutant strain of a bacterium belonging to the genus Lactobacillus, which exhibits low temperature sensitivity and causes poor growth when the microbial cells are inoculated into a nutrient medium and cultured at low temperatures. 2. The microorganism according to claim 1, wherein the culture temperature condition is about 15° C. or lower, and the mutant strain is Lactobacillus plantarum. 3. Lactobacillus plantarum is Lactobacillus plantarum.
plantarum LTSM119 strain and Lactobacillus
The microorganism according to claim 2, which is strain Plantarum LTSM175. 4. Fermentation by inoculating at least one type of microorganism according to claims 1 to 3 as a starter during fermentation of a plant material for fermentation, fermenting at a temperature suitable for the growth of the bacterial species, and then storing at a low temperature. A method for producing a fermented vegetable product, which is characterized by suppressing the growth of microorganisms in the product. 5. The manufacturing method according to claim 4, wherein the fermentation temperature is about 28°C to 35°C, and the storage temperature is about 15°C or less. 6. A fermented plant product obtained by the production method according to claim 4 or 5.