JPH0549471A - New microorganism, lactic fermentation food prepared by using the microorganism and its preparation - Google Patents

Abstract

PURPOSE:To obtain a new microbial strain capable of performing vigorous lactic acid fermentation even in an environment having high salt concentration compared with the fermentation attainable by the separate use of the original strains by fusing a cell of a microbial strain belonging to genus Lactobacillus to a cell of a microbial strain belonging to Pediococcus halophilus. CONSTITUTION:The objective microorganism F-6 strain, etc., is produced by forming protoplast of preferably Lactobacillus plantarum No.14 strain and Pediococcus halophilus No.8 strain, fusing both protoplast cells with each other, regenerating the cell wall in a hypertonic medium and selecting a microbial strain exhibiting vigorous growth in a medium having high salt concentration (e.g. sodium chloride concentration of 10-20%).

Description

Detailed Description of the Invention

BACKGROUND OF THE INVENTION

FIELD OF THE INVENTION The present invention relates to a technique for producing a lactic acid fermented product. Specifically, the present invention relates to a technique for producing a fermented product obtained by lactic acid-fermenting an arbitrary raw material of a plant substance and an animal substance under a high salt concentration, which is difficult for an ordinary microorganism to grow. More specifically, a fused microorganism obtained by cell-fusing a strain belonging to the genus Lactobacillus and a strain belonging to Pediococcus halophilus, which is a fused microorganism showing vigorous growth under high salt concentration, The present invention relates to a method for producing a lactic acid fermented food using a microorganism and a lactic acid fermented food produced by the method.

[0002]

[Outline of Invention]

[0003]

If good lactic acid fermentation can be carried out under a high salt concentration, there will be no spoilage due to various bacteria,
A new lactic acid fermented food product that makes the best use of the rawness of the raw materials can be produced without the need for addition of antibacterial agents or heat sterilization. Furthermore, if good lactic acid fermentation can be performed in a state of high salt concentration, a new fermented food which has never existed will be produced. The present invention, a raw material for any fermentation of animal substances and plant substances, under conditions of high salt concentration, which is difficult to grow with ordinary microorganisms, a technology that enables lactic acid fermentation,
That is, it is an object of the present invention to provide a novel microorganism for this purpose, a method for producing a lactic acid fermented food using the same, and a lactic acid fermented food.

[0004]

[Means for Solving the Problems] As a result of intensive studies, the present inventors have found that Lactobacillus plantarum and Pediococcus halophilus can perform vigorous lactic acid fermentation even at a salt concentration of around 15%. Succeeded in obtaining a fusion strain with, and by inoculating this fusion strain as a starter of fermentation, found that the fermentation under the high salt concentration of the above purpose is possible, and completed the present invention. It was That is, the microorganism according to the present invention is formed by fusing a strain belonging to the genus Lactobacillus and a strain belonging to Pediococcus halophyllus, and shows vigorous growth than both strains before fusion in an environment of high salt concentration. The fused microorganism shown is a preferable example of the fused microorganism obtained by cell fusion of Lactobacillus plantarum and Pediococcus halophilus. Further, the method for producing a lactic acid fermented food according to the present invention, at least one of the above-mentioned fused microorganisms is inoculated as a starter during fermentation of the fermentation raw material, other microorganisms cannot grow vigorously, and the fused microorganisms grow. Is carried out under a high salt concentration suitable for, while suppressing the growth of other microorganisms in the fermented product. Furthermore, the lactic acid fermented food according to the present invention is produced by the above production method.

[Detailed Description of the Invention] <Fusion Microorganism> The fusion microorganism according to the present invention includes a strain belonging to the genus Lactobacillus ( L. ) and a strain belonging to the genus Pediococcus ( P. ). It has been described above that it is formed by fusing, and shows a stronger growth than both strains before fusion in an environment of high salt concentration. The environment of high salt concentration, the normal microorganisms present during fermentation are killed or unable to reproduce but the fusion microorganism of the present invention contains a high concentration of salt showing vigorous growth, specifically, For example, about 10
It contains about 20% (W / V) sodium chloride (salt). Preferred examples of strains belonging to the genus Lactobacillus include, for example, Lactobacillus plantarum ( L. pl.
antarum ), Lactobacillus casei ( L. casei ),
Lactobacillus carbatas ( L. curvatus ) and the like. A preferred specific example of the fused microorganism according to the present invention is a fusion of Latoku Bacillus plantarum and Pediococcus halophilus. As a typical preferred example thereof, for example, F-6 strain and A-9 strain described later are can give. The present inventors have found that Lactobacillus plantarum NO. Pediococcus halophilus NO. Isolated from 14 strains and soy sauce moromi
Using the 8 strains as the parent strains, a fusion strain showing vigorous growth in a high salt concentration environment, specifically, for example, when about 15 to 20% concentration of salt is added, that is, the F-6 strain, by an ordinary cell fusion method. And A-9 strain were produced and isolated. This F
The strains -6 and A-9 are two types of fused microorganisms obtained from the same cell fusion strain producing system and having different physiological properties, as shown in the Examples below (see Table 1 below).

Lactobacillus plantarum NO. 14
Stock (parent stock) and Pediococcus halophilus N
O. 8 strains (parent strain) and fusion strains F-6 strain and A-9
The mycological properties of the strain are as shown in Table 1 below. These mycological properties are based on the Bergey's Manual of Systematic Bacteriology.
f Systematic Bacteriology) Vol. 2 (1986) and the world of intestinal bacteria-classification and identification of anaerobic bacteria- (written by Michioka Tomohushi).

_{Table 1} NO. 14 NO. 8 F-6 A-9 a) Morphology (1) Shape and size of cells Bacillus cocci Staphylococcus coccus (2) Motility − − − − (3) Presence of spores − − − − (4) Gram stain positive Positive Positive Positive b) Physiological properties (1) Reduction of nitrate − − − − (2) MR reaction + + + + (3) VP reaction − − − − (4) Indole formation − − − − (5) Sulfide Production of hydrogen − − − − (6) Hydrolysis of starch − − − − (7) Utilization of citric acid − − − − (8) Formation of pigment − − − − (9) Urease − − − − (10) Oxidase − − − − (11) Catalase − − − − (12) OF test Fermentation Fermentation Fermentation Fermentation (13) Salt tolerance test (W / V) -10% -22% -22% -22% (14) Utilization of inorganic nitrogen source − − − − (15) Liquefaction of gelatin − − − − (16) Optical activity of lactic acid DL L L L (17) Litmus milk Milk acidification Weak reduction Weak reduction Weak return (18) Growth range Growth pH 4.0 to 8.0 6.0 to 9.0 5.0 to 9.0 5.0 to 9.0 Optimum pH 7.0 7.0 to 8.0 7.0 to 8.0 7.0 to 8.0 Growth temperature 15 to 38 ° C 15 to 38 ° C 15 to 38 ° C 15 to 38 ℃ Optimum temperature 33 to 35 ℃ 33 to 35 ℃ 33 to 35 ℃ 33 to 35 ℃ (19) Production of gas from glucose (20) Production of acid from sugar L-arabinose-* ++ D -Xylose-*---D-Glucose + + + + D-mannose + + + + D-fructose + + + + D-galactose + + + + + maltose +-+ +-sucrose +-+-lactose +-- -Trehalose + --- D-Sorbit + --- D-mannitol +++ + inosit-* --- Glycerin-* +++ + starch + --- *: indicates that it cannot grow.

The production of the fused microorganisms or cell fusion strains according to the present invention is basically carried out by first converting two types of cells having high proliferative activity into protoplasts by a conventional method and then fusing the protoplast cells with each other to produce cells. In contrast, the cell wall is regenerated in a hypertonic medium. From the fused cells thus regenerated, a high salt concentration, for example, 10 to 20% salt,
The desired cell fusion strain can be easily obtained by selecting one showing a vigorous growth in a medium of preferably about 15% (gram percent concentration). As the strain for fusion used for protoplast formation, vegetative cells cultured in an ordinary lactic acid bacterium medium or various media in which the strain for fusion can grow can be used. The culture medium used is Ro
Any medium capable of growing a strain for fusion, such as various lactic acid bacterium typified by gosa medium, GPY medium or MRS medium, can be used. The vegetative cells cultured in these media are prepared by preparing a bacterial cell suspension by washing and removing the media components with a commonly used hypertonic buffer solution (eg, Tris-maleic acid buffer solution, phosphate buffer solution, etc.). ,
Protoplasts can be formed by treating the cell suspension with a lactic acid bacterium cell wall lytic enzyme. Specific examples of the cell wall lysing enzyme include egg white lysozyme (manufactured by Seikagaku Corporation, hereinafter abbreviated as lysozyme) or
N-acetyl-muramidase (manufactured by Seikagaku Corporation, hereinafter abbreviated as NAM) and the like can be mentioned. By using the above-mentioned two kinds of enzymes in combination, protoplasts can be obtained more efficiently. The fused microorganism or cell fusion strain can be easily obtained by a usual cell fusion method such as the electric pulse method or the polyethylene glycol method using the mixed solution of protoplast cells prepared as described above. For a general description of the above-mentioned various methods for cell fusion, see, for example, "Cell fusion and gene transfer method by electric pulse and its present state" (Shimadzu, Vo1.46, No4, 1989.
12) can be referred to.

Deposit of microorganisms Lactobacillus plantarum NO. The 14 strains have been deposited in the Institute of Microbial Engineering, National Institute of Industrial Science and Technology as Micromachine Research Institute No. 11550 (FERM P-11550). Pediococcus halophilus NO. Eight strains were sent to the Institute of Microbial Science and Technology of the Agency of Industrial Science and Technology, Microtechnical Institute No. 12224 (FERM P-122
24) has been deposited. The cell fusion strains F-6 strain and A-9 strain were obtained from the Institute of Microbial Science and Technology, the Institute of Industrial Science and Technology, by the Institute of Microbiology and Industry, No. 12226 (FERM P-12226) and the No. 12225, Microtechnical Institute. 12225).

<Method for producing lactic acid fermented food> The method for producing a lactic acid fermented food according to the present invention comprises inoculating at least one of the above-mentioned fused microorganisms of the present invention as a starter during fermentation of a fermentation raw material, and other microorganisms vigorously. Fermentation that can not grow in and under high salt concentration suitable for growth for the fusion microorganism while suppressing the reproduction of other microorganisms in the fermented product,
And enables active lactic acid fermentation under high salt concentration. Therefore, the production method according to the present invention is characterized in that it is not necessary to sterilize the microorganisms in the raw material by heating or a sterilizing agent because the fermentation is carried out under a high salt concentration. (1) Raw Material for Fermentation The raw material for the fermented product (fermented food) may be any of plant substances or animal substances that can be used by the fused microorganism of the present invention. For example, in the case of plant substances, cucumber, Chinese cabbage, radish , Vegetables such as carrots, fruits such as apples, pears, bananas, strawberries, cereals such as soybeans, wheat, adzuki beans, etc., and general examples of animal and animal substances include various meats and fishes. (2) Fermentation The above-mentioned fermentation raw material is usually washed with water or sterilized with a sterilizing agent such as sodium hypochlorite (in order to usually protect the rawness of the raw material). Is not subjected to heat sterilization, but if necessary, this heat treatment may be performed), a state of high salt concentration, preferably salt 10
% To 20%, more preferably 15% to 18% (W / V). As the salt, other than the salt, if necessary, another salt such as KCl or MgCl ₂ can be added in a necessary amount. If desired, seasonings such as amino acids and nucleic acids can be added in desired amounts. A culture solution of the fusion microorganism of the present invention (usually at a concentration of 1 × 10 ⁶ to 1 × 10 ⁹ cells / ml, preferably 1 × 10 ⁸ cells / ml) was added to the raw material for fermentation in the high salt concentration state. 1% to 10
%, Preferably about 1% (each by volume). Then, usually 10 ° C to 40 ° C, preferably 28 ° C to 35 ° C
The fermentation can be carried out at the temperature of. Fermentation time may vary depending on the raw materials or conditions such as the desired degree of fermentation.
Generally about 4 weeks. Note that the high-concentration salt can be reduced to a desired concentration after fermentation by ordinary desalting, if necessary. By the production method of the present invention as described above, it is possible to obtain a completely new lactic acid fermented food by vigorous lactic acid fermentation under a high salt concentration, which does not require sterilization of microorganisms in the raw material by heating or a bactericide.

[0011]

The following are examples of the present invention.
The present invention is not limited to this. Example 1 Acquisition of cell fusion strain of Lactobacillus plantarum and Pediococcus halophilus (1) Culture (i) Lactobacillus plantarum NO. 14 shares (te
One platinum loop was inoculated into 10 ml of GPY medium (Table 2) from the t +, NaCl-) storage medium and cultured at 30 ° C for 1 day.
The culture was inoculated into the same medium, and subculture at 30 ° C. for 1 day was repeated several times to stabilize the growth, which was used as a preculture. Next, 0.3 ml of the preculture liquid was inoculated into 30 ml of the same medium and cultured at 30 ° C. for 10 hours to give a main culture. (ii) Pediococcus halophilus NO. 8 shares (te
t-, NaCl +) storage medium 1 platinum loop, NaCl 7%
(W / V) -added Rogosa medium (Table 3) (10 ml) was inoculated and cultured at 30 ° C for 2 days. The culture medium was inoculated into the same medium, and the subculture was repeated several times at 30 ° C. for 2 days to stabilize the growth, and this was used as a preculture. Next, 0.3 ml of the preculture liquid was inoculated into 30 ml of the same medium and cultured at 30 ° C. for 24 hours to give a main culture. Table 3 Rogosa medium tryptic case 10 g yeast extract 5 g tryptose 3 g K ₂ HPO ₄ 3 g KH ₂ PO ₄ 3 g ammonium citrate 2 g salt solution * 5 ml Tween 80 1 g sodium acetate / 3H ₂ O 1.7 g glucose 20 g cysteine (hydrochloride) 0.2 g (agar 15 g) distilled water 1000 ml (pH 6.8) * salt _{solution: MgSO 4 · 7H 2 O 11.5g} , FeSO 4 · 7H 2 O 0.68g, water MnSO ₄ · nH 2 O 2.4g Dissolved in 100 ml In addition, (tet +, NaCl in (i) and (ii) above
-) And (tet-, NaCl +) have the following meanings. Tetracycline (20 μg / ml) NaCl (10% (W / V)) Lactobacillus plantarum No.14 strain + (resistance)-(susceptibility) Pediococcus halophilus No.8 strain- + (2) Protoplast formation The cells after the main culture are centrifuged (7500 xg / 5 min / 4
Then, the cells were collected. The bacterial cells collected according to the conventional method are
Washing was performed twice with MS buffer (Table 4), and after washing, O. D. _The bacterial suspension was adjusted so that the value at _{660 nm} was 0.7. Next, 4 ml of bacterial suspension and 1 enzyme solution
ml was added to bring the total volume to 5 ml. The enzyme reaction was carried out at 35 ° C. with slight shaking. The reaction was started by adding the enzyme, and the reaction time of the enzyme was set to 60 minutes.
The respective enzyme concentrations are shown in Table 5. Table 5 Enzymes used for protoplast formation and enzyme concentration * L. plantarum NO.14 α-amylase 50 μg / ml N-acetyl-muramidase 10 μg / ml P.halophilus NO.8 α-amylase 250 μg / ml lysozyme 25 μg / ml *: After the final concentration protoplast reaction, washing was performed twice using the electric pulse solution ¹⁾ . After washing, a suspension of protoplast cells was prepared using the same solution, and two kinds of cells were mixed in equal amounts to prepare a cell fusion sample. ¹⁾ Solution for electric pulse: 0.3 M sucrose solution (3) Fusion operation Inject 0.2 ml of the adjusted cell fusion sample into a fusion chamber (Shimadzu FTC-01) and let it stand for 1 minute. The fusion operation was performed under the conditions shown. After performing the fusion operation, the cells were left for 2 minutes to recover the cell fusion treated cells. (4) Cell fusion device: Shimadzu Corporation: SSH-1 Fusion chamber: FTC-01 (5) Reaction conditions Fusion was performed under the following conditions. FREQ 1.0MHz VAC (PRI) 40V INITIAL TIME 120s VAC (SEC) 40V REPEAT INTERVAL 1s n 5 VDC DEC RATE 100% FINAL TIME 1s VAC DEC RATE 300μ pulse width TEMP AMB
sec. Number of pulses 5 times Electric field strength 1.0 to 14.
0kV / cm (6) Regeneration Selective medium ²⁾ to which 0.8% (W / V ⁾ of agar was added, 0.1 ml of the protoplast mixture after the fusion reaction was mixed and regenerated in a 30 ° C incubator. The fusion strain was allowed to colonize. Colonies that appeared in the selective solid medium were treated with 7% Na
Bacteria are sown in a Rogosa medium containing Cl and cultured at 30 ° C. for growth. 0.1 ml of medium in which the bacterial cells grew
Is inoculated into 10 ml of the same medium and cultivated at 30 ° C., which is referred to as preculture. Further, the same operation was performed to preculture the parent strains. Ro containing 0.1 ml of preculture liquid containing 7% NaCl
Rogos containing 10 ml of gosa medium and 15% NaCl
Each of 10 ml of the medium a was inoculated and cultured at 30 ° C. Rog containing 15% NaCl from these strains
Those that grew faster than the parent strain in osa medium were selected. ²⁾ Selection medium: 10% (W / V) NaCl in Rogosa medium
And tetracycline 20 μg / ml (final concentration) plus 0.3 M sucrose.

Example 2 Colonies appearing in a selective solid medium were treated with 7% NaCl.
Bacteria were added to the Rogosa medium supplemented with and cultured at 30 ° C. for growth. Then, the medium 0.
1 ml was inoculated into 10 ml of the same medium and cultured at 30 ° C., which was used as preculture. Further, the same operation was performed to preculture the parent strains. Pre-cultured solution 0.1 ml Rogosa medium 10 ml containing 7% NaCl and Rog containing 15% NaCl
Each 10 ml of osa medium was inoculated and cultured at 30 ° C. O.O. D. The value at _{660 nm} was measured and used as the growth rate (Figs. 1 and 2).

Example 3 Rogos with 13%, 15% and 18% NaCl
a. The parent strain Pediococcus halophilus N on medium
O. Equal amounts of precultured strains of 8 strains, F-6 strain, and A-9 strain were inoculated and cultured at 30 ° C. for 1 week. The amount of lactic acid produced in the culture broth after the culturing was compared to compare the production of lactic acid at a high salt concentration (FIG. 3).

Example 4 Cucumber was washed and cut into slices each having a width of about 1.5 cm. It was chased with salt, left for 10 minutes, washed thoroughly with water, and drained. 200pp of cucumber after draining
Immerse in m 3 hypochlorous acid solution for 3 minutes, thoroughly wash with water and drain to 90 g of cucumber at a final concentration of 15% salt (W /
100 ml of saline solution of V) was added. The parent strain and the fusion strain were inoculated to each of these to a concentration of 1% (V / V).
Culturing was performed in an incubator at 0 ° C. As a control, the same treatment was carried out with a final concentration of 15% (W / V) without inoculation of bacteria. The results were compared by the state of pickles and the turbidity of pickles in fungi (see Tables 6 and 7). Table 6 Pickling solution Smell 15% NaCl bacteria Not inoculated No cloudiness Blue odor peculiar to cucumber L. Plantanum No cloudiness Blue odor peculiar to cucumber P. halophilus, lightly cloudy, weak cucumber odor Fusion strain (F-6) White turbid pickled odor

Example 5 Lactobacillus plantarum NO. 14 strains, Pediococcus halophilus NO. 8 strains, F- which is a fusion strain
The cells of strain 6 and strain A-9 were treated with Rogosa medium and 7 strains.
Culture was carried out in a Rogosa medium containing% NaCl. The cultured cells are collected, washed and diluted to 1 × 10 ⁸ cells /
Adjusted to ml. This adjusted bacterium was used as a starter. Next, Rakukyo was peeled off, the roots were cut off and washed with water. 60 g of drained Rakukyo and 60 ml of distilled water containing NaCl were combined to adjust the final salt concentration to 15% (W / V). The adjusted bacterial cells were added to the final concentration of 1 × 10 ⁶ ce.
The test was started by inoculating to lls / ml and fermenting at 30 ° C. As a control test section, a non-inoculated cell was prepared and the same treatment was performed. Fermentation period is 4 weeks, test items are lactic acid, pH, number of lactic acid bacteria,
The measurement was carried out for four items of the number of general bacteria (see FIGS. 4 to 7).

[0016]

EFFECTS OF THE INVENTION The fused microorganism of the present invention has the characteristic of exhibiting vigorous growth under high salt concentration, which is difficult for ordinary microorganisms to grow. By using this fused microorganism of the present invention as a starter at the time of fermentation of a fermentation raw material under a high salt concentration, which is difficult to grow with a normal microorganism, vigorous lactic acid fermentation under a high salt concentration becomes possible. Therefore, since the fermentation is carried out under a high salt concentration, it is possible to obtain a completely new lactic acid fermented food that does not require sterilization of microorganisms in the raw material by heating or a bactericide. The present invention can also be applied to fermented foods having a high salt concentration, such as miso, soy sauce, and anchovy.

[Brief description of drawings]

FIG. 1 is an explanatory view showing the growth properties of the cell fusion strain of the present invention and a parent strain in a 7% NaCl-added Rogosa medium.

FIG. 2 15% NaCl of the cell fusion strain of the present invention and the parent strain
Explanatory drawing which shows the growth property in addition Rogosa culture medium.

FIG. 3 is an explanatory view showing the production amount of lactic acid in the high-concentration medium of the cell fusion strain of the present invention and the parent strain.

FIG. 4 is an explanatory view showing a change with time in pH of a fermentation broth in the Rakukyo fermentation test of the cell fusion strain of the present invention and the parent strain.

FIG. 5 is an explanatory diagram showing a time-dependent change in the amount of lactic acid in the fermentation broth in the Rakukyo fermentation test of the cell fusion strain of the present invention and the parent strain.

FIG. 6 is an explanatory view showing changes over time in the number of each lactic acid bacterium in the fermentation broth in the Rakukyo fermentation test of the cell fusion strain of the present invention and the parent strain.

FIG. 7 is an explanatory diagram showing the time-dependent changes in the number of general bacteria in the fermentation broth in the Rakukyo fermentation test of the cell fusion strain of the present invention and the parent strain.

Continuation of front page (51) Int.Cl. ⁵ Identification number Office reference number FI technical display location C12N 15/03 // (C12N 15/03 C12R 1:25 7804-4B 1:10) (72) Inventor Mori Akihiro Ta, 410, Doman, Amaguma, Kasukabe, Saitama Prefecture Inside Momoya Research Institute, Inc.

Claims (8)

[Claims] 1. A fusion which is formed by fusing a strain belonging to the genus Lactobacillus and a strain belonging to Pediococcus halophyllus, and shows more vigorous growth than both strains before fusion in an environment of high salt concentration. Microorganisms. 2. The fused microorganism according to claim 1, wherein the high salt concentration is at least about 10% to 20% (W / V) of sodium chloride. 3. The fused microorganism according to claim 1, wherein the strain belonging to the genus Lactobacillus is Lactobacillus plantarum. 4. The fused microorganism according to claim 3, wherein the fused microorganism is strain F-6. 5. The fused microorganism according to claim 3, wherein the fused microorganism is strain A-9. 6. At least one of the fused microorganisms according to any one of claims 1 to 5 is inoculated as a starter during fermentation of a fermentation raw material, other microorganisms cannot grow vigorously, and the fused microorganisms are highly suitable for growth. A method for producing a lactic acid fermented food, which comprises fermenting while suppressing the growth of other microorganisms in the fermented product under a salt concentration. 7. The method according to claim 6, wherein the high salt concentration contains at least about 10% to 20% (W / V) sodium chloride. 8. A lactic acid fermented food obtained by the production method according to claim 6.