JPH099954A - New lactic acid strain - Google Patents

Abstract

(57) [Abstract] [Purpose] Cell membrane-bound adenosine triphosphatase
Provided is a lactic acid bacterium strain having a decreased (ATPase) activity. From the parent strain of the structure Lactobacillus lactis ssp lactis (Lactococcuslactis subsp. Lactis), cell membrane bound ATPase activity can select the lactic acid bacteria strains having decreased. [Effect] This lactic acid bacterium strain grows when the pH of the medium becomes low, and the amount of lactic acid produced decreases. You can

Description

Detailed Description of the Invention

[0001]

FIELD OF THE INVENTION The present invention relates to Lactococcus lactis subspecies lactis (Lac ) having a decreased activity of cell membrane-bound adenosine triphosphatase (ATPase).
tococcus lactis subsp. lactis ).

[0002]

2. Description of the Related Art ATPase is an enzyme that hydrolyzes a phosphate at the γ-position of adenosine triphosphate in the presence of magnesium ion or calcium ion to decompose adenosine diphosphate and inorganic phosphate. It is known that the energy generated during the decomposition reaction is important for the living body. And lactic acid bacterium Lactococcus lactis subsp.
It is known that lactis ) has a cell membrane-bound ATPase as in Escherichia coli. However, not been elucidated about the impact of this cell membrane-bound ATPase is on the sugar metabolism and energy production of Lactococcus lactis ssp lactis (Lactococcus lactis subsp. Lactis). Furthermore, Lactococcus lactis subspecies
No strain of Lactococcus lactis subsp. Lactis is known.

On the other hand, a cell membrane-bound ATPase-deficient mutant strain was induced by transduction from an Escherichia coli K-12 strain pyruvate-producing bacterium, and its fermentation pattern was compared with that of the parent strain. It is known that the glycolytic system is activated due to the decrease in the energy level of the cells, and the sugar consumption per cell and the pyruvic acid-producing ability are remarkably increased.

Further, a Lactobacillus strain having a decreased cell membrane-bound ATPase activity is selected, and a fermented dairy product in which an increase in lactic acidity is suppressed by using this strain has been proposed [JP-A-7- No. 123977].

[0005]

DISCLOSURE OF THE INVENTION The present inventors have found that Lactococcus lactis subspecies lactis (Lacto
coccus lactis subsp.lactis ), various researches were underway, and as a result, Lactococcus lactis subspecies lactis (L
actococcus lactis subsp. lactis ). Accordingly, the present invention is Lactococcus lactis ssp lactide <br/> scan the cell membrane bound ATPase activity is decreased (Lactococcus lactis subsp. Lactis) and to provide a mutant strain.

[0006] Cell membrane bound Lactococcus lactis ssp La <br/> Kuchisu the ATPase activity is decreased in the present invention (Lactococcus lactis subsp. Lactis) mutant, rapidly lactic acid production and growth in acidic Because it will decrease
It can be used when producing fermented dairy products such as cheese.

[0007]

[Means for Solving the Problems] Lactococcus lactis sub, which has a decreased cell membrane-bound ATPase activity of the present invention.
The sp. lactis ) mutant strain can be obtained as follows.

[0008] Lactococcus lactis ssp lactis (Lactococcus lactis subsp. Lactis) to get the neomycin natural resistant strains from the parent strain, the inside of neomycin natural resistant strains to obtain a growth decline strain growth was lower than the parent strain. Then, the ATPase activity of this growth-decreased strain is measured to obtain an ATPase activity-decreased strain having an ATPase activity reduced by 30% or less as compared with the ATPase activity of the parent strain.

The thus obtained lactococcus
Lactis subspecies lactis (Lactococcus la
ctis subsp. lactis ATPase activity decrease strain), when the pH in the fermentation process is reduced their growth is suppressed, since the amount of lactic acid is decreased over time, such as dairy products it is necessary to suppress the generation of lactic acid It can be used for manufacturing.

Hereinafter, the present invention will be described in detail with reference to Examples.

【Example】

(1) Acquisition of neomycin resistant strain Lactococcus lactis, which was purchased from L. Mackay
Lactococcus lactis subs
A neomycin naturally resistant strain was obtained using the p. lactis ) C-2 strain as a parent strain. That is, M17L whose composition is shown in Table 1 for the parent strain
Inoculate in 5 ml of liquid medium and incubate at 30 ℃ overnight.
Cultures on M17L plate medium containing neomycin at μg / ml concentration
0.5 ml was plated and cultured at 30 ° C for 2 days. Then, 1500 grown neomycin resistant strains were obtained.

[0011]

[Table 1] ─────────────────────────
── Polypepton 5g (per liter of medium) Phytone peptone 5g Beef extract 5g Yeast extract 2.5g Ascorbic acid 0.5g β-Disodium GP 19.0g 1.0M MgSO ₄ / 7H ₂ O 1.0ml Lactose Monohydrate 5.26g ────── ────────────────────
──

(2) Acquisition of growth-decreased strains The above neomycin-resistant strains were shake-cultured in an M17L liquid medium to obtain 20 growth-decreased strains whose growth was lower than that of the parent strain.

(3) Measurement of ATPase activity The ATPase activity of the above growth-decreased strain was measured. ATPa
The se activity was measured by the following method.

First, 4% of the cells were inoculated into M17L liquid medium, cultured at 30 ° C. for 3 to 4 hours, and then centrifuged at 4 ° C. (8,0).
The cells were collected at 00 × g for 10 minutes). The cells were washed twice with 100 mM Tris-HCl buffer (pH 8), and 1 g of the cells were suspended in 5 ml of the same Tris-HCl buffer and sonicated.
The supernatant was recovered by centrifugation (20,000 xg, 10 minutes) at ℃. Then, the supernatant was ultracentrifuged at 4 ° C (100,000 x
(g, 1 hour) to recover the precipitate, which was suspended in the same Tris-hydrochloric acid buffer solution to give a crude enzyme solution. On the other hand, 5 mM sodium adenosine triphosphate as a substrate was dissolved in 100 mM Tris-HCl buffer (pH 8) containing 2.5 mM magnesium chloride to prepare a substrate solution.

550 μl of the above substrate solution and 50 μl of the crude enzyme solution
Were mixed and reacted at 37 ° C. for 10 minutes, and then 300 μl of ice-cooled 0.1N hydrochloric acid was added to stop the reaction. Then, 2.1 ml of the color developing solution having the composition shown in Table 2 was added and color was developed at 18 ° C. for 10 minutes, then, the mixture was centrifuged at 3,000 rpm for 10 minutes to remove the precipitate, and the OD was measured at 660 nm.

[0016]

[Table 2] ────────────────────────────────── 5N sulfuric acid 10ml 2.5% ammonium molybdate 10ml 3% sulfuric acid Sodium hydrogen-1% Paramethylaminophenol sulfuric acid 10ml Water 40ml ───────────────────────────────────

(4) Acquisition of low ATPase activity strain As shown in FIG. 1, ATPa having 30% or less of the ATPase activity of the parent strain is used.
9 strains showing se activity were obtained. The strain with the lowest ATPase activity was 12% of the ATPase activity of the parent strain. This ATPa
Lactococcus lactis subsp. la is the strain with the lowest se activity.
ctis ) SBT 10439 was deposited at the Institute of Biotechnology, Institute of Industrial Science and Technology (accession number: FERM P-14940).

The Lactococcus lactis subsp.
lactis ) SBT 10439 strain is a parent strain of Lactococcus lactis (Lactococcus lactis s).
Since the ubsp. lactis ) C-2 strain had the properties shown in Table 3, Lactococcus lactis subspecies
Lactis (Lactococcus lactis subsp. Lactis ).

[0019]

[Table 3] ─────────────────── Growth at 10 ℃ ＋ Growth at 39.5 ℃ ＋ Growth at 45 ℃ －2% Growth with NaCl ＋ 4% Growth on NaCl + Growth on 6.5% NaCl-Hydrolysis of arginine + CO ₂ from citrate − Growth at pH 9.2 + Form of lactic acid produced CO ₂ − from glucose L (+) glucose − − ──────────────────

[0020]

[Test Example] A 500 ml jar fermenter filled with the medium shown in Table 4 was added to the parent strain Lactococcus lactis s.
ubsp. lactis) C-2 strain and Lactococcus lactis ssp. lactis as ATPase activity reducing strain (Lactoco
ccus lactis subsp. lactis) SBT 10439 strain were each inoculated, stirring speed 100 rpm, by controlling the respective pH at incubation temperature 30 ° C.,
Fermentation characteristics were compared by jar culture.

[0021]

TABLE 4 ───────────────────── Polypepton 8 (g / _{l) Yeast extract 5 KH 2 PO 4} 2 NaCl 0.1 MgSO 4 · 7H 2 O 0.5 Glucose 50 ─────────────────────

The growth, the amount of lactic acid produced, and the amount of lactic acid produced per bacterium of the ATPase activity-reduced strain at each pH 12 hours after the start of the culture are shown in FIG. . When the pH increases, both lactic acid production and growth decrease, and the lactic acid production per cell is always 80% of that of the parent strain.
%, But both growth and lactic acid production decrease when pH decreases, especially when lactic acid production significantly decreases.
In 4.5, the amount of lactic acid produced per cell decreased to 60% of the parent strain.

[0023]

[Effect of the invention] Lactococcus lactis subsp. La of the present invention
Strains with reduced ATPase activity of ctis ) are useful in the production of dairy products and the like that require suppression of lactic acid production over time because their growth is suppressed when pH is lowered during the fermentation process.

[Brief description of drawings]

[Figure 1] AT of low ATPase activity strain against ATPase activity of parent strain
Shows Pase activity.

FIG. 2 shows growth of strains with reduced ATPase activity, lactic acid production, and lactic acid production per bacterium.

Claims (2)

[Claims] 1. A Lactococcus lactis (Lactococcus lactis ) having a decreased activity of cell membrane-bound adenosine triphosphatase (ATPase).
subsp. lactis ) mutant strain. 2. Lactococcus lactis (Lactococcus lactis ) having a decreased activity of cell membrane-bound adenosine triphosphatase (ATPase).
subsp. lactis) SBT 10439 (FERM P-14940).