JP3018165B2 - Method for determining subspecies of lactic acid bacteria Lactococcus lactis - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a method for determining a lactic acid bacterium subspecies, and more particularly, to detecting γ-aminobutyric acid in a microorganism culture solution to accurately, quickly and easily determine a lactic acid bacterium subspecies. About the method.

[0002]

2. Description of the Related Art Lactococcus lactis ( Lactococcu s lactis) is a kind of L. lactis subsp. Lactis (hereinafter sometimes abbreviated as lactis) and L. lactis s.
ubsp. cremoris (hereinafter sometimes abbreviated as "cremoris"). Moreover, L. those that produce a diacetyl in lactis lactis subsp. Lactis
biovar diacetylactis (hereinafter sometimes abbreviated as diacetylactis). In the field of dairy products, especially cheese production, a fermentation starter containing L. lactis as a main bacterial species is added to raw milk to cause lactic acid fermentation. As a starter for cheese, Cremoris, which has properties such as suitable acid generating ability, generation of cheese flavor, and non-growth at high temperature (40 ° C.), is suitable for cheese production. Diacetylactis may also be used as a starter to impart a unique flavor.

[0003] Since the formation of flavor by microorganisms varies from strain to strain even for the same strain, lactic acid bacteria are still isolated from traditional fermented milk production sites around the world in search of strains with even higher utility. Is being done. The isolated bacteria are
Since the properties related to production can be estimated to some extent depending on the bacterial species, identification of the bacterial species is indispensable. L. lactis can be identified by microscopic observation and by examining properties such as Gram stain (+), catalase reaction (-), motility (-), lactic acid rotation (L), and sugar fermentation. Is being done. L.
The determination of lactis subspecies, ie, lactis (including diacetylactis) and cremoris, is based on the ability to produce ammonia from arginine, ie, the presence or absence of arginine deiminase activity.

[0004]

The arginine deiminase activity is determined by culturing the test bacteria in a liquid medium, collecting the cells by a solid-liquid separation operation such as centrifugation, washing the cells, and then washing the cells with physiological saline. This is carried out by mixing a suspension with a constant concentration with a reagent for measuring enzyme activity and observing the presence or absence of activity 4 hours later. In this method, it is necessary to select a medium that is suitable for collecting bacteria and has good growth of the bacteria.
In addition, laborious operations such as adjusting the concentration of the cell suspension and dispensing reagents are required. As a medium for lactic acid bacteria, MR
S medium, M17 medium, etc. have been developed, but all L.
There is no medium in which lactis grows well. for that reason,
It is necessary to select a suitable medium for each strain.

[0005] An object of the present invention is to provide a method for accurately, quickly and easily determining a subspecies of a lactic acid bacterium L. lactis .

[0006]

That SUMMARY OF THE INVENTION The present invention is lactic acid bacteria Lactococcu s la, characterized in that check for γ- aminobutyric acid in the culture of lactic acid bacteria Lactococcu s lactis
Related to ctis subspecies determination method.

[0007]

BEST MODE FOR CARRYING OUT THE INVENTION Although γ-aminobutyric acid is one kind of amino acids, it is not an α-amino acid constituting a protein but a γ-amino acid generated from L-glutamic acid by the action of glutamate decarboxylase. This γ-aminobutyric acid is well known as a neurotransmitter in animals, but its physiological function has not been clarified in plants and bacteria. In general, bacteria have glutamic acid decarboxylase, and produce γ-aminobutyric acid by decarboxylating glutamic acid in the medium as it grows. In L. lactis , γ-
There is no example that the presence or absence of aminobutyric acid generating ability was examined.
Therefore, this property has never been used for subspecies determination.

[0008] The γ-aminobutyric acid contained in the test bacterial culture can be detected by an existing analytical method such as an amino acid analyzer or thin layer chromatography. When the ability of L. lactis to produce γ-aminobutyric acid was examined, it was completely consistent with the presence or absence of arginine deiminase activity. Then, γ-
L. lactis subspecies is determined by the presence or absence of aminobutyric acid-producing ability. That is, a strain that produced γ-aminobutyric acid was determined to be lactis (including diacetylactis), and a strain that did not produce γ-aminobutyric acid was Cremoris.

[0009] Since L. lactis is a lactic acid bacterium isolated from milk and milk products, it can grow well on a skim milk medium. The skim milk medium cannot be used for cultivation for collecting bacteria because casein precipitates with the growth of lactic acid bacteria.
In the present invention, most of L. la
A skim milk medium in which ctis can grow is suitable. Since skim milk contains almost no γ-aminobutyric acid, the background upon detection is kept low, and γ-aminobutyric acid
The presence or absence of aminobutyric acid production can be clearly determined.
The skim milk medium is prepared by converting commercially available skim milk powder with water to 8 to 10% (w /
Prepare by dissolving in v). If necessary, glucose and / or yeast extract (commercially available product, for example, powdered yeast extract D-3 manufactured by Wako Pure Chemical Industries, Ltd.) can be added. Although yeast extract contains γ-aminobutyric acid, adding yeast extract to about 0.1% (w / v) does not affect the background of γ-aminobutyric acid detection. When the sensitivity of the method for detecting γ-aminobutyric acid is low, for example, by thin layer chromatography, it is desirable to add L-glutamic acid to the medium to increase the γ-aminobutyric acid concentration after culture. The concentration of L-glutamic acid is usually appropriately 0.1 M or less.

[0010]

EXAMPLES Next, the present invention will be described in more detail with reference to examples, but the present invention is not limited thereto. Example 1 A strain isolated from a cheese starter in which lactis (including diacetylactis) and cremoris were mixed was 8%
The cells were cultured at 30 ° C. for 4 days using a skim milk medium. After completion of the culture, 1 ml of 30% trichloroacetic acid was added to 5 ml of the culture, centrifuged (3000 rpm, 20 minutes) to obtain a supernatant, and 1 ml of 0.1 N hydrochloric acid was added thereto, followed by filtration. Was used to analyze the amount of γ-aminobutyric acid. The analysis was performed using an amino acid analyzer, and the ability of the strain to produce γ-aminobutyric acid was examined. In addition, the presence or absence of arginine deiminase activity by a conventional method was also examined. Table 1 shows the results. As is clear from the table, the results of both measurements were completely in agreement, indicating that the method of the present invention can accurately determine the lactic acid bacteria subspecies.

[0011]

[Table 1]

Example 2 A known L. lactis strain was examined for its ability to produce γ-aminobutyric acid by the method of the present invention shown in Example 1. The results are shown in Table 2. ATCC19435, ATCC13675 and
The ATCC19257 strain is a standard strain of lactis, diacetylactis and cremoris, respectively. As shown in the table, all of lactis and diacetylactis produced γ-aminobutyric acid, and none of Cremoris strains.

[0013]

[Table 2]

Example 3 TYG containing 0.5% tryptone, 0.5% yeast extract, 1.0% glucose and 0.5% sodium succinate
100 ml of a medium (pH 6.8) was inoculated with 1% of L. lactis and cultured at 30 ° C. for 16 hours. Then, centrifugation (3
(000 rpm, 20 minutes) to collect the cells, and the cells were washed once with PBS, and then centrifuged again. After suspending the obtained cells in distilled water, the cells were freeze-dried to 50 mM.
An enzyme solution was obtained by suspending in an acetate buffer (pH 4.7). A 50 mM acetate buffer (pH 4.7), 2 mM glutamic acid and 0.1 mM pyridoxal phosphate were added to the enzyme solution to prepare 1 ml of the enzyme reaction solution, and reacted at 30 ° C. for 20 hours.
Glutamate decarboxylase activity was measured by measuring the amount of γ-aminobutyric acid produced. As a result, the activity of lactis was 5.1 to 58.0 kat / kg (dry cell weight),
The activity of diacetylactis is 5.7 to 58.9 kat / k.
g (dry cell weight), but in the case of Cremoris, the activity was not detected (<0.01).

[0015]

According to the present invention, L. lactis subspecies can be determined accurately, quickly and simply by examining the culture of L. lactis for the production of γ-aminobutyric acid. it can. The conventional method for observing arginine degradation requires that the cultured strain be collected once, so that an appropriate medium must be selected, and the concentration of the bacterial solution must be adjusted.
It takes time to work such as dispensing reagents. However, in the present invention, a skim milk medium in which most L. lactis grows can be used, and selection of the medium is unnecessary. In addition, the present invention is practical because it can reduce operations such as sample preparation and reagent dispensing.

──────────────────────────────────────────────────続 き Continuation of front page (72) Inventor Yukio Someya 5-16, Matsushiro, Tsukuba-shi, Ibaraki 520-204 (58) Fields investigated (Int. Cl. ⁷ , DB name) C12Q 1/00-3/00 BIOSIS (DIALOG) WPI (DIALOG)

Claims (2)

(57) [Claims] 1. A variant judgment method of lactic acid bacteria Lactococcu s lactis, characterized in that check for γ- aminobutyric acid in culture of lactic acid bacteria Lactococcu s lactis. 2. The method according to claim 1, wherein the lactic acid bacterium Lactococcu s lactis is cultured in a skim milk medium, and the presence or absence of γ-aminobutyric acid in the culture solution is examined.