JPH09121896A - Early judgment of lactic acid bacteria growing in beer and kit for the judgment - Google Patents

Abstract

PROBLEM TO BE SOLVED: To enable early judgment whether or not a detected lactic bacterium grows in beer for quality control in beer production by allowing a lactic bacterium to desirably be judged to react with an organic acid, then determining the ATP in the bacterial cells according to the luciferin-luciferase technique. SOLUTION: A lactic bacterium to be desirable judged is anaerobically cultured in a medium for detecting lactic bacteria at a pH of 4.5-6.0 at 25-28 deg.C for 2-3 days. Then, the bacterial cells are collected, rinsed with a buffer solution, then cultured in a beer with a pH of 4.0-5.2 for 6-24 hours, centrifuged to collect the cells, washed, then allowed to react with an organic acid such as malic, citric or pyruvic acid and the ATP in the cells of this lactic bacterium is determined according to the luciferin-luciferase technique whereby whether the detected lactic bacterium can grow in beer or not and whether the bacterium damages the quality of the beer or not can be efficiently judged at an early stage.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to an early determination method and determination kit for lactic acid bacteria grown in beer.

[0002]

2. Description of the Related Art When harmful microorganisms are mixed in foods and drinks, the quality of the products is significantly impaired due to turbidity and rancidity. In beer, the gram-positive bacterium Lactobacillus (Lact
Lactic acid bacteria such as obacillus), Pediococcus genus (Pediococcus), and Leuconostoc genus (Leuconostoc) are typical bacteria detected as beer-contaminating bacteria. However, all of these strains do not necessarily grow in beer and impair beer quality, and include those that can grow even in the same genus, those that can grow only after acclimatizing to beer, or those that cannot grow in beer at all. Has been. Therefore, when strains belonging to these are detected in beer products and beer manufacturing processes, it is extremely important to determine whether the strains can grow in beer.

At present, lactic acid bacteria detected in the manufacturing process or product beer are subcultured into beer again from the agar medium for detection, and the turbidity and the number of bacteria are measured to judge the growth. In this case, Not all lactic acid bacteria grow, and it takes a very long time to judge the growth (about 1 to 2 months). On the other hand, it is known to use ATP or the luciferin-luciferase method for determining beer harmful lactic acid bacteria. As the documents describing these uses, the main ones are "Lactic acid bacterium quantification method and its kit" in JP-A No. 54-123094, "Measurement method of fire-fall bacterium" in JP-A No. 07-195, JAM SOC BREW CHEM, VOL.
52, NO.1, 19-23 (1994) "ATP Bioluminescence Measurement of Potential Beer Hazardous Bacteria", J INST BRE
W, VOL.95, NO.5, 317-319 (1989), "A rapid method for detecting yeast and lactic acid bacteria by utilizing bioluminescence of ATP" and the like are available. The luciferin-luciferase method emits light, and the luminescence intensity is used to determine whether or not the corresponding lactic acid bacterium exists, and if so, the number of viable bacteria.

[0004]

However, the conventionally used method for measuring ATP in lactic acid bacteria by the luciferin-luciferase method is to extract ATP existing in the lactic acid bacteria in the first place and measure the ATP to detect lactic acid bacteria. However, it is not possible to determine whether the measured lactic acid bacteria can grow in beer.

Therefore, the present invention provides a method and a judging kit for judging early whether or not the detected lactic acid bacteria grow in beer.

[0006]

In order to achieve the above-mentioned object, the present invention comprises culturing a lactic acid bacterium for a specimen, and then adding at least one organic acid selected from pyruvic acid, malic acid and citric acid. After reacting for a certain period of time, ATP in the bacterial cells
A method and kit for measuring and determining the amount by the luciferin-luciferase method.

Lactic acid bacteria that grow in beer consume pyruvic acid, malic acid or citric acid in beer and newly generate lactic acid, acetic acid or carbon dioxide gas. On the other hand, for lactic acid bacteria that do not grow in beer, the pH of the microbial cells is lowered due to the ionophore action of isohumulone in beer, growth is inhibited, and death occurs. In this regard, the amount of ATP produced based on decarboxylation of organic acids such as bilvic acid, malic acid or citric acid was measured using lactic acid bacteria that grow with beer and lactic acid bacteria that cannot grow. Was confirmed to be significantly higher than the amount of non-viable bacteria.

Therefore, the present invention utilizes this phenomenon to cause an organic acid such as malic acid to act on a lactic acid bacterium of a sample to change the amount of ATP in the lactic acid bacterium to be judged, and the changed AT
The amount of P is measured by a known method, the luciferin-luciferase method, to determine the growth ability of lactic acid bacteria in beer. At this time, the amount of ATP in the microbial cells increases in beer-grown lactic acid bacteria, but the amount of ATP in the microbial cells does not increase in lactic acid bacteria that cannot grow.

This method makes it possible to predict early (6 to 24 hours) the growth ability of beer products or bacteria detected in the beer production process in beer.

[0010]

BEST MODE FOR CARRYING OUT THE INVENTION The present invention can be implemented as follows.
The lactic acid bacterium to be determined is first cultured in a lactic acid bacterium detection medium. The culture conditions are anaerobic culture, pH 4.5 to 6.0, and the culture temperature is 25 to 28 ° C. for 2 to 3 days. The medium for detecting lactic acid bacteria is not limited as long as beer-grown lactic acid bacteria can grow properly. However, 10 to 50% of beer is added to the medium, or hop components (2
10 ppm, Isohumurone), organic acids (pyruvic acid, malic acid, citric acid) and metal (0.5 to 1.5 mm MgSO _4, MnSO
It is better to add ( ₄ etc.) because it grows faster.

The precultured lactic acid bacteria are then harvested and washed with a buffer solution. In addition, in order to increase the detection efficiency, pH after harvesting 4.
A step of culturing with beer of 0 to 5.2 for 6 to 24 hours may be added. The washed lactic acid bacteria are suspended in an organic acid solution and reacted for 2 to 30 minutes. The reaction time may be set to an optimum time depending on the temperature condition at the time of implementation. As the organic acid, malic acid, citric acid, pyruvic acid and the like can be used. The concentration of the organic acid is preferably 1-20 mM. The appropriate amount of lactic acid bacteria added to the organic acid solution is in the range of 10 ³ to 10 ¹⁰ cells / ml. Known luciferin
It is measured by the luciferase method, and the amount of ATP in the cells is examined. The luciferin / luciferase method is easy to use using an existing measurement kit or the like.

On the other hand, the amount of ATP in the cells is measured in the same manner by using a system containing no organic acid as a control. The obtained ATP amount measured value was applied to the following formula: ATP amount in the organic acid-added microbial cells / ATP amount in the microbial cells without an organic acid (control test value), and if the obtained value was 2 or more, it was grown in beer. Judged as capable.
A flow chart of the method of the present invention is shown in FIG.

It is also possible to construct a kit for detecting beer-growing lactic acid bacteria based on the above-mentioned detection method. The constitution is shown in Table 1.

[0014]

[Table 1]

[0015]

According to the present invention, beer-growing lactic acid bacteria can be determined early by a simple method.

[0016]

EXAMPLES The present invention will be described below with reference to examples, but the invention is not limited thereto. Example 1 7 strains of beer grown strains (Lactobacillus brevis Asahi Breweries, Ltd. stock strains (hereinafter referred to as ABCC) 45, Lactob)
acillus brevis ABC46, Lactobacillus brevis A
BBC65, Lactobacillus brevis ABBC69, Lacto
bacillus sp. ABC70, Lactobacillus brevis AB
BC99, Lactobacillus brevis ABBC104) and 7 beer non-growing strains (Lactobacillus brevis ABBC2)
16, Lactobacillus buchneri ABC219, Lactobacil
lus buchneri ABC220, Lactobacillus buchneri
ABC221, Lactobacillus paracasei ABC223,
Lactobacillus sp. ABC229, Lactobacillus sp.
ABBC232) is a medium for detecting lactic acid bacteria (Merck, trade name: MRS broth) and a medium for detecting lactic acid bacteria MRS containing 25% beer added to beer-added MRS medium (hereinafter, B
MRS medium)) at 25 ℃,
Anaerobic culture was performed for 3 days. 0.1% of the culture solution after the culture
Add the solution to 10 ml of beer adjusted to pH 5.0 and add 25
Cultured for hours. 10 times each bacterial suspension at 10,000 rpm
The cells were collected by centrifugation for 1 minute and washed with a tartrate buffer solution having a pH of 4.2. Then, centrifuge to remove the supernatant, re-suspend in a tartaric acid buffer of pH 4.2, add 20 mM malic acid and 1 mM citric acid as a substrate to the suspension, and allow to react at 25 ° C for 20 minutes. , Intracellular ATP was produced.

The intracellular ATP was measured by the luciferin-luciferase method using a kit (trade name: Luciferase LU Plus) manufactured by Kikkoman Corporation. As a control, those without addition of organic acid were reacted in the same system, and the growth ability was determined by the following formula. ATP containing organic acid
Amount / organic acid-free intracellular ATP amount> 2, judged to be viable.

When the amount of intracellular ATP produced was examined, the beer-grown strain and the non-grown strain could be clearly distinguished in a short time. The results are shown in Table 2.

[0019]

[Table 2]

[Brief description of the drawings]

FIG. 1 is a flowchart of a method for early determination of lactic acid bacteria grown in beer.

Claims (5)

[Claims] 1. A method for early determination of beer-grown lactic acid bacteria, which comprises reacting a lactic acid bacterium to be determined with an organic acid, and then measuring the amount of intracellular ATP by the luciferin-luciferase method. 2. The determination method according to claim 1, wherein the organic acid is at least one selected from pyruvic acid, citric acid, and malic acid. 3. A lactic acid bacterium detection medium is added with a lactic acid bacterium to be determined, the mixture is cultivated, and at least one organic acid selected from pyruvic acid, citric acid and malic acid is added to react with the lactic acid bacterium.
A method for early determination of beer-growing lactic acid bacteria, which comprises measuring the amount of P by the luciferin-luciferase method. 4. The determination method according to claim 3, wherein the medium for detecting lactic acid bacteria is a medium to which beer or hop components are added. 5. A luciferin-luciferase luminescent solution,
A kit for detecting beer-grown lactic acid bacteria, which comprises an ATP extract, an organic acid reaction solution, a beer solution for culture, and a medium for detecting lactic acid bacteria.