JPH0779798A - Detection of bacterium - Google Patents

Abstract

PURPOSE:To provide an effective method for readily and sensitively detecting whether or not a yeast suspension contains a bacterium including a lactic acid bacterium by using the suspension to be examined. CONSTITUTION:This detection method comprises adding a yeast cell wall digesting enzyme solution to a bacterium-containing yeast suspension, dissolving the cell wall of the yeast and subsequently applying the resultant solution to a bacterium detection medium.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a method for easily detecting the presence of bacteria mixed in a yeast suspension.

[0002]

2. Description of the Related Art In the field of brewing beer and sake using yeast and in the field of food manufacturing, contamination with wild bacteria during the manufacturing process greatly affects the quality of the product. Therefore, it is necessary to collect a sample at a key point of the manufacturing process and confirm whether or not bacteria are mixed therein. However, when yeast and bacteria are mixed, it is difficult to efficiently and accurately confirm the presence or absence of bacteria due to the presence of yeast.

At present, as a method corresponding to this, taking lactic acid bacteria as an example, the presence or absence of lactic acid bacteria is confirmed by examining a sample with a microscope. There is a method of centrifuging a sample by utilizing the difference in specific gravity between yeast and lactic acid bacteria to separate both bacteria, and then inoculating the lactic acid bacteria fraction into a lactic acid bacteria detection medium and observing the grown colonies. However, the former method has a problem that the detection sensitivity is low, and the latter method has a problem that the work is complicated and there is a risk of contamination by other bacteria.

Also, Japanese Patent Publication No. 63-26870 and Japanese Patent Publication No. 59-10726.
3, JP-A-62-10100 is a method for detecting lactic acid bacteria using an antigen-antibody reaction, and JP-A-61-257200 and JP-A-5-15400 are for detecting lactic acid bacteria using a gene sequence. Kaisho 63-146799, JP-A-3-91493, JP-A-3-259095, JP-A
4-20283 discloses a lactic acid bacterium detection medium by adding a specific substance, but the technique is different from the lactic acid bacterium detection method of the present invention.

[0005]

Therefore, a method for enhancing the detection sensitivity was investigated while aiming at simplification of the method for detecting bacteria under the condition that yeast is present. as a result,
Evaluate whether or not bacteria are contained.A yeast cell wall lysing enzyme solution is added to the yeast suspension to react, and then the reaction solution is applied to a bacteria detection medium and cultured to easily and sensitively detect bacteria. The present invention was completed with good detection.

[0006]

In carrying out the present invention, the concentration of the yeast suspension for examining whether or not bacteria are contained is adjusted so that the yeast cell wall lysing enzyme can sufficiently react.
In addition, it is necessary to dilute the yeast suspension so that bacteria can be properly detected. For example, it is advisable to adjust the number of yeasts in the yeast suspension to 1.5 to 2.0 × 10 ⁹ cells / ml or less.
It is preferably 10 ⁷ to 10 ⁹ cells / ml.

As the yeast cell wall lysing enzyme, any enzyme can be used as long as it can lyse the yeast cell wall. For example, as a commercially available enzyme, YL-15 (manufactured by Amano Pharmaceutical Co., Ltd.), Tunicase (manufactured by Daiwa Kasei Co., Ltd.), Zymolyase (manufactured by Seikagaku Corporation), Kitarase (manufactured by Wako Pure Chemical Industries, Ltd.) and the like are used. it can. When a commercially available enzyme is not used, microorganisms that produce yeast cell wall lysing enzymes, such as Arthrobacter sp. (Japanese Patent Publication No. 48-2790) and Oerskophia sp. (Japanese Patent Publication No. 63-5167).
6), a desired yeast cell wall lysing enzyme may be obtained from Streptomyces sp. (Japanese Patent Publication No. 60-22916) and used in the present invention.

In the case of a commercially available enzyme, the reaction conditions for yeast are generally 30 minutes to 1 hour at a temperature of about 30 ° C. Furthermore, in order to promote the action of these cell wall lysing enzymes, the enzyme conventionally used for lysing the cell wall of yeast and used for protoplast formation, for example, 0.2 to 1% by weight of mercaptoethanol or 10 to 100 mM of EDTA solution is used as an enzyme. It may be added before the treatment or simultaneously with the enzyme treatment.

The solution treated to dissolve the yeast cell wall is applied to a conventionally used bacterial detection medium and cultured to confirm whether or not bacterial colonies appear on the medium. Anaerobic bacteria or aerobic bacteria are used as the bacteria, and lactic acid bacteria are particularly preferable.

[0010]

EXAMPLES The present invention will be specifically described below with reference to Examples and Comparative Examples. Example 200 ml of yeast suspension from the fermentor in the beer brewing process
Collect and add sterile water to obtain a yeast cell count of 2.0 × 10 ⁹ cells / m 2.
The sample A was obtained by adjusting to l. 200 ml of yeast suspension was collected from the same fermentor and stored at 4 ℃ for 7 days.
^It was adjusted to ⁹ cells / ml to obtain sample B. For each of Samples A and B, 5 lactic acid bacteria of the genus Lactobacillus
0 ² cells / ml was added.

1 ml of a Zymolyase 100T (manufactured by Seikagaku Corporation) solution was prepared by dispensing 4 ml of each of samples A and B to a test tube and dissolving them in a phosphate buffer of pH 7.0 to a concentration of 5 mg / ml. And β-mercaptoethanol 5μ
l was added. After allowing to stand for 1 hour at 30 ° C. to dissolve the cell wall of yeast, 0.8 ml of each sample was surface-coated on a plate medium prepared by adding cycloheximide at a ratio of 10 mg / l to MRS medium which is a lactic acid bacteria detection medium. The appearance of colonies was examined by anaerobic culture at 25 ° C.

On the other hand, 5 lactic acid bacteria of the genus Lactobacillus added to Samples A and B were suspended in sterilized water in the order of 10 ² cells / ml to prepare Sample C, 0.8 ml of which was surface-coated on MRS medium. The number of colonies was examined by anaerobic culture at 25 ° C. The detection rates calculated by comparing the number of colonies appearing in samples A and B with the number of colonies appearing in sample C are shown in Table 1.

Comparative Example The same sample as the sample B of the example was prepared and designated as sample D. Lactobacillus bacterium No. 74 of the genus Lactobacillus was inoculated into each well at the order of 10 ² cells / ml, and then 0.8 ml of each sample was surface-coated on the same MRS medium as in Example without treatment with yeast cell wall lysing enzyme. The number of colonies was examined by anaerobic culture at 25 ° C. The results are shown in Table 1. Table 1 Detection Rate of Lactic Acid Bacteria from Yeast Suspension Detection Rate of Additive Lactic Acid Bacteria Strain No. Sample A Sample B of Sample B Detection Rate Detection Rate 38 132 104 104 46 123 130 130 58 110 110 90 72 107 113 113 74 104 104 0 80 (Yeast (Not detectable due to bacterial growth ) (Note) The detection rates of samples A, B, and D are the rates when the number of colonies of sample C was 100.

From the above results, when lactic acid bacteria were detected without using yeast cell wall lysing enzyme, lactic acid bacteria could not be detected at all in sample D of Table 1, whereas lactic acid bacterium was detected using yeast cell wall lysing enzyme. In the case of detecting lactic acid, lactic acid bacteria could be confirmed with the same detection rate as that of sample C in the absence of yeast.

[0015]

EFFECTS OF THE INVENTION Therefore, the present invention is an effective method for easily detecting with high sensitivity whether or not bacteria such as lactic acid bacteria are present in a yeast suspension from a suspension to be confirmed.

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[Procedure amendment]

[Submission date] March 18, 1994

[Procedure Amendment 1]

[Document name to be amended] Statement

[Correction target item name] 0010

[Correction method] Change

[Correction content]

[0010]

EXAMPLES The present invention will be specifically described below with reference to Examples and Comparative Examples. Example 1 200 yeast suspension from fermenter in beer brewing process
Collect ml and add sterile water to increase the yeast cell count to 2.0 x 10
^The sample A was obtained by adjusting to ⁹ cells / ml. Recover 200 ml of yeast suspension from the same fermentor, then
After storing at 7 ° C. for 7 days, sterilized water was added in the same manner as above to adjust the number of yeast cells to 2.0 × 10 ⁹ cells / ml to obtain sample B. Samples A and B were each treated with 10 ² cells / ml of five Lactobacillus lactic acid bacteria.
Was added in the order of.

[Procedure Amendment 2]

[Document name to be amended] Statement

[Correction target item name] 0013

[Correction method] Change

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Comparative Example 1 Sample D was prepared by preparing the same sample as sample B of the example. Lactobacillus No. of Lactobacillus was added to this. 74
Each of them was inoculated at the order of 10 ² cells / ml, and 0.8 ml of each sample was surface-coated on the same MRS medium as in Example without yeast cell wall lysing enzyme treatment, and anaerobically cultured at 25 ° C. for colony formation. I checked the number of. The results are shown in Table 1.

[Procedure 3]

[Document name to be amended] Statement

[Correction target item name] 0014

[Correction method] Change

[Correction content]

From the above results, when lactic acid bacteria were detected without using yeast cell wall lysing enzyme, lactic acid bacteria could not be detected at all in sample D of Table 1, whereas lactic acid bacterium was detected using yeast cell wall lysing enzyme. In the case of detecting lactic acid, lactic acid bacteria could be confirmed with the same detection rate as that of sample C in the absence of yeast. Example 2 200 yeast suspensions from the yeast tank in the beer brewing process
ml, and then stored at 4 ° C for 7 days, then sterilized water
And the yeast cell number is added to 1.5 to 2.0 × 10 ⁹ cells.
A sample E was obtained by adjusting to s / ml or less. Sample E
2 types of pectinatus (strain No. DSM6306, DS
M20467) and one acetic acid bacterium (strain No.IF03)
284) in the order of 10 ¹ to 10 ² cells / ml
Added in. Dispense 4 ml each of sample E into a test tube,
Diluted solution (4.5 g of KH ₂ PO ₄ , Na ₂ HPO ₄
6.0 g, L-cysteine / HCl / H ₂ O 0.5
g, Tween 80 0.5 g in distilled water 1000 ml
Dissolved) to a concentration of 20 mg / ml.
Add 1 ml of Thalase (Wako Pure Chemical Industries, Ltd.) solution
did. Let stand at 30 ° C for 1 hour to dissolve the yeast cell wall.
And then Pectinateus detection medium MRSpH5.5
Medium and acetic acid bacteria detection medium containing 1% glucose
Cycloheximide at a ratio of 10 mg / 1 was added to the broth medium.
Each sample was added to the
Apply 0.8ml each to the surface and pectinate 30
℃ anaerobic culture, acetic acid bacteria aerobically cultivated at 25 ℃
I checked the present. On the other hand, the pectinator added to sample E
2 types of strains and 1 type of acetic acid bacteria were treated with 10 ¹ to 10 ² cells /
What was suspended in sterilized water in the order of ml was designated as sample F.
, 0.8 ml MRS medium and 1% glucose added
Surface-applied to normal broth medium, and pectinate is 3
Number of colonies after anaerobic culture at 0 ℃ and aerobic culture at 25 ℃ for acetic acid bacteria
I checked. Sample the number of colonies that appeared in sample E
The detection rate calculated by comparing with the number of colonies that appeared in F is displayed.
Shown in 2. From the above results, yeast suspension using yeast cell wall lysing enzyme
Can detect pectinatus and acetic acid bacteria present in liquid
It was confirmed.

Claims (3)

[Claims] 1. A method for detecting the presence or absence of bacteria by adding a yeast cell wall lysing enzyme solution to a yeast suspension containing bacteria to dissolve the yeast cell wall and then applying the solution to a bacteria detection medium. 2. The method according to claim 1, wherein the bacterium is an anaerobic bacterium or an aerobic bacterium. 3. The method according to claim 1, wherein the bacterium is a lactic acid bacterium.