JP3163432B2 - Simple method for short-term determination of turbid bacteria - 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, in a short time, whether a microorganism to be tested is a cloudy bacterium that grows in a brew such as beer.

[0002]

2. Description of the Related Art There are several methods for detecting the presence of microorganisms such as lactic acid bacteria. At present, as a method for detecting such lactic acid bacteria and other microorganisms, a colony counting method in which colonies are grown and detected in a medium is generally used. As a colony counting method used in the beer industry, there are two international standard methods, ASBC (Analytical Method of American Society of
Brewing Chemists (EBC) method and EBC (European Brewing Con
gres. ANALYTICA MICROBIOLOGICA) law. These features include the convenience and simplicity of operation without the need for special equipment, the wide range of applications, and other advantages, but it takes time to detect colonies,
It has disadvantages such as low cost of equipment, but complicated operation, and the detection sensitivity is easily influenced by various factors.

As applications for detecting microorganisms such as lactic acid bacteria, Japanese Patent Publication No. 63-26870 and Japanese Patent Application Laid-Open No.
3, JP-A-62-10100 discloses a method for detecting lactic acid bacteria using an antigen-antibody reaction, and JP-A-61-257200 and JP-A-5-1540.
0 contains a lactic acid bacteria detection method using gene sequences,
JP-A-63-146799, JP-A-3-91493, JP-A-3-25909
5. Japanese Patent Application Laid-Open No. 4-20283 discloses a lactic acid bacteria detection medium by adding a specific substance, but the technique is different from the turbid bacteria determination method of the present invention.

[0004] On the other hand, in the brewing process of beer and the like, study of measures for preventing turbidity of the product by microorganisms such as lactic acid bacteria is important for quality control. However, there is a method for detecting the presence or absence of microorganisms such as lactic acid bacteria as in the above-described conventional technology, but there is no method for short-term determination of whether it grows and becomes turbid in brewed products.

[0005]

As described above, in the production of brews, short-term detection of turbid bacteria is required for more reliable quality control. Therefore, the present invention has been studied and completed in order to solve this problem, and establishes a method for quickly and reliably determining turbid bacteria growing in brewery, particularly in beer.

[0006]

SUMMARY OF THE INVENTION The present invention relates to a method for converting a brew to pH.
Adjust to 4.7-5.5, sterilize, add test specimen containing test bacteria, perform anaerobic culture, measure turbidity during culture period to determine presence of turbid bacteria in test specimen It is a short-term simple determination method of bacteria. The brew used in the present invention is not particularly limited as long as it is widely and generally produced in beer, sake, etc. which require detection of turbid bacteria, particularly lactic acid bacteria, in the quality control step, but is very effective in beer brewing. .

[0007] In adjusting the pH of the brewed product, it is advisable to degas the brewed product in advance, especially for those containing carbon dioxide gas, in consideration of the operability thereafter. Brew pH
Adjust to the range of 4.7 to 5.5. The pH can be adjusted using a reagent commonly used for pH adjustment, for example, NaOH or the like. The setting range of the pH varies depending on the microorganism to be tested, but in the case of lactic acid bacteria (Lactobacillus), the pH is 4.8 to 5.2, preferably pH 5.0.

[0008] The brew is sterilized. Sterilization can be performed before or after pH adjustment. However, in order to prevent contamination, sterilization is preferably performed after pH adjustment. The test substance can be a test bacterium or a brew containing the test bacterium, and the brew in this case may be the same as or different from the brew used as the medium.

The method of determination will be described in a concrete example. A sterilized brew is dispensed into a pre-sterilized test tube, and a test bacterial solution as a test substance has an initial concentration of 10 ⁴ to 10 ⁵ cells / ml. After inoculating this, anaerobic culture is performed and the turbidity is measured. It is preferable to measure the turbidity over time. In the case of beer, good results are obtained when measured at OD ₆₆₀ , and the determination can be made at about 25 ° C. in a short period of about one week. In addition, when turbidity can be visually determined, the opacity may be visually determined.

Hereinafter, the present invention will be described specifically with reference to examples.

[0011]

【Example】

Example 1 Test strains 5 Lactobacillus lactic acid bacteria growing in beer (A-
1, A-2, A-3, A-4, A-5) and 5 strains of lactobacilli of the genus Lactobacillus that do not grow on beer (B-1, B-2, B-3,
B-4, B-5) were used.

2. Preculture The test strain was 10 ml of MRS medium (manufactured by Merck, 10 g of peptone, 5 g of meat extract, 5 g of yeast extract in 1 liter).
g, glucose 20 g, Tween 80 1 g, dipotassium hydrogen phosphate 2 g: ammonium citrate 2 g, sodium acetate 5 g, magnesium sulfate 0.1 g, manganese sulfate 0.05 g)
For 3 days at 25 ° C. in an anaerobic jar.

3. Preparation of culture medium Super Dry, Asahi Breweries trade name, Pure Gold and Zet were degassed, and the pH was adjusted to 4.
After adjusting the pH to 12 to 6.0 (hereinafter, in the case of pH 5.0, for example, SD pH 5.0, PG pH 5.0 and Z pH 5.0 according to the adjusted pH of each beer), a membrane filter (pore size 0.45) was used. The solution was sterilized by filtration through a filter (μm), and dispensed in 10 ml portions into test tubes (18 mmφ) which had been sterilized in advance.

4. Method The pre-cultured bacterial solution is appropriately diluted using physiological saline, and inoculated into SD, PG and Z adjusted to each pH so that the initial bacterial concentration is 10 ⁴ to 10 ⁵ cells / ml, and then OD ₆₆₀ is _added . It was measured. After anaerobic culture in an anaerobic culture device at 25 ° C for 7 days,
The OD ₆₆₀ was measured again, and the value of ΔOD ₆₆₀ was calculated.

[0015] 5. Growth determined in decision grow, Super Dry after 7 days of anaerobic culture, when the value of .DELTA.OD ₆₆₀ of each pH beer pure gold and jet is 0.1 or more, or obvious turbidity it at less was also visually occurred Was determined to have grown. 6. Results Table 1 and Table 2 show the presence or absence of growth of 5 strains of beer growing strain and 5 strains of non-beer growing strain in the super-dry adjusted to each pH.
It was shown to. For beer growing strains, all five strains have a pH of 5.
Growth was observed at any of pH 0, pH 5.5 and pH 6.0. On the other hand, for pH4.12 and pH4.5, A-
Only 4 and A-5 grew by day 7, but A-
No growth was observed for 1, A-2 and A-3. On the other hand, in the case of beer non-growing beer, all five strains have pH 5.0,
No growth was observed at pH 4.5 and pH 4.12.

[0016] Even when the varieties are Pure Gold and Zet, all of the five beer growing strains have pH 5.0 and pH 5.5.
And growth was observed at any pH of pH6.0 (Tables 3 and 5). On the other hand, the pH of the five non-growing strains of beer was pH
No growth was observed at 5.0, pH 4.5 and pH 4.12 (Tables 4 and 6). From these results, the presence or absence of beer growth of lactic acid bacteria can be determined within one week by inoculating beer with increased pH.

Table 1 Growth or absence of growth of 5 strains of beer growing strains in super dry adjusted to each pH A-1 A-2 A-3 A-4 A-5 pH4.12 ^* ---+++ pH4.5 ---++ pH5.0 +++++++ pH5.5 ++++++ pH6.0 ++++++ *: Original pH Culture conditions: 25 ° C, anaerobic culture, 7 days +: Growth-: Do not grow

Table 2 Presence or absence of growth in superdry adjusted to each pH of 5 strains of non-growth beer strains B-1 B-2 B-3 B-4 B-5 pH4.12 ^* -----pH4 .5 − − − − − pH5.0 − − − − − pH5.5 − − − − − pH6.0 − + + + − *: Original pH Culture conditions: 25 ° C, anaerobic culture, 7 days +: Growth -: Does not grow

Table 3 Growth of Pure Beer Grown at Five pHs of Beer Growth Strains at Each pH A-1 A-2 A-3 A-4 A-5 pH 4.30 ^* ---+ + pH 4. 5---++ pH5.0 +++++++ pH5.5 ++++++ pH6.0 ++++++ *: Original pH Culture conditions: 25 ° C, anaerobic culture, 7 days +: Growth- : Does not grow

Table 4 Presence or absence of growth on pure gold adjusted to each pH of 5 strains of beer non-growing strains B-1 B-2 B-3 B-4 B-5 pH 4.30 ^* -----pH 4 .5-----pH5.0-----pH5.5--+-- pH6.0-+ + +- *: Original pH Culture conditions: 25 ° C, anaerobic culture, 7 days +: Growth -: Does not grow

Table 5 Growth of 5 strains of beer growing strains on Zet adjusted to each pH A-1 A-2 A-3 A-4 A-5 pH 4.19 ^* -----pH 4.5 ---++ pH5.0 +++++++ pH5.5 ++++++ pH6.0 ++++++ *: Original pH Culture conditions: 25 ° C, anaerobic culture, 7 days +: Growth-: Do not grow

Table 6 Growth or non-growth of the five non-growing beer strains on ZET adjusted to each pH B-1 B-2 B-3 B-4 B-5 pH 4.19 ^* -----pH 4. 5-----pH5.0-----pH5.5 -----pH6.0-+ + +- *: Original pH Culture conditions: 25 ° C, anaerobic culture, 7 days +: Growth- : Does not grow

Embodiment 2 1. Test strain 10 Lactobacillus lactic acid bacteria growing in beer (A-1
To A-10, of which A-1 to A-5 are the same as those used in Example 1.)

2. Pre-culture was performed in the same manner as in Example 1. 3. Preparation of culture medium Asahi Breweries Super Dry (hereinafter abbreviated as SD) was degassed, the pH was adjusted to 5.0 with 5N NaOH (hereinafter abbreviated as SD pH 5.0), and then passed through a membrane filter (pore diameter 0.45 μm). The solution was sterilized by filtration, and each 10 ml was dispensed into a test tube (18 mmφ) previously sterilized.

4. Method The pre-cultured bacterial solution was appropriately diluted using a physiological saline solution, and the initial pH was adjusted to 10 ⁴ to 10 ⁵ cells / ml. After inoculation, the OD ₆₆₀ was measured for SD pH 5.0, anaerobic culture was performed at 25 ° C. using an anaerobic culture device, and OD ₆₆₀ was measured every day as much as possible to calculate the value of ΔOD ₆₆₀ . The SD (small bottle) was cultivated at 25 ° C. in a stationary state, and the growth of the bacteria was visually observed.

[5] Growth judgment judgment of the growth in SD pH5.0 beer, the value of ΔOD ₆₆₀
Even when the concentration was 0.1 or more, or less than 0.1, it was determined that the plants grew when clear turbidity was visually observed. As for the SD (small bottle), as a result of visual observation, it was determined that the beer grew when it clearly became cloudy.

6. Results Table 7 shows the growth days of the 10 beer growing strains at SD pH 5.0 and SD (small bottle). As a result, all 10 shares were SD
Growth at pH 5.0 was within 7 days, all of which grew faster than when SD was inoculated directly. From this result, inoculation of lactic acid bacteria to beer whose pH has been raised can make the determination of the presence or absence of beer growth of lactic acid bacteria faster than inoculation of beer whose pH is not adjusted.

Table 7 SD pH5.0 of beer growing strain and the number of days until it is determined to grow on SD (small bottle) SD pH5.0 SD (small bottle) A-1 410 A-2 317 A-3 210 A-4 2 8 A-5 2 4 A-6 3 30 A-7 3 43 A-8 3 16 A-9 2 12 A-10 4 17

[0029]

According to the present invention, turbid bacteria are determined by turbidity using a medium in which the pH of the brew is adjusted to a constant value. The presence or absence of bacteria can be determined.

Claims (3)

(57) [Claims] 1. Using a sterilized brew whose pH has been adjusted to 4.7 to 5.5 as a culture medium, anaerobic culturing is performed by adding a test substance, and the presence or absence of turbid bacteria in the test substance is determined by measuring the turbidity. Short-term simple method for determining turbid bacteria. 2. The short-term simple determination method according to claim 1, wherein the brew is beer. 3. The short-term simple determination method according to claim 1, wherein the turbid bacterium is a lactic acid bacterium.