JP4145073B2 - Method for removing malt fermented beverage remaining in membrane filter and method for rapid detection of microorganisms in fermented malt beverage - Google Patents

Description

【００４８】
【表２】

【図面の簡単な説明】
【図１】メンブランフィルタ上に泡が残存しないサンプル(左)と泡が残存しているサンプル（右）を示す図である。
【図２】本発明の洗浄処理を行って実施した発光量の測定結果を示す。図２−１（Ａ），（Ｂ）は実施例１による測定結果、図２−２（Ａ），（Ｂ）は実施例２の測定結果を示す。
【図３】実施例３の測定結果を示す図である。
【図４】実施例４の測定結果を示す図である。
【図５】実施例５の実験結果を示す図である。
【図６】実施例５の有機酸について実施他測定結果を示す図である。
【図７】実施例６の測定結果を示す図である。
【図８】実施例６の無機塩類及び微量元素について実施した測定結果を示す図である。
【図９】実施例７の測定結果を示す図である。
【図１０】実施例８の測定結果を示す図である。[0001]
BACKGROUND OF THE INVENTION
The present invention relates to a method for measuring the number of viable microbes mixed in a beverage without culturing, and more specifically, using a method for measuring the number of viable bacteria using the amount of ATP derived from microorganisms as an index, The present invention relates to a method for measuring the number of viable bacteria of about 1 to 100 cells present in malt fermented beverages such as beer and sparkling liquor without culturing.
[0002]
[Prior art]
As a method for quickly measuring the number of viable microorganisms present in a beverage, a luciferin-luciferase reaction is carried out using adenosine triphosphate (hereinafter referred to as “ATP”) specifically present in living cells as a coenzyme, and ATP The ATP-luciferase method, which confirms the presence of microorganisms by detecting minute luminescence generated in proportion to the content with a high-sensitivity detector, has attracted attention as a rapid microorganism testing method, and embodies this. As an apparatus that combines a membrane filtration method and an ATP bioluminescence method, that is, a beverage that is a specimen is filtered with a membrane filter, microorganisms are captured on the filter, and then an extractant and then a luminescent reagent are sprayed, and the luciferin- It is a method that counts the number of microorganisms by performing a luciferase reaction and using a highly sensitive luminescent spot counting system. . As a convenient device for embodying this method, there is a microorganism rapid detection device (Nippon Millipore, hereinafter referred to as RMDS), which has already been put into practical use in the beverage industry such as beer (antibacterial and antibacterial, 27, 759-764). (1999)). RMDS can detect microorganisms with high ATP content per cell such as brewer's yeast without culturing, but it can detect bacteria such as Lactobacillus brevis known as lactic acid bacteria that rot beer. In order to do so, a culture time of about 16 to 24 hours on an agar medium was required after membrane filtration [Biosci. Biotechnol. Biochem., 64, 1032-1037 (2000)].
[0003]
The microbial inspection of beer products by the agar plate method, which has been performed for a long time, required 2 to 4 days, but the above-mentioned microbial inspection is completed within 24 hours due to the practical application of RMDS. Although it has become possible, there is a desire to further reduce the inspection time.
[0004]
In order to detect bacteria in beer in a shorter time using RMDS, by increasing the activity of luciferin-luciferase reagent (RMD reagent, Nihon Millipore), which has been used to emit ATP in microorganisms. There is a method for enhancing the luminescence reaction and detecting a smaller number of microbial cells (Japan Air Cleaners Association Symposium, 29-38, (1997)). A highly sensitive reagent (Nihon Millipore) is commercially available as a reagent for achieving this method. On the other hand, ATP consumed by luminescence reaction by ATP-luciferin-luciferase is recycled from ADP to ATP by pyruvate orthophosphate dikinase to continue the luminescence reaction for a long time, thereby detecting microbial cells with higher sensitivity. A method has been reported (Japanese Patent Laid-Open No. 11-69994).
[0005]
These methods are very effective for detecting microbial cells mixed in a solvent that does not contain ATP.However, if microbial cells mixed in a solvent containing ATP in a solution such as beer are detected, they are contained in the cells. Since the amount of ATP produced was too small for the amount of ATP contained in the solvent, there was a problem that the luminescence of the solvent became an obstacle and microbial cells could not be detected. That is, when the luminescence reaction is enhanced and the RMDS detection sensitivity is increased, the presence of ATP in the solvent becomes a problem as the amount of ATP in the microorganism to be examined becomes smaller, and a method for removing this is necessary. It becomes.
[0006]
As a method for removing ATP remaining in a filter obtained by subjecting drinking water or the like to membrane filtration, distilled water, ion-exchanged water, pH buffer solution, aqueous sugar solution, aqueous polyalcohol solution (Japanese Patent Laid-Open No. 11-89592, Japanese Patent Laid-Open No. 02-163098), ethanol-containing solutions, washing methods such as saline (Patent -02812338), adenosine phosphate deaminase solution, adenosine phosphate deaminase and other enzymes (eg, apyrase, afukali phosphatase, acid phosphatase, There are methods for decomposing ATP on a filter with a mixed solution of hexokinase, adenosine triphosphatase, and glucokinase) (JP-A-11-69994, JP-A-11-89592, JP-A-02905727, JP-A-10-165200, JP2000-2000A). -189197, JP-A-11-56393, JP-A-09-182600, JP2001.340100).
[0007]
However, the cleaning with the above-described cleaning liquid does not sufficiently remove the beer components, and the luminescence derived from beer is strong, so that the signal / noise (S / N) necessary for detecting the luminescence of bacterial cells cannot be obtained. That is, since the amount of light emission around the microorganism (corresponding to S of S / N) is larger than that of the microorganism itself (corresponding to N of S / N, hereinafter referred to as “background noise”), It becomes impossible. The data shown in Table 1 shows the results of a survey measuring the effect of the cleaning agent. The method for producing the used sample (filter) is as follows. That is, 350 ml of commercially available beer (aseptic beer) was poured aseptically (in a clean room) into a cup-shaped filter equipped with an isopore filter with a 0.4 μm ring (manufactured by Nihon Millipore), and suction filtration was performed. After the beer is completely filtered by suction, the membrane filter is washed with a sterilized washing solution (10 ml of distilled water, 300 ml of distilled water, 300 ml of 0.1% Tween80, 300 ml of 5% ethanol, 1% maltose), and an agar medium (for example, a special medium) (Disclosed in Kai 08-224096). Next, luminescence was accumulated for 90 seconds by RMDS by the above-described operation. With respect to the obtained image data, the light emission amount in the range of 300 pixels in diameter was measured and converted to the light emission amount per second. According to the inventor's experiment, as one of the conditions for detecting bacterial cells without culture, when the solvent such as beer and happoshu is almost removed, the luminescence amount (background noise) of the membrane filter is 100 RLU. / It was confirmed to be less than sec. Therefore, as shown in Table 1, for the purpose of washing with water, surfactant, alcohol, sugar solution, pH buffer, etc. as known until now, lactic acid bacteria etc. can be detected by RMDS within 24 hours. In the method of placing the membrane filter on the developed nutrient medium, it can be seen that the solvent or the luminescent material derived from the nutrient medium remains on the membrane filter, and the background noise is too high.
[0008]
If the emphasis is on washing beer components such as alkaline solutions and surfactants, cells may be damaged, and ATP in the cells may be eluted by washing operations. Therefore, when removing a beer component by washing | cleaning, the washing | cleaning agent which can wash | clean a beer component is required, without damaging the cell used as a measuring object.
[0009]
In addition, when treatment with ATP-degrading enzyme is performed, the amount of beer-derived ATP remaining on the filter is too large compared to the amount of ATP contained in the bacterial cells. In the processing conditions using the titer of ATP-degrading enzyme, it is difficult to sufficiently remove or inactivate ATP-degrading enzyme, and ATP contained in cells is degraded.
[0010]
Therefore, there is a need for a method for removing ATP derived from malt fermented beverages such as beer and happoshu remaining in the filter without affecting the detection of ATP contained in bacterial cells.
[0011]
In addition, in order to detect microorganisms without culture, it is necessary to increase S of S / N, and it is necessary to increase ATP derived from microorganisms to be detected in a short time.
[Problems to be solved by the invention]
The present invention has been made from the above viewpoint, and culture operation is performed on a viable count of minute bacterial cells of about 1 to 100 cells mixed in a malt fermented beverage such as several hundred ml to several liters of beer and sparkling liquor. It is an object to provide a method of measurement without any problem.
[Means for Solving the Problems]
As a result of intensive research in order to solve the above problems, the present inventor has developed an ATP derived from a malt fermented beverage remaining on the filter by devising a washing method for a filter obtained by membrane-filtering a malt fermented beverage and selecting a medium to be used. The present invention was completed by finding that it can be measured without being affected and without culturing the number of viable bacterial cells on the membrane filter.
[0012]
That is, as one of the conditions for solving the above-mentioned problem, the sample (malt fermented drinks such as beer and happoshu) remaining in the membrane filter is removed to reduce background noise.
[0013]
This residue is broadly divided into foam and other beverage ingredients. As for the foam, when performing a membrane filtration in a fermented malt beverage such as beer or sparkling liquor, if foam generated when poured into a filter remains in the filter, ATP contained in the foam becomes background noise. When a large amount of foam is generated by foaming when pouring into the filter, the foam remains on the filter even if suction filtration is performed thereafter. As a countermeasure, the filter is gently poured so as not to foam as much as possible, and suction filtration is started when the liquid surface is observed from the foam layer formed on the surface of the sample liquid.
[0014]
Further, the membrane after filtration using a lower alcohol such as ethanol, a surfactant such as turpentine (trade name: Tween 80) or a silicon-based antifoaming agent that has an antifoaming effect and does not adversely affect the microorganism to be detected. It was found that the foam could be removed by washing the filter, and the S / N was greatly improved.
[0015]
Furthermore, reducing the liquid temperature of the sample beer to 10 ° C. or less makes it difficult for foaming to occur. However, when the temperature is 4 ° C. or lower, filtration congestion is likely to occur, and therefore 4 ° C. <liquid temperature ≦ 10 ° C. is appropriate.
[0016]
Furthermore, the beverage component remaining on the filter could be removed by washing with a weak alkaline aqueous solution such as sodium hydrogen carbonate.
[0017]
By using the countermeasure against the remaining foam and the countermeasure against the remaining beverage component, the filter residue is almost removed, and the background noise (corresponding to N of S / N) is greatly reduced.
[0018]
After removing the ATP contained in the specimen by rinsing the specimen from the filter as described above, the specimen is further placed in the ATP / FREE medium for 30 minutes to 2 hours, and the ATP remaining slightly on the filter is diffused to the medium side to remove it from the membrane filter. By further removing ATP, the background noise could be further reduced.
[0019]
Furthermore, in the said invention, ATP in microorganisms was increased by adding the component which activates the metabolism of microorganisms to ATP * FREE culture medium, and S of detection S / N was able to be raised.
[0020]
In the measurement of the amount of ATP luminescence, the presence / absence of microorganisms and the number of bacteria can be detected in a short time without culture by significantly improving the S / N.
DETAILED DESCRIPTION OF THE INVENTION
The sample solution can be filtered by attaching a membrane filter, for example, an isopore filter with a 0.4 μm ring (Nippon Millipore) to a cup-shaped filtration container (for example, 47 mm polysulfone holder made by Advantech), and beer (sparkling liquor) Aseptically, the microorganism is sucked and filtered to trap microorganisms on the membrane filter. Pour beer into a cup-shaped filtration container and perform suction filtration. After the beer has been completely filtered by suction, the membrane filter is transferred to another cup-type filter, and 0.1-1.0%, preferably 0.1% sodium bicarbonate, 100-500 ml, preferably 100 ml, is added to the cup-type filter. Pour into 1 and hold for 1 to 10 minutes, preferably 5 minutes, and perform suction filtration and washing. In addition, it is more preferable that the washing with the sodium hydrogen carbonate is repeated more than once.
[0021]
After completely aspirating, the sample is sprayed with an ATP extraction reagent and a luminescent reagent, and a process of causing ATP of microorganisms present on the filter of the sample to emit light is performed. That is, the filter taken out from the agar plate is dried at 25 to 45 ° C., preferably 35 ° C. for 1 to 20 minutes, preferably 2 minutes. After drying, spray 60 μl of ATP extraction reagent (high sensitivity reagent kit, manufactured by Nihon Millipore). Next, the filter is dried at 25 to 45 ° C., preferably 35 ° C. for 1 to 20 minutes, preferably 1 minute, and then sprayed with 60 to 100 μl of a luminescence reagent composed of luciferin-luciferase (high sensitivity reagent kit, manufactured by Nihon Millipore). To do.
The amount of luminescence of the sample processed as described above is measured by RMDS.
[0022]
Moreover, after filtering the malt fermented drink which is a test substance with a membrane filter beforehand, and using the obtained membrane filter as a sample, it wash | cleans using the processing method of the membrane filter which substantially removes the ATP derived from the malt fermented drink, and further samples The membrane filter was placed on an agar plate (containing 0.5 to 1.5% agar, preferably 1.0%, hereinafter referred to as “ATP / FREE agar plate”) for 30 minutes to 2 hours. Then, perform ATP extraction and luminescence treatment, and measure the amount of luminescence with RMDS. In addition, nutrients (saccharides, organic acids, inorganic salts, vitamins) that activate the metabolism of microorganisms are added to the agar plate, and the membrane filter as a sample is placed on the agar plate for about 1 hour to several hours. To increase ATP in microorganisms remaining in the sample. Thereby, S of S / N is raised and S / N is increased.
[0023]
[Example 1]
When beer is poured into a cup-shaped filter, it foams and forms a foam layer at the beer interface. When suction filtration is started in this state, the liquid layer of beer is sucked, but the foam remains on the membrane filter, although there is a difference depending on the thickness of the foam layer and the state of the foam (FIG. 1, right). Even if the membrane filter is washed with sterile water or the like in this state, the bubbles float on the interface of the sterile water and the sterile water is sucked first, so that it is difficult to completely remove the bubbles.
[0024]
On the other hand, (FIG. 1 left) is a membrane filter surface washed with 10 ml of 5% ethanol after suction filtration of beer. Ethanol is known as an antifoaming agent, and it can be seen that the foam layer on the sample has disappeared (washed) as shown in FIG. Here, the concentration of ethanol used is equivalent to or close to the alcohol content of the specimen (beer / happoshu). Since beer and happoshu generally have an alcohol content of 4.0 to 5.5%, the alcohol content of ethanol used for these is preferably 4.O% or more and 10% or less.
[0025]
In addition to the lower alcohol such as ethanol, a surfactant such as turpentine or a silicon-based antifoaming agent can be used as the cleaning agent to be used.
[0026]
In addition to washing with the above antifoaming agent for the treatment of the foam layer, after pouring beer into the cup-shaped filter, wait until the foam is repelled and the foam layer is lowered and the beer liquid level can be visually observed. Suction filtration is performed. This makes it possible to obtain a sample state equivalent to that in FIG.
[0027]
Next, 100ml of 0.1-1.0% sodium bicarbonate solution is poured into the cup-shaped filter with the filter on the left of Fig. 1 and held for 5 minutes. After suction, 100ml of sodium bicarbonate solution with an equal concentration is added and aspirated. It was. This washing process reduces beer components remaining in the filter. The obtained washed sample was subjected to ATP extraction and luminescence treatment, and the amount of luminescence was measured by RMDS. The measurement results are shown in FIG. The amount of light emission is below 100RLU / sec, clearing the target background noise amount.
[0028]
In addition, the sample which does not perform the foam washing | cleaning and beer component washing | cleaning process of the right side of FIG. 1 performed the ATP extraction and the light emission process, without wash | cleaning by sodium hydrogencarbonate, and measured the light-emission quantity by RMDS. As a result, the light emission amount was 13,451 RLU / sec. Therefore, the background noise of the sample subjected to the cleaning process on the left in FIG. 1 is reduced to 1/134 or less compared to the sample not subjected to the cleaning.
[0029]
[Example 2]
Sample used in Example 1 (after suction filtration of the filter on the left in FIG. 1, 100 ml of 0.1 to 1.0% sodium hydrogen carbonate solution was poured into a cup-shaped filter, held for 5 minutes, sucked, and further 100 ml of an equal concentration of 100 ml. A sodium bicarbonate solution was added and aspirated.This cleaning process reduced the beer components remaining on the filter) and placed on an ATP / FREE agar plate for several hours, followed by ATP extraction and luminescence treatment. The amount of luminescence was measured. The result is shown in FIG. Even compared with FIG. 2-1 (A) above, the background noise was further improved, and was 50-60 RLU / sec.
Further, the sample on the right side of FIG. 1 used in Example 1 was subjected to liquid washing (washing twice with sodium hydrogen carbonate), and then the amount of luminescence of the sample placed on the ATP / FREE agar plate for several hours was measured. However, the result of FIG. 2-2 (A) was obtained. Compared with the measurement results of FIG. 2-1 (B), all measured values greatly exceed 100 RLU / sec. From this, it can be seen that if the beer foam is not removed, the foam remains even if the beer liquid is washed, and the influence of background noise due to this remains.
[0030]
The graph shown in FIG. 2-2 (B) is obtained by measuring the amount of luminescence of the sample after being placed in a conventional modified A medium for several hours after washing with residual foam and residual liquid.・ It can be seen that the background noise is significantly worse than when using a FREE agar plate.
[0031]
This is because the sample membrane filter absorbed ATP contained in the culture medium, and the ATP increased despite the drastic reduction in beer-derived ATP by washing.
[Example 3]
The concentration of sodium bicarbonate for washing beer components remaining in the filter was examined. The membrane filter obtained by filtering the sample beer was washed with 10 ml of 5% ethanol to eliminate the remaining foam layer, and then the filter was transferred to another cup-shaped filter and prepared 0.1%, 0.2 Wash 100% of each sodium bicarbonate solution with 1%, 0.5%, 0.8%, and 1.0% sodium bicarbonate solution, pour into a cup-shaped filter, hold for 5 minutes, suck, and then 100 ml of sodium bicarbonate solution with the same concentration. The solution was added and aspirated. Next, after placing on an ATP / FREE agar plate for several hours, ATP extraction and luminescence treatment were performed, and the amount of luminescence was measured by RMDS. FIG. 3 shows the measurement results. The lowest noise was obtained when 0.5% sodium bicarbonate was used, but within this range, in all cases, the emission was less than 100 RLU / sec.
[Example 4]
The beer was filtered through a membrane filter, and the filter surface was washed with 20 ml of 10% ethanol. After washing, the filter was transferred to a new cup-shaped filter, 100 ml of 0.1-1.0% sodium hydrogen carbonate solution was poured into the cup-shaped filter, held for 5 minutes, and aspirated to give sample 1. Further, 100 ml of an equal concentration sodium hydrogen carbonate solution was added and aspirated to obtain sample 2. Both samples were placed on ATP / FREE agar plates for several hours, and then measured by RMDS.
[0032]
The measurement results are shown in FIG. The single washing of the sodium bicarbonate solution resulted in a value exceeding 100 RLU / sec, whereas the two washings resulted in all measured values of 85 RLU / sec or less.
[0033]
Therefore, it was concluded that the washing with the sodium hydrogen carbonate solution needs to be carried out twice or more.
In summary of the above results, the most desirable procedure for reducing background noise is as follows.
[0034]
A membrane filter, for example, an isopore filter with a 0.4 μm ring (Nihon Millipore) is attached to a cup-shaped filtration container, and the product beer (happoshu) is poured aseptically. After pouring the beer into the cup-shaped filtration container, the beer is allowed to stand until the beer foam decreases and the liquid level of the beer can be visually confirmed, and then suction is started. Alternatively, foam remaining on the filter after beer membrane filtration is washed with a lower ethanol solution such as 5% ethanol or a surfactant such as 0.1% Tween 80. As mentioned above, it is set as the state which cannot confirm the bubble which remain | survives on a membrane filter by any operation visually. Thereafter, the membrane filter is transferred to a new cup-shaped filter, and 100-500 ml, preferably 100 ml, of 0.1-1.0% or preferably 0.1% sodium hydrogen carbonate is poured into the cup-shaped filter. Hold for 5 minutes, and perform suction filtration. After complete suction, 100 to 500 ml, preferably 100 ml, of 0.1 to 1.0%, preferably 0.1% sodium hydrogen carbonate is poured into a cup-shaped filter and suction filtration is performed. After filtration, place the membrane filter on an ATP / FREE agar plate (agar concentration: 1.0%) from 30 minutes 2 Put the time.
[Example 5]
According to the present inventor, when a non-culture test of brewer's yeast is carried out using a conventional luminescent reagent, it has been experienced that the amount of luminescence increases by being brought into contact with the medium once (holding within 30 minutes). This is probably because the yeast cells did not grow, but the nutrients in the medium were taken up into the cells and metabolized, resulting in an increase in intracellular ATP.
[0035]
For example, lactic acid bacteria are fermented using various sugars as nutrient sources to produce lactic acid, acetic acid, formic acid, alcohol, etc. (EMP pathway, HMP pathway, ED pathway, bifism pathway), as well as wine production. Malolactic fermentation is known in which an organic acid (malic acid) is converted into lactic acid and carbon dioxide to reduce acidity. Therefore, by adding a substrate such as sugar or organic acid to the microorganisms remaining in the sample (natural product components including ATP increase the background noise and cannot be used without completely decomposing and removing ATP). It is thought that metabolism is activated and ATP in microorganisms can be increased. Moreover, since it is not culture, the time required for these treatments is considered to be several hours. According to this, even if the number of microorganisms is several, the ATP emission amount, that is, S / N, can be increased, and the background noise, that is, the reduction of N of S / N described above is also combined. Significant S / N improvement is achieved and ATP luminescence may be measured without culturing.
[0036]
Therefore, in order to confirm the possibility of an increase in the amount of ATP luminescence, samples were prepared as follows and the amount of ATP luminescence was measured.
[0037]
Lactic acid bacteria were suspended in beer and subjected to membrane filtration. Lactic acid bacteria were diluted to about 50 cells per filter. The obtained sample is washed to remove background noise due to beer-derived ATP as described above. Next, prepare an ATP / FREE agar plate and perform the following process. That is, as a basic composition of the agar plate, 0.2% malic acid as organic acid, Bacto Yeast Nitrogen Base (hereinafter referred to as “YNB”. Trade name, used as a base of a medium manufactured by DIFCO, Inc. Yes), 1% agar, pH 5.2, (1) 1% glucose added as a saccharide to this agar plate (2) No saccharide added (agar plate itself with the above basic composition) (3) ATP as a comparative example・ After adding 1% glucose to a FREE agar plate and placing each sample on the above three types of agar plates for about 1 hour, add ATP extraction / luminescence reagent to the sample, and then measure the amount of luminescence with RMDS It was measured.
[0038]
The result is shown in FIG. When an agar plate containing a nutrient source of sugar (glucose), organic acid (malic acid), and YNB was used, it was revealed that the number of detected bacteria was the largest and the increase in ATP was large. It can be seen that if any of the above three nutrient sources is missing, the ATP increase is small.
[0039]
Next, when examining organic acids, experiments were conducted using citric acid, malic acid, and lactic acid, respectively. The result is shown in FIG.
[0040]
The number of bacteria detected when lactic acid was used was clearly smaller than when citric acid and malic acid were used. This is considered that metabolism was inhibited by the presence of lactic acid which is the main final metabolite of lactic acid bacteria. What can be considered from this result is that acetic acid is also a final metabolite of lactic acid bacteria, so that the number of detected bacteria is expected to decrease. On the other hand, from the results of using citric acid and malic acid, it is presumed that addition of fumaric acid or oxaloacetic acid involved in malolactic fermentation is also effective.
[0041]
The agar plate used in this experiment was basically 1% glucose, YNB, agar 1%, pH 5.2. Moreover, the mounting time on the agar pool was set to 2 to 5 hours.
[Example 6]
Although the usefulness of saccharides and organic acids has been confirmed by previous investigations, the usefulness of other components was investigated here. YNB is used as a nutrient source for microorganisms, and the component composition of YNB is shown in Table 2. Among these components, (7B) YNB with N source (ammonium sulfate) and amino acids removed (7C) YNB with amino acids removed (7C) YNB with vitamins removed from each agar plate (1 % Glucose, 0.2% malic acid, agar 1%, PH 5.2) and the sample was placed for 1 hour. The measurement results of each sample are shown in FIG. For comparison, a sample added with YNB was also measured in FIG. 7 (7D). From this measurement result, no significant difference was found. That is, it was confirmed that each of the above components does not activate ATP metabolism of microorganisms.
[0042]
Next, the influence of inorganic salts and trace elements (both see Table 1) contained in YNB was investigated. The agar brate used was the same as the above, and (8A) YNB added (8B) inorganic salts + trace elements (8C) inorganic salts (8D) trace elements (8E) YNB-free agar plates were easily prepared respectively. did. Moreover, the mounting time on each said agar plate was 1 hour. The prepared sample was subjected to ATP extraction and luminescence treatment, and the amount of luminescence was measured by RMDS. The result is shown in FIG. FIG. 8 clearly shows that the number of counted bacteria was small in the cases (8D) and (8E). From this, it became clear that among the components of YNB, the components that activate ATP metabolism are inorganic salts.
[Example 7]
The pH of the agar plate was examined. The agar plate used was prepared by adding 1% glucose, 0.2% malic acid, and YNB, and prepared an agar plate with varying pH. On the other hand, a sample was prepared by placing on the agar plate for 1 hour, and the number of bacteria was measured. FIG. 9 shows the results. From these results, it can be seen that there is no hindrance in measurement if PH is in the range of 3.8 to 5.5.
[Example 8]
The placement time on the agar plate was examined. The same agar plate used in Example 7 was used. FIG. 10 shows the measurement results. From this result, if it was 1 hour or more, the substantially same result was stably obtained. From this, it was confirmed that about 1 hour is sufficient for the mounting time on the agar plate.
[0043]
From the results examined in Examples 5 to 8 above, when measuring the presence or number of microorganisms by measuring the amount of ATP luminescence, the metabolism of microorganisms to be measured is activated at the time of preparation of the measurement sample. By reducing the ATP emission S / N (improvement of S) by adding a nutrient source that increases the ATP of the sample, the background noise of the sample described above is reduced (the N of ATP emission is reduced). In addition, ATP luminescence can be measured sufficiently even when the number of bacteria present in the sample is very small, about 1 to 100, and placed on the agar plate for about 1 hour without culturing microorganisms for a long time. It was confirmed that measurement was possible by increasing the ATP. In order to reduce background noise, the effect was obtained by placing it on an ATP / FREE agar plate for a minimum of 30 minutes. However, when used together, it takes about an hour to increase the amount of ATP. It was confirmed that it was necessary to place the cells on an agar plate for about 1 hour in order to enable non-cultured ATP luminescence measurement.
[0044]
In particular, for microbial counts of lactic acid bacteria, reduction of background noise as described above, sugars (glucose, galactose, fructose, etc.) and organic acids (malic acid, citric acid) that serve as nutrient sources for lactic acid bacteria during sample preparation In addition, by adding at least inorganic salts (magnesium sulfate, potassium phosphate, sodium chloride, calcium chloride, etc.), the S / N ratio of ATP emission can be improved.
【The invention's effect】
As is clear from the detailed description above,
According to the present invention, a lower alcohol such as ethanol, a surfactant such as turpentine (trade name: Tween 80) or a silicon-based antifoaming agent that has an antifoaming action and does not adversely affect the microorganism to be detected. By washing the membrane filter after filtration, bubbles can be removed, and the S / N can be greatly improved.
[0045]
Further, the beverage component remaining on the filter can be removed by washing with a weak alkaline aqueous solution such as sodium hydrogen carbonate.
[0046]
By using the countermeasure against the remaining foam and the countermeasure against the remaining beverage component in combination, the filter residue is almost removed, and the background noise (corresponding to N of S / N) is greatly reduced.
[0047]
[Table 1]

[0048]
[Table 2]

[Brief description of the drawings]
FIG. 1 is a view showing a sample (left) in which bubbles remain on a membrane filter and a sample (right) in which bubbles remain.
FIG. 2 shows the results of measurement of the amount of light emitted by performing the cleaning treatment of the present invention. FIGS. 2-1 (A) and (B) show the measurement results of Example 1, and FIGS. 2-2 (A) and (B) show the measurement results of Example 2. FIGS.
3 is a graph showing the measurement results of Example 3. FIG.
4 is a graph showing measurement results of Example 4. FIG.
FIG. 5 is a diagram showing experimental results of Example 5.
6 is a graph showing results of measurements performed on the organic acid of Example 5. FIG.
7 is a graph showing measurement results of Example 6. FIG.
8 is a graph showing the results of measurement performed on inorganic salts and trace elements of Example 6. FIG.
9 is a graph showing the measurement results of Example 7. FIG.
10 is a graph showing measurement results of Example 8. FIG.

Claims (7)

  After filtering the malt fermented beverage with a membrane filter, the membrane filter is washed with a defoaming agent solution, and further washed with a weakly alkaline aqueous solution. A method for rapidly detecting microorganisms in a fermented malt beverage, characterized by measuring   When filtering the malt fermented beverage with a membrane filter, the beverage liquid level is confirmed through the foam layer formed on the upper surface of the malt fermented beverage to be filtered, and then suction filtration is performed. A method for rapidly detecting microorganisms in a fermented malt beverage, characterized by spraying an ATP extraction reagent and a luminescent reagent on a sample obtained by washing in step 1 and measuring the amount of luminescence.   2. The microorganism in a malt fermented beverage according to claim 1, wherein a lower alcohol such as ethanol having a content equivalent to the alcohol content of the malt fermented beverage to be filtered is used as the antifoam solution of the membrane filter. Rapid detection method.   2. The malt according to claim 1, wherein a surfactant solution or a silicon-based antifoaming agent solution that does not adversely affect microorganisms mixed in the malt fermented beverage to be filtered is used as an antifoaming solution for the membrane filter. A method for rapid detection of microorganisms in fermented beverages. The method for rapidly detecting microorganisms in a fermented malt beverage according to claim 1 or 2, wherein the membrane filter is washed with a weakly alkaline aqueous solution a plurality of times .   After filtering the malt fermented beverage with a membrane filter, the membrane filter is washed with an antifoaming solution, and further washed with a weakly alkaline aqueous solution. The sample obtained from saccharides, organic acids, inorganic salts and vitamins with ATP / FREE After being placed on an agar plate containing a microbial ATP activator that activates the metabolism of microorganisms for 1 to several hours, the amount of luminescence is measured by spraying an ATP dispensing reagent and a luminescent reagent. A method for rapid detection of microorganisms in a malt fermented beverage.   When filtering the malt fermented beverage with a membrane filter, the beverage liquid level is confirmed through the foam layer formed on the upper surface of the malt fermented beverage to be filtered, and then suction filtration is performed. Samples obtained by washing with ATP / FREE are selected from the group consisting of saccharides, organic acids, inorganic salts and vitamins, and 1 to several agar plates containing microbial ATP activators that activate microbial metabolism A method for rapidly detecting microorganisms in a fermented malt beverage, characterized by spraying an ATP pouring reagent and a luminescent reagent and measuring the amount of luminescence after being placed for a period of time.

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