JP2010011759A - Method for measuring microorganism in tea-based beverage - Google Patents

Abstract

The present invention provides a rapid measurement method for microorganisms in tea-based beverages based on the ATP method, which has been difficult in the past.
By mixing and processing a tea-based beverage and a specific buffer, ATP derived from the raw material in the tea-based beverage is extracted and released without destroying microorganisms, and then ATP is obtained by reaction with an ATP scavenger. In addition to increasing the efficiency of the erasing process, chemiluminescence is reduced, and microorganism-derived ATP in tea-based beverages can be measured quickly.
[Selection] Figure 1

Description

  The present invention relates to a rapid method for measuring microbial contamination in tea-based beverages.

  Conventionally, as a method for measuring microbial contamination in beverages, a sample is cultured for 2 to 3 days using an agar medium, and the sample is filtered using an agar plate method for counting the number of colonies produced, or a membrane filter, and then agar. A membrane filter method is generally used, which is applied to a medium and cultured in the same manner on a filter. However, these microorganism measurement methods require a long time to obtain measurement results, and lack rapidness as a detection method / process control method for microbial contamination in beverages. In addition, since the time required to determine shipping becomes longer, it is necessary to expand the warehouse space with the increase in inventory, resulting in financial and spatial losses.

  In order to solve such problems, a bioluminescence method (hereinafter referred to as “ATP method”) is known as one of the rapid measurement methods for microbial contamination that has been widely adopted. The “ATP method” is a method for measuring microbial contamination based on the amount of luminescence based on the fact that the amount of luminescence produced by oxidation of luciferin is proportional to the amount of ATP in the presence of luciferin, luciferase and ATP. If the “ATP method” is used, microbial contamination can be measured in a short time, so that the measurement time can be greatly shortened.

  On the other hand, in beverages, free ATP derived from raw materials and ATP attached to somatic cells, proteins, fibers, etc. (hereinafter collectively referred to as “raw material-derived ATP”) are also included. At the time of ATP measurement, the amount of microorganisms may not be accurately measured because “raw material-derived ATP” emits light. Therefore, when measuring ATP derived from microorganisms in a beverage (hereinafter referred to as “microorganism-derived ATP”), a pretreatment method for reducing the influence of “raw material-derived ATP” is required.

As a technology for erasing the “raw material-derived ATP” reported so far,
(1) A method of extracting ATP derived from somatic cells (hereinafter referred to as “somatic cell ATP”) with a “somatic cell ATP” extractant and erasing these ATPs with an “ATP scavenger” (hereinafter referred to as “somatic cell treatment method”). ")
(2) Method of performing centrifugation after sufficiently reacting “somatic cell ATP” extractant and “ATP eliminator” to somatic cells in a plant beverage (hereinafter referred to as “plant beverage method”)
(For example, refer to Patent Documents 1 to 3).

  In the “somatic cell treatment method” of (1) above, after extracting ATP from the somatic cells using the “somatic cell ATP” extractant, the “ATP scavenger” is added to act to erase the “raw material-derived ATP”. However, for tea-based beverages, the effect of eliminating “raw material-derived ATP” is insufficient, and an accurate measurement result of “microbe-derived ATP” cannot be obtained.

  In the “plant beverage method” of (2) above, ATP is extracted from a somatic cell derived from a plant beverage using a “somatic cell ATP” extractant, then “raw material-derived ATP” is eliminated, and then these are not required by centrifugation. The remaining reagent and degradation products are removed, and only the remaining “microbe-derived ATP” is detected. However, with tea-based beverages, good results cannot be obtained using the “plant beverage method”. In addition, the “plant beverage method” has a problem in that it requires complicated processes such as centrifugation and dedicated equipment.

  Furthermore, it is known that components such as gallic acid and polyphenol contained in components in tea-based beverages generally generate chemiluminescence in the presence of active oxygen under alkaline conditions (for example, non-patent documents). 1 and Patent Document 4). The extraction of ATP from microorganisms and the luciferase-luciferin reaction are carried out under a pH range from near neutral to alkaline conditions (see, for example, Non-Patent Document 2). When the pH is alkaline, the aforementioned chemiluminescence occurs, and it is considered that non-ATP-derived noise is generated. This makes it difficult to rapidly measure microorganisms in tea-based beverages based on the “ATP method”. .

JP 2002-168859 A Japanese Patent No. 2867181 Japanese Patent No. 2905727 JP 2003-238495 A “Biochemical Dictionary”, 3rd edition, Tokyo Chemical Doujin, October 1998, p. 274 Hattori et al., Anal. Biochem. (2003) 319, 287-295

  The present invention has been made to solve the above-mentioned problems, and provides a rapid measurement method for microorganisms in tea-based beverages based on the “ATP method”, which has been difficult in the past with a simple operation. Objective.

  As a result of intensive studies to solve the above-mentioned problems, the present inventors extracted “raw material-derived ATP” in tea-based beverages without destroying microorganisms by mixing and treating the sample and a specific solution. In addition to increasing the efficiency of the ATP elimination step by the subsequent reaction with the “ATP elimination agent”, it is possible to reduce chemiluminescence that does not originate from “raw material-derived ATP”. The present inventors have found that it can be measured quickly by the “ATP method” and completed the present invention.

That is, the present invention
(1) Method for measuring microorganisms in tea-based beverages including the following steps: ATP-erasing pretreatment reagent that releases “raw material-derived ATP” in tea-based beverages without reducing microorganisms and reduces chemiluminescence derived from tea-based beverage components is added to tea-based beverages and allowed to react for a certain period of time. First step 2. 2. Second step of adding “ATP erasing agent” and reacting to eliminate “raw material-derived ATP” in the reaction solution. 3. ATP extraction agent is added and reacted to release ATP in the microorganism into the reaction solution. Fourth step of measuring released “microorganism-derived ATP” with a luciferin-luciferase reagent (2) The microorganism measuring method according to (1) above, wherein the pH of the ATP elimination pretreatment reagent is 7.0 to 9.0.
(3) The microorganism measuring method according to the above (1) to (2), wherein the buffer in the ATP elimination pretreatment reagent is one or two or more buffers selected from phosphoric acid, Tricine or HEPES.
(4) The microorganism measuring method according to the above (1) to (3), wherein the concentration of the buffer in the ATP elimination pretreatment reagent is 40 to 150 mM.
(5) The microorganism measuring method according to the above (1) to (4), wherein the reaction time after adding the ATP elimination pretreatment reagent to the tea-based beverage is 1 to 30 minutes.
It is.

Definitions In the present invention, “tea-based beverages” used for measurement samples are non-fermented tea (green tea), semi-fermented tea (Oolong tea), and fermented tea (black tea). Specifically, green tea, sencha, gyokuro, kabuse tea, bancha, tamago green tea, tencha, matcha tea, hojicha, brown rice tea, semi-fermented tea, sorghum tea, oolong tea, fermented tea as straight tea, (Including milk tea, lemon tea, and flavor tea) and other tea-based beverages such as turmeric tea, buckwheat tea, barley tea, kombu tea, sweet tea, Tochu green tea, kashiwanoha tea, dodomi tea, genkosho tea tea, gymneme tea, herbal tea, guava tea , Fennel tea, koku tea, loquat tea, mate tea, rooibosti, pearl barley tea, ketsushi tea and corn tea.
Moreover, the blend tea which mixed tea type | system | group drinks, the drink containing these dried products, an extract, a powdery thing, and the thing which added milk, raw milk, powdered milk, cream, fruit juice, or saccharides to tea type | system | group drinks are also included.

  Of these, green tea, oolong tea, black tea, and green tea-containing beverages (mixed beverages with milk and tea-based beverages added with green tea) are particularly preferred as tea-based beverage samples.

  In a broad sense, “chemiluminescence” is also referred to as chemiluminescence, and refers to a phenomenon in which atoms or molecules are excited by energy generated by a chemical reaction to emit light (“Biochemical Dictionary”, 3rd edition, Tokyo Chemical Dojin, 1998 10 Month, p.274). In the present invention, chemiluminescence refers to luminescence caused by a reaction other than the luciferin-luciferase reaction, which is thought to be caused by gallic acid and polyphenols contained in tea beverages.

  In the present invention, “raw material-derived ATP” is included in free ATP contained in tea-based beverages, ATP physicochemically attached to tea leaf-derived fibers, proteins or other insoluble particles, and tea leaf-derived somatic cells. ATP derived from ingredients of the beverage such as ATP or cell components derived from tea leaves (chlorophyll etc.). The more “raw material-derived ATP” brought into the measurement without being completely erased, the more the background luminescence generated during the luciferin-luciferase reaction increases and the S / N (signal / noise) ratio decreases. Measurement becomes difficult.

  In the present invention, “background light emission” refers to light emission of noise. The noise is a value obtained by subtracting the luminescence generated by the “microbe-derived ATP” due to the luciferin-luciferase reaction from the luminescence measured during the luminescence measurement. Luminescence of these noises (background) includes chemiluminescence considered to be derived from components in tea-based beverages, and luminescence generated by luciferin-luciferase reaction of “raw material-derived ATP” contained in components in tea-based beverages.

  In the present invention, the present inventors have found that “background luminescence” can be effectively reduced by reacting a tea-based beverage with a buffer having an alkaline pH prior to the ATP elimination step. Regarding the “background light emission” reduction effect, the following two events can explain this effect.

ATP has a phosphate group and an amino group, and each pKa (dissociation constant) is 6.5 for the phosphate group and 4.5 for the amino group. That is, ATP is negatively charged near a neutral pH. ATP is widely known to be very unstable and easily hydrolyzed in an acidic solution. The pH of tea-based beverages is generally pH 5.0 to 7.0, which is a weakly acidic condition, but is not considered to be a suitable condition because ATP exists stably.
For example, in a green tea beverage having a pH of 6.5 as used in the examples of the present invention, the amino group of ATP is neutral, and the phosphate group may exist in a state close to neutrality. Therefore, “raw material-derived ATP” in such tea-based beverages is adsorbed nonspecifically or physicochemically to components in tea-based beverages such as tea leaf-derived fibers and proteins, other insoluble particles or tea leaf-derived somatic cells. Alternatively, it is considered that they are encapsulated in these components and exist in a form that avoids hydrolysis.
When a buffer solution having an alkaline pH and a tea beverage are mixed, ATP adsorbed on the components in the tea beverage is negatively charged. The electrostatic repulsion between the negatively charged ATP and the ingredients in the tea beverage, or a substitution reaction occurs between these ingredients and the ATP, OH ^- ion, and the buffer, and the ATP in the ingredients in the tea beverage is It is possible to release effectively.

On the other hand, gallic acid and polyphenol contained in tea-based beverage components generally emit chemiluminescence in the presence of active oxygen under alkaline conditions. In the measurement of microorganisms by typical bioluminescence methods (see Hattori et al., Anal. Biochem. (2003) 319, 287-295), extraction of ATP from microorganisms is carried out in alkaline conditions, and luciferase- Since the luciferin reaction is also performed under alkaline conditions of pH 7.0 or higher, chemiluminescence may occur.
With the ATP erasing pretreatment reagent that is an alkaline buffer solution, once the tea-based beverage is made alkaline to cause chemiluminescence, chemiluminescence can be made difficult to occur in a later step.

Composition of ATP Erasing Pretreatment Reagent In the present invention, “ATP elimination pretreatment reagent” means a pH of neutral or higher, and without degrading microorganisms, “raw material-derived ATP” is converted to protein or fiber in tea-based beverages. Alternatively, it has the property of being liberated from raw material-derived components such as insoluble particles and reducing chemiluminescence.
That is, the ATP elimination pretreatment reagent is adjusted to pH 7.0 to 10.0. In order to release the “raw material-derived ATP” from the raw material-derived components in the beverage and destroy chemiluminescence without destroying microorganisms, pH 7.0 to 9.0 is particularly preferable, and pH 8.0 is more preferable. A range of 9.0 is preferred.

  As the buffer solution, a phosphate buffer or a HEPES buffer or Tricine buffer having an optimum buffer capacity in the range of pH 7.0 to 9.0, more preferably pH 8.0 to 9.0, among the Good buffers. A liquid is used, and the concentration is preferably 40 to 150 mM.

  The ATP elimination pretreatment reagent may contain one or more surfactants. As the surfactant, those having a function of liberating “raw material-derived ATP” from the raw material-derived components in the tea-based beverage are preferable, and nonionic surfactants are particularly preferable. Examples of the nonionic surfactant include Tween 20, Tween 80, and Triton X-100. Preferably, Tween 80 is used and the concentration is about 0.1-2.0%.

  The ATP elimination pretreatment reagent may also contain one or more chelating agents. Examples of the chelating agent include ethylenediaminetetraacetic acid (EDTA) and ethylene glycol tetraacetic acid (EGTA). Preferably, EDTA or EGTA is used as a chelating agent, and its concentration is 10 to 40 mM.

Reaction conditions of ATP elimination pretreatment reagent In the present invention, as the first step, by reacting the above ATP elimination pretreatment reagent with the sample, ATP is extracted and released from the ingredient derived from the raw material in the tea-based beverage, And it reduces chemiluminescence.
The reaction temperature and reaction time between the ATP elimination pretreatment reagent and the sample are sufficient to extract and liberate ATP from the ingredients derived from the ingredients in the tea-based beverage and reduce chemiluminescence, and also destroy microorganisms. It must be possible to implement without Since the reaction temperature and reaction time are determined by these balances, the reaction temperature is affected by the target sample and microbial species, but the reaction temperature is 20 to 55 ° C, preferably 30 to 37 ° C, and the reaction time is within 60 minutes, preferably Is preferably carried out in the range of 1 to 30 minutes.

"ATP eraser"
In the present invention, as a second step, after the pretreatment with the above ATP elimination pretreatment reagent, the sample is reacted with a reagent containing an ATP degrading enzyme (referred to as “ATP elimination agent”), and extracted / released in the first step. The “raw material-derived ATP” is decomposed and eliminated. By treating with “ATP scavenger”, “raw material-derived ATP” is decomposed, background luminescence in the luciferin-luciferase reaction in the subsequent step can be reduced, and the detection sensitivity of microorganisms can be improved. .

  As the “ATP scavenger”, one kind of conventionally known ATP degrading enzyme such as adenosine phosphate deaminase, adenosine phosphate deaminase and other enzymes (eg, apyrase, alkaline phosphatase, acid phosphatase, hexokinase, adenosine triphosphatase) is used. Or the mixed solution containing multiple types is preferable. The ATP-degrading enzyme contained in the “ATP scavenger” is preferably deactivated before entering the next step. If ATP-degrading enzyme remains in the sample, this “ATP” is also decomposed when “microbe-derived ATP” is extracted in a later step, resulting in a decrease in the amount of luminescence and a decrease in detection sensitivity. . The method for deactivating the enzyme activity is not particularly limited, and examples thereof include a method of adding an activity inhibitor or a method of adding an acid or alkali to change the pH of the sample to deactivate.

  The reaction temperature and reaction time of the “ATP scavenger” and the sample must be sufficient for decomposing “raw material-derived ATP” and can be carried out without destroying microorganisms. Although it is affected by the target sample and microbial species, the reaction temperature is 20 to 55 ° C, preferably 30 to 37 ° C, and the reaction time is within 60 minutes, preferably 1 to 30 minutes.

As a third step, the sample that has undergone the ATP degradation elimination reaction is reacted with an appropriate ATP extractant to extract “microorganism-derived ATP”. As a method for extracting ATP, a method of adding a known ATP extractant is suitable. As the ATP extractant, a surfactant, a mixed solution of ethanol and ammonia, methanol, ethanol, trichloroacetic acid, and perchloric acid can be used. Of these, the surfactant is preferably higher in the extraction efficiency of ATP.
Surfactants include sodium dodecyl sulfate (SDS), potassium lauryl sulfate, sodium monolauroyl phosphate, sodium alkylbenzenesulfonate, benzalkonium chloride (BAC), benzethonium chloride (BZC), cetylpyridinium chloride, cetyltrimethyl bromide Ammonium and myristyldimethylbenzylammonium chloride are mentioned.

As the fourth step, the extracted “microorganism-derived ATP” is measured. As a method for measuring “microorganism-derived ATP”, a method using luciferin and luciferase as a bioluminescent reagent is used. In this case, the sample containing the extracted ATP is reacted with a luciferin-luciferase bioluminescence reagent, and the amount of luminescence produced is measured to determine the amount of “microorganism-derived ATP”. Bioluminescent reagents include luciferin, luciferase and magnesium ions.
The amount of luminescence produced is a luminometer, for example, Lumitester C-100, C-100N, C-110, K-100, K-200, K-210 (all manufactured by Kikkoman), Luminescence Reader BLR-201 It can be measured by a mold (manufactured by Aroka) and Lumat LB9501 (manufactured by Bertoled).
Hereinafter, the present invention will be described in more detail with reference to examples, but the present invention is not limited thereto.

(Preparation of ATP elimination pretreatment reagent)
In order to examine the pH of the ATP elimination pretreatment reagent, a total of 8 types of ATP elimination pretreatment reagents having a pH of 6.0 to 8.5 were prepared. As buffers, 80 mM MES was used for the ATP elimination pretreatment reagent at pH 6.0, 80 mM HEPES was used for pH 7.0 to 8.0, and 80 mM Tricine was used for pH 8.2 to 8.5, respectively. Dissolved in. The pH was adjusted with a 1N aqueous sodium hydroxide solution, and this was used as a pretreatment reagent for ATP elimination.

(Elimination process of “raw material-derived ATP”)
A commercial green tea having a pH of about 6.5 was prepared as a sample. 0.1 ml of commercially available green tea was dispensed into Lumitube (manufactured by Kikkoman) using a micropipette. Add 0.1 ml of the ATP erasing pretreatment reagent of each pH obtained in Example 1 (preparation of ATP erasing pretreatment reagent) to the tube in which the sample is collected, and stir with a tube mixer or the like. ATP erasing pretreatment reaction was carried out by leaving it still in an air-conditioning thermostat set to ° C (manufactured by ASONE, IC-300A) for 30 minutes. After the reaction, 0.1 mL of a lucifer treatment liquid (manufactured by Kikkoman Corporation, including Tween 80 and a chelating agent) was dispensed into each tube and stirred. After adding 0.1 ml of ATP elimination reagent (Kikkoman Co., Ltd., dissolved in the attached solution) and 0.7 ml of lucifer diluted solution (Kikkoman Co., Ltd.), the mixture was stirred and kept at a constant temperature of 35 ° C. as above. The elimination reaction of “raw material-derived ATP” was carried out by leaving it in the vessel for 30 minutes.
At the same time, a measurement method not using an ATP elimination pretreatment reagent (described in Table 1 as “no pretreatment”) was also examined. In that case, the above ATP erasing pretreatment reagent was not added, and instead the lucifer diluted solution was added, the addition amount was increased from 0.7 ml to 0.8 ml to make the solution amount uniform. Other reactions were carried out in the same manner as in the case of using the ATP elimination pretreatment reagent.

(Extraction process of “microorganism-derived ATP”)
A 0.1 ml sample from which “raw material-derived ATP” had been erased was dispensed into a new lumitube. ATP extraction reagent (Kikkoman Co., Ltd.) 0.1ml was added there, and ATP was extracted by stirring.

(Measurement process of light emission)
0.1 mL of Lucifer Luminescent Reagent-HS (Kikkoman Co., Ltd., dissolved in the attached lysing solution) was added to 0.2 ml of ATP-extracted sample and stirred, and immediately after that Lumitester C-100 (Kikkoman Co.) The amount of luminescence was measured. The amount of luminescence of the sample measured above is shown in Table 1.

  From Table 1, when the ATP elimination pretreatment reagent having a pH of 6.0 was used, a decrease in background luminescence was not observed as compared to the case where the ATP elimination pretreatment reagent was not used. In the range of pH 7.0 to 8.5, a significant decrease in background light emission was observed, and in particular, it was shown that the pH was reduced to 1/10 or less at pH 7.5 or higher.

(Preparation of ATP elimination pretreatment reagent)
In order to investigate the influence of the ATP elimination pretreatment reagent on microorganisms, an ATP elimination pretreatment reagent having a pH of 7.0 to 9.0 was prepared. As buffering agents, 20 to 200 mM HEPES was used at pH 7.0, and 20 to 200 mM Tricine was used at pH 8.0 to 9.0, and dissolved in ultrapure water. The pH was adjusted with a 1N aqueous sodium hydroxide solution, and this was used as a pretreatment reagent for ATP elimination.

(Elimination process of “raw material-derived ATP”)
Enterobacter cloacae NBRC3320 and Staphylococcus aureus ATCC 6538 were prepared as additional bacteria. These added bacteria are one of the contaminating bacteria detected in tea-based beverages. Each bacterium was cultured on a standard agar medium at 35 ° C. for 24 hours.
Colonies generated after the culture were suspended in PBS, diluted appropriately with the same PBS, and 0.1 mL was fractioned into a Lumitube (manufactured by Kikkoman Corporation) using a micropipette.
Add 0.1 ml of ATP elimination pretreatment reagent of each pH obtained in Example 2 into the tube from which the bacterial solution was collected, and stir with a tube mixer etc. ATP elimination pretreatment reaction was performed by leaving it in the container for 30 minutes.

After the reaction, 0.1 mL of a lucifer treatment liquid (manufactured by Kikkoman Corporation, including Tween 80 and a chelating agent) was dispensed into each tube and stirred.
ATP elimination reagent (Kikkoman, dissolved in the attached solution) 0.1 ml, Lucifer diluted solution (Kikkoman) 0.6 ml was added and stirred, and the temperature was set to 35 ° C. as above. The elimination reaction of “raw material-derived ATP” was carried out by leaving it in the vessel for 30 minutes.
At the same time, a measurement method not using an ATP elimination pretreatment reagent (described in Table 2 as “no pretreatment”) was also examined. In this case, the above ATP erasing pretreatment reagent was not added, and instead the lucifer diluted solution was added, the addition amount was increased from 0.6 ml to 0.7 ml to adjust the solution amount. Other reactions were carried out in the same manner as in the case of using the ATP elimination pretreatment reagent.

(Extraction process of “microorganism-derived ATP”)
A 0.1 ml sample from which “raw material-derived ATP” had been erased was dispensed into a new lumitube. ATP extraction reagent (Kikkoman Co., Ltd.) 0.1ml was added there, and ATP was extracted by stirring.

(Measurement process of light emission)
After adding and stirring 0.1 ml of Lucifer Luminescent Reagent-HS to 0.2 ml of ATP-extracted sample, it was immediately put into Lumitester C-100 (manufactured by Kikkoman Corporation), and the amount of luminescence was measured.

(Calculation of “emission ratio”)
Based on the following formula, in Table 2, the ratio of the luminescence amount of each bacterial suspension reacted with each ATP elimination pretreatment reagent to the luminescence amount of each suspension at “no pretreatment” (hereinafter referred to as “luminescence ratio”). ").

Formula: “Luminescence ratio” = (Emission amount of each bacterial suspension reacted with each ATP elimination pretreatment reagent) ÷ (Emission amount of various bacterial suspensions when “no pretreatment”) × 100

The “luminescence ratio” of the sample measured above is shown in Table 2.

In Table 2, the case where the “light emission ratio” is 80% or more is underlined. E. The “luminescence ratio” of cloacae was 100% or more except when reacted with an ATP elimination pretreatment reagent of 200 mM HEPES, pH 7.0, and all were 80%.
S. The “luminescence ratio” of Aureus was 80% or more in the range of 40 to 150 mM Tricine, pH 8.0 to 9.0. All were less than 80% at pH 7.0.
Based on the above, if it is specified that the decrease in the amount of luminescence of the microorganism when using the ATP erasing pretreatment reagent can be tolerated up to 80%, pH 7.0 to 9.0 is desirable, more preferably pH 8. It was 0 to 9.0, and it was considered that the buffer concentration was preferably 40 mM or more and 150 mM or less.

(Preparation of ATP elimination pretreatment reagent)
Using ultrapure water, each reagent was dissolved to 80 mM Tricine and 0.2% Tween 80. The pH was adjusted to 8.2 with 1N aqueous sodium hydroxide solution, and this was used as a pretreatment reagent for ATP elimination.

(Sample adjustment)
The same commercially available green tea, lemon tea and oolong tea as above were prepared as samples. All used what was filled in the PET bottle. Using these commercially available tea-based beverages, the samples in the non-microbe-added section were used as blanks for the following luminescence measurements. As a microorganism-added section, Enterobacter cloacae NBRC3320 was added to each tea beverage so as to be about 10 ⁶ cfu / mL, and a sample diluted 10-fold, 100-fold, and 1,000-fold was added. Prepared.

(Elimination process of “raw material-derived ATP”)
0.1 ml of each commercial tea-based beverage in the non-microbe-added group and the microorganism-added group was dispensed into a Lumi tube using a micropipette. Add 0.1 ml of the ATP elimination pretreatment reagent obtained in Example 3 (Preparation of ATP elimination pretreatment reagent) into the tube in which the sample is separated, and after stirring, an air-conditioning type set to 35 ° C. ATP erasing pretreatment reaction was performed by leaving it in a thermostat for 30 minutes. After the reaction, 0.1 ml of a lucifer treatment solution (manufactured by Kikkoman Corporation, including Tween 80, chelating agent) was dispensed into each tube and stirred. After adding 0.1 ml of ATP elimination reagent and 0.6 ml of lucifer diluted solution, the mixture was stirred and left in a thermostat set at 35 ° C. for 30 minutes in the same manner as described above to eliminate the “raw material-derived ATP”. went.

(Extraction process of “microorganism-derived ATP”)
A 0.1 ml sample from which “raw material-derived ATP” had been erased was dispensed into a new lumitube. ATP extraction reagent 0.1ml was added there, and ATP extraction was performed by stirring.

(Measurement process of light emission)
After adding and stirring 0.1 ml of Lucifer Luminescent Reagent-HS to 0.2 ml of ATP-extracted sample, it was immediately put into Lumitester C-100, and the amount of luminescence was measured.

(Confirmation of the number of bacteria in the sample)
The microorganism-added sample was appropriately diluted with each tea-based beverage to which no microorganism was added, and 0.1 ml was smeared on a standard agar medium. After culturing at 35 ° C. for 48 hours or more, the number of colonies produced was counted, and the bacterial concentration contained in each sample was calculated. The bacterial concentration and the amount of luminescence obtained from the samples of green tea, lemon tea and oolong tea are shown in FIGS.

(Comparative Example 1)
The sample described in Example 3 was used. In addition, in the measurement method described in Example 3, in the “step of erasing“ raw material-derived ATP ””, without using the ATP erasing pretreatment reagent, instead of adding 0.6 ml of lucifer diluted solution, Other reactions were carried out in the same manner except that the amount added was increased to 0.7 ml and the liquid volume was made uniform. The bacterial concentration and the amount of luminescence obtained from the samples of green tea, lemon tea and oolong tea are shown in FIGS.

(Comparative Example 2)
Using the sample described in Example 3, the amount of luminescence was measured by a method according to Japanese Patent No. 2905727 which is a so-called “plant beverage method”.
In the “plant beverage method”, ATP is extracted from somatic cells derived from plant beverages using a “somatic cell ATP” extractant, then “raw material-derived ATP” is erased, and then these reagents and decomposition products that have become unnecessary by centrifugation are removed. This is a method of removing the substance and detecting only the remaining “microbe-derived ATP”.

(Elimination process of “raw material-derived ATP” by centrifugation)
0.1 ml of each commercially available tea-based beverage in the non-microbe-added group and the microorganism-added group was dispensed into 1.5 ml microtubes using a micropipette. In each tube, 0.1 ml of a lucifer treatment liquid (manufactured by Kikkoman Corporation, Tween 80, containing a chelating agent) was dispensed and stirred. After adding 0.1 ml of ATP elimination reagent and stirring, it was allowed to stand in a thermostat set at 35 ° C. for 30 minutes. This was centrifuged at 15,000 × g for 5 minutes using a centrifuge, and the resulting supernatant was removed by suction with a micropipette. 1.0 ml of lucifer diluted solution was added and the precipitate collected on the bottom of the microtube was redissolved by stirring with a tube mixer to obtain a suspension.

(Extraction process of “microorganism-derived ATP”)
0.1 ml of the above suspension was dispensed into a new lumitube. ATP extraction reagent 0.1ml was added there, and ATP extraction was performed by stirring.

(Measurement process of light emission)
The amount of luminescence was measured by the method described in Example 3 to calculate the bacterial concentration. The bacterial concentration and the amount of luminescence obtained from the samples of green tea, lemon tea and oolong tea are shown in FIGS.

From the results shown in FIG. 1, the amount of luminescence of the sample having a bacterial concentration of 3.4 × 10 ³ cfu / ml in the green tea microorganism non-addition group (blank) and the microorganism addition group was measured using the ATP elimination pretreatment reagent. In the case of Example 3, they were 75 RLU and 148 RLU, respectively. In contrast, in the case of Comparative Example 1, when the ATP erasing pretreatment reagent was not used, they were 846 RLU and 636 RLU. In the case of Comparative Example 2, the values were 400 RLU and 417 RLU. The signal / noise ratio of the example was 148/75 = about 2 times, and microorganisms could be measured. In Comparative Example 1 and Comparative Example 2, microorganisms of the order of 10 ³ cfu / ml contained in green tea were used. Measurement was difficult.

From the results of FIG. 2, the amount of luminescence of the sample with the bacterial concentration of 5.2 × 10 ³ cfu / ml and the sample with the bacterial concentration of 5.2 × 10 ⁴ cfu / ml in the microbial addition group of lemon tea is ATP. In the case of Example 3 using the erase pretreatment reagent, they were 456 RLU and 1,225 RLU, respectively. On the other hand, when the ATP erasing pretreatment reagent was not used, in the case of Comparative Example 1, they were 9,053 RLU and 10,522 RLU. In Comparative Example 2, they were 1,474 RLU and 1,181 RLU. The signal / noise ratio of the example was 1,225 / 456 = about 2.7 times, and microorganisms could be measured. In Comparative Example 1 and Comparative Example 2, 10 ⁴ cfu / ml contained in lemon tea Measurement of microorganisms on the order was difficult.

From the results shown in FIG. 3, the luminescence amount of the sample having a microbial concentration of 4.8 × 10 ³ cfu / ml in the oolong tea blank and the microorganism-added group was as in Example 3 using the ATP elimination pretreatment reagent. 63 RLU and 122 RLU. On the other hand, when the ATP erasing pretreatment reagent was not used, in the case of Comparative Example 1, they were 1,851 RLU and 964 RLU. In the case of Comparative Example 2, they were 235 RLU and 334 RLU. The signal / noise ratio of Example was 122/63 = about 2 times, and microorganisms could be measured. In Comparative Example 1 and Comparative Example 2, microorganisms of the order of 10 ³ cfu / ml contained in green tea were used. Measurement was difficult.

Moreover, from the result of FIGS. 1-3, compared with the case where it measures by the method of the comparative examples 1-2 which is a conventional method, when measured in Example 3, linearity is obtained from the sample of a lower concentration microorganism-added section. It is clear that it shows.
As described above, by using the ATP erasing pretreatment reagent of the present invention, it is possible to reduce the background luminescence of various tea-based beverages, and the microorganisms in the beverage can be measured with higher sensitivity than conventional methods. Is possible.

The correlation between the amount of luminescence of green tea and the number of bacteria measured by the method described in Example 3 and the comparative example which is a conventional method is shown. The correlation between the amount of luminescence of lemon tea and the number of bacteria measured by the method described in Example 3 and the comparative example which is a conventional method is shown. The correlation between the amount of luminescence of oolong tea and the number of bacteria measured by the method described in Example 3 and the comparative example which is a conventional method is shown.

Claims (5)

A method for measuring microorganisms in a tea-based beverage, comprising the following steps.
(1) An ATP erasing pretreatment reagent that releases ATP from raw materials in tea-based beverages and reduces chemiluminescence derived from tea-based beverage components without destroying microorganisms is added to tea-based beverages and reacted for a certain period of time. First step (2) ATP elimination agent is added and reacted, and second step (3) ATP extractant is added and reacted to eliminate ATP in the reaction solution, and ATP in the microorganism is added to the reaction solution. (4) Fourth step of measuring the released microorganism-derived ATP with a luciferin-luciferase reagent   The microorganism measuring method according to claim 1, wherein the pH of the ATP elimination pretreatment reagent is 7.0 to 9.0.   The microorganism measuring method according to claim 1 or 2, wherein the buffer in the ATP elimination pretreatment reagent is one or two or more buffers selected from phosphoric acid, Tricine or HEPES.   The method for measuring microorganisms according to claims 1 to 3, wherein the concentration of the buffering agent in the ATP elimination pretreatment reagent is 40 to 150 mM.   The microorganism measuring method according to claim 1, wherein the reaction time after adding the ATP elimination pretreatment reagent to the tea beverage is 1 to 30 minutes.

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