JPH0576393A - Determination of yeast activity - Google Patents

Abstract

PURPOSE:To easily, simply and accurately determine yeast activity such as proliferativity or fermentativity in a short time by leaving yeast to stand in a low-pH environment followed by measuring the pH value in the cells. CONSTITUTION:Firstly, yeast is left to stand in a low-pH (<=6, pref. 2-3) environment for a specified time (pref. 30-100min). Thence, the pH value in the yeast cells is measured, for example, by such a means that 5,5-dimethyl-2,4- oxazolidinedione (DMO) is added to a suspension of the above cells followed by equilibration, and the DMO concentrations in both the inside and outside of the cells are determined; from the resultant partition degree, the pH value in the cells is calculated. Based on the results, yeast with higher pH value is determined as more active than that with lower pH value. For realizing the low-pH environment, it is recommended that an inorganic or organic acid be added to an aqueous suspension of the yeast.

Description

Detailed Description of the Invention

BACKGROUND OF THE INVENTION

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention provides an easy and simple method for measuring the intracellular pH of yeast to easily and simply measure the activity of yeast, for example, the proliferative power or fermentative power, or the viable or dead ratio. It relates to a method for determining the activity of yeast.
It is desirable to know the yeast activity in a short time in process control or product quality control in the fermentation industry.

[0002]

2. Description of the Related Art Conventionally, there has been a method for measuring the growth ability of microorganisms such as a plate culture method or a slide culture method in which a microorganism is actually cultured and its growth ability is known. This method is the most reliable method because it actually forms colonies on the agar medium and knows the actual growth ability from the number of colonies. However, the fact that culturing is required means that it takes about 1 to 6 days to obtain the results,
There is a drawback that the measurement result cannot be obtained quickly.

On the other hand, other than this, a dyeing method using methylene blue, a fluorescein derivative or the like is known. In this method, between live and dead yeast, only live bacteria decolorize methylene blue incorporated into cells, or an ester derivative of fluorescein (which is usually non-fluorescent) is incorporated into yeast. And a living cell, which is decomposed by esterase in yeast to regenerate fluorescein to generate fluorescence, which is a simple method of counting stained cells under a microscope after staining. Is. However, the obtained measurement results do not always match the proliferative potential of the cells.

[Outline of the Invention]

As described above, the prior art requires a long period of time and a result can be obtained in a short period of time, but the proliferative power may not always match. There were technical challenges. The present invention is directed to the activity of the cells in the shortest possible time, such as the proliferative potential,
It is intended to provide a new method for grasping the fermentative power and the like.

[0005]

[Means for Solving the Problems] The present invention is to measure the intracellular pH of a yeast cell after allowing it to stand in a solution of a low pH environment for a certain period of time to determine the activity of the cell, specifically, the proliferative power and fermentative power. , Or the viable cell rate, based on the finding that it is possible to grasp.

<Summary> That is, the method for measuring yeast activity according to the present invention is characterized by comprising the following steps. (1) Yeast is placed in a low pH environment of pH 6 or less, and after a lapse of a predetermined time, the intracellular pH of the yeast is measured. (2) The obtained yeast having a high intracellular pH value is judged to have higher activity than yeast having a low intracellular pH value.

[0007] Regarding another embodiment of this step (2), the method for measuring yeast activity according to the present invention comprises the following steps:
It is characterized by. (1) Yeast is placed in a low pH environment of pH 6 or less, and after a lapse of a predetermined time, the intracellular pH of the yeast is measured. (2) Obtained intracellular pH value and intracellular p value unique to the yeast
Yeast with a small difference from the H value is judged to have greater activity than yeast with a large difference.

Another method for measuring yeast activity according to the present invention is characterized by comprising the following steps. (1) A step of incorporating a pH-sensitive fluorescent substance into yeast cells. (2) Yeast containing a fluorescent substance has a low pH of 6 or less.
It is placed in an H environment, and after a lapse of a predetermined time, the yeast cells are irradiated with excitation light to generate fluorescence, and two types of fluorescence intensities are measured. (3) The intracellular pH value corresponding to the ratio of the measured fluorescence intensities is obtained from the calibration curve prepared in advance. (4) The obtained yeast having a high intracellular pH value is judged to have higher activity than yeast having a low intracellular pH value.

[0009] Regarding the other embodiment of this step (4), the method for measuring yeast activity according to the present invention is characterized by comprising the following steps. (1) A step of incorporating a pH-sensitive fluorescent substance into yeast cells. (2) Yeast containing a fluorescent substance has a low pH of 6 or less.
It is placed in an H environment, and after a lapse of a predetermined time, the yeast cells are irradiated with excitation light to generate fluorescence, and two types of fluorescence intensities are measured. (3) The intracellular pH value corresponding to the ratio of the measured fluorescence intensities is obtained from the calibration curve prepared in advance. (4) Obtained intracellular pH value and intracellular p value unique to the yeast
Yeast with a small difference from the H value is judged to have greater activity than yeast with a large difference.

<Effect> The present invention falls within the scope of the dyeing method. According to the present invention, first, by placing the yeast in a low pH environment for a predetermined time, the activity of individual cells of the target yeast is first confirmed. Since the difference is amplified and the "predetermined time" is usually up to several hours, it is possible to enjoy the effect of measurement in a short time that is inherent to the staining method.

The fact that the intracellular pH measured after placing yeast in a low pH environment correlates with the activity of yeast cells is considered to be a phenomenon first discovered by the present inventors. It is speculated that the phenomenon is due to the H pumping action of yeast cells (although such a presumption imposes no limitation on the present invention).
That is, highly active yeast cells have a strong H pumping action and have a large ability to maintain the intracellular pH at the original pH even when the environment has a low pH. On the other hand, since cells having low activity do not maintain the original intracellular pH value, the intracellular pH after a predetermined time elapses is lowered to a certain level lower than the original pH value.

Therefore, there is a significant difference in intracellular pH between a highly active yeast cell and a yeast cell that is inferior in a low pH environment after a predetermined period of time. The activity of a given yeast can be detected with high sensitivity and in a short time by detecting it by the emission of a sensitive fluorescent substance.

Since the activity of yeast can be grasped by a simple method, the present invention greatly benefits the quality control and process control of products relating to fermentation in the yeast industry, for example, in the food industry.

[Detailed Description of the Invention] <Yeast> The yeasts whose activity is to be measured according to the present invention are all yeasts that fall into the category of yeasts in terms of classification. Typical of them are brewer's yeast, baker's yeast, wine yeast, sake yeast, alcohol yeast and the like, which belong to Saccharomyces microbiologically. These yeasts include, for example, those bred by a brewer depending on the purpose of use, and such "variants" are also the subject of the present invention.

<Low pH environment> Intracellular p of yeast according to the present invention
The measurement of H is performed after the yeast cells are placed in a low pH environment for a predetermined time. The low pH environment in the present invention means a condition of pH 6 or less, preferably 5 or less. There is no particular lower limit to the pH value, but it is preferably about pH 2 to 3.

Such a low pH environment can be easily realized by adding an inorganic acid or an organic acid to an aqueous suspension of yeast. Further, in order to maintain a predetermined pH value, it is usual to use a buffering agent, specifically an acidic buffering agent.

The time for which the yeast cells are placed in such a low pH environment is preferably, functionally, the time until the intracellular pH of the yeast cells does not substantially change. On the other hand, quantitatively, this is 1 minute or longer, preferably 30 minutes or longer, and hardly exceeds 200 minutes. A preferable time is 5 to 100 minutes, and a particularly preferable time is 30 to 100 minutes.
Minutes. The temperature of this low pH environment is the pH of the yeast cells.
It goes without saying that it should be the temperature at which the maintenance capacity (H pump capacity according to the above-mentioned estimated mechanism) operates. This is generally not lower than the metabolic resting temperature of yeast, while not so high as to kill the yeast, but the preferred temperature is minus several degrees to room temperature.

<Measurement of intracellular pH (No. 1)> The intracellular pH of yeast cells placed in such a low pH environment for a predetermined time can be measured by any purposeful method or means. it can. The measurement of intracellular pH is generally known, and specifically, for example, the science of life 39 (5):
386-397 (1988), an appropriate one can be selected and used in the present invention.

One of the methods for measuring intracellular pH is as follows:
Method using partitioning of weak acid / base (J.Clin.Invest. 38 : 720
-729 (1959)), and specifically, for example, DMO
(5,5-dimethyl-2,4-oxazolidinedione)
Is added to the cell suspension, after equilibration, the intracellular and extracellular DMO concentrations are measured, and the intracellular pH is calculated from the degree of partitioning.

Another method for measuring the intracellular pH of yeast utilizes the fact that the chemical shift of inorganic phosphate changes depending on the intracellular pH (J. Biol. Chem. 248 : 7).
276 (1973)), the cells are directly subjected to NMR analysis, and the intracellular pH is measured by measuring the chemical shift of the inorganic phosphate.

<Measurement of intracellular pH (part 2)> Another method of measuring the intracellular pH of yeast cells, and a preferred method of the present invention, is to incorporate a pH-sensitive fluorescent substance into yeast cells,
It consists of measuring the fluorescence generated corresponding to the intracellular pH.

Fluorescent substances that are pH-sensitive, that is, fluorescent substances that produce a difference in fluorescent color in response to the pH conditions in which they are placed, are well known, and any substance that can be incorporated into yeast cells is known. It can be used in the present invention.

Incorporation of the pH-sensitive fluorescent substance into yeast cells can be carried out by any purposeful method or means. Generally, yeast cells may be contacted with the pH-sensitive fluorescent substance in the aqueous suspension thereof at room temperature for a predetermined time (for example, several seconds to 60 minutes). However, the pH-sensitive fluorescent substance is specifically as follows,
In many cases, the necessary amount of these is not taken up within a reasonable time even if they are contacted with yeast cells as described above. Since many such fluorescent substances have an ionic group such as a phenolic hydroxyl group or a carboxyl group, they are converted into an ester (for example, ester with lower carboxylic acid such as acetic acid or lower alcohol such as methanol). Then, it will be easily taken up by the yeast cells.

Some specific examples of pH sensitive phosphors suitable for use in the present invention are fluorescein and its derivatives, quene 1 and its derivatives,
1,4-dihydroxy-phthalonitrile and its derivatives, umbelliferone and its derivatives, hydroxypyrene and its derivatives, 5-dimethylaminonaphthalene-1-sulfonate (dansyl) color-forming group, carboxy-
Semi-naphthofluor (Fluoro-seminaphthorhodafl)
uor) (carboxy SNARF), carboxy-seminaphthofluorescein (carboxy SNAFL), etc. (further details can be found in, for example, Science of Life 39
(5): 386-389 (1988), see FIG. 1). As described above, it is usual that the actual form when these are taken up by yeast cells is the ester derivative thereof. Typical pH-sensitive fluorescent substances in the present invention include, for example, 5 (6) -carboxyfluorescein diacetate and 2 ′, 7′-bis (carboxyethyl)-
It is 5 (6) -carboxyfluorescein tetraacetoxymethyl ester.

The pH-sensitive fluorescent substance taken into the yeast cell as an ester derivative is destroyed by the esterase in the cell to destroy the ester structure and regenerate the ionic fluorescent substance. Since the ionic fluorescent substance is difficult to be taken out of the cell as well as being difficult to be taken up by the yeast cell, it is accumulated in the cell. When the pH-sensitive fluorescent substance thus incorporated into the yeast cell is irradiated with excitation light, it emits fluorescence according to a given intracellular pH value.

A calibration curve is prepared by previously obtaining the relationship between the fluorescence intensity obtained by measuring the fluorescence generated at a predetermined excitation wavelength at a predetermined wavelength and the pH condition in which the pH-sensitive fluorescent substance is placed, The yeast intracellular pH can be known by measuring the intensity of fluorescence generated by the yeast cells by the excitation light.

However, even with a given yeast cell preparation,
The amount of pH-sensitive fluorescent substance incorporated may vary depending on individual cells, and the intensity of fluorescence generated by excitation light irradiation depends on the intracellular amount or concentration of the incorporated pH-sensitive substance. The absolute value of the strength may not be able to indicate its exact pH dependence. Therefore, it is preferable to eliminate the influence of the intracellular concentration of the fluorescent substance by adopting the ratio of the two types of fluorescence intensity instead of one type of fluorescence intensity, that is, the absolute value of the fluorescence intensity.

A specific example of such two types of fluorescence intensity is as follows:
It is obtained by measuring the fluorescence intensity at a predetermined wavelength using two kinds of excitation light having different wavelengths. Specifically, for example, in the case of 5 (6) -carboxyfluorescein diacetate, Wavelengths of 441 nm and 488 nm,
There are two types of fluorescence intensity obtained under the condition that the fluorescence measurement wavelength is 518 nm. One of the other specific examples of the two types of fluorescence intensity for eliminating the influence of the intracellular concentration of the fluorescent substance is obtained by measuring the fluorescence at two predetermined wavelengths using one type of excitation light. Specifically, for example, carboxy-S
In the case of NARF, there are two types of fluorescence intensity obtained under the conditions of an excitation wavelength of 534 nm and fluorescence measurement wavelengths of 604 nm and 634 nm.

Whichever method is used, the obtained 2
A calibration curve may be prepared by using the ratio of the fluorescence intensities of the species as a function of the pH-sensitive substance. Fluorescence intensity may be measured with a cell suspension fluorometer (Biochemistry 18: 221).
0-2218 (1979)), the cells may be placed on a slide glass and measured and measured under a fluorescence microscope (Nature 32).
5 : 447-450 (1987)).

Intracellular pH that can be used in the present invention
As a specific example of the measuring device, the one described in JP-A-3-24442 (Japanese Patent Application No. 1-158708) can be mentioned.

The method according to the present invention comprises the intracellular p
It can be used to measure H as well as to measure the average intracellular pH value of a yeast preparation, that is, a fixed population. In the former case, the fluorescence emission status of each yeast cell will be examined. In such a case, it is convenient to use an image processing or flow cytometry technique. The above-mentioned Japanese Patent Laid-Open No. 3-24442 discloses information necessary for an example of image processing.

<Determination of Yeast Activity> The fact that the intracellular pH of the yeast thus measured has a correlation with the activity of the yeast cell will be clarified by the experimental facts described below.
Yeast with a higher intracellular pH value has more active activity. Therefore, if information on the intracellular pH value and yeast activity is prepared in advance for the target yeast, the activity of the yeast can be grasped from the intracellular pH value.

As mentioned above, the method according to the present invention is useful not only for measuring the average intracellular pH value of yeast preparations as described above, but also for measuring the intracellular pH value of each yeast. However, by measuring the activity of individual cells in a given yeast sample, it is possible to know the distribution of the cell activity of the sample, and thus the yeast preparation, which is the yeast population.

[0034]

【Example】

<Example 1> Beer yeast was cultivated in wort until a stationary phase (8
C). The cells were washed with water and stored aseptically in water at 2 ° C. By changing the number of storage days, cell populations with different viable cell rates were obtained. The viable cell rate of each yeast was determined by the plate culture method using malt agar medium. On the other hand, intracellular pH
Was measured as follows. Collect about 2 ml of yeast,
MES buffer (pH 6.2, 50 mM 2- (N-morpholino) ethanesulfonic acid, NaCl 110 mM, KC
5 mM, MgCl ₂ 1 mM), and then final concentration 1 mM 5
(6) -Carboxyfluorescein diacetate was added and left at 0 ° C. for 30 minutes. Then, pH 3 citric acid / phosphate buffer (50 mM, NaCl 110 mM, KCl
After washing with 5 mM and MgCl ₂ 1 mM), the cells were suspended in the same buffer. After 90 minutes at 0 ° C., fluorescence (518 nm) for two excitation wavelengths (441 nm and 488 nm) was measured with a fluorescence photometer (Spectrofluorimeter “RF-5000” manufactured by Shimadzu Corporation).
The strength was measured. The ratio of the fluorescence intensities was calculated, and the intracellular pH (pH as a cell population) was determined from the calibration curve prepared in advance.

The results were as shown in FIG. As is clear from the results in FIG. 1, there is a correlation between the intracellular pH value and the viable cell rate (obtained by the plate culture method). The calibration curve used here was created by plotting the data in the table below.

_{PH 5.99 5.78 5.58 5.37 5.27 5.16 4.95} I ₄₈₈ ln 1.641 1.430 1.194 1.051 0.867 0.770 0.519 ^I 441 In addition, the same yeast as used above was suspended in MES buffer in the same manner, and a final concentration of 1 mM 5 (6) -carboxyfluorescein diacetate was added. Incubated for minutes. The suspension was observed under a fluorescence microscope (excited with B), and the stained cells were regarded as living cells and the unstained cells were regarded as dead cells, and the viable cell ratio was measured. The result was as shown in FIG.

Example 2 Baker's yeast was mixed with 2% peptone,
Yeast having different viable cell ratios are cultured in a medium containing 1% yeast extract and 2% glucose to a stationary phase (25 ° C), washed with water, and aseptically stored at 2 ° C, 8 ° C, 20 ° C and 25 ° C. Got The same experiment as in Example 1 was performed. The result is shown in Figure 3.
It was as shown in.

<Example 3> Beer yeast was cultivated in wort to a stationary phase (8 ° C), washed with water, and the cells were aseptically stored (4).
C.) and various storage days were changed to obtain various yeasts having different fermentation abilities. The relationship between the intracellular pH of these yeasts and the wort fermenting power (8 ° C) was determined. The result was as shown in FIG.

In this experiment, the fermenting power of the yeast decreases as it ages, but it was examined whether or not it was accurately grasped by the intracellular pH. Figure 4
From the results, it can be seen that the fermentative power of yeast is accurately grasped by the intracellular pH value.

<Example 4> Beer yeast was cultivated in wort until a stationary phase (8 ° C). The cells were washed with water and stored aseptically in water at 2 ° C. By changing the number of storage days, cell populations having different yeast activities (specifically, growth ability and fermentation ability) were obtained. The intracellular pH of each yeast was measured in Example 1 except that the yeast cells were suspended in a citric acid / phosphate buffer of pH 3 except that they were suspended in the following buffers.
It carried out similarly to.

Buffer pH6.66, 50mM Mops, 110mM NaCl, 5mMKCl, 1mM MgCl ₂ pH6.20, 50mM MES, 110mM NaCl, 5mMKCl, 1mMMgCl ₂ pH5.60, 50Mm citrate-disodium phosphate, 110mM NaC
l, 5mMKCl, 1mM MgCl ₂ pH5.30, 50Mm citric acid-sodium phosphate, 110mM NaC
l, 5mM KCl, 1mM MgCl ₂ pH4.60, 50Mm citric acid-sodium phosphate, 110mM NaC
l, 5mM KCl, 1mM MgCl ₂ pH4.00, 50Mm citric acid-sodium phosphate, 110mM NaC
l, 5mM KCl, 1mM MgCl ₂ pH3.40, 50Mm citric acid-sodium phosphate, 110mM NaC
l, 5mMKCl, 1mM MgCl ₂ pH3.00, 50Mm citric acid-sodium phosphate, 110mM NaC
l, 5 mM KCl, 1 mM MgCl _{2 The} results were as shown in FIG.

Example 5 Brewer's yeast was cultivated in wort until the stationary phase (8 ° C.). After washing with water, cells aseptically stored in water at 2 ° C. were used for the experiment. Citrate cells at pH 3 /
Phosphate buffer (50 mM, NaCl 110 mM, KCl
After washing with 5 mM, MgCl ₂ 1 mM), the cells were suspended in the same buffer, 5 (6) -carboxyfluorescein diacetate at a final concentration of 5 μM was added, and the mixture was left at room temperature for 30 minutes, then 2
C./90 minutes left under slight stirring. 3% glucose,
0.67% East Nitrogen Base (Difco), 1
The cells were mixed with a% carrageenan (Sigma Type II) medium, and slide culture was performed using a hemacytometer. Immediately, intracellular pH and cell position of individual cells were measured using a fluorescence microscope image processor. Then, it incubated at 20 degreeC. After 16 hours, the number of cells after the proliferation of the cells whose intracellular pH was measured was counted. The result was as shown in FIG.

[0043]

EFFECTS OF THE INVENTION By measuring the intracellular pH of a yeast cell when it is placed in a low pH environment for a predetermined time, the activity of the yeast cell, that is, the proliferative power, the fermentative power, the viable cell rate, etc. can be accurately measured in a short time. The fact that the above can be grasped is as described above in the section of "Means for solving problems".

[Brief description of drawings]

FIG. 1 is a graph showing the relationship between the intracellular pH value of brewery yeast and the viable cell ratio.

FIG. 2 is a graph showing the relationship between the viable cell rate of brewer's yeast by the fluorescence method and the viable cell rate by the plate culture method.

FIG. 3 is a graph showing the relationship between the intracellular pH value of baker's yeast and the viable cell ratio.

FIG. 4 is a graph showing the relationship between the intracellular pH value of brewery yeast and the fermentative power.

FIG. 5 is a graph showing the correspondence between intracellular pH values when brewer's yeast cells are placed under various extracellular pH conditions.

FIG. 6 is a graph showing the number of cells after a predetermined culture time for each intracellular pH, when brewer's yeast was cultured in a low pH environment for a predetermined time and then cultured.

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[Procedure amendment]

[Submission date] July 29, 1992

[Procedure Amendment 1]

[Document name to be amended] Statement

[Name of item to be corrected] Claim 2

[Correction method] Change

[Correction content]

[Procedure Amendment 2]

[Document name to be amended] Statement

[Name of item to be corrected] Claim 9

[Correction method] Change

[Correction content]

[Procedure 3]

[Document name to be amended] Statement

[Correction target item name] 0007

[Correction method] Change

[Correction content]

[0007] Regarding the other embodiment of this step (2), the method for measuring yeast activity according to the present invention is characterized by comprising the following steps. (1) Yeast is placed in a low pH environment of pH 6 or less, and after a predetermined time has elapsed, the intracellular pH of the yeast is measured. (2) A yeast having a smaller difference between the obtained intracellular pH value and the intracellular pH value specific to the yeast is determined to have higher activity. Here, the "intracellular pH value peculiar to the yeast" is used in the step (1) when the yeast has a peculiar activity, in other words, an activity not deteriorated, that is, a high activity. The intracellular pH value shown in a low pH environment.

[Procedure amendment 4]

[Document name to be amended] Statement

[Correction target item name] 0009

[Correction method] Change

[Correction content]

[0009] Regarding the other embodiment of this step (4), the method for measuring yeast activity according to the present invention is characterized by comprising the following steps. (1) A step of incorporating a pH-sensitive fluorescent substance into yeast cells. (2) The yeast into which the fluorescent substance has been incorporated is placed in a low pH environment of pH 6 or less, and after a predetermined time has elapsed, the yeast cells are irradiated with excitation light to generate fluorescence, and the two types of fluorescence intensity are measured. (3) The intracellular pH value corresponding to the ratio of the measured fluorescence intensities is obtained from the calibration curve prepared in advance. (4) A yeast having a smaller difference between the obtained intracellular pH value and the intracellular pH value specific to the yeast is determined to have higher activity. Here, the "intracellular pH value peculiar to the yeast" is used in the step (1) when the yeast has a peculiar activity, in other words, an activity not deteriorated, that is, a high activity. The intracellular pH value shown in a low pH environment.

[Procedure Amendment 5]

[Document name to be amended] Statement

[Correction target item name] 0011

[Correction method] Change

[Correction content]

The fact that the intracellular pH measured after placing yeast in a low pH environment correlates with the activity of yeast cells is considered to be a phenomenon first discovered by the present inventors. It is presumed that the phenomenon is due to the H pumping action of yeast cells (however, the present invention is not restricted by such estimation).
That is, highly active yeast cells have a strong H pumping action and have a large ability to maintain the intracellular pH at the original pH even when the environment has a low pH. On the other hand, since cells having low activity do not maintain the original intracellular pH value, the intracellular pH after a predetermined time elapses is lowered to a certain level lower than the original pH value.

[Procedure Amendment 6]

[Document name to be amended] Statement

[Name of item to be corrected] 0032

[Correction method] Change

[Correction content]

<Determination of Yeast Activity> The fact that the intracellular pH of the yeast thus measured has a correlation with the activity of the yeast cell will be clarified by the experimental facts described below.
Yeast with a higher intracellular pH value has more active activity. Therefore, if information on the intracellular pH value and yeast activity is prepared in advance for the target yeast, the activity of the yeast can be grasped from the intracellular pH value. More specifically, the measured intracellular pH value is the intracellular p-value peculiar to the yeast.
It is possible to determine that the activity is higher as the difference is smaller than that of the H value. Here, the intracellular pH value unique to the yeast used when determining the yeast activity means that the yeast having an inherent activity, in other words, the activity is not deteriorated, that is, the activity is high, is measured. It is an intracellular pH value obtained when the plate is placed in a low pH environment used for a predetermined time.
This value varies depending on the type of yeast and the low pH value used, but is usually 5.5 to 6.5. When such a determination method is used, for example, when yeast is used for fermentation or the like for industrial purposes, the yeast activity measurement according to the present invention is performed, and the difference between the cell pH value specific to the yeast and the measured value is obtained. When the value exceeds a certain value, the yeast is not used, so that only the yeast having high activity can always be used. When the yeast is used for fermentation or the like, the activity of the target yeast is grasped by the measured intracellular pH value itself instead of the difference with the intracellular pH value specific to the yeast, that is, the measured cell It goes without saying that it can be carried out depending on whether or not the internal pH value is higher than a predetermined level.

[Procedure Amendment 7]

[Document name to be amended] Statement

[Correction target item name] 0038

[Correction method] Change

[Correction content]

Example 3 Beer yeast was cultivated in wort to a stationary phase (8 ° C.), washed with water, and the cells were stored aseptically (4 ° C.), and the fermentation ability was changed by changing the number of storage days. Various yeasts were obtained. The relationship between the intracellular pH of these yeasts and the wort fermenting power (8 ° C) was determined. The yeast suspension is suction-filtered to squeeze the yeast, added to hop-added wort prepared at 11 ° P (0.35% (wet weight / v)), thoroughly aerated, and then allowed to stand at 8 ° C. Let it ferment. A part of the fermented liquor was collected over time, filtered through a filter (Toyo Filter Paper No. 7), and then the extract content (sugar content) of the filtrate was measured by a vibrating densitometer. The amount of extract consumed for 3 days from the start of fermentation was determined as the fermentation ability of yeast. The result was as shown in FIG.

[Procedure Amendment 8]

[Document name to be amended] Statement

[Correction target item name] 0041

[Correction method] Change

[Correction content]

Buffer pH 6.66, 50 mM Mops, 110 mM NaC
1, 5 mM KCl, 1 mM MgCl_Two  pH 6.20, 50 mM MES, 110 mM NaC
1, 5 mM KCl, 1 mM MgCl_Two  pH 5.60, 50 mM citric acid-phosphate 2 sodium
System, 110 mM NaCl, 5 mM KCl, 1 mM MgC
l_Two  pH 5.30, 50 mM citric acid-phosphate 2 sodium
System, 110 mM NaCl, 5 mM KCl, 1 mM MgC
l_Two  pH 4.60, 50 mM citric acid-phosphate 2 sodium
System, 110 mM NaCl, 5 mM KCl, 1 mM MgC
l pH 4.00, 50 mM citric acid-phosphoric acid 2 sodium salt
System, 110 mM Nacl, 5 mM KCl, 1 mM MgC
l_Two  pH 3.40, 50 mM citric acid-phosphate 2 sodium
System, 110 mM NaCl, 5 mM KCl, 1 mM MgC
l_Two  pH 3.00, 50 mM citric acid-phosphate 2 sodium
System, 110 mM NaCl, 5 mM KCl, 1 mM MgC
l_Two  The result was as shown in FIG.

[Procedure Amendment 9]

[Document name to be amended] Statement

[Correction target item name] 0042

[Correction method] Change

[Correction content]

Example 5 Brewer's yeast was cultivated in wort until the stationary phase (8 ° C.). After washing with water, cells aseptically stored in water at 2 ° C. were used for the experiment. Citrate cells at pH 3 /
Phosphate buffer (50 mM, NaCl 110 mM, K
After washing with Cl 5 mM and MgCl ₂ 1 mM), the cells were suspended in the same buffer, 5 (6) -carboxyfluorescein diacetate with a final concentration of 5 μM was added, and the mixture was allowed to stand at room temperature for 30 minutes, followed by slight stirring at 2 ° C./90 minutes. I left it at. 3% glucose, 0.67% yeast nitrogen base (Di
fco), 1% carrageenan (Sigma Type I)
I) The medium and this cell were mixed, and slide culture was carried out using a hemacytometer. Immediately, intracellular pH and cell position of individual cells were measured using a fluorescence microscope image processor. Then, it incubated at 20 degreeC. After 16 hours, the number of cells after the proliferation of the cells whose intracellular pH was measured was counted. The result was as shown in FIG. <Example 6> The method of <Example 1> was further simplified, and a method of measuring yeast activity more rapidly was examined. Brewery yeast was cultivated in wort until stationary phase (8 ° C). After washing with water, 2
Cells that were aseptically stored in water at ℃ were used for the experiment. Cells were adjusted to pH 3, citrate / phosphate buffer (50 mM, N
aCl 110 mM, KCl 5 mM, MgCl ₂ 1
After washing with the same buffer, 5 (6) -carboxyfluorescein diacetate having a final concentration of 1 mM was added, and the mixture was left at 0 ° C. for 15, 30 or 60 minutes. Then, with a fluorometer, 2 excitation wavelengths (441,488 nm)
The fluorescence (518 nm) intensity was measured. The ratio of the fluorescence intensities was calculated, and the pH was obtained from the calibration curve prepared in advance. Further, the same yeast was analyzed by the same method as in <Example 1>. The analysis was performed 5 times for each analysis method using the same yeast (AC) in order to see the variation in the analysis value. The results are shown in Tables 1, 2 and 3. It can be seen that also by this simplified method, the same results as in the case of the method of <Example 1> can be obtained with good reproducibility.

Claims (12)

[Claims] 1. A method for measuring yeast activity, which comprises the following steps. (1) Yeast is placed in a low pH environment of pH 6 or less, and after a lapse of a predetermined time, the intracellular pH of the yeast is measured. (2) The obtained yeast having a high intracellular pH value is judged to have higher activity than yeast having a low intracellular pH value. 2. A method for measuring yeast activity, which comprises the following steps. (1) Yeast is placed in a low pH environment of pH 6 or less, and after a lapse of a predetermined time, the intracellular pH of the yeast is measured. (2) Obtained intracellular pH value and intracellular p value unique to the yeast
Yeast with a small difference from the H value is judged to have greater activity than yeast with a large difference. 3. The low pH environment is an environment having a pH of 5 or less,
The method for measuring yeast activity according to claim 1 or 2. 4. The method for measuring yeast activity according to any one of claims 1 to 3, wherein the yeast activity is yeast growth ability or fermentation ability. 5. The method for measuring yeast activity according to claim 1, wherein the yeast activity is the viability of yeast. 6. The method for measuring yeast activity according to claim 1, wherein the measured yeast intracellular pH value is an individual yeast intracellular pH measurement value. 7. The method for measuring yeast activity according to claim 1, wherein the measured yeast intracellular pH value is an average measured value of the yeast population. 8. A method for measuring yeast activity, which comprises the following steps. (1) A step of incorporating a pH-sensitive fluorescent substance into yeast cells. (2) Yeast containing a fluorescent substance has a low pH of 6 or less.
It is placed in an H environment, and after a lapse of a predetermined time, the yeast cells are irradiated with excitation light to generate fluorescence, and two types of fluorescence intensities are measured. (3) The intracellular pH value corresponding to the ratio of the measured fluorescence intensities is obtained from the calibration curve prepared in advance. (4) The obtained yeast having a high intracellular pH value is judged to have higher activity than yeast having a low intracellular pH value. 9. A method for measuring yeast activity, which comprises the following steps. (1) A step of incorporating a pH-sensitive fluorescent substance into yeast cells. (2) Yeast containing a fluorescent substance has a low pH of 6 or less.
It is placed in an H environment, and after a lapse of a predetermined time, the yeast cells are irradiated with excitation light to generate fluorescence, and two types of fluorescence intensities are measured. (3) The intracellular pH value corresponding to the ratio of the measured fluorescence intensities is obtained from the calibration curve prepared in advance. (4) Obtained intracellular pH value and intracellular p value unique to the yeast
Yeast with a small difference from the H value is judged to have greater activity than yeast with a large difference. 10. The method according to claim 8, wherein the two types of fluorescence intensity are fluorescence intensities at a predetermined wavelength among the fluorescence generated by the two types of excitation light having different wavelengths. 11. The method according to claim 8, wherein the two types of fluorescence intensities are the fluorescence intensities at two predetermined wavelengths among the fluorescence generated by one type of excitation light. 12. The pH-sensitive fluorescent substance has a phenolic hydroxyl group and / or a carboxyl group, and this functional group is esterified with a lower carboxylic acid and / or a lower alcohol to be incorporated into a yeast cell, The method according to any one of claims 8 to 11, wherein the pH-sensitive fluorescent substance is generated in yeast cells.