JP2890128B2 - Yeast viable cell count method - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION [Object of the invention]

[Industrial Application Field] The present invention provides a method for quickly and simply measuring the viable cell count of yeast in a sample containing yeast, for example, a food containing bacteria and yeast or in a fermentation medium in a food production process using yeast. About. [Prior Art] (1) Background Problems in quality control of foods are deterioration of quality due to microbial contamination and food poisoning. Conventional testing has focused primarily on pathogenic bacteria from the perspective of food poisoning prevention,
In the future, it is necessary to strengthen the inspection system for non-pathogenic yeast. In particular, raw materials and foods containing a large amount of carbohydrates undergo fermentation by yeast during storage or transport, and the container expands due to carbon dioxide gas generated by the fermentation, or the flavor deteriorates due to alcohol or the like. (2) Problems of the prior art However, in general microbiological tests in food companies, it takes 24 hours or more for coliform bacteria and 5 days for molds and yeasts. Even a recently developed method for measuring the yeast activity of microorganisms requires 6 to 7 hours, and the operation is not simple. In addition, the method of measuring the number of viable cells using a microscope that does not require cultivation requires a six-step operation of filtration and concentration of bacteria in the solution → tannic acid treatment → washing with water → staining → washing with water → microscopic observation. Not an operation. Similar problems also occur in the process control in the food manufacturing process using yeasts such as alcoholic beverages, but as described above, microbial testing requires complicated aseptic operations, culture, etc. It has the character that it is difficult to be efficient. [Problems to be Solved by the Invention] In view of the circumstances described above, the present invention has developed a novel method for measuring the number of viable yeast cells, whereby (1) shortening the working time, 2) Multi-sample processing, (3)
It is an object of the present invention to provide a simple and quick method having an advantage of improving inspection accuracy and (4) requiring no skill.

Configuration of the Invention

[Means for Solving the Problems] (1) Overview The method for determining the viable cell count of yeast according to the present invention comprises adding quinone to coexisting bacteria and yeast, and determining the amount of viable yeast cells based on the amount of hydrogen peroxide generated. It is characterized in that the number is measured. Hereinafter, the principles of the method of the present invention and other matters related to the configuration of the present invention will be described separately. (2) Principle Bacteria, yeasts and molds are all intracellular NADs
(P) Life is maintained using H (reduced nicotinamide amide adenine dinucleotide (phosphate)) and ATP (adenosine triphosphate) as energy sources, but when oxidized quinone is added to these microorganisms, only yeast Produces hydrogen peroxide. The mechanism of hydrogen peroxide production is that oxidized quinone added from the outside is reduced by NAD (P) H: quinone redox yeast in cells and then reacts with dissolved oxygen to produce hydrogen oxide. Conceivable. On the contrary, it is thought that bacteria either lack a membrane enzyme that reduces external oxidized quinone or have a system that reoxidizes quinone. For example, compared to bacteria such as Escherichia coli, Bacillus, and Proteus, yeast has nearly 100 times the ability to generate hydrogen peroxide. In addition, in the presence of 0.5 mM menadione, bacteria stop producing hydrogen peroxide in about one minute, while yeast produces sustained hydrogen peroxide production. Therefore, by utilizing such a biochemical difference between yeast and bacteria, it is possible to specifically measure the viable cell count of only yeast from a mixture of bacteria and yeast. (3) Quinone In the present invention, quinone acts as a carrier for hydrogen atoms at the cell membrane interface, and changes extracellular dissolved oxygen into hydrogen peroxide. The quinone used herein includes quinones such as benzoquinone, naphthoquinone, diphenoquinone and anthraquinone and derivatives thereof,
Those having no oxidizable portion other than the quinone group are preferred. Representative quinones mentioned above can be used in all invention purposes, the be particularly preferred in that they are physiologically substance is a 2-methyl-1,4-Nafunokinon known as vitamin K ₃ (menadione) . (4) Quantification of Hydrogen Peroxide In the present invention, the production of reduced quinone can be measured by various known analytical techniques, but what seems to be pointed out at present is a method of quantifying hydrogen peroxide. Hydrogen peroxide generated in the reaction system reacts with an excitation source material such as an aryl ester of oxalic acid to change into an excitation material such as 1,2-dioxetanedione, and the latter is a π electron such as perylene. By increasing the energy level of the fluorescent material rich in light, it emits light. The reaction when diphenyl oxalate is used as the excitation source material and perylene is used as the fluorescent substance is schematically shown by the following formula. The quinone in the reaction system changes cyclically from the oxidized form to the reduced form and from the reduced form to the oxidized form, and acts as a catalyst for producing hydrogen peroxide. Quantitative accuracy of hydrogen peroxide by more luminescent reaction, 10 ⁷
1010 ⁻³ mol / l, and the required time is about 10 seconds. On the other hand, the time required for the production of hydrogen peroxide by cells depends on the physiological state of individual cells, but in yeast (10 μg / ml)
Detectable hydrogen peroxide is produced in 30 seconds. Therefore, about 40
It is possible to measure the number of viable yeast cells within seconds. [Action] According to the present invention, the viable cell count of yeast can be simply and rapidly measured in a food containing yeast alone or yeast and bacteria. That is, as long as the correlation between the number of viable yeast cells and the ability to produce hydrogen peroxide is initially determined as a calibration curve, the number of viable yeast cells in a large number of samples can be measured based on this calibration curve. is there. In particular, if the luminescence reaction is used for the determination of hydrogen peroxide,
Automatic measurement can be easily performed. [Examples] Hereinafter, embodiments of the present invention will be described with reference to examples. However, the examples are for explanation, and are not intended to limit the inventive concept. Example 1 (Quantitative determination of hydrogen peroxide) In 100 ml of acetonitrile, 60 mg of TCPO (bis (1,4,6-
Trichlorophenyl) oxalate) and 10 mg of pyrene were dissolved, and this was used as a luminescence reaction solution. 1 ml of the test material was mixed with 1 ml of this TCPO solution, and the luminescence count for 5 seconds was measured. Example 2 (Measurement of viable yeast cell count) Malt extract 1%, yeast extract 0.5% and glucose 2
Baker's yeast (IFO 2044) was added to a culture solution (pH 5.8) containing the same, and cultured at 28 ° C. for 12 to 18 hours. The obtained culture solution was diluted with 50 mM imidazole / nitric acid (pH 7.
After dilution at 0), menadione was added to a concentration of 0.25 mM, and the mixture was incubated at 30 ° C. for 3 minutes.
1 ml of the TCPO solution was added to 1 liter, and the luminescence count for 5 seconds was measured immediately. As shown in FIG. 1, the cell number of 10 ³ cells / ml to 10 ⁷ cells / ml could be measured quickly. As a precaution, even if E. coli or spore bacilli were added in a 10-fold amount, no effect was observed. If it is desired to further increase the detection sensitivity, the yeast suspension is filtered and concentrated using a membrane filter, and the membrane is immersed in 50 mM imidazole / nitric acid (pH 7.0, followed by adding menadione). In this way, for example, when the yeast is concentrated 100 times, it is possible to measure 10 cells / ml Example 3 (Method for increasing the ability of yeast to produce hydrogen peroxide) Leaving the yeast in 50mM imidazole nitrate (pH 7.0) at 25 ° C for 3 days reduces the capacity of yeast to produce hydrogen peroxide, but when glucose is added to this system, the ability to produce hydrogen peroxide is quickly restored. It is considered that the glycolysis of yeast was activated by the addition of glucose, and sufficient NAD (P) H was generated, and thus, by utilizing this phenomenon, glucose was added to produce hydrogen peroxide. After enhancing the performance, hydrogen peroxide By quantifying the ability of quantity, it is possible to increase the detection sensitivity. In other words, suspended 1.4 × 10 ⁷ cells / ml of the yeast with 18-hr cultured yeast culture solvent (50 mM imidazole nitrate pH 7.0) A suspension was prepared, left at 25 ° C. for 3 days, added with the same volume of TCPO solution, and immediately measured for luminescence count, as shown in FIG. It can be seen that is increased.

【The invention's effect】

As described above, according to the present invention, the number of viable yeast cells in food and other specimens can be measured easily and quickly, so that labor saving in quality control of food and rationalization in the fermentation industry using yeast can be achieved. Can contribute to

[Brief description of the drawings]

FIG. 1 is a graph showing the relationship between the number of yeasts and the amount of hydrogen peroxide generated, and FIG. 2 is a graph showing the relationship between the passage of time after the addition of glucose to the left yeast solution and the amount of glucose hydrogen peroxide generated. It is.

Claims (1)

(57) [Claims] A method for determining the number of living yeast cells by adding quinone to a sample containing yeast and measuring the number of viable yeast cells from the amount of hydrogen peroxide generated. A method for measuring the viable cell count of yeast, wherein glucose is added to increase the production capacity of hydrogen peroxide, thereby increasing the detection sensitivity.