JP3214156B2 - Optical isomer measurement method - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a D
The present invention relates to a method for measuring optical isomers such as lactic acid and L-lactic acid.

[0002]

2. Description of the Related Art Lactic acid fermentation has been widely used in the food industry. For example, lactic acid fermentation by microorganisms not only produces lactic acid and milk drinks, but also cheese, butter, sake,
It is used for the production of soy sauce, miso, pickles, etc. Microorganisms used in these lactic acid fermentations include lactic acid bacteria and filamentous fungi. Among them, lactic acid bacteria that produce only D-lactic acid, those that produce only L-lactic acid,
Those producing a mixture of D-lactic acid and L-lactic acid are known, and measurement of D-lactic acid and L-lactic acid in a fermentation process is indispensable for fermentation control, discovery of contamination, and the like.

Conventionally, D-lactic acid has been measured using D-lactate dehydrogenase (hereinafter abbreviated as D-LDH) and a coenzyme nicotinamide adenine dinucleotide (hereinafter abbreviated as NAD) to form reduced nicotinamide. Adenine dinucleotide (hereinafter abbreviated as NADH) or pyruvic acid was measured by a spectrophotometric method or a fluorescent intensity method. However, this measurement method had various problems due to the nature of D-LDH. First, since the reaction efficiency is low, the amount of enzyme required at a practical level is too large, and the cost for measurement is high. In order to reduce the amount of the enzyme used, it is conceivable to increase the reaction time or adjust the reaction temperature, but this has been a negative factor such as more complicated operation and extension of the measurement time. Second, since the equilibrium of the reaction involving D-LDH is inclined toward the generation of D-lactic acid from NADH and pyruvic acid, the reaction stops before all the D-lactic acid is completely changed. . Therefore, it is necessary to take measures such as consuming the produced pyruvic acid with glutamate pyruvate transaminase, which complicates the reaction step and increases the analysis cost.

In order to measure not only D-lactic acid but also L-lactic acid, the same measurement as that for D-lactic acid is performed using L-lactate dehydrogenase (hereinafter abbreviated as L-LDH). It is necessary to analyze D-lactic acid once more. Therefore, the operation becomes complicated, and the measurement time and cost are doubled. Furthermore, in the measurement method using only lactate dehydrogenase, the general detection means of the reaction is NAD or N
This is a change in absorbance in the ultraviolet region of ADH, and has a drawback that the sample is susceptible to turbidity and coloring substances.

[0005]

SUMMARY OF THE INVENTION The present invention solves the above-mentioned problems and provides a method for accurately measuring two components of D-lactic acid and L-lactic acid in a short time using D-LDH and L-LDH. The purpose is to provide. Further, the present invention relates to D-
Glutamic acid and L-glutamic acid, D-leucine and L-
Provided is a method for measuring other optical isomers such as leucine, D-galactose and L-galactose.

[0006]

SUMMARY OF THE INVENTION The present invention provides an L-form and / or a D-form in a sample containing an L-form and / or a D-form of an optical isomer.
A method for measuring an L-isomer of an optical isomer in a sample, and in the presence of an L-dehydrogenase and a D-dehydrogenase of the optical isomer and an oxidized coenzyme. A method for measuring the L-form and the D-form of an optical isomer in a sample, comprising a step of performing a conversion reaction between the L-form and the D-form in the sample, and then measuring the L-form after the conversion reaction. . Also optical
L-form in sample containing L-form and / or D-form of isomer
And a method for measuring the D-form, wherein the optical isomer in the sample is
Measuring the D-isomer; and L-deprotection of the optical isomer
Hydrogenase, D-dehydrogenase, and oxidized coenzyme
In the presence, a conversion reaction between the L-form and the D-form in the sample is carried out.
In the sample including the step of measuring the D-form after the conversion reaction in
This is a method for measuring the L-form and the D-form of the optical isomer.

The step of measuring the L-form of the optical isomer in the sample comprises oxidizing the L-form with an L-form oxidase,
The above-mentioned measurement method comprising detecting an electrode active substance which increases or decreases by the reaction of the oxidase is disclosed. A step of measuring the D-form of the optical isomer in the sample;
Oxidizes the D-form with a D-form oxidase,
Electrode active material that increases or decreases due to elemental reactions.
The above-described measuring method comprising detecting is disclosed.

[0008] The L-form and the D-form of the optical isomer are each L-form.
Lactic acid and D-lactic acid, L-glutamic acid and D-glutamic acid, L-leucine and D-leucine, or L-galactose and D-galactose, and L-dehydrogenase and D-dehydrogenase are L-lactic acid dehydrated, respectively. Enzyme and D-lactate dehydrogenase, L-glutamate dehydrogenase and D-glutamate dehydrogenase, L-leucine dehydrogenase and D-leucine dehydrogenase, or L-galactose dehydrogenase and D-galactose dehydration The above-mentioned measuring method which is an enzyme is disclosed.

The present invention relates to a step of measuring L-lactic acid in a sample, the equilibrium between D-lactic acid and L-lactic acid in the sample in the presence of L-lactic acid dehydrogenase, D-lactic acid dehydrogenase, and NAD. Performing a reaction and measuring L-lactic acid after the equilibrium reaction. A method for measuring D-lactic acid and L-lactic acid in a sample.

Further, the present invention provides the above-mentioned measuring method, wherein the step of measuring L-lactic acid comprises detecting the amount of an electrode active substance which increases or decreases L-lactic acid in a sample by oxidizing L-lactic acid in the sample. Disclose. Further, in the present invention, the step of measuring L-lactic acid comprises the steps of: bringing a sample into contact with an L-lactic acid oxidase-immobilized body, and electrochemically detecting a decrease in oxygen or an increase in hydrogen peroxide. Is disclosed.

Further, the present invention relates to L-lactate dehydrogenase,
-D-lactic acid and L in the presence of lactate dehydrogenase and NAD
-L-lactate dehydrogenase for performing an equilibrium reaction of lactic acid,
A measurement kit containing D-lactate dehydrogenase and NAD is disclosed.

Incidentally, the L-form (or D-form) is an expression in which only one of the L-form and the D-form is read in the same sentence. It is described in two sentences.

[0013]

The reaction between D-LDH and L-LDH is shown in the following equation. D-LDH reaction: D-lactic acid + NAD ⁺ = pyruvic acid +
NADH + H ⁺ L-LDH reaction: L-lactic acid + NAD ⁺ = pyruvic acid +
Both the NADH + H ⁺ reactions are reversible, but the equilibrium of both enzymes is tilted towards the production of lactic acid and NAD. for that reason,
Even when D-LDH is brought into contact with a sample in a large amount in the presence of NAD, it hardly changes to pyruvate. The same applies to L-LDH.

However, the present inventors have studied a more advantageous reaction which can measure D-lactic acid using D-LDH. As a result, when D-LDH and L-LDH act on D-lactic acid, It was found that L-lactic acid was produced from the produced pyruvic acid and NADH. In addition, D-LD is added to L-lactic acid.
When H and L-LDH act, L-lactic acid decreases.

The activity of LDH is generally defined by measuring the rate at which lactic acid is formed from pyruvate. D-
The ratio of L-LDH to LDH can be used from about 1/10 to about 20 times in terms of activity.
Is preferably in the range of 1/2 to 10 times, more preferably 1/2.
５5 times, more preferably 1 to 5 times. Even if the ratio of L-LDH to D-LDH is increased from about 1-fold to about 5-fold, the rate of generation of L-lactic acid from D-lactic acid does not change much. In this reaction, the equilibrium reaction always proceeds until the ratio of D-lactic acid to L-lactic acid becomes constant, regardless of whether D-lactic acid or L-lactic acid is used or even if they are mixed. That is, D
-Lactic acid: L-lactic acid = 1: 1.

Although the reactions of D-LDH and L-LDH have been known, it is quite unexpected that the combination of the two reactions can efficiently convert, for example, D-lactic acid to L-lactic acid. . Based on this new fact, it is possible to convert L-lactic acid from D-lactic acid.
Also, if this step is combined with a selective method for measuring L-lactic acid, it becomes possible to quantify D-lactic acid and L-lactic acid. still,
As described above, the measurement was mainly performed in a state where the equilibrium reaction progressed to a substantially constant state. However, even during the conversion reaction between D-lactic acid and L-lactic acid, the reaction conditions were accurately adjusted to obtain a calibration curve. Is prepared, D-lactic acid and L-lactic acid of the present invention can be measured even if L-lactic acid is measured during the reaction.

Next, the method for quantifying D-lactic acid and L-lactic acid in combination with the method for selective measurement of L-lactic acid will be further described. The produced L-lactic acid is L-lactic acid or LAD such as NAD.
-It must be measured in a way that does not affect the presence of substances other than lactic acid. Then, a measuring method using L-lactic acid oxidase can be used.

The reaction of L-lactate oxidase is shown by the following formula. L-lactic acid + O ₂ → pyruvic acid + H ₂ O ₂ This reaction is not affected even if D-lactic acid and NAD coexist. L-lactic acid can be detected by detecting generated hydrogen peroxide or reduced oxygen, or by interposing an electron carrier such as dichloroindophenol, potassium ferricyanide, or benzoquinone, a so-called mediator.

According to this method, the L-lactic acid of the sample is measured in advance, and then the L-lactic acid is measured after the conversion of D-lactic acid to L-lactic acid. Can be measured. Even if the reaction is not completed until a certain ratio of D-lactic acid and L-lactic acid, D-lactic acid per unit time
Since the production rate of L-lactic acid from lactic acid and the reduction rate of L-lactic acid are constant, if the reaction time of the enzyme is kept constant, it is possible to quantify D-lactic acid and L-lactic acid by comparison before and after the reaction. is there.

For the determination of L-lactic acid using L-lactic acid oxidase, it is sufficient to detect decreased oxygen or increased hydrogen peroxide. This includes, for example, peroxidase and 2,
2'-azino-di (3-ethylbenzthiazoline) -6
-A method using sulfonic acid or 4-aminoantipyrine (Trinder method) can be used to measure the absorbance in the visible region. The absorbance measurement in the visible region is less susceptible to turbidity than the ultraviolet absorbance measurement.

The electrochemical measurement method in which the increase or decrease of oxygen or hydrogen peroxide is converted into a current value by an electrode and measured is compared with a measurement using a spectrophotometer, in which the sample contains turbidity and a coloring substance. It is not affected even if it is, and the operation is simple and preferable. D-LDH, L-LDH and L-lactate oxidase can be used in the form of a solution or immobilized. When a solution is used, the amount of the enzyme required for the reaction can be added to the sample, but the measurement cost increases because the enzyme is disposable. The use of the immobilized product has advantages that the enzyme can be used repeatedly and that the reaction conditions of the enzyme can be easily adjusted.

The method of immobilizing the enzyme includes an adsorption method, a chemical bonding method,
Any of the inclusive methods may be used. As a carrier used for immobilization, diatomaceous earth, silica gel, glass beads, alumina, ceramic, carbon, activated carbon, molecular sieve, silicon rubber, cellulose, agarose, amino acid-based polymer and the like can be used. The immobilized enzyme constitutes a reactor, for example, packed in a column or the like.

Particularly, by immobilizing L-lactate oxidase having a relatively high reaction rate, a highly sensitive and simple measuring apparatus can be constructed. It can be detected by the colorimetric method as well as the increase and decrease of oxygen and hydrogen peroxide. However, the electrochemical measurement method has no influence even if the sample contains turbidity or contains a coloring substance, so that the operation is simple and excellent. Usually, an electrode for measuring hydrogen peroxide is incorporated in a cell. As the material of the cell, a non-conductive material such as acrylic, fluororesin, vinyl chloride resin, and glass, a conductive material such as stainless steel, gold, and platinum, or a combination thereof can be used. When using a conductive material, care must be taken, such as performing electrical insulation treatment with the electrode system. Examples of the electrode include a two-electrode system including a working electrode and a counter electrode, and a three-electrode system including a working electrode, a reference electrode, and a counter electrode. The electrodes can be formed by, for example, embedding a conductive substance in the bottom surface of the measurement cell, depositing a metal on the inner wall surface, solution plating, electroless plating, printing, or the like. The counter electrode and the reference electrode are desirably provided near the working electrode in order to reduce the influence of the resistance between the solutions.

As the working electrode, a metal electrode such as gold, platinum, silver or the like, or a material usually used in electrochemical measurement such as glassy carbon or carbon paste can be used. For the counter electrode, the materials exemplified above for the working electrode or a conductive material such as stainless steel can be used, and the liquid contact part of a cell formed using a conductive material such as stainless steel can be used as the counter electrode. Examples of the reference electrode include a general electrode such as a silver / silver chloride reference electrode and a saturated calomel reference electrode. Based on Table 1 below, D-lactic acid, L-lactic acid, D- and L-lactate dehydrogenase, NAD, L-lactate oxidase are replaced with those described in the second and subsequent columns of Table 1, Can be measured in the same manner as in the case of lactic acid.

[Table 1]

[0025]

EXAMPLES The present invention will be described in more detail with reference to the following Examples, but it should be understood that the present invention is by no means restricted thereto.

Experiment A NAD, D-LDH and LL were added to various concentrations of L-lactic acid.
DH was contacted, and L-lactic acid concentration was measured after a certain period of time.
The measurement of L-lactic acid was carried out by a method of measuring the generated hydrogen peroxide with a platinum electrode using an immobilized L-lactic acid oxidase column.

(1) Method for producing L-lactic acid oxidase-immobilized column Refractory brick (30 to 60) which is a diatomaceous earth-based silicate carrier
(Mesh) 150 mg is thoroughly dried, immersed in an anhydrous toluene solution of 10% γ-aminopropyltriethoxysilane for 1 hour, thoroughly washed with toluene, and dried. After the aminosilane-treated carrier is immersed in 5% glutaraldehyde for 1 hour, the carrier is thoroughly washed with distilled water and finally replaced with a 100 mM sodium phosphate buffer having a pH of 7.0 to remove this buffer as much as possible. A solution obtained by dissolving L-lactate oxidase of Pediococcus at a concentration of 50 units / ml in 100 mM sodium phosphate buffer at pH 7.0 in this formylated refractory brick was 200 μl.
1 and allowed to stand at 0-4 ° C. for 1 day to immobilize. This enzyme-immobilized carrier was packed in a column having an inner diameter of 3.5 mm and a length of 30 mm to obtain an L-lactic acid oxidase-immobilized column.

(2) Method for producing hydrogen peroxide electrode A side surface of a platinum wire having a diameter of 2 mm is covered with a heat-shrinkable Teflon,
One end of the line is smoothed with a file and 1500 emery paper. The platinum wire was used as a working electrode, a 1 cm square platinum plate was used as a counter electrode, and a saturated calomel electrode was used as a reference electrode.
Perform an electrolytic treatment at +2.0 V for 10 minutes in sulfuric acid. After thoroughly washing the platinum wire with water, drying at 40 ° C. for 10 minutes,
After being immersed in an anhydrous toluene solution of 10% γ-aminopropyltriethoxysilane for 1 hour, the substrate is washed. 20 mg of bovine serum albumin (Fraction V, manufactured by Sigma) is dissolved in 1 ml of distilled water, and glutaraldehyde is added to the solution so that the concentration becomes 0.2%. 5 μl of this mixed solution was quickly placed on the previously prepared platinum wire, and was dried and cured at 40 ° C. for 15 minutes to form a permselective membrane that eliminated the adverse effects of measurement interfering substances such as ascorbic acid. This was used as the working electrode of the hydrogen peroxide electrode.

An Ag / AgCl reference electrode was used as a reference electrode, and a conductive pipe was used as a counter electrode. Working electrode,
The hydrogen peroxide electrode (6) including the reference electrode and the counter electrode was housed in a cell and incorporated in an L-lactic acid measuring device.

(3) L-lactic acid measuring device L-lactic acid is measured by the flow type L-lactic acid measuring device shown in FIG. The buffer solution is sent from the buffer solution tank (1) by the pump (2), and the sample 5 is sent from the autosampler (3).
Inject μl. From L-lactic acid in the sample, thermostat (4)
Hydrogen peroxide is generated by the L-lactic acid oxidase-immobilized column (5) therein, and a change in current value is captured by the hydrogen peroxide electrode (6) and detected by the detector (7). Further, a signal can be sent to a personal computer (10).

The composition of the buffer is 100 mM sodium phosphate, 50 mM potassium chloride, 1 mM sodium azide, and pH 7.0.

(4) Measuring method The concentrations in the reaction system were 5 mM NAD, 54.3 units / ml D-LDH, 54.3 units / ml L-LDH, pH 7.0, and the samples were 1 mM L-lactic acid and 5 mM L-lactic acid. , L-lactic acid 10 mM, and the total volume is 2 ml. The L-lactic acid concentration of this sample was determined in a thermostat at 30 ° C. after 5 minutes, 10 minutes, and 20 minutes after the reaction, and the ratio of L-lactic acid detected to the L-lactic acid before the reaction was determined. And

The calibration curve was prepared using distilled water as a blank, 1, 2,
It was prepared by measuring 5, 10 mM L-lactic acid. In addition, a reaction solution containing no substrate was measured, and it was confirmed that L-lactic acid was not detected. Further, the measurement was performed on a sample containing no enzyme.

(5) Results The detection rates after 5 minutes for each of 1 mM, 5 mM and 10 mM were 0.56, 0.56 and 0.54, respectively, and the detection rates after 10 minutes were 0.50 and 0.5 respectively. 51 and 0.49. In addition, the detection rates after 20 minutes were 0.51 and 0.5, respectively.
51 and 0.50. Even if the concentration was different, the reaction rate after a certain time was the same. It can be seen that the detection rate becomes approximately constant at 0.5 when the reaction is performed for about 10 minutes or more.

Experiment B: D-lactic acid, L-lactic acid and D, having the same total lactic acid concentration
NAD, D-LDH, and L-LDH were brought into contact with the L-mixed lactic acid, and the L-lactic acid concentration was measured after a certain period of time. L-lactic acid was measured by a method in which L-lactic acid oxidase was immobilized and the generated hydrogen peroxide was measured with a platinum electrode as in Experiment A described above. The L-lactic acid oxidase-immobilized column, hydrogen peroxide electrode using a platinum electrode as a working electrode, and an L-lactic acid measuring device used in the measurement are the same as those in Experiment A.

(1) Measurement method The concentrations in the reaction system were 5 mM NAD, 54.3 units / ml D-LDH, and 54.3 units / ml L-LDH. The pH was adjusted to pH 7.0. Lactic acid concentration is all 5
In mM, only D-lactic acid, only L-lactic acid, a mixture of 2.5 mM D-lactic acid and 2.5 mM L-lactic acid, and the total volume is 2 ml. The L-lactic acid concentration of this sample was determined in a thermostat at 30 ° C., 5 minutes after and 10 minutes after the reaction. The calibration curve was prepared by measuring 1, 2, 5, and 10 mM L-lactic acid using distilled water as a blank. Also, measure the reaction solution without the substrate,
It was confirmed that no detection value of L-lactic acid could be obtained.

Further, the measurement was performed on a sample containing no enzyme.

(2) Results Five minutes after the start of the reaction, the detection rate from the sample containing only D-lactic acid was 0.48, and the detection rate from the sample containing only L-lactic acid was 0.5.
6, for the mixed sample of D-lactic acid and L-lactic acid, the ratio of the amount of L-lactic acid produced to the total amount of D, L-lactic acid was 0.51. After 10 minutes, the detection rate from the sample containing only D-lactic acid was 0.51, the detection rate from the sample containing only L-lactic acid was 0.51, and the D, L-lactic acid mixed sample had D, L-lactic acid. The ratio of the amount of L-lactic acid produced to the total amount of L-lactic acid was 0.51. Thus, the detection rate after 10 minutes was constant at approximately 0.5, regardless of the composition. That is, it was found that the reaction rate after a certain time was constant in each of D and L.

Example 1 NAD, D-LDH and L-LDH were brought into contact with a standard solution of D-lactic acid and L-lactic acid and a mixed sample of D-lactic acid and L-lactic acid, and the L-lactic acid concentration was measured after a certain period of time. . The L-lactic acid concentration was also measured for the sample before the reaction without addition of the enzyme. As in Experiment A, L-lactate oxidase was immobilized, and the generated hydrogen peroxide was measured using a platinum electrode. The L-lactic acid oxidase-immobilized column, hydrogen peroxide electrode using a platinum electrode as a working electrode, and an L-lactic acid measuring device used in the measurement are the same as those in Experiment A.

(1) Measuring method The concentration in the reaction system was 5 mM NAD, 54.3 units / ml D-LDH, 54.3 units / ml L-LDH, pH 7.0, and no enzyme was added to one sample. Two points were measured, namely, the enzyme was added and reacted at 30 ° C. for 10 minutes or more (about 20 minutes). In the measurement, distilled water was used as a blank, and a calibration curve was prepared from the current values of the standard solutions of 1, 2, 5, and 10 mM D-lactic acid and L-lactic acid without the enzyme and after the enzyme reaction. The sample is
(A) 2.5 mM D-lactic acid, 5 mM L-lactic acid, (B) D
-Lactic acid 5 mM, L-lactic acid 2.5 mM, (c) D-lactic acid 5
mM and 5 mM L-lactic acid.

(2) Result

[0042]

[Table 2]

The current value after the enzyme reaction without the enzyme in the standard solution was as shown in [Table 2], and the following calibration curve was obtained. Here, Y is the current value (nA), and X is the lactic acid concentration (mM) in the sample before the reaction. Calibration curve 1, L-lactic acid calibration curve without enzyme Y = 55.6
6X-2.44 calibration curve 2, L-lactic acid calibration curve after enzyme reaction Y = 27.5
4X-0.52 calibration curve 3, after enzymatic reaction D-lactic acid calibration curve Y = 27.3
6X-0.39 From this calibration curve, the concentrations of D-lactic acid and L-lactic acid in the samples (a), (b), and (c) are determined with high accuracy from the obtained current values as shown in [Table 3]. I was able to. An example of the calculation method is shown below.

[0044] and the current value before the enzymatic reaction (no enzyme), a calibration curve 1 (no enzyme, L- lactic acid calibration curve) than, are obtained L- lactic quantities x ₁ which were present from the beginning in the sample. Next, the amount x ₂ of L-lactic acid after the enzyme reaction can be calculated from the current value after the enzyme reaction and the calibration curve 1 (without enzyme, L-lactic acid calibration curve). According to Experiments A and B, after enzymatic reaction, both D and L lactic acid have a value obtained by multiplying the amount before the reaction by about 0.5, so that x ₂ = 0.5 × ₁ +
0.5 · (D-lactic acid amount), and the D-lactic acid amount can be obtained.

[0045]

[Table 3]

Example 2 Various commercially available lactic acid drinks A (yogurt manufactured by Kansai Luna), B
D-lactic acid, L-lactic acid, and total lactic acid of (fermented milk manufactured by Yakult Co., Ltd.), C (sterilized lactic acid bacteria beverage manufactured by Kyodo Dairy), D (yogurt manufactured by Snow Brand Milk Industry), and E (fermented milk manufactured by Snow Brand Milk Industry) The concentration was determined. Dilute the sample 20- to 40-fold, and add NAD, D-L
DH and L-LDH were contacted, and L-lactic acid concentration was measured after a certain period of time. L-lactic acid measurement was performed in the same manner as in Experiment A described above.
-Lactate oxidase was immobilized, and the generated hydrogen peroxide was measured using a hydrogen peroxide electrode. L- used for measurement
The lactate oxidase-immobilized column, the hydrogen peroxide electrode using a platinum electrode as the working electrode, and the L-lactic acid measuring device were the same as those in Experiment A.

(1) Measurement method The L-lactic acid concentration was determined by using the sample diluent as it was for the measurement of L-lactic acid. L-lactic acid measurement after enzyme reaction: NAD 10 mM, D-LDH1
0 unit / ml, L-LDH 50 unit / ml, and a phosphoric acid concentration of 200 mM, and a pH 7.5 reaction solution.
Add 5 ml. The reaction was allowed to stand at room temperature for 2 hours or more, the reaction was completed, and the L-lactic acid concentration was determined. The calibration curve was prepared by measuring 1, 2, and 5 mM L-lactic acid using distilled water as a blank. The lactic acid concentration% in the original concentration was converted from the obtained measured value. (2) Results The concentration% of D-lactic acid, L-lactic acid, and total lactic acid of the five lactic acid beverages was as shown in Table 4 .

Comparative Example 1 The concentrations of D-lactic acid, L-lactic acid and total lactic acid of various commercially available lactic acid drinks A to E were determined using an F kit (Boehringer Yamanouchi). (1) Measuring method In this comparative example, since the absorbance in the ultraviolet region (340 nm) is measured, the turbidity of the sample has a great effect. Therefore, centrifugation (18,000 rpm 2) is performed for the purpose of removing the precipitate of the sample diluent.
0 minutes). Therefore, pretreatment was required. The lactic acid concentration was determined from the obtained absorbance and converted to the lactic acid concentration% of the original concentration. That is, NAD, glutamic acid, and glutamate pyruvate transamylase were added to and mixed with the sample using the F kit, and the absorbance at 340 nm was measured with a spectrophotometer to obtain a blank. Add D-LDH and mix.
After standing for 0 minutes, the absorbance was measured. The D-lactic acid concentration in the sample was calculated from the change in absorbance due to D-LDH. For the measurement of L-lactic acid, L-LDH was further added to the reaction solution, mixed, allowed to stand for 20 minutes, and the absorbance was measured. The L-lactic acid concentration in the sample was calculated from the change in absorbance due to L-LDH.

(2) Results Table 5 shows the concentration% of D-lactic acid, L-lactic acid and total lactic acid of the five kinds of lactic acid beverages. Based on this value, the correlation between the measured value (Y) of the present invention according to Example 2 and the measured value (X) of the F kit according to Comparative Example 1 was examined. D-lactic acid measurement Y = 1.02X + 0.00r
= 1.000 L-lactic acid measured value Y = 0.98X + 0.02 r
= 0.998 Total lactic acid measurement Y = 1.03X-0.01 r
= 0.999 D-lactic acid, L-lactic acid, and total lactic acid are in good agreement, and accurate measurement is possible by the method according to the present invention. Further, in the measuring method of the present invention, there was no influence of the sample turbidity, and the pretreatment could be omitted.

[0050]

[Table 4]

[Table 5]

[0051]

According to the method for measuring lactic acid of the present invention, D-lactic acid,
L-lactic acid can be easily and accurately quantified. In the present invention, the number of pitting operations is smaller than that of the F kit method, so that the method can be easily performed. Since there are few types of reagents and enzymes to be used, the operation can be performed easily and the cost is low.
The measurement concentration range is wide, and can be changed by changing the injection amount of the L-lactic acid measuring device. If a sample injection device is used for the measurement, labor can be saved. Etc. Further, according to the present invention, optical isomers such as amino acids can be measured.

[Brief description of the drawings]

FIG. 1 is a diagram of a flow-type L-lactic acid measuring device used in Examples.

[Explanation of symbols]

 Reference Signs List 1 buffer tank 2 pump 3 sampler 4 constant temperature bath 5 L-lactate oxidase immobilized column 6 hydrogen peroxide electrode 7 detector 8 single board computer 9 RS232C code 10 personal computer 11 sampler control signal 12 liquid pump control signal 13 drain liquid

Claims (7)

(57) [Claims] 1. A method for measuring an L-form and / or a D-form in a sample containing an L-form and / or a D-form of an optical isomer, wherein the L-form of the optical isomer in the sample is measured. And a conversion reaction between the L-form and the D-form in the sample in the presence of an L-dehydrogenase and a D-dehydrogenase of the optical isomer and an oxidized coenzyme. A method for measuring the L-form and the D-form of an optical isomer in a sample, comprising the step of measuring the L-form. 2. The L-form and / or D-form of an optical isomer
A method for measuring the L-form and the D-form in a sample, comprising: measuring the D-form of an optical isomer in the sample; and
L-dehydrogenase and D-dehydrogenase of the optical isomers,
Of the L- and D-forms in the sample in the presence of the oxidized form of the coenzyme
Perform a conversion reaction and then measure the D-isomer after the conversion reaction
L- and D-forms of optical isomers in a sample including the step of
Measurement method. 3. The electrode active substance, wherein the L-form of the optical isomer in the sample is measured by oxidizing the L-form with an L-form oxidase and increasing or decreasing by the reaction of the oxidase. The measuring method according to claim 1, comprising detecting 4. The D-isomer of an optical isomer in a sample is measured.
The step of oxidizing the D-form with a D-form oxidase,
Electrode activity increased or decreased by the enzyme reaction
3. The method according to claim 2, comprising detecting the quality. 5. The L-form and the D-form of the optical isomer are each L
-Lactic acid and D-lactic acid, L-glutamic acid and D-glutamic acid, L-leucine and D-leucine, or L-galactose and D-galactose, and L-dehydrogenase and D-dehydrogenase are L-lactic acid, respectively. Dehydrogenase and D-lactate dehydrogenase, L-glutamate dehydrogenase and D-glutamate dehydrogenase, L-leucine dehydrogenase and D-leucine dehydrogenase, or L-galactose dehydrogenase and D-galactose 3. The method according to claim 1, which is a dehydrogenase. 6. A step of measuring L-lactic acid in a sample,
Performing an equilibrium reaction between D-lactic acid and L-lactic acid in a sample in the presence of lactate dehydrogenase, D-lactic acid dehydrogenase, and NAD, and measuring L-lactic acid after the equilibrium reaction. Method for measuring D-lactic acid and L-lactic acid in a sample. 7. The method according to claim 6 , wherein the step of measuring L-lactic acid comprises detecting the amount of an electrode active substance which increases or decreases L-lactic acid in the sample by oxidizing L-lactic acid in the sample.