JP6933511B2 - Acetoin measurement method - Google Patents

Description

The present invention relates to a simple method for measuring acetoin in a fermented product sample such as mash.

In many fermented foods and drinks, the diacetyl odor is the odor that affects the properties of the food. In fermented dairy products such as yogurt, the diacetyl odor is considered to be a good odor. However, in alcoholic beverages, it has a butter-like odor and is recognized as an unfavorable odor. Therefore, it is important to monitor fermented foods and drinks and diacetyl in the manufacturing process thereof.

As a method for measuring diacetyl and acetoin, an aqueous solution of creatine and α-naphthol dissolved in 2.5N sodium hydroxide are prepared, and these reagents are rapidly reacted with a sample containing diacetyl or acetoin to measure the change in absorbance. The foges-proscavell reaction (hereinafter referred to as "VP reaction") is known (Non-Patent Document 1).

However, since the threshold value for humans to perceive diacetyl in alcoholic beverages (200 to 300 ppb for people who are sensitive to odors and about 500 ppb for most people) is low, it cannot be measured accurately by the above colorimetric method. For the measurement, GC / MS capable of quantitative measurement at a low concentration is required. Therefore, it is difficult to measure at the site such as a factory where GC / MS is not installed, and it is largely judged by a sensory test.

In the production of alcoholic beverages, diacetyl is also present at the mash stage, but increases after the mash is mashed or filtered. Before upper tank or filtration, acetolactic acid, which is also a precursor of diacetyl, is converted to other metabolites by yeast. However, the presence of α-acetolactic acid gradually becomes non-enzymatic reaction products during the storage period, such as diacetyl and acetoin, because yeast is separated by upper tank or filtration and the flow to other metabolites is blocked. Changes to. Generally, the diacetyl content is low immediately after filtration in the upper tank or immediately after filtration, and it is often difficult to distinguish the diacetyl odor by a sensory test unless a storage period is set. Quantitative monitoring of diacetyl by sensory tests during fermentation poses an even more difficult problem.

Since it is difficult to directly measure diacetyl, a method for quantifying diacetyl precursors acetolactic acid and acethydroxybutyric acid has been reported (for example, Patent Document 1).
In addition, in order to prevent the generation of diacetyl in the manufacturing process of alcoholic beverages, it is common to measure the concentration of pyruvic acid (on the order of ppm), which is said to have a correlation with the generation of diacetyl, and use 100 ppm as a control index. Has been done.

Japanese Unexamined Patent Publication No. 2008-263977

J.Biol. Chem. 1945, 161: 495-502

However, in the method of Patent Document 1, since the amount of diacetyl precursors such as acetolactic acid contained in alcoholic beverages is very small, the measurement sensitivity is poor and the formation of diacetyl cannot be predicted accurately.
In addition, when the results of diacetyl analysis were verified by the method of measuring pyruvic acid, it was found that diacetyl was high even if the pyruvic acid level was low. Therefore, it was considered that monitoring of pyruvate alone was not sufficient to control the risk of developing diacetyl.

Therefore, an object of the present invention is to provide a simple and highly accurate method capable of predicting the amount of diacetyl produced in a fermented food or drink or the production process thereof before obtaining a final product.

Therefore, when the present inventor analyzed the accumulated diacetyl analysis data, acetoin emerged as a candidate compound as a compound having a correlation with diacetyl.
In the case of yeast, acetoin is produced from α-acetolactic acid by a non-enzymatic decarboxylation reaction like diacetyl, but in the case of lactic acid bacteria, acetoin is produced by α-acetolactic decarboxylase.
Diacetyl is on the order of ppb, whereas acetoin is on the order of ppm, and is generally produced in an amount about 30 to 100 times that of diacetyl.
Further, as described above, since the amount of acetoin is larger than that of diacetyl, 2,3-pentanedione, etc., the colorimetric measurement can be performed approximately as the amount of acetoin, and it can be easily measured at the site of quality control.
Based on this finding, we have found that the amount of diacetyl can be predicted by measuring acetoin contained in fermented foods and drinks and their production intermediates and using this as an index, and applied for a patent (Japanese Patent Application No. 2015-248467).

However, components such as glucose and ethanol are contained in fermented food and drink production intermediates such as mash, and depending on the VP method, which is a simple colorimetric method, even acetoin contained in the ppm order may be contained. It turned out that it could not be measured accurately. In fermented food and drink production intermediates such as mash, the amounts of glucose and ethanol change over time, and if they are affected by these components, the accuracy of predicting the amount of diacetyl will decrease, which is not preferable.
Therefore, we further investigated to develop a means for accurately and easily measuring acetoin and accurately predicting the amount of diacetyl. As a result, the amount of glucose contained in the fermented food and drink production intermediate such as 醪 is diluted to a certain value or less, treated with a certain concentration of alkali hydroxide in advance under low temperature conditions, and then α-naphthol. It was found that the amount of acetoin can be measured accurately and easily without being affected by glucose or ethanol, and the amount of diacetyl can also be predicted by performing colorimetric measurement using creatin. It was also found that if α-naphthol and creatine are pulverized in advance in a container used for the reaction separately from the alkaline hydroxide aqueous solution, acetoin can be easily measured without preparing a reagent at the time of measurement. ..

That is, the present invention provides the following [1] to [12].

[1] (a) Dilution step of reducing the amount of glucose contained in a fermented food or drink or its production intermediate to 1% by mass or less.
(B) A step of adding an alkaline hydroxide aqueous solution to the fermented food or drink or its manufacturing intermediate to oxidize acetoin in the fermented food or its manufacturing intermediate to diacetyl under the conditions of 0 ° C. or higher and 15 ° C. or lower, and ( c) A step of measuring diacetyl in the sample subjected to steps (a) and (b) by a colorimetric method.
A method for measuring acetoin in a fermented food or drink or an intermediate for producing the same.
[2] The method for measuring acetoin according to [1], wherein step (a) and step (b) are carried out simultaneously or in the order of step (a) and then step (b).
[3] The acetoin according to [1] or [2], wherein the reaction solution obtained by performing steps (a) and (b) and the reaction solution in step (c) have an alkali concentration of 0.3N to 2N. Measurement method.
[4] The acetoin according to [1] or [2], wherein the reaction solution obtained by performing steps (a) and (b) and the reaction solution in step (c) have an alkali concentration of 0.5N to 1N. Measurement method.
[5] The method for measuring acetoin according to any one of [1] to [4], wherein the colorimetric method in step (c) is a method for measuring the absorbance by reacting a sample with α-naphthol and creatine.
[6] The method for measuring acetoin according to any one of [1] to [5], wherein the production intermediate is mash.
[7] The method for measuring acetoin according to any one of [1] to [6], wherein step (a) is a step of diluting a fermented food or drink or a production intermediate thereof by a factor of 2 or more.
[8] The content of diacetyl in the fermented food or drink or the production intermediate thereof, which comprises measuring acetoin in the fermented food or drink or the production intermediate thereof by the method according to any one of [1] to [7]. How to predict the amount.
[9] A method for producing alcoholic beverages, which comprises producing alcoholic beverages by using the method according to any one of [1] to [7] or the prediction method of [8].
[10] A kit for measuring acetoin containing powdered creatine and α-naphthol in a container.
[11] The kit for measuring acetoin according to [10], which has a protocol capable of measuring acetoin by the method according to any one of [1] to [5].
[12] The kit for measuring acetoin according to [10] or [11], which further comprises an acetoin standard solution used for measuring acetoin and an aqueous alkali hydroxide solution used for the reaction.

According to the acetoin measuring method of the present invention, acetoin in a fermented food or drink containing glucose or ethanol or its production intermediate can be accurately measured by a simple operation, and the amount of diacetyl can be predicted easily and accurately. can. In addition, the acetoin measurement kit of the present invention can easily measure acetoin without preparing a reagent at the time of measurement as in the conventional VP method.

It shows the reaction delay due to glucose. Shows reaction suppression by ethanol. It shows a gradual increase in color development due to 2,3-butanediol. The effect of suppressing the gradual increase in color development due to 2,3-butanediol by setting the reaction temperature at a low temperature (comparison between 40 ° C. and 30 ° C.) is shown. A comparison of the reactivity of diacetyl and acetoin in the presence of glucose is shown. The measurement operation procedure introduced in the pretreatment is shown. A comparison of pretreatment temperatures is shown. A comparison of glucose concentrations at a pretreatment temperature of 15 ° C. is shown. The glucose concentration distribution of sake mash is shown. The ethanol concentration distribution of sake mash is shown. The acetoin concentration distribution of sake mash is shown. The fluctuation of the calibration curve due to the influence of glucose and ethanol is shown. The stabilization of the calibration curve by the introduction of pretreatment (0 ° C., 0.5N NaOH, 10 minutes) is shown. The 2,3-butanediol concentration distribution of sake mash is shown. Shows alkali strength and color development. The improvement of the measurement operation procedure is shown. Shows the prototype conditions and finished state of the kit. The calibration curve creation by the prototype kit is shown. The 40 ° C storage test of the prototype kit is shown. The 4 ° C storage test of the prototype kit is shown. The correlation between diacetyl and acetoin contained in sake is shown.

The method for measuring acetoin in a fermented food or drink or a production intermediate thereof of the present invention is characterized by having the following steps (a), (b) and (c).
(A) A dilution step of reducing the amount of glucose contained in a fermented food or drink or a production intermediate thereof to 1% by mass or less.
(B) A step of adding an aqueous alkali hydroxide solution to a fermented food or drink or a production intermediate thereof to oxidize acetoin in the fermented food or drink or a production intermediate thereof to diacetyl under the conditions of 0 ° C. or higher and 15 ° C. or lower.
(C) A step of measuring diacetyl in a sample obtained by performing steps (a) and (b) by a colorimetric method.

Examples of the fermented food and drink as the sample to be measured include fermented food and drink that may contain diacetyl and / or acetoin, and specific examples thereof include fermented dairy products (yogurt and the like) and alcoholic beverages. Of these, it is preferably applied to alcoholic beverages whose diacetyl odor is not preferable. In the present invention, alcoholic beverages refer to beverages having an alcohol content of 0.01% or more. The liquor in the present invention includes five types of liquor: sparkling liquor, brewed liquor, distilled liquor, mixed liquor, and salty soy sauce.
As the production intermediate for fermented foods and drinks, it is preferable to apply it to the production intermediate for alcoholic beverages, especially mash. By measuring the acetoin concentration in the mash, the diacetyl concentration can be predicted and the subsequent fermentation process can be controlled.
Moromi is a product that has been fermented into an article that is a raw material for alcoholic beverages (limited to those that can be used for the production of alcoholic beverages) and has not been strained or distilled (alcoholic beverages that are not strained or distilled). Refers to those before the end of main fermentation).

When the mash is used as a sample, it is preferable to measure the acetoin contained in the mash between 10 days before the scheduled end date of fermentation of the mash and the scheduled end date of fermentation. In order to avoid the generation of diacetyl in alcoholic beverages, it is preferable to measure acetoin at the earliest possible stage of mash, but if the measurement of acetoin is too early, the prediction accuracy will be low, so there is a trade-off relationship. It is in. It is more preferable to measure the acetoin contained in the mash from 10 days before the scheduled fermentation end date to 3 days before the scheduled fermentation end date, and from 10 days before the scheduled fermentation end date to 5 days before the scheduled fermentation end date. It is more preferable to carry out. In the case of sake mash, it is preferable to measure the acetoin contained in the sake mash between about 10 days after the addition and the scheduled fermentation end date, and from about 10 days after the addition to 3 days before the scheduled fermentation end date. It is more preferable to carry out the fermentation in the above, and it is more preferable to carry out the treatment from about 10 days after the addition to 5 days before the scheduled completion date of fermentation.
The scheduled fermentation end date means the scheduled date for straining or distilling the mash (the date when the main fermentation ends for alcoholic beverages that are not strained or distilled), which is set in advance when producing a specific liquor. When industrially producing alcoholic beverages, the approximate fermentation days are determined by the target alcoholic beverages, their raw materials, fermentation tanks, etc., although it depends on the fermentation conditions.
If acetoin is measured 11 days or more before the scheduled completion date of fermentation, the accuracy of acetoin measurement may decrease because diacetyl contains a large amount of components such as sugars and amino acids, depending on the raw material of alcoholic beverages. This is because the accuracy of predicting the occurrence of diacetyl may decrease.
It is also possible to measure acetoin beyond the expected fermentation end date. However, in this case, since the mash is usually strained or distilled on the scheduled end date of fermentation, it is not possible to take measures to reduce the diacetyl odor by increasing the number of fermentation days.

According to the study of the present inventor, it has been found that glucose contained in a fermented food or drink or an intermediate for its production (hereinafter, also referred to as a sample) delays the reaction of oxidizing acetoin to diacetyl under alkaline conditions. .. This reaction delay due to glucose is eliminated by diluting the amount of glucose contained in the step (a) sample to 1% by mass or less. On the other hand, it was also found that there is no problem in quantifying acetoin even if the sample is diluted in this way. Dilution can be carried out using water or the alkaline hydroxide aqueous solution of step (b).
In addition, ethanol contained in the sample suppresses the VP reaction. In the present invention, by diluting the sample so that the glucose concentration is 1% by mass or less, the content of ethanol is also reduced, and as a result, the influence of ethanol on the VP reaction can be reduced. That is, by diluting the sample so that the glucose concentration is 1% by mass or less, the influence of glucose and ethanol contained in the sample on the acetoin measurement value can be reduced. The dilution ratio may be 2 times or more in consideration of the amount of glucose normally contained in mash or the like, but is preferably 2 to 4 times, and particularly preferably about 3 times. The dilution in step (a) may be at room temperature (5 to 35 ° C.), but is preferably performed at 0 ° C. or higher and 15 ° C. or lower.

It was also found that the delay in the reaction of glucose from acetoin to diacetyl in the sample can be eliminated by setting the alkaline conditions in a certain range and lowering the reaction temperature. That is, the reaction delay due to glucose can be eliminated by adding an aqueous alkali hydroxide solution to the sample in step (b) and oxidizing acetoin in the sample to diacetyl under the conditions of 0 ° C. or higher and 15 ° C. or lower. Here, the step (a) and the step (b) may be performed by the step (a) and then the step (b), or may be performed at the same time. When the step (a) and the step (b) are performed at the same time, the sample is diluted with an aqueous alkali hydroxide solution so that the glucose concentration in the sample is 1% by mass or less and the alkali concentration is 0.5N to 1N. It is preferable to do so.

The alkali concentration of the reaction solution obtained by adding an aqueous alkali hydroxide solution to the sample can be set in a wide range, but from the viewpoint of the progress of the reaction from acetoin to diacetyl and the stable transition of the absorbance of the obtained coloration, it is 0. .3N to 2N is preferable, and 0.5N to 1N is more preferable. Specifically, in order to complete the step (b) within 10 minutes and the step (c) in about 30 minutes to obtain a stable color reaction, the alkali concentration of the reaction solution is 0.3N to 2N. It is preferable that the value is 0.5N to 1N, and more preferably 0.5N to 1N.
Examples of the alkali hydroxide include sodium hydroxide and potassium hydroxide, but sodium hydroxide is preferable.

The sample mixed with the alkaline hydroxide aqueous solution may be cooled by immediately immersing the container in which they are mixed in a water tank at 0 ° C. or higher and 15 ° C. or lower and allowing it to stand for 1 minute or longer for cooling treatment. As an easy method for surely setting the treatment temperature to 0 ° C. or higher and 15 ° C. or lower, it is more preferable to prepare ice-cooled water and immerse it in the treatment. In addition, it is preferable that the reaction time is short in order to proceed with the work quickly, but if it is too short, there will be no margin and the accuracy of the work will deteriorate. Therefore, this reaction time is set somewhere between 1 minute and 10 minutes. It is preferable to set it to about 5 minutes in order to maximize both the speed and accuracy of the work.

By the dilution and the alkali treatment at a low temperature, the influence of glucose and ethanol contained in the sample on the acetoin measurement value can be avoided. Further, since acetoin in the sample is oxidized by the alkali treatment and changed to diacetyl, if the pretreated sample is reacted with a colorimetric reaction (step (c)), for example, α-naphthol and creatine. , A red dye is produced according to the following reaction formula.

The reaction of the pretreated sample in step (c) with α-naphthol and creatine may be carried out at 30 to 40 ° C. for 30 minutes. Since a red pigment is produced by this reaction, the acetoin concentration in the sample can be quantified by measuring the absorbance of the sample. As the absorbance, the absorbance in the vicinity of 530 nm may be measured.

In the method for measuring acetoin of the present invention, by using an acetoin measuring kit containing powdered creatin and α-naphthol in a container for carrying out the reaction, α- is used for each measurement as in the conventional VP method. It is not necessary to prepare an aqueous alkali hydroxide solution of naphthol, and further, there is no need to worry about the influence on the measured value due to the deterioration of the prepared aqueous alkaline hydroxide solution of α-naphthol over time. Further, the powdered mixture of creatine and α-naphthol can be dissolved in 30 to 40 seconds by stirring after adding the alkaline hydroxide aqueous solution mixed with the sample. That is, by using the kit of the present invention, the steps (a), (b) and (c), preferably the steps (a) and (b) are simultaneously performed, and then the step (c) is performed. Acetoin in a sample can be measured easily and accurately.

Powdered creatine and α-naphthol can be obtained by adding an aqueous solution of creatine and an ethanol solution of α-naphthol to a container and freeze-drying. Further, the kit of the present invention may be accompanied by an acetoin standard solution used for preparing a calibration curve for quantification, or may be accompanied by an aqueous alkali hydroxide solution. Further, the kit of the present invention may be accompanied by a protocol describing the measurement procedure. The attached alkali hydroxide aqueous solution may have any alkali concentration, but if it is 1.25 N or less, it is excluded from the designation as a deleterious substance and the burden of handling management can be reduced, which is more preferable. It is possible to lower the alkali concentration of the attached alkaline hydroxide aqueous solution to 0.1N, but a concentration capable of reducing the alkali concentration in the reaction solution to 0.5N to 1.0N is preferable.

By the above method, the amount of acetoin in the fermented food or drink or its production intermediate can be measured easily and accurately. On the other hand, the amount of diacetyl in the sample in which the amount of acetoin was measured is lower than the amount of acetoin, but it is unknown. However, according to the accumulated data of the present inventor, it was found that the amount of acetoin and the amount of diacetyl have a certain relationship in the manufacturing process of a specific fermented food or drink. Therefore, if the relationship between the amount of diacetyl and the amount of acetoin in the production process of the same fermented food and drink is measured in advance by GC / MS analysis, the amount of diacetyl can be predicted from the amount of acetoin simply measured by the method of the present invention. can.

Next, the present invention will be described in more detail with reference to examples.

<Measurement method>
In the quantitative measurement of diacetyl and acetoin color development using the VP reaction, a 0.5% creatine aqueous solution (reagent solution 1) and a 5% α-naphthol / 2.5N sodium hydroxide solution (reagent solution 2) are usually prepared in advance. Prepared according to the above, and perform the procedure as shown in Table 1. First, the absorbance of Examples was measured in this system.

Example 1
(1) Components that affect the color development reaction system When commercially available sake was measured as a sample by the above VP reaction, it was suggested that the color development reaction may be affected by the components contained in the sake. And examined to identify the content of the impact.
As a result, it was clarified that the reaction was delayed by glucose (see FIG. 1), the reaction was suppressed by ethanol (see FIG. 2), and the color development was gradually increased by 2,3-butanediol (see FIG. 3). FIG. 1 shows the result of dissolving a predetermined amount of glucose in an aqueous solution containing 5 ppm of acetoin, and FIG. 2 shows the result of dissolving a predetermined amount of ethanol in an aqueous solution containing 5 ppm of acetoin. Sake A and B in FIG. 3 indicate different commercially available sake.
It was also clarified that the rate of gradual increase in color development due to 2,3-butanjir was suppressed by lowering the reaction temperature (see FIG. 4). In the case of the reaction at 30 ° C., it was found that the color development due to 2,3-butanjir was considerably suppressed within the reaction time of 40 minutes, so the reaction temperature was decided to be 30 ° C. in future studies. Sake C to E in FIG. 4 show different sakes on the market.

(2) Examination of the effects of glucose and ethanol In the measurement of acetoin by the VP reaction, as shown in the reaction formula below, the reaction step in which acetoin is converted to diacetyl under alkaline conditions and diacetyl reacts with the reagent under alkaline conditions to develop color. Two stages of the steps to be performed are proceeding in parallel.

We investigated which of these steps glucose affected. As a result, it was found that there is no effect of glucose on step 2 in which diacetyl reacts with the reagent to develop color, and it affects the oxidation reaction (step 1) in which acetoin is converted to diacetyl, thereby delaying the reaction. (See Fig. 5). Glucose tends to have a chain structure under alkaline conditions, which exposes aldehyde groups at the ends and exhibits reducing properties. It was speculated that this reducing property interfered with the oxidation reaction in which acetoin was converted to diacetyl. It was also clarified that diacetyl rapidly reacts with the reagent to develop color, but acetoin has a lag time to be converted to diacetyl, and it takes about 30 minutes in the presence of 3% glucose. As a result of various studies on measures to avoid the influence of glucose based on these findings, it was devised to separately carry out the step of converting acetone to diacetyl under the condition that the reducing property of glucose can be avoided.
In FIG. 5, "diacetyl 0% glucose" is an aqueous solution containing 5 ppm diacetyl, "acetoin 0% glucose" is an aqueous solution containing 5 ppm acetoin, and "diacetyl 3% glucose" is an aqueous solution containing 5 ppm diacetyl with 3% glucose added. "Acetoin 3% glucose" refers to an aqueous solution containing 5 ppm of acetoin supplemented with 3% glucose.

As shown in FIG. 6, before step 2 in which diacetyl reacts with the reagent to develop color, a measurement procedure in which pretreatment is introduced is newly constructed, and the conditions under which glucose reducibility can be avoided are examined using the measurement method. bottom. As a result, by lowering the pretreatment temperature to 15 ° C. or lower, color development is stabilized even in the presence of 3% glucose (see FIG. 7), and further, by lowering the glucose concentration to 1% or lower, color development suppression is avoided. Was confirmed (see FIG. 8). This is because the ring opening of glucose is suppressed at a low temperature of 15 ° C. or lower under alkaline conditions with 0.5N sodium hydroxide, and further, if the glucose is 1% or less, the ring is opened and glucose having a reducing power is obtained. Is a very small amount, and it is presumed that it has almost no effect.
In FIG. 7, "G0% 6 minutes" is an aqueous solution containing 5 ppm acetoin pretreated at a predetermined temperature for 6 minutes, and "G0% 42 minutes" is an aqueous solution containing 5 ppm acetoin pretreated at a predetermined temperature for 42 minutes. In "G3% 6 minutes", 3% of glucose was added to an aqueous solution containing 5 ppm acetoin and treated at a predetermined temperature for 6 minutes, and in "G3% 42 minutes", 3 glucose was added to an aqueous solution containing 5 ppm acetoin. % And treated at a predetermined temperature for 42 minutes, respectively.
In FIG. 8, an aqueous solution containing 5 ppm of acetoin and 1, 3, and 5% glucose added to the aqueous solution were measured at a pretreatment temperature of 15 ° C. and treatment times of 6 minutes and 42 minutes.

It was also clarified that when the pretreatment temperature is 15 ° C., there is no difference between the pretreatment time as short as 6 minutes and the pretreatment time as long as 42 minutes (FIGS. 7 and 8). ).

As a method for reducing the glucose concentration in sake mash or sake to 1% or less, a method of treating with glucose oxidase was examined, but good results were not obtained. Therefore, we investigated the distribution of glucose concentration in the sake mash to be analyzed, and decided to deal with it by diluting it so that the glucose concentration was 1% or less of the upper limit. As a result of analyzing and investigating the concentration distributions of glucose, ethanol, and acetoin in the sake mash to be measured 10 days after the addition of sake mash, the results shown in FIGS. 9, 10 and 11, respectively, were obtained.
From the results, it was confirmed that the maximum glucose concentration was 3%, and it was found that in order to reduce the glucose concentration of sake mash to 1% or less, it should be diluted 3-fold. With this dilution, the ethanol concentration becomes 6.4% or less and the acetoin concentration becomes 9 ppm or less.

Therefore, using a model sample dissolved in water so that the glucose concentration was 1% and the ethanol concentration was 6.4%, a calibration curve was prepared by the measurement in which the pretreatment at 15 ° C. or lower was introduced. In addition, in order to set the pretreatment temperature to 15 ° C. or lower without preparing a special cooling constant temperature bath, the temperature was set to 0 ° C. using ice-cold water. As a result of the test, the slope of the calibration curve has been fluctuated due to the influence of glucose and ethanol (see FIG. 12), but the calibration curve has a constant slope without being affected even in the presence of glucose and ethanol. The line was able to be created (see FIG. 13). That is, it was found that when the pretreatment at 0 ° C. was performed with the glucose concentration set to 1% or less and the ethanol concentration set to 6.4% or less, not only the influence of glucose but also the influence of ethanol could be avoided. That is, after diluting sake mash or sake with water 2 to 4 times, pretreatment at 15 ° C. or lower is performed and a VP reaction is carried out, so that the influence of glucose and ethanol can be avoided.

(3) Examination of the effect of 2,3-butanediol 2,3-butanediol (hereinafter referred to as "BD") is a component that is inevitably present in sake mash and is a final metabolite. It has been reported that it accumulates as fermentation progresses, and in sake mash it reaches an average of around 500 ppm. Therefore, the concentration of BD contained in the mash 10 days after the addition of sake mash was measured, and the concentration distribution was investigated. As a result, it was found that the concentration distribution was as shown in FIG. 14 in the range of 93 to 656 ppm. The BD concentration detected in most of the mash is 211 to 250 ppm, and it was confirmed that the BD concentration is equivalent to 0.8 ppm of acetoin. Therefore, it can be sufficiently dealt with by subtracting the added portion from the measured value. It was considered. However, since it is considered that the time for predicting the production of diacetyl is the time when the accumulation of BD is still small, it seems that there is no problem in ignoring the amount of color development due to BD.

(4) Examination of improvement of measurement sensitivity It was examined whether the reactivity could be increased and the measurement sensitivity could be improved by increasing the alkaline strength of the reaction solution. As a result, it was found that by increasing the normality of sodium hydroxide in the reaction solution from 0.5N to 0.75N, the color development of acetoin can be improved without changing the color development due to the blank and BD. (See FIG. 15). In other words, the sensitivity can be improved by increasing the alkaline strength of the reaction solution as much as possible, so the measurement operation procedure was examined again. As shown in FIG. 16, the measurement operation procedure devised so that the sample is automatically diluted 3-fold and the alkali strength can be increased as much as possible by simply mixing the sample and the 1N sodium hydroxide solution at a constant ratio has been improved as shown in FIG. ..

Example 2
(1) Preparation of a kit A kit suitable for direct use in carrying out the measurement method of the present invention was prepared as shown in Table 2. The conditions for freeze-drying are shown in FIG.

(2) Sample measurement using the prototype kit The measurement using the kit was performed by the procedure shown after the modification on the right side of FIG. 16, and a commercially available 1N sodium hydroxide aqueous solution (excluded from the designation as a deleterious substance) was used.

(5) Performance test of prototype kit Two types of kits with different freeze-drying conditions were prototyped as shown in Fig. 17, and a test was conducted to confirm their performance. In the test, it was decided to perform measurements with an improved operating procedure and confirm by creating a calibration curve in the presence of glucose and ethanol.
There are three types of glucose and ethanol concentrations set for preparing the calibration curve, 3% and 15.2%, 2% and 16.1%, 1% and 17. The combination was 0%.
As a result of the test, there was almost no difference in the prepared calibration curves for both kits A and B in the three types of glucose and ethanol concentration settings, but kit A was superior in linearity. On the other hand, it was shown that the absorbance of Kit B decreased slightly as the concentration of acetoin increased. Regarding the concentration of glucose and ethanol in the reference solution used when preparing the calibration curve, we decided to set it to an intermediate concentration of sake to be measured, and set it to 2% glucose and 16.1% ethanol. bottom.

(6) Kit storage test A storage test at 40 ° C was performed using the prototype kit. Looking at the calibration curve on the 10th day of storage at 40 ° C, which corresponds to 2 months of storage at 25 ° C, Kit A maintained linearity and was almost the same as that stored at 4 ° C, but Kit B was linear. However, an upward shift was observed due to reagent coloring (see FIG. 19). This difference between kits A and B was thought to be due to the fact that the dry state of kit B was slightly inferior to that of kit A. Therefore, in future kit production, the freezing time should be 12 hours or more according to the production conditions of kit A. It was decided that the vacuum drying time would be 3 hours. It was also found that if stored at 4 ° C, it is stable even for a long period of one and a half years (see FIG. 20). If the above kit is prepared in advance and stored at 4 ° C or lower, it is possible to quickly respond to the measurement of mash and sake.

Reference example 1
Diacetyl and acetoin contained in 319 kinds of commercially available sake (if they are the same brand, different lots, 111 kinds of which are pure rice sake type) were analyzed by GC / MS.
For GC / MS analysis, 0.5 mL of 0.6 M potassium phosphate buffer (pH 7.0) containing 0.5 mL of the above sake and 1 ppm of 4-methyl-2-pentanol and 0.5 mL of ethyl acetate were placed in a 2 mL microtube. In addition, the mixture was stirred and extracted with a vortex mixer for 2 minutes, and centrifuged at 12,000 g for 1 minute. 200 μL of the upper ethyl acetate layer was transferred to a sample tube for GC / MS analysis, and GC / MS analysis was performed under the following conditions.
(Measurement condition)
Equipment: Shimadzu GC / MS QP-2010plus / Shimadzu AOC-5000
Column: GL Sciences INERT CAP Pure WAX (60m x 0.25mm ID x 0.25μm)
Column temperature: 40 ° C (10 min) → (4 ° C / min) → 55 ° C (0 min) → (40 ° C / min) → 200 ° C (2 min) → (60 ° C / min) → 230 ° C (30 min)
Carrier gas: He, total flow rate 50 mL / min Linear velocity 30.0 cm / sec Constant barge flow rate 3.0 mL / min
Injection: 250 ℃ Splitless sample time 1min
Injection amount: 1 μL
Ion source: EI, 200 ° C Interface: 230 ° C

The obtained results are shown in FIG. As shown in FIG. 21, a positive correlation (correlation index 0.75) was observed between diacetyl and acetoin contained in sake. It was confirmed that the amount of diacetyl that causes a butter-like odor in sake is on the order of ppb, while that of acetoin is on the order of ppm. It was suggested that acetoin could be measured by the colorimetric method.
A diacetyl odor was observed in all sakes having diacetyl of 500 ppb or more. Calculated from the correlation coefficient shown in FIG. 21, acetoin is 18 ppm or less to control diacetyl contained in sake to less than 500 ppb, acetoin is 10 ppm or less to control diacetyl to less than 300 ppb, and diacetyl is less than 200 ppb. It was suggested that the amount of acetoin should be 5 ppm or less.

Claims (9)

(A) Dilution step of reducing the amount of glucose contained in a fermented food or drink or its production intermediate to 1% by mass or less.
(B) A step of adding an alkaline hydroxide aqueous solution to the fermented food or drink or its manufacturing intermediate to oxidize acetoin in the fermented food or its manufacturing intermediate to diacetyl under the conditions of 0 ° C. or higher and 15 ° C. or lower, and ( c) A step of measuring diacetyl in the sample subjected to steps (a) and (b) by a colorimetric method.
A method for measuring acetoin in a fermented food or drink or an intermediate for producing the same. The method for measuring acetoin according to claim 1, wherein step (a) and step (b) are carried out simultaneously or in the order of step (a) and then step (b). The method for measuring acetoin according to claim 1 or 2, wherein the reaction solution obtained by performing steps (a) and (b) and the reaction solution in step (c) have an alkali concentration of 0.3N to 2N. The method for measuring acetoin according to claim 1 or 2, wherein the reaction solution obtained by performing steps (a) and (b) and the reaction solution in step (c) have an alkali concentration of 0.5N to 1N. The method for measuring acetoin according to any one of claims 1 to 4, wherein the colorimetric method in step (c) is a method for measuring the absorbance by reacting a sample with α-naphthol and creatine. The method for measuring acetoin according to any one of claims 1 to 5, wherein the production intermediate is mash. The method for measuring acetoin according to any one of claims 1 to 6, wherein step (a) is a step of diluting a fermented food or drink or a production intermediate thereof in a factor of 2 or more. A method for predicting the diacetyl content in a fermented food or drink or a production intermediate thereof, which comprises measuring acetoin in the fermented food or drink or a production intermediate thereof by the method according to any one of claims 1 to 7. .. A method for producing alcoholic beverages, which comprises producing alcoholic beverages by using the method according to any one of claims 1 to 7 or the prediction method according to claim 8.

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