WO2009096102A1 - Method for measuring yeast aggregation activity of malt using crystal oscillator - Google Patents

Abstract

A method for measuring yeast aggregation activity of malt according to the invention comprises bringing a macromolecular polysaccharide obtained by separation from malt to be tested followed by concentration into contact with a crystal oscillator immobilized with concanavalin A in a buffer, and measuring a change in oscillation frequency of the crystal oscillator caused by binding of the macromolecular polysaccharide to concanavalin A. According to the invention, the yeast aggregation activity of malt serving as a raw material can be promptly and simply measured without actually performing a fermentation test using yeast.

Description

Method for measuring yeast aggregating activity of malt using crystal oscillator Reference to related applications

The present application is based on Japanese Patent Application No. 2008-018890 (filing date: January 30, 2008), which is a prior Japanese patent application, and claims the benefit of its priority. The entirety is hereby incorporated by reference.

Background of the Invention

The present invention relates to a method for measuring yeast aggregating activity of malt using a crystal oscillator. More specifically, the present invention relates to a method for measuring agglutination activity that causes agglutination and precipitation of yeast in malt used for producing alcoholic beverages such as beer, happoshu and whiskey without actually performing fermentation.

Related Art When producing alcoholic beverages made from malt as a raw material, if fermentation is carried out using a type of yeast called bottom fermentation yeast, a phenomenon in which yeasts form aggregates and settle immediately before the end of the fermentation process can be observed. . By the sedimentation of the yeast agglomerates, yeast necessary for the next fermentation of alcoholic beverage production can be obtained. The mechanism of such a phenomenon is considered as follows.

That is, there are lectins and sugar chains on the surface of yeast cells, and sugars such as glucose contained in the fermentation liquid inhibit the binding between lectins and sugar chains between adjacent yeast cells. Yeast is floating. However, when fermentation progresses, that is, when sugars such as glucose are taken into yeast and metabolized to alcohol, the inhibitor of binding between the lectin and sugar chains decreases, and as a result, between adjacent yeast cells It is considered that the binding between the lectin and the sugar chain proceeds to gradually form aggregates and sedimentation occurs.

However, it has been reported that a phenomenon called “early yeast agglutination phenomenon” (hereinafter sometimes referred to as “early coagulation phenomenon”) is observed separately.

"Early yeast agglomeration phenomenon" is a phenomenon in which yeast agglomerates and settles in the fermentation process of yeast, especially in the latter stage of fermentation, even though the sugars that can be assimilated by yeast remain in the fermentation broth. If the yeast aggregates and settles due to this early aggregation phenomenon, the progress of fermentation stops. Therefore, if this phenomenon is observed, the manufactured product becomes out of specification, leading to a decrease in the alcohol content due to lack of fermentation and a significant decrease in quality, such as poor disappearance of immature components such as acetaldehyde and diacetyl. In the brewing of fermented malt beverages, etc., it will also suffer great damage.

In order to solve the problem of precoagulation during the production of alcoholic beverages made from malt, many studies have been conducted for a long time, and the causative agent of premature aggregation (Premature Yeast Flocculation Factor, PYF). ) Is reported to be a kind of high molecular polysaccharide contained in raw barley or malt (Rept. Res. Lab. Kirin Brewery Co., Ltd., No.19,45-53,1976) . In the presence of this high molecular polysaccharide, yeast cell surface lectin-precoagulant factor (high molecular polysaccharide) -yeast, despite the presence of saccharides such as glucose that inhibit lectin-sugar chain binding on the yeast cell surface. It is believed that cell surface lectins bind and promote yeast aggregate formation.
Therefore, it can be said that malt containing a large amount of a certain kind of high molecular polysaccharide, which is an early coagulation factor, has strong yeast aggregating activity and is likely to cause an early aggregation phenomenon.

Conventionally, in brewing fermented malt beverages made from barley, in order to avoid the early agglomeration phenomenon in the fermentation process, the yeast agglomeration activity of malt and barley is confirmed, and the malt that does not cause the early agglomeration phenomenon is selected in advance. However, it has been desired to be used.

In order to evaluate the strength of yeast aggregating activity such as malt and barley, conventionally, after actually preparing wort from the target malt, add yeast and conduct a fermentation test, and evaluate by the progress of fermentation There has been a way to do it.

However, in any of these methods, the fermentation test is a test in which the actual brewing scale (fermentation test scale) is made small, and it is necessary to actually ferment the wort. A long time was needed. In actual fermentation, the progress of the fermentation may be greatly affected by the components other than the precoagulant factor contained in the test malt (for example, trace metals such as amino acids and zinc) and the activity of the yeast used. It was not easy to accurately measure the yeast aggregating activity.

In order to shorten the period of the fermentation test, an enzyme is added to the test raw wheat and the raw wheat is subjected to an enzyme treatment. The resulting enzyme-treated product or the polymer fraction separated from the enzyme-treated product is converted into a synthetic wort. A method for determining the presence or absence of an early cohesive factor in raw wheat by measuring the turbidity of the fermentation test raw material after 48 hours was added (Patent No. 31215552). Although this method has greatly shortened the test period, this method is based on a fermentation test, and there is still room to consider the above-mentioned problems associated with the actual fermentation process. .

A crystal oscillator is a structure in which a crystal thin film is cut into a thin plate and a metal thin film is attached to both sides of the slice. When an alternating electric field is applied to the metal thin film, the crystal oscillator vibrates at a constant frequency. It is known. It is also known that when a small amount of substance is adsorbed on a metal thin film, the vibration frequency decreases in proportion to the mass of the substance, and the mass can be obtained using this (QCM (Quartz Crystal Microbalance: quartz Oscillator microbalance).

As a sensor or technology using such QCM, for example, a carbohydrate sensor (Japanese Patent Laid-Open No. 63-196932) having a structure in which concanavalin A is bonded to a polyvinyl butyral film on the surface of a crystal oscillator via a dialdehyde compound. ), A microorganism sensor in which an antimicrobial antibody is immobilized via protein A on the surface of an electrode of a crystal oscillator (Japanese Patent Laid-Open No. 2002-340766), a living cell is immobilized on the surface of an electrode of a crystal oscillator, and a receptor- A method for analyzing the interaction between ligands (Japanese Patent Laid-Open No. 2003-83974) and a method for evaluating the taste of alcoholic beverages (Japanese Patent Laid-Open No. 2000-183333 and Japanese Patent Laid-Open No. 2000-183333) JP 2001-215183 A) and the like.

However, as far as the present inventors know, no study has been made on the use of a crystal oscillator for measuring the yeast aggregating activity in malt.

Summary of the Invention

The present inventors examined a method for measuring yeast aggregating activity of malt without performing a fermentation test. At this time, attention was paid to the fact that the precoagulant factor that promotes yeast aggregation contained in the malt is a high molecular weight polysaccharide, which binds to the lectin on the surface of the yeast cell. Therefore, first, the yeast cells themselves were immobilized on a quartz oscillator, and the yeast cells were used to capture and measure precoagulant factors in the malt. However, when immobilizing yeast cells themselves, it is difficult to immobilize yeast cells and it is difficult to supply yeast cells with a constant amount of lectin on the surface of yeast cells. The inconvenience that the sensitivity fluctuated due to the difference in the amount of lectin was considered. Next, the yeast cell surface lectin itself was obtained, immobilized on a crystal oscillator, and the precoagulation factor in the malt was captured and measured by the lectin. However, since yeast cell surface lectins are immobilized, it was practically difficult to obtain a large amount of the purified lectins.

Here, the present inventors form a dimer or tetramer depending on concanavalin A (subunit molecular weight 25,572, pH), which is a lectin derived from pea ( Canavalia ensiformis ), as a precoagulant factor contained in malt. It was found to bind specifically to “ConA”. Therefore, the present inventors further produced a sensor in which ConA is immobilized on the surface of the crystal oscillator, immersed the sensor in a buffer solution, added a polymer fraction separated from the malt in the buffer solution, And the rapid-flocculation factor contained in the polymer fraction were reacted, and then the change in the vibration frequency of the crystal oscillator resulting from the combination of ConA and the rapid-flocculation factor was successfully measured. It was confirmed that there was a good correlation between the strength of the yeast aggregating activity and the obtained frequency change. Furthermore, a high concentration of high molecular weight polysaccharides can be obtained by combining high molecular weight polysaccharides from malt with ethanol precipitation and ion exchange column chromatography. It was very effective to preliminarily concentrate the high-molecular-weight polysaccharide when measuring. The present invention is based on these findings.

Therefore, an object of the present invention is to provide a method for measuring yeast aggregating activity contained in malt as a raw material quickly and easily without actually performing a fermentation test using yeast.

The method for measuring the yeast aggregating activity of malt according to the present invention is as follows.
The vibration of the crystal oscillator produced by the contact between the polymer polysaccharide separated from the test malt and concentrated with the crystal oscillator having immobilized concanavalin A in a buffer solution, and the binding of the polymer polysaccharide and concanavalin A. Measuring a change in frequency.

According to a preferred embodiment of the present invention, the measurement method according to the present invention includes a step of obtaining a concentrated polymer polysaccharide from a test malt,
Ethanol is added to the wort obtained by saccharifying the test malt to produce an ethanol precipitate containing a high molecular polysaccharide, and the resulting precipitate is dissolved in a buffer solution. The method further includes a step of subjecting the concentrated fraction containing the high molecular weight polysaccharide to an exchange column chromatography.

According to one preferable aspect of the present invention, in the measurement method according to the present invention, the high molecular weight polysaccharide and the crystal oscillator on which concanavalin A is immobilized are brought into contact in an acidic buffer solution.

According to one more preferred aspect of the present invention, in the measurement method according to the present invention,
The method further comprises obtaining the value of the yeast agglutination activity of the test malt from the obtained change in the frequency based on the correlation between the previously obtained value of the yeast agglutination activity and the change in the frequency.

According to another aspect of the invention,
A method for quantifying yeast early aggregation factors in malt,
Using the method for measuring yeast aggregating activity of malt according to the present invention, and
Obtaining the amount of yeast early aggregation factor in the raw malt from the obtained change in frequency based on the correlation between the amount of yeast early aggregation factor and the change in frequency obtained in advance. A method is provided.

According to yet another aspect of the present invention, the method for measuring the yeast aggregating activity of the malt according to the present invention is used to determine the yeast aggregating activity of the malt raw material, the malt during production, or the produced malt, thereby producing a malt production process. There is provided a method for producing malt, characterized in that

According to still another aspect of the present invention, the malt raw material to be used is selected and adjusted by measuring the yeast aggregating activity in the malt using the method for measuring malt yeast aggregating activity according to the present invention. A method for producing a fermented alcoholic beverage is provided.

As described above, conventionally, the evaluation of the strength of the yeast aggregating activity contained in the malt must use yeast in any method, and therefore the influence of the yeast to be used could not be avoided. In addition, the conventional fermentation test method requires several days of fermentation using a large dedicated fermentation apparatus. However, according to the method of the present invention, the yeast aggregating activity of the desired malt as a raw material can be obtained without using a large-scale fermentation apparatus, in the same or less days as the conventional fermentation test method, and without using yeast. Evaluation can be performed. In addition, according to the method of the present invention, since fermentation using yeast is not performed, not only malt or barley before malting, but also barley in the middle of malting, or other cereals and extracts used for brewing, etc. It can also be applied to the evaluation of yeast aggregating activity. As a result, it not only contributes to precise process control and quality control of liquor production according to the fermentation characteristics of brewing raw materials including malt, but also contributes to elucidation of the production conditions of malt causing early yeast agglomeration phenomenon. .

The figure shows the measurement results in Example 2. The figure shows the measurement results in Example 3. The figure is a graph showing the correlation between the yeast aggregating activity strength (DPF) and the vibration frequency change 1 minute after addition of the malt polymer concentrate obtained in Example 4.

Detailed description of the invention

As described above, the measurement method according to the present invention is a method for measuring the yeast aggregating activity of malt, comprising: a high molecular weight polysaccharide separated from the test malt and concentrated; and a crystal oscillator having concanavalin A immobilized thereon. It is made to contact in a buffer solution, and the change of the vibration frequency of the crystal oscillator produced by the coupling | bonding of a high molecular weight polysaccharide and concanavalin A is measured.

In the present invention, the high molecular weight polysaccharide separated and concentrated from the test malt sample is used.
That is, in the present invention, yeast agglutination factor contained in malt is measured. When concanavalin A immobilized on a crystal oscillator is reacted with precoagulation factor, precoagulation factor is purified and concentrated in advance. is important.

In the present invention, the separation and concentration of the high molecular weight polysaccharide from the test malt sample is preferably performed, for example, as follows. That is, first, the test malt is pulverized and extracted with water as necessary according to a conventional method, then saccharified to obtain wort, and ethanol is added thereto. At this time, it is preferable to add about twice as much ethanol to wort, and the ethanol concentration here is preferably about 60 to 70%, for example. When ethanol is added, a polymer component is precipitated to form a precipitate (hereinafter sometimes referred to as “malt polymer extract”). This is separated and collected by means such as centrifugation. In this way, the fast coagulation factor, which is a high molecular acidic polysaccharide, can be separated from the raw material malt as a precipitate.

Next, the resulting precipitate (malt polymer extract) containing the rapid setting factor is concentrated.
The malt polymer extract is dissolved in a phosphate buffer and subjected to anion exchange column chromatography to fractionate a fraction enriched in the early coagulation factor, including the early coagulation factor. Presence / absence of a fast-coagulation factor for each fraction can be confirmed by a separately conducted fermentation test, or a fraction to be fractionated can be specified based on a previously conducted fermentation test. Specifically, each fraction obtained by column chromatography is freeze-dried, and these are added to synthetic wort prepared with components other than malt (sugar, amino acid, inorganic salts, etc.), and yeast is further added. In addition, it is fermented at a temperature of 20 ° C. for 48 hours. After fermentation, the absorbance (OD800) at a wavelength of 800 nm was measured using a turbidimeter, and a value DPF (Degree of Premature Flocculation) 48 obtained by subtracting OD800 of the test group from OD800 of the control group was compared. The intensity of the aggregating activity can be determined. Based on this result, it is possible to identify a fraction that contains a large amount of precoagulant factors and is concentrated. A fraction having a high yeast agglutination activity and containing a large amount of precoagulant factors can be collected and lyophilized as necessary to obtain a desired high molecular polysaccharide content (hereinafter sometimes referred to as “malt polymer concentrate”). it can.
In addition, the preparation method of synthetic wort can be performed according to description of patent document 1. FIG.
As anion exchange column chromatography used here, a Mono-Q column or a Q Sepharose column manufactured by GE Healthcare Biosciences can be used.

Therefore, according to a preferred embodiment of the present invention, as described above, the measurement method according to the present invention includes a step of obtaining a concentrated high molecular polysaccharide content from the test malt.
Ethanol is added to the wort obtained by saccharifying the test malt to produce an ethanol precipitate containing a high molecular polysaccharide, and the resulting precipitate is dissolved in a buffer solution. The method further includes a step of subjecting the concentrated fraction containing the high molecular weight polysaccharide to an exchange column chromatography.

Here, as mentioned above, concanavalin A is a lectin derived from pea (Canavaliaformensiformis), has a subunit molecular weight of 25,572, and forms a dimer or tetramer depending on pH.

In the measurement method of the present invention, a crystal resonator on which concanavalin A is immobilized is used.
Here, the crystal oscillator constitutes a crystal oscillator sensor, and a commercial product can be used as the sensor. Specifically, the crystal oscillator sensor is available from, for example, Initium Co., Ltd.

Here, the fixation of concanavalin A to the crystal oscillator is allowed to stand after dropping a suspension of concanavalin A on the electrode portion of the crystal oscillator sensor. Thereafter, concanavalin A that is not immobilized with distilled water or the like is washed away, and a crystal oscillator in which concanavalin A is immobilized can be prepared.
A specific example of immobilizing concanavalin A on a crystal oscillator is as follows. Concanavalin A is suspended in water to a concentration of 0.5 to 1.0 mg / L, and 50 μL is dropped onto the surface of the crystal oscillator sensor chip. Leave for about 30 minutes. Thereafter, the concanavalin A that has not been immobilized with distilled water is washed away, and a crystal oscillator having the concanavalin A immobilized thereon can be obtained.

In the measurement method of the present invention, the high molecular weight polysaccharide separated and concentrated from the test malt is brought into contact with a crystal oscillator on which concanavalin A is immobilized in a buffer solution. Preferably, at this time, the buffer solution is an acidic buffer solution. And the change of the vibration frequency of the crystal oscillator which arises by the coupling | bonding of high molecular weight polysaccharide and concanavalin A is measured.

A specific procedure here will be described as an example as follows.
As described above, the quartz oscillator sensor on which concanavalin A is immobilized is immersed in a phosphate buffer (for example, 10 mM phosphate buffer containing 0.1 % Tween 20, 1 mg / mL mannose). At this time, the phosphate buffer is desirably adjusted to the acidic side, and preferably has a pH value of around 5.0. A predetermined amount (for example, 5 μL) of a high molecular weight polysaccharide (malt polymer concentrate) adjusted to a predetermined concentration (equivalent amount of mannose, distilled water) is added to the buffer solution, so that concanavalin A and the high molecular weight polysaccharide are added. It reacts with the early coagulation factor contained in. At this time, although there is no restriction | limiting in particular in reaction temperature, For example, it can carry out at room temperature (for example, about 25 degreeC). On the other hand, using a molecular interaction measuring device (for example, AFFINIX Q4 manufactured by Initiam Co., Ltd.) connected to a crystal oscillator sensor, the crystal oscillator generated by the binding of a high molecular weight polysaccharide (particularly precoagulant) and concanavalin A Measure the change (decrease) in vibration frequency. The measurement disclosure time and measurement time can be appropriately selected and set in consideration of the progress of the reaction from the start of the binding reaction and the frequency attenuation state. For example, for 60 seconds after the addition of the high molecular polysaccharide content Measure frequency change. In the present invention, the experiment is performed on the assumption that the frequency change for 60 seconds after the addition is the binding activity with ConA characteristic of the original malt.

According to another aspect of the invention, the measurement method according to the invention comprises:
The method further comprises obtaining the value of the yeast agglutination activity of the test malt from the obtained change in the frequency based on the correlation between the previously obtained value of the yeast agglutination activity and the change in the frequency.
That is, a malt containing a fast-coagulation factor having a known concentration is prepared in advance, and a correlation (for example, a calibration curve) between the value of the yeast aggregating activity and the change in the frequency is obtained. When the unknown test malt is measured, the value of the yeast aggregating activity in the test malt can be calculated from the obtained frequency change result. Conventionally, the method for measuring yeast aggregating activity of malt was able to determine the presence or absence of yeast agglutinating activity and its strength, but it was not sufficient for quantification. According to the present invention, since the value of the yeast aggregating activity of the test malt can be obtained, precise measurement and discrimination can be performed with higher accuracy. This can be said to be extremely effective from the viewpoints of malt production, production of alcoholic beverage products, process control there, and the like.

Similarly, according to another aspect of the present invention, as described above,
A method for quantifying yeast early aggregation factors in malt,
Using the method for measuring yeast aggregating activity of malt according to the present invention, and
From the obtained change in frequency, further comprising obtaining the amount of yeast early aggregation factor in the raw malt based on the correlation between the amount of yeast early aggregation factor obtained in advance and the change in frequency. A method is provided.

In this specification, expression of a value using “about” or “degree” includes a variation in a value that can be allowed by those skilled in the art to achieve the purpose by setting the value. Meaning. For example, it means that a variation within 20%, preferably within 10%, more preferably within 5% of a predetermined value or range can be tolerated.

The present invention will be described in detail by the following examples, but the present invention is not limited thereto.

Outline of Experimental Method 1) Preparation of wort from malt Congress wort (European Brewery Convention, Analytica-EBC (4th ed.), Method 4.4, p.E59, Braurei-) und Getrauke-Rundschau, CH-8047).

2) Extraction of polymer component containing polymer polysaccharide from wort About twice as much ethanol was added to wort, and the precipitated polymer component was separated and recovered as a precipitate (malt polymer extract). This method is called ethanol precipitation. The fast-coagulation factor, which is a high-molecular acid polysaccharide, can be separated as a precipitate by this method.

3) Method for evaluating yeast aggregating activity by fermentation test To synthetic wort prepared with ingredients other than malt (sugar, amino acids, inorganic salts, etc.), polymer components separated from wort and yeast are added and the temperature is 20 ° C. The turbidity after 48 hours of fermentation was evaluated. In this case, absorbance (OD800) at a wavelength of 800 nm was measured using a turbidimeter, and a value DPF (Degree of Premature Flocculation) 48 obtained by subtracting OD800 of the test group from OD800 of the control group was compared. The method for preparing the synthetic wort was as described in Patent Document 1.

4) Concentration of malt polymer extract The malt polymer extract obtained in 2) above was dissolved in a phosphate buffer, and a fraction containing yeast aggregating activity was fractionated by anion exchange column chromatography. The presence or absence of yeast aggregating activity was determined by lyophilizing the fraction and conducting a fermentation test with the addition of the fraction, and determining the progress of fermentation in the same manner as in the above method 3). Fractions containing yeast aggregating activity were collected and lyophilized.

5) Preparation of crystal oscillator sensor with immobilized ConA ConA was suspended in a concentration of 0.5 to 1.0 mg / L, and 50 μL was dropped onto the surface of the crystal oscillator sensor chip and left for 30 minutes. Thereafter, Con A that was not immobilized with distilled water was washed away.

6) Measurement of the binding reaction between the ConA-immobilized crystal oscillator sensor and the malt polymer concentrate (polymer polysaccharide content) It was immersed in 10 mM phosphate buffer containing 1 % Tween 20, 1 mg / mL mannose. Into this, 5 μL of malt polymer concentrate previously prepared to a predetermined concentration (equivalent to mannose, distilled water) was added to initiate the binding reaction. Frequency change (decrease) was measured with an intermolecular interaction measuring device.

The specific experimental procedure and results are described below.

Example 1: Evaluation of yeast aggregating activity contained in malt (conventional method)
Nine malt samples of various origins were prepared as test samples.
First, 320 mL of water was put into a saccharification beaker, and the water temperature was kept at 45 ° C. 50 g of pulverized malt (test sample) pulverized using a disk mill was added thereto, and the mixture was stirred well so as to be uniform. Next, an extraction process of precoagulant factors from the pulverized malt was performed. The temperature program setting for extraction was 45 ° C., hold for 30 minutes → temperature rise at 1 ° C./minute→70° C., hold for 2 hours. After the extraction treatment, this was thoroughly stirred and filtered using filter paper (Toyo Filter Paper No. 2), and 200 mL of this filtrate was accurately collected using a 200 mL graduated cylinder.

The 200 mL of the collected filtrate was boiled by high heat until the liquid volume became half or less, adjusted to 100 mL using distilled water, and filtered again in the same manner. To the obtained filtrate, 200 mL of ethanol was gradually added while stirring, and after stirring for 5 minutes, the mixture was centrifuged and the supernatant was discarded. After boiling water was added to the resulting precipitate to adjust the total amount to 45 mL, the mixture was centrifuged again, and the supernatant (hereinafter sometimes referred to as “malt polymer fraction extract”) was subjected to a fermentation test.

To 160 mL of synthetic wort, 40 mL of a malt polymer fraction extract was added to adjust the pH to 5.7 to obtain a test group. On the other hand, distilled water was used in place of the malt polymer fraction extract, and a pH of 5.7 was prepared as a control.
After adding 0.70 g of brewer's yeast to each of these, put it in a fermentation tube (100 mL volume) with a diameter of 27 mm and ferment at 20 ° C., and after 48 hours, take 2 mL each from 5 cm from the liquid level. OD800 was measured for each. The value of the test group was subtracted from the value of the control group to obtain a DPF (Degree of Premature Flocculation) 48 indicating the degree of yeast aggregation. The DPF 48 was an average value of duplicate tests.

The results were as shown in Table 1.
From the results, the yeast aggregating activity of the malt sample numbers “No. 1” and “No. 3” is weak, and the yeasts of the malts of “No. 2”, “No. 8” and “No. 9” Aggregation activity was found to be significantly stronger.

Example 2: Preparation of a high molecular polysaccharide content (malt polymer concentrate) As a result of use in actual alcohol production, malt having a strong yeast aggregating activity was prepared.
First, 320 mL of water was put into a saccharification beaker, and the water temperature was kept at 45 ° C. 50 g of pulverized malt (test sample) pulverized using a disk mill was added thereto, and the mixture was stirred well so as to be uniform. Next, an extraction process of precoagulant factors from the pulverized malt was performed. The temperature program setting for extraction was 45 ° C., hold for 30 minutes → temperature rise at 1 ° C./minute→70° C., hold for 2 hours. After the extraction treatment, this was thoroughly stirred and filtered using filter paper (Toyo Filter Paper No. 2), and 200 mL of this filtrate was accurately collected using a 200 mL graduated cylinder.

The 200 mL of the collected filtrate was boiled by high heat until the liquid volume became half or less, adjusted to 100 mL using distilled water, and filtered again in the same manner. To the obtained filtrate, 200 mL of ethanol was gradually added while stirring, and after stirring for 5 minutes, the mixture was centrifuged and the supernatant was discarded. Boiling water was added to the resulting precipitate to adjust the total amount to 45 mL. This was transferred to a dialysis tube (molecular weight 6000-8000 cut), and dialyzed against 50 mM phosphate buffer (pH 7.0). The dialysate in the tube was collected and centrifuged to obtain a supernatant. This supernatant was treated with an anion exchange column (Mono-Q, manufactured by GE Healthcare Bioscience), flow rate: 1 mL / min, solution A: 50 mM phosphate buffer (pH 7.0), solution B: 50 mM phosphate buffer. Liquid-Use 0.5M sodium chloride to fractionate B liquid from 0% to 100% and a total elution volume of 20 mL, fraction the first 4 mL of eluate as a flow-through fraction, and then use a fraction collector. 1 mL was used to fractionate into 34 sections. 34 sections were grouped by 2 to 5 sections and reconfigured into 10 samples “Fr.1” to “Fr.10”.

Next, in the same manner as in the fermentation test using synthetic wort in Example 1, except that 1 mL each of “Fr.1” to “Fr.10” was used instead of the malt polymer fraction extract. Then, DPF48 indicating the degree of yeast aggregation was determined.

The results were as shown in Table 2 and FIG.
From the results, it was revealed that the yeast aggregating activity was concentrated to “Fr.4” or “Fr.5” by using the conditions in Example 2 here. In the following, “Fr.4” or “Fr.5” was used as a high molecular polysaccharide content (malt polymer concentrate).

Example 3: Measurement of binding reaction between ConA-immobilized quartz crystal sensor and malt polymer concentrate (polymer polysaccharide content) As a result of the fermentation test of Example 1, the yeast agglutination activity was strong (DPF 48 value: 1. 364 (sample number 2)) and two types of malt samples known to be weak (DPF48 value: 0.202 (sample number 1)) were selected from the samples of Example 1. (In FIG. 2, “cohesion strong” and “cohesion weak” are indicated).
From these two selected malt samples, a polymer polysaccharide (malt polymer concentrate) having a concentration of 10 mg / mL (equivalent to mannose, distilled water) was prepared according to Example 2.

Next, a concanavalin A (ConA) immobilized crystal oscillator sensor was prepared by the following procedure.
Piranha solution (hydrogen peroxide: concentrated sulfuric acid = 1: 3 mixed solution) or 1% SDS solution was dropped on the gold electrode portion of a commercially available quartz oscillator sensor (ceramic sensor chip 27 MHz manufactured by Inishham) to clean the surface. Thereafter, it was washed away with a sufficient amount of distilled water, and the water remaining on the surface was blotted and removed by paper. Next, 50 μL of Con A (obtained from Wako Pure Chemical Industries, Ltd., catalog number 594-02833) suspension prepared to a concentration of 0.5 to 1.0 mg / mL distilled water was dropped onto the gold electrode portion, and this was completely dried. After standing for 30 minutes in a box where the humidity can be maintained (for example, a petri dish with moistened filter paper), the ConA suspension remaining on the gold electrode portion was washed away with a sufficient amount of distilled water. . As a result, a ConA-fixed crystal oscillator sensor was obtained.

Next, the ConA-immobilized crystal oscillator sensor was attached to an intermolecular interaction measuring apparatus (AFFINIX Q4 manufactured by Inishham). Using the thus obtained apparatus, the frequency change of the crystal oscillator resulting from the combination of ConA and the malt polymer concentrate was measured according to the following procedure.

First, it was confirmed that the ConA-immobilized sensor portion was immersed in 500 μL of 10 mM phosphate buffer prepared at a predetermined pH (pH 5.0 or pH 8.0) to stabilize the frequency. Next, malt polymer concentrate Fr. was adjusted to 10 mg / mL (equivalent to mannose, distilled water). 5 5 μL was added, and the change in frequency was measured at 25 ° C. until 3 minutes after the addition.
The measurement results were as shown in FIG.

As shown in the results, it is observed that when the pH of the phosphate buffer is adjusted to 8.0, the vibration frequency of the crystal oscillator sensor is hardly reduced by adding any malt polymer concentrate. It was done.
On the other hand, when the pH of the phosphate buffer is adjusted to 5.0, the vibration frequency rapidly decreases immediately after the addition of the malt polymer concentrate. It was observed that the polymer concentrate obtained from was larger than the polymer concentrate obtained from malt with weak yeast aggregating activity.

These results indicate that the decrease in the vibration frequency is an increase in weight due to the binding of some substance to the sensor surface, so that the binding between ConA and the polysaccharide fractionated from the malt in an acidic environment close to the actual alcoholic fermentation state. Is concentrated in the polymer concentrate extracted from malt having a strong yeast aggregating activity, and contains a large amount of polysaccharides that bind to ConA, and the amount of the polymer concentrate is separated from the malt having a weak yeast aggregating activity. It was thought that more were included. In addition, the relationship between the elapsed time after addition and the frequency change (decrease) is that the frequency decreases rapidly within 1 minute of addition and then gradually decreases. It was considered that the specific binding reaction was completed within 1 minute, and then nonspecific binding proceeded. From this, the vibration frequency change 1 minute after addition of the malt polymer concentrate was defined as ConA binding activity.

Example 4: Correlation between yeast agglutination strength by fermentation test and amount of binding of malt polymer concentrate to ConA-immobilized crystal oscillator sensor Intermolecular interaction measurement apparatus equipped with ConA-immobilized crystal oscillator sensor (Inishham) Of the malt polymer concentrate separated from the malt sample using 9 AFFINIX Q4) and 9 malt samples that were found to have various yeast cohesive strengths from the results of the fermentation test of Example 1. The change in vibration frequency resulting from binding to ConA was measured.
The experimental procedure followed Example 3.
The vibration frequency change was measured 3 to 4 times for each malt polymer concentrate, and the average value was used. The correlation between the yeast aggregating activity strength (DPF) by the fermentation test and the vibration frequency change 1 minute after addition of the malt polymer concentrate was determined.

The result was as shown in FIG. As shown in the results, there was a good correlation between the two.

Claims (7)

1. A method for measuring yeast aggregating activity of malt,
   The vibration of the crystal oscillator produced by the contact between the polymer polysaccharide separated from the test malt and concentrated with the crystal oscillator having immobilized concanavalin A in a buffer solution, and the binding of the polymer polysaccharide and concanavalin A. Measuring the change in frequency.
2. As a process of obtaining a concentrated high molecular polysaccharide content from the test malt,
   Ethanol is added to the wort obtained by saccharifying the test malt to produce an ethanol precipitate containing a high molecular polysaccharide, and the resulting precipitate is dissolved in a buffer solution. The method according to claim 1, further comprising a step of subjecting the concentrated fraction containing a high molecular weight polysaccharide to an exchange column chromatography.
3. The method according to claim 1 or 2, wherein the high molecular weight polysaccharide and a crystal oscillator having immobilized concanavalin A are contacted in an acidic buffer solution.
4. From the obtained change in the frequency, further comprising obtaining the value of the yeast agglutination activity of the test malt based on the correlation between the value of the yeast agglutination activity obtained in advance and the change in the frequency, The method according to any one of claims 1 to 3.
5. A method for quantifying yeast early aggregation factors in malt,
   Using the method according to any one of claims 1 to 3, and
   Obtaining the amount of yeast early aggregation factor in the raw malt from the obtained change in frequency based on the correlation between the amount of yeast early aggregation factor and the change in frequency obtained in advance. ,Method.
6. The malt production process is managed by determining the yeast aggregating activity of the malt raw material, the malt during production, or the produced malt using the method according to any one of claims 1 to 5. , A method for producing malt.
7. A method for producing a fermented alcoholic beverage, comprising: selecting and adjusting a malt raw material to be used by measuring yeast aggregating activity in malt using the method according to any one of claims 1 to 5. Method.

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