JP3357744B2 - Method for measuring beer foam protein and kit used for the method - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to the measurement of a foam protein component, which is one of the main components defining the foam stability of beer, using an antibody specific to the foam protein, thereby obtaining the final product of beer. The present invention relates to a method for evaluating foam retention and foam retention stability in a production process, and also for evaluating beer malt and beer stabilizers.

[0002]

2. Description of the Related Art Beer is obtained from wort by saccharification using malt as a main raw material, and the wort is subjected to main fermentation using yeast. Then, the young beer is subjected to a post-fermentation (sake) process, followed by filtration and bottling. It is manufactured through a process.

[0003] Among the appearance qualities of the beer thus produced, one of the most important properties is foam. In defining this property, it can be roughly classified into two types of foaming and foam persistence (foam retention).

[0004] As a method of evaluating the foam retention, a method utilizing the physicochemical properties of the foam, that is, a method based on the disappearance rate of the foam, a method based on the adhesion to the glass surface, and the like are used. However, since these methods measure the bubbles themselves, the measurement varies widely, and a standard method for completely expressing the bubbles due to the complicated properties of the bubbles has not yet been proposed.

[0005]

Therefore, the present invention focuses on a protein component (foam protein), which is one of the main components that regulate the beer's foam durability, and specifically applies it by applying an immunological technique (ELISA). The purpose of the present invention is to provide a method for quantifying, quickly and accurately measuring foam retention and foam retention stability, and completed the present invention.

[0006]

SUMMARY OF THE INVENTION The present invention uses beer foam protein as an antigen, and performs immunoassay using an antibody obtained from the antigen to produce a final product of beer and a beer sample in a beer production process. A method for measuring the foam retention and foam stability of beer, comprising measuring the foam protein content of beer.

[0007] Foam protein used as an antigen in the assay method of the present invention can be prepared according to the method of Asano et al. (Rept. Res.
Lab. Kirin Brewery Co., Ltd., No.23, p1-13 (198
0)), salting out the final beer product immediately after production using ethanol, ammonium sulfate, etc., to obtain a precipitate, and by various column chromatography methods (reverse phase column chromatography, ion exchange chromatography, gel filtration, etc.) It can be obtained as a foam protein fraction. By SDS-polyacrylamide gel electrophoresis and isoelectric focusing, it was found that the obtained foam protein had a molecular weight of about 41,000 and an isoelectric point of 4.4 to 4.7. Was.

[0008] In addition, in preparing an antibody specific to a foam protein, a conventional method (Shinsei Kagaku Jikken Kozai 1 Protein I, p3
89-397, 1992), antigen (foam protein)
Can be obtained by immunizing an animal and collecting antibodies produced in the living body. The titer of the polyclonal antibody obtained in the present invention was 2000 units / mg.
Met.

[0009] The foam protein contained in the final beer product and the beer sample in the beer production process is measured by an immunoassay using the antibody thus obtained.

[0010] The immunological assay used in the present invention includes:
ELISA (Enzyme-linked Immun)
Ossorbent Assay), including an enzyme immunoassay, a radioimmunoassay, an immunofluorescence method, an immunoturbidimetry, etc., and particularly preferably an ELISA method. As the ELISA method, a standard ELISA method using an enzyme-labeled antibody can be used. In particular, an ELISA method using a solid phase method using avidin / biotin (Protein Nucleic Acid Enzyme, Supplement No. 31, p13-26, 198)
7) is preferred. At this time, avidin-conjugated alkaline phosphatase is used as the labeling enzyme, and biotinylated goat anti-rabbit immunoglobulin is used as the second antibody.

The method for measuring the foam retention of beer according to the present invention will be described below. First, a target antigen (beer sample) is dispensed into a microplate using a sample adsorption buffer, incubated, and the supernatant is removed. Similarly, dispensing the blocking solution, the antibody, the second antibody, and the labeling enzyme in this order, incubating,
Repeat washing. Finally, after dispensing the substrate solution and keeping it at room temperature, the absorbance at 405 nm was measured, and the concentration of the foam protein contained in the beer sample was determined by using a calibration curve separately prepared from a standard dilution series of the foam protein. Can be calculated.

As a sample adsorption buffer, for example, bovine serum albumin (BSA), a PBS (-) buffer containing gelatin or ovalbumin, or the like can be used. Examples of the washing solution include a PBS (−) buffer solution containing NaN ₃ and Tween-20, NaCl and Tween-2.
Tris-buffered saline (TBS) containing 0 can be used. Further, as the blocking solution, for example, 1 to
A PBS (-) buffer containing 3% BSA, a PBS (-) buffer containing 1 to 5% skim milk and the like can be used. The antibody can be used after being diluted to 0.01 to 1.0 μg / ml with a blocking solution. As the labeling enzyme for the second antibody, for example, peroxidase, acid phosphatase, alkaline phosphatase, β-galactosidase and the like are used. In addition, the substrate solution may be used in combination with a labeling enzyme such as p-nitrophenyl phosphate disodium salt (PNPP) when the labeling enzyme is alkaline phosphatase, o-nitrophenyl β-D-galactosidase when β-galactosidase is used. Can be selected appropriately.

[0013] In this way, by measuring the foam protein content in the beer final product and the beer sample in the beer production process, the foam retention and foam retention stability of the beer can be evaluated. In the beer production process, various stabilizers are added in order to maintain the quality, but it is important that such stabilizers do not adversely affect foam retention. The measurement method of the present invention can also be used for determining the efficacy of such a stabilizer.

Since the method of the present invention does not measure the foam itself, but measures the foam protein content in the beer sample, there is little variation due to the measurement. In addition, the method of the present invention uses the foam retention measurement method (SHV) described in Reference Example 1 below.
The results showed good correlation with the measurement results obtained by the method, and it was clarified that the method can be quickly, simply and accurately used for beer retention and foam retention stability tests instead of the conventional method.

Further, the present invention provides a kit used for the method. The kit of the present invention contains an antibody obtained using a foam protein as an antigen. The antibody may be in the form of an antibody diluent diluted in the above diluent, or a lyophilized antibody. The kit of the present invention includes, in addition to the antibody, a 96-well plate, a buffer for sample adsorption, a washing solution, a blocking solution, a substrate solution, a second antibody diluent, and a calibration curve graph.

[0016]

【Example】

Example 1 Preparation of Foam Protein Foam protein was fractionated by the method of Asano et al. (Rept. Res. Lab. K.
irin Brewery Co., Ltd., No. 23, p1-13, (1980)). That is, the final beer product (Suntory Ltd.)
) And ethanol and ammonium sulfate salting out to precipitate the foam protein fraction. After dialysis, reverse phase column chromatography was performed first, and the obtained active fractions were sequentially purified in the order of ion exchange chromatography, gel filtration, and reverse phase HPLC. The purity of the final purified product was evaluated by a single peak in this reverse phase HPLC. As a method for evaluating the active fraction after column chromatography, the method of Asano et al. (Rept. Res. Lab. Kirin Brewery Co., Ltd., No. 2)
3, p1-13, (1980)). That is, from 100 to 3
A 00 mg sample was treated with a 3.6% aqueous ethanol solution (pH 4.
2) Dissolve in 1 L, put 20 ml of this solution into a graduated tube, and shake at 20 ° C. for 5 seconds (400 times / minute). The volume of the foam after shaking is measured, and only the highly active fraction is collected.

Example 2 Preparation of Polyclonal Antibody The foam protein obtained in Example 1 was suspended in distilled water, and an appropriate protein concentration (1 mg / ml) was added using physiological saline (0.9 w / w% NaCl aqueous solution). ). This is mixed with complete Freund's adjuvant at a volume ratio of 3: 2 to prepare a water-in-oil emulsion. Japanese White Rabbit (SPF (special
As an initial immunization, 2 mice (pathogen free), 12 weeks old, female) are injected with 0.5 mg / rabbit equivalent of the antigen into the footpad and subcutaneous flank, respectively. The booster was prepared in the same manner using incomplete Freund's adjuvant to prepare an emulsion containing 0.25 antigen.
A dose of mg / rabbit is injected subcutaneously into the back of the rabbit in several places. Booster immunizations are given at intervals of 1 week to 10 days.
Perform it twice. About 10 days after the final immunization, whole blood is aseptically collected from the ear or carotid artery, and the blood is separated by centrifugation.

Next, the obtained plasma was heated at 56 ° C.
Heat treatment for 0 minutes, and add 0.01M P
BS (-) 0.01 M containing buffer (0.1% NaN ₃
Dilute by adding an equal volume of phosphate buffered saline, pH 7.4). To this, an aqueous solution of saturated ammonium sulfate (pH 7.4) previously prepared with aqueous ammonia is added in an equal volume to the diluent. This was centrifuged at 4 ° C., 30 ° C.
Remove the supernatant for 1 minute at 14000 rpm; 1000 × G). A physiological saline solution is added to the precipitate to completely dissolve the solution, and then the solution is dialyzed or applied to a Sephadex G-25 column to remove the remaining ammonium sulfate. Confirmation of removal of ammonium sulfate
This is performed using a Nessler reagent (manufactured by Nacalai Tesque).

Next, a cooled clearing agent (Friegen, Behrin)
Using gwerke (trichlorotrifluoroethane), an inner volume of the dialysate and an equal amount of a clearing agent are mixed, and the mixture is shaken and centrifuged to separate the inner liquid layer. This defatting operation is repeated three times to obtain a crude IgG fraction (polyclonal antibody fraction).

Example 3 Enzyme immunoassay (ELISA)
Determination of Foam Protein in Beer Samples by the Method) The polyclonal antibody obtained in Example 2 was applied to an ELISA method by a solid phase method using avidin / biotin (Protein Nucleic Acid Enzyme, Supplement No. 31, P13-26, 1
987), and the amount of foam protein contained in beer is determined.

First, a buffer solution for sample adsorption (D containing BSA)
ulbecco's PBS (-) buffer)
The beer sample was dispensed into a 96-well microplate and 3
Incubate at 7 ° C for 1 hour. Separately, for the purpose of preparing a calibration curve of foam protein, a standard dilution series of foam protein is prepared, dispensed into a 96-well microplate using a sample adsorption buffer in the same manner as described above, and incubated.

Next, the supernatant of the standard dilution series and the beer sample was removed, and the washing solution (NaN ₃ and Tween-20) was removed.
Was washed three times with a 0.01-0.1 M PBS (-) buffer solution containing a blocking solution (1-3% BS).
0.01～0.1M PBS containing A and NaN ₃
(-) Buffer solution, pH 7.5 to 8.5) at 200 µl / well
Incubate at 37 ° C. for 30 minutes.

On the other hand, a diluent (0.01M PB containing 0.1-0.5% BSA and 0.05-0.5% NaN ₃₎
S (−), pH 7.5 to 8.5) is used to dilute the polyclonal antibody obtained in Example 2 (antibody diluent).

After washing the above microplate three times with a washing solution, the antibody diluting solution was dispensed at 100 μl / well at 37 ° C.
And incubate for 1 hour.

This was washed three times with a washing solution, and a biotinylated goat anti-rabbit immunoglobulin (1000-fold diluted, manufactured by Amersham) was dispensed as a second antibody in 100 μl / well portions.
Incubate for 1 hour at ° C.

Further, this was similarly washed three times with a washing solution, and avidin-conjugated alkaline phosphatase (diluted 1000-fold, manufactured by Dakopatts) was dispensed in 100 μl / well portions.
Incubate at 7 ° C for 30 minutes.

Subsequently, the plate was further washed with a washing solution for 3 hours.
After washing twice, a substrate solution (p-nitrophenyl phosphate disodium salt (PNPP) diluted with a diethanolamine solution (pH 9.0 to 9.5) containing MgCl ₂ ) was dispensed at 100 μl / well, Room temperature or 37 ° C
And then hold for 10-15 minutes, then 1-3N NaOH 5
The reaction is stopped by dispensing 0 μl, and the absorbance at 405 nm is measured using an automatic absorbance meter.

The measured absorbance values of the standard dilution series are plotted to prepare a calibration curve for foam protein (FIG. 1). By using this calibration curve, the concentration of foam protein contained in each beer sample can be calculated.

Example 4 Absorption Experiment of Polyclonal Antibody Next, an absorption experiment of a foam protein-specific antibody is performed to verify whether the result of the ELISA method is due to an antigen-antibody reaction.

First, the dilution series of the foam protein solution (100 to
0.05 μg / ml), dispensed 100 μl each into a 96-well microplate, and incubate at 37 ° C. for 1 hour. After blocking using a blocking solution, 100 μl of the diluted solution (0.1 μg / ml) of the foam protein polyclonal antibody obtained in Example 2 is dispensed.
As a control negative sample (control), a non-immunized rabbit antibody is diluted to the same concentration as the foam protein polyclonal antibody, and 100 μl is similarly dispensed into a 96-well microplate after blocking. The diluent is
Use a PBS (-) buffer containing 0.1% BSA.

Next, the reacted antibody is detected by ELISA according to Example 3, and the absorbance at 405 nm is measured. The results are shown in FIG.

The control group shows a constant value irrespective of the dilution concentration of the foam protein (antigen), whereas the test group using the foam protein polyclonal antibody shows a lower value from the higher foam protein (antigen) dilution series. The absorbance clearly decreases as one moves. Therefore, it was found that there was an antigen-antibody reaction between the foam antigen and this polyclonal antibody fraction.

Example 5 Comparison of ELISA Method and Conventional Method Evaluating various beer end products purchased from the market by ELI
SA method and conventional foam retention measurement method (SHV method (Reference Example 1)
Reference) and a comparison was made. Furthermore, two types of protein quantification methods (T-N method (Reference Example 2) and high-molecular protein quantification method (Reference Example 3)) which were conventionally performed were performed, and compared with the SHV method.

FIG. 3 shows a comparison result between the ELISA method and the SHV method.
Shown in The ELISA method and the SHV method have a correlation coefficient of 0.85.
7, indicating a very good correlation. Also,
FIG. 4 shows a comparison result between the TN method and the SHV method, and FIG. 5 shows a comparison result between the polymer protein quantification method and the SHV method. TN method and S
The correlation coefficient of the HV method was 0.492, and the correlation coefficient of the high-molecular-weight protein determination method and the SHV method was 0.638, and a high correlation was not obtained. It showed that it could not be quantified specifically.

Example 6 Evaluation of Foam Protein in Beer Malt by ELISA Method Malt (referred to as T malt and C malt), which is used as a raw material of beer and has two different origins, is brewed under the same conditions, and a product beer obtained therefrom Was measured by the ELISA method. At the same time, the foam stability of the product beer was evaluated using the SHV method.

Further, each malt is measured by the TN method and the SN method (the malt is crushed, extracted with warm water, and performed according to the TN method), and the TN of the product beer is measured. The measurement by the method was also performed. The results are shown in Table 1 below.
Shown in

[0037]

[Table 1] From Table 1, the foamed protein amount measured by the ELISA method and the foam retention measured by the SHV method showed that T malt was 50% and about 40% of C malt, respectively. On the other hand, in the TN method and the SN method of malt and product beer, no difference was observed between C / T. Therefore, in the evaluation of foam protein and foam retention based on the difference in beer malt, the present method has higher evaluation accuracy than conventional methods such as the TN method and the SN method, and correlates with the SHV method. It proved to be useful for protein evaluation.

Example 7 Evaluation of Stabilizing Agent by ELISA Method Beers prepared under the same brewing conditions were treated with various stabilizing agents (A to J) under certain conditions, and the resulting beer was subjected to foam protein by ELISA method. Was determined and the stabilizer was evaluated. Table 2 below shows the residual foam protein ratio of each stabilizer when the residual foam protein ratio of Stabilizer A was set to 100.

[0039]

[Table 2] From Table 2, excellent stabilizers (B and H), which do not adversely affect the beer foam retention, could be selected accurately.

Reference Example 1 Foam retention measurement method (SHV-Schaumh
aftvermogen-method This is a method to evaluate the retention of beer foam by measuring the degree of foaming and the adhesion of foam to the glass surface. That is, the entire amount of beer is poured into the measuring cylinder at average for 20 seconds at a time, and the amount of foam remaining on the measuring cylinder wall 30 minutes after the pouring is measured using a planimeter to quantitatively evaluate the foam retention. I do. At this time, the unit of foam retention is expressed in cm ² (see Chemistry and Biology, p. 1354, (1975)).

Reference Example 2 Nitrogen determination method (TN method; semi-micro Kjeldahl method) The nitrogen content of a beer sample is analyzed with a Keltec total nitrogen analyzer.

When the sample is decomposed with concentrated sulfuric acid, the nitrogen content changes to an ammonia state, and is converted into ammonium sulfate by the reaction with sulfuric acid. When a NaOH solution is added thereto and then heated, ammonia is liberated, and this is trapped with boric acid. The amount of nitrogen is determined by titrating this with sulfuric acid of known concentration (see European Brewery Convention, Analytica-EBC, 4th edition, (1987)).

Reference Example 3 High-molecular protein assay (Bradford
Method) Add Bradford reagent (Bio-Rad) to the sample and add 595 n
The absorbance at m is measured to determine the amount of protein. That is, Bradfo
Coomassie Brilliant Blue G-25 in rd reagent
The 0 (CBBG-250) anion binds to the ammonia group in the sample, and the absorption maximum of triphenylmethane in the CBBG-250 molecule shifts from 465 nm to 595 nm. The amount of protein in the sample is calculated using a calibration curve drawn from the absorbance at 595 nm of the protein standard solution (ASBC J
ournal, 40 (2), pp. 46-50, (1981)).

[0044]

EFFECT OF THE INVENTION Specific Determination of Foam Protein in Beer
By applying the LISA method, it has become possible to quickly, easily and accurately measure one of the most important properties of the appearance quality of beer, such as foam protein. as a result,
It has become possible to measure foam proteins in beer samples in the final beer product and in the beer production process, and to very accurately predict the selection of the best beer malt and stabilizer.

[Brief description of the drawings]

FIG. 1 shows a calibration curve of foam protein by an ELISA method.

FIG. 2 shows a control (antibody of a non-immunized rabbit)
2 shows the results of an experiment on the absorption of foam protein with the use of two rabbits, respectively. The horizontal axis is the concentration of the foam antigen expressed on a logarithmic scale (mg protein
/ l), and the vertical axis indicates the absorbance (405 nm) after 10 minutes.

FIG. 3 shows the relationship between the ELISA method and the SHV method.

FIG. 4 shows the relationship between the TN method and the SHV method.

FIG. 5 shows the relationship between the high molecular weight protein quantification method and the SHV method.

──────────────────────────────────────────────────続 き Continuing on the front page (72) Kazuo Nakatani 1-1-1 Wakayamadai, Shimamoto-cho, Mishima-gun, Osaka Prefecture Beer Research Laboratories, Suntory Limited (72) Inventor Yugo Terano Wakayama, Shimamoto-cho, Mishima-gun, Osaka No. 1-1, Daiichi Suntory Ltd. Biomedical Research Laboratory (56) References JP-A-7-318558 (JP, A) JP-A-5-268955 (JP, A) JP-A-6-46881 (JP, A) JP-A-6-105698 (JP, A) JP-A-6-311894 (JP, A) JP-A-10-306100 (JP, A) J Am Soc Brow Chem, Vol. 51, No. 1 (1993) p. 21-28 (58) Fields investigated (Int. Cl. ⁷ , DB name) G01N 33/53 G01N 33/14

Claims (4)

(57) [Claims] The present invention relates to a method for measuring the content of foam protein in a beer final product and a beer sample in a beer production process by an immunoassay using a foam protein as an antigen and an antibody obtained from the antigen. A method for measuring the foam stability and foam stability of beer. 2. The method according to claim 1, wherein the immunological assay is an ELISA method. 3. A molecular weight of about 41,000 and an isoelectric point of 4.4 to 4.
7. The method according to claim 1, wherein the foam protein of No. 7 is used as an antigen. 4. A method using a foam protein as an antigen, an antibody obtained from the antigen, a 96-well plate, a buffer for adsorbing a sample,
A kit for measuring beer retention and foam retention stability, comprising one or more selected from a washing solution, a blocking solution, a substrate solution, a second antibody diluent, and a calibration curve graph.