JPS63164877A - Preparation of brewed liquor and apparatus therefor - Google Patents

Abstract

PURPOSE:To prepare a high-quality brewed liquor having excellent storage stability, by contacting brewed liquor with an immobilized protease after filtering the liquor to remove dregs from the liquor, thereby inactivating the enzyme in a short time without deteriorating the excellent flavor and taste of refined liquor. CONSTITUTION:One or more proteases selected from pepsin, trypsin, papain and heat-resistant protease are immobilized on a polymeric insoluble carrier having nonionic hydrophilic functional groups on the surface e.g. by physical adsorption process and the carrier supporting the enzyme is packed in a small- capacity column. A brewed liquor finished with brewing step and filtration step for the removal of dregs and, if necessary, with aseptic treatment step with a filter for precise filtration, aseptic and deproteinizing step by ultrafiltration or sterilization step with ultraviolet ray is passed through the column and made to contact with the carrier for a short time.

Description

DETAILED DESCRIPTION OF THE INVENTION (Industrial Field of Application) The present invention relates to a method and apparatus for producing brewed liquor that improves the stability of the quality of brewed liquor.

(Prior Art) Sake, which is a brewed alcoholic beverage, has traditionally been pasteurized after the brewing process and the filtering process.

This pasteurization is done to inactivate enzymes (α-amylase, glucoamylase, acidic protease, acidic carboxypeptidase, etc.) remaining in the sake and to sterilize yeast, lactic acids, and especially hiochi bacteria in order to maintain the storage stability of sake. It has been done.

However, since this pasteurization process heats the raw sake, it is stable in storage, but it has the drawback of not degrading the flavor of the raw sake and causing a change in taste. If the temperature of the pasteurization is low, as with raw sake storage, glucose and amino acids may increase due to residual enzymes, impairing the quality of the sake, and may lead to spoilage due to hiochi bacteria.

Against this background, in recent years, unpasteurized sake with a mild taste has become commercially available.
Since the remaining enzymes are not inactivated, low-temperature storage and low-temperature distribution are recommended.

(Problems to be Solved by the Invention) In view of the current situation, the present invention deactivates enzyme activity without impairing the flavor of sake, unlike enzyme deactivation methods such as heat treatment, and improves the quality of brewed sake. The purpose is to increase the stability over time.

(Means for Solving the Problems) In order to solve the above problems, the method for producing brewed liquor of the present invention is characterized by including a step of contact-treating brewed liquor with an immobilized protease. .

Furthermore, the column according to the present invention used in the above production method is
The following is characterized in that a cylindrical container with an inlet at one end and an outlet at the other end is filled with a polymeric carrier having a nonionic and hydrophilic functional group on the surface of which a protease is immobilized. , the method for producing brewed liquor of the present invention and the column used therein will be specifically explained.

The proteolytic enzymes to be immobilized include α-amylase derived from koji, glucoamylase, and glucoamylase, which are used during the sake brewing process.
A substance with low substrate specificity that is capable of deactivating acidic protease, acidic carboxypeptidase, and other enzymes is also used. In addition, in order to reduce process costs, it is preferable to use a material that is inexpensive and can be stably supplied. Pepsin, trypsin.

Papain and the like satisfy these conditions.

Furthermore, proteases derived from bacteria are also included, but especially Penicillium dupontii, which has excellent stability in degrading activity.
Thermostable proteases such as 1014 are preferred.

Immobilized proteolytic enzymes have the enzymes immobilized on polymer-insoluble carriers.

The insoluble carriers used here include synthetic polymers, polymer polysaccharides, and inorganic carriers. Examples of synthetic polymer carriers include nylon, polyvinyl alcohol, and polyacrylamide. Examples of polymeric polysaccharide-based carriers include Sephadex, Sepharose [Pharmacia Co., Ltd.: trade name Kotserofin [Seikagaku Kogyo Co., Ltd.: trade name], cross-linked dextran-based, cross-linked agarose-based, cellulose-based, Glass, silica gel, etc. can be used as the inorganic carrier.

A carrier that does not adsorb flavor components and does not change the flavor is required, and carriers having nonionic and hydrophilic surfaces such as glucosyl groups and hydroxyl groups are desirable.

The proteases can be immobilized on these polymeric carriers using conventional methods such as physical adsorption, ionic bonding, and covalent bonding (“Protein φ Nucleic Acid/Enzyme J V
ol, 31. No. 3 (198B), P220-229
reference). In the physical adsorption method, enzymes are adsorbed onto a carrier with a hydrophobic surface using hydrophobic interactions, and in the ionic bonding method, cationic or anionic functional groups are bonded to the carrier surface, and enzymes are absorbed into ions by The covalent bonding method is
This is a method in which an epoxy group, a saluccinyl group, a tosyl group, etc. are bonded to a carrier using a reactive reagent, and an enzyme is further bonded covalently.

Sake is generally pasteurized to be stored in tanks, and then reheated during the bottling process to sterilize it in what is called a bottle can. Since it changes, it is necessary to approve the approval process again.

According to the present invention, sake is passed through a column packed with a polymer carrier on which proteolytic enzymes are immobilized. This allows the enzyme to be deactivated without losing aroma. Moreover, since the reaction time required for deactivating the enzyme using the column is only a short time, a large-capacity column is not required.

It is also possible to pass through the column after heating to 30°C to 60°C. In this case, the higher the temperature of the treated sake, the faster the enzyme reaction will proceed, so there is an advantage that the capacity of the immobilized enzyme column can be even smaller. however,
The activity of the immobilized enzyme decreases more quickly than when the heating is not performed.

Further, according to the present invention, the sake after the brewing process and the filtering process can be passed through the column after the sterilization process using ultraviolet rays. Even in this case, it is possible to deactivate the enzyme without losing the aroma. It is preferable to use the ultraviolet light having a wavelength that is particularly effective against bacteria.

Furthermore, according to the present invention, the sake after the brewing process and the strain filtration process can be passed through the column after the sterilization process using a filter that performs microfiltration, or after the sterilization and protein removal process using ultrafiltration. It is. In this case as well, the enzyme can be deactivated without losing the aroma.

In addition, it is also possible to perform the above-mentioned sterilization step, sterilization step, and sterilization/removal protein step after the column passage step.

(Example) Figure 1 is a diagram for explaining the present invention in detail, and Figure (1) shows that the process of contact-treating brewed liquor for enzyme deactivation consists of passing through an enzyme immobilization column. Examples are shown in (2), (3), and (4) in the same figure.

(5) shows an example including a sterilization process by ultraviolet irradiation, a sterilization process by microfiltration, a sterilization and protein removal process by ultrafiltration, and a pasteurization process before the protease-immobilized column passage process. Figure 2 shows, for reference, a conventional process that does not include the step of passing through a protease-immobilized column.

Sake is fed by a pump to a column packed with a polymer carrier on which proteolytic enzymes are immobilized, and the enzyme components contained therein are deactivated.

LLI A 0.1% glutaraldehyde aqueous solution pH 7.0 of 100100O was added to 100100O of Cellulofine GC-700 (trade name), which is a polymeric material, and the mixture was gradually stirred with a stirrer at room temperature for 24 hours. The polymer carrier thus obtained was filtered through a glass filter, and the procedure of pouring into 2000 m of pure water and washing with stirring was repeated until there was no glutaraldehyde in the filtrate. This polymer carrier was added to 5r1Mole of pH 8,9.
Pepsin 10, a proteolytic enzyme, is added to the phosphate buffer solution of
The protease was immobilized on the polymer carrier by mixing the protease into a solution containing 0 mg of the protease and gradually stirring with a stirrer at 5° C. for 20 hours.

The polymer carrier on which the protease thus produced was immobilized was placed in a cylindrical container with an inner diameter of 22 mm and a length of 750 mm, with an inlet at one end and an outlet at the other end, as shown in Figure 3. Fill a column consisting of the following and wash with pure water until there is no protein in the effluent.

In order to investigate the deactivation effect of glycolytic enzymes using the column packed with the polymeric carrier on which the proteases immobilized prepared as described above were used, we used a commercially available pre-heated
When samples IA and B are passed through the column (
The enzyme activity was measured after passing through the column) and without passing through the column (before passing through the column).

Il) Sample A without passing through the column of this example.

B was dialyzed with a dialysis tube, 0.1 mA of each sample was added to 5 m+1 of a 0.1% starch solution at pH 15, and the amount of glucose produced was measured by reacting at 40°C for 20 minutes. Activity was calculated.

Further, after heating samples A and B to 36° C., they were each passed through the above column at a flow rate of 10 mQ/win. The amount of glucose produced was measured by the same method as above for Samples A and H that were not passed through the column, and the glycolytic enzyme activity was calculated. Furthermore, the peptidase activity of the above samples was measured as described below in Table 1. Glycolytic enzyme activity, peptidase activity (unit "unit").

As shown in Table 1, when the column of this example is not passed, a considerable amount of glycolytic enzyme activity and peptidase activity remains, whereas when the column of this example is passed, , glycolytic enzyme activity and peptidase activity were not detected. The unit of glycolytic enzyme activity is "unit", and 1 unit is 1 sake
It is the amount of activity that liberates 1 μMol of glucose per minute at 40° C. from a 00.1% starch solution in 11Q. However, the unit of peptidase activity is Tyr, μMol.
/h, and the following method was used for its measurement. That is, in+Mol of bebutide (Cbz-L-Glu-
Sake 1.5+ dialyzed to 1.5 mN containing L-Ty
wl was added, and the reaction was carried out in an L-shaped test tube for 30'C11 hours with gentle shaking using a mono-set shaker, and tyrosine liberated in the supernatant was quantified by the ninhydrin method. The activity unit was expressed as tyrosine μ gate 01 produced by a 30'C, 1 hour leap reaction.

Practical Example J- A column packed with a polymer carrier on which a protease was immobilized was produced in the same manner as in Example 1 except that trypsin was used instead of pepsin as the protease.

In this case, the enzymatic activity of the glycolytic enzyme was measured in the same manner as in Example 1, without passing sample C after pasteurizing the sake into the rum and sample C' before pasteurizing it through the column of this example. did.

Further, after heating samples c and c' to 35° C., they were each passed through the above column at a flow rate of 10 ml/s+in. Samples c and c' that are not allowed to pass through the column
The amount of free glucose was measured by the same method as above for measuring, and the enzyme activity was calculated.

Table 2 Glycolytic enzyme activity (unit "unit") As shown in Table 2, the results show that the polymeric materials with no enzyme activity detected were passed through the column of this example. 1000a of Cellulofine GC-700 (trade name) + ICZ pH 9 (7) 0.1% aqueous solution of Ehychlorohydrin 1000% was added and left at room temperature for 2 hours. The polymer carrier thus obtained was washed in the same manner as in Example 1. 1) 8 on this polymer carrier
A solution in which 10011 g of papain, a protease, was dissolved in 5mM phosphate buffer solution of No. 9 was added, and the solution was gradually stirred with a stirrer at 5° C. for 20 hours to immobilize the protease on the polymer carrier. This was packed into a column and washed repeatedly with pure water until no protein was detected in the effluent.

Sample C after burning used in Example 2 was added to the protease-immobilized polymer carrier-packed column produced in this way.
1.Sample C'1 before pasteurization 1Sample C'' which was further pasteurized (80'C5 minutes) of Sample C, and Sample CIl+ which was obtained by irradiating Sample C with ultraviolet rays were passed through each sample to measure the glycolytic enzyme activity and to evaluate its taste. A panel of 10 people examined their taste and impressions, and the results shown in Table 3 were obtained.
5: Excellent, 4: Slightly excellent, 3: Average, 2:
The average value of the evaluation (slightly inferior, 1: inferior) is shown.

As is clear from these results, it can be seen that the enzymes were completely deactivated by passing each sample through the column. In addition, Sample C', which does not include the pasteurization process after H-making, has excellent taste and aroma, while Sample C11, which includes the pasteurization process, has less aroma.
The taste is also slightly lower than that of '. Sample CI 1 1, which was irradiated with ultraviolet rays without pasteurization, was slightly inferior to sample C'', but it had a fragrance, and the method using ultraviolet rays was superior to the sterilization method using pasteurization.

In the above embodiments, the case of refined sake was explained as the brewed liquor, but it goes without saying that the present invention can also be applied to other brewed liquors that require enzyme deactivation.

(Effects of the Invention) As detailed above, according to the present invention, the brewed liquor after the brewing process and the filtering process is passed through a column packed with a polymer carrier on which a proteolytic enzyme is immobilized. By doing so, it is possible to deactivate the enzyme without losing the aroma. Moreover, since enzyme deactivation using a column requires only a short time to close during the reaction, a large-capacity column is not required, and the apparatus can be constructed in a small size.

In addition, when passing through the above column after heating the sake to 30°C to 50°C, the higher the temperature of the sake to be processed, the faster the enzyme reaction proceeds, so the capacity of the immobilized protease column is even smaller. There is an advantage that it can be done. However, the activity of the immobilized protease decreases more quickly than in the case without heating.

Furthermore, the present invention performs a sterilization treatment using ultraviolet rays, a sterilization treatment by microfiltration, or a sterilization and protein removal treatment by ultrafiltration on the brewed liquor after the brewing process and the filtering process, and passes it through the above column after the filtration process. In this case, it is also possible to deactivate the remaining enzyme without losing the aroma.

Furthermore, when raw sake is distributed to the market, it has conventionally required low-temperature storage, which has been complicated to handle and expensive to distribute, but with the present invention, transportation has become easier and costs have also been reduced. It has the advantage of being cheaper.

Furthermore, since the present invention can omit the pasteurization step, the cost required for pasteurization is reduced.

[Brief explanation of the drawing]

Fig. 1 is a diagram for explaining the present invention in detail, and Fig. 1 (1) shows an example in which the step of contact-treating brewed liquor for enzyme deactivation consists of a step of passing through a protease-immobilized column. (2) and (3) in the same figure. (4) and (5) are, respectively, a sterilization process by ultraviolet irradiation, a sterilization process by microfiltration, and a sterilization/removal protein removal process by ultrafiltration before the process of passing through the protease-immobilized column in (1) of the same figure. An example including a process and a pasteurization process is shown. FIG. 2 shows a conventional process that does not include the step of passing through a protease-immobilized column. FIG. 3 shows an embodiment of a column according to the invention. Patent Applicant 1) Satoshi Tan Figure 1 Figure 2 Figure 3 Proteolytic enzyme-immobilized polymer carrier procedural amendment (1st chapter) April 22, 1988 Commissioner of the Japan Patent Office Black 1) Akira Yu 1, Indication of the case Patent Application No. 61-315764 2, Title of Invention Method and Apparatus for Producing Brewed Alcohol 3, Relationship with the Amendment Case Patent Applicant Address Mr. 5-2-15 Kihirocho, Wakayama City
Name 1) Satoshi Tan 4, Deputy
Man

Claims (10)

[Claims] (1) A method for producing brewed liquor, which comprises a step of contact-treating brewed liquor with an immobilized protease. (2) The brewed liquor according to claim (1), wherein the step of contact-treating the brewed liquor is a step of passing the brewed liquor through a column filled with an immobilized protease. manufacturing method. (3) The method for producing a brewed liquor according to claim (1), wherein the step of contact-treating the brewed liquor is a contact treatment with an immobilized protease using a batch method. (4) Claims (1) and (2) characterized in that the protease to be immobilized is any one of pepsin, trypsin, papain, thermostable protease, or a combination thereof. or (3). (5) Claims (1), (2), or (2) include a filtration step either before or after the contact treatment with the immobilized protease, or both. (3) The method for producing brewed liquor described in section (3). (6) Claim No. (1) characterized in that a sterilization step using a microfilter is included either before or after the contact treatment with the immobilized proteolytic enzyme, or both.
2. The method for producing brewed liquor according to item 2, item 2, or item 3. (7) Claims (1), (2), or 3) The method for producing brewed liquor described in section 3). (8) Claim (1) characterized in that a sterilization and protein removal step by ultrafiltration is included either before or after the contact treatment with the immobilized proteolytic enzyme, or both.
2. The method for producing brewed liquor according to item 2, item 2, or item 3. (9) A cylindrical container with an inlet at one end and an outlet at the other end has a protease immobilized on its surface and is filled with a polymeric carrier having nonionic and hydrophilic functional groups. A column that deactivates enzymes in brewed liquor. (10) The column according to claim (9), wherein the polymer carrier contains a polymer polysaccharide as a main component.