JPH07170A - Enzymatic agent for brew - Google Patents

Abstract

PURPOSE:To obtain the subject enzymatic agent giving a raw material-utilizing rate and fermentation speedsame as or higher than those of a rice wine(sake) koji, respectively, reduced in the amino acid content, coloration degree and iron content of the produced liquor, and improved in storability and qualities. CONSTITUTION:This enzymatic agent for brew comprises alpha-amylase, glucoamylase, an acidic protease and an acidic phosphatase.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a brewing enzyme agent used in place of koji in the main fermentation mash during the brewing of sake or shochu.

[0002]

2. Description of the Prior Art About 16 tons of brewing enzyme agent is used in sake brewing annually, but the main purpose of the application is the four-step enzyme agent used for the four-step preparation for sweetening sake. . This four-stage enzyme preparation is prepared by mixing the liquefying enzyme α-amylase and the saccharifying enzyme glucoamylase to adjust the activity. Some of them were mixed with α-amylase alone or transglucosidase instead of glucoamylase to produce non-fermentable oligosaccharides (Japanese Patent Publication No. 55-26).
835) The product exists. However, these four-stage enzyme preparations are intended to dissolve steamed rice at a weak acidity of pH 5 to 6 and at a high temperature of 55 ° C.
-Inappropriate balance of amylase and glucoamylase, and α-amylase of bacterial origin that does not work under acidic conditions of pH 4.0 to 4.5 is used in some cases. I can't do that.

There are 1 and 2 commercial products as enzyme agents for addition to mash, which are sold as substitutes or reinforcements for koji. However, these products are simply a mixture of α-amylase and glucoamylase. When sake is brewed using koji instead of koji during fermentation, dissolution of steamed rice is extremely poor, and fermentation in the early stage of mash is delayed, making it impractical.

Recently, due to a shortage of successors to brewing, it has become an urgent task to make brewing with a weekly work system. In order to realize this problem, it is necessary to use an enzyme preparation for beer having a function equivalent to that of koji, and development of an enzyme preparation for brewing that meets this purpose is desired.

[0005]

[Problems to be Solved by the Invention] In sake brewing, the enzyme balance of various enzymes produced by Aspergillus oryzae affects dissolution of steamed rice starch, saccharification and protein degradation in the main fermentation mash, which is indirect to fermentation of yeast. Affect As a result, the enzyme balance of koji has a great relationship with the quality of sake. Therefore, the present inventors have conducted a study for the purpose of clarifying the role of various enzymes produced by Aspergillus or the amount of activity required for the production of excellent sake, as a result, α-amylase, glucoamylase, acid protease and acid phosphatase. When an attempt was made to brew sake by using an enzyme agent for brewing which was mixed in an optimum balance, it was found that the fermenting agent for brewing had a fermentation rate equal to or higher than that of koji, and the present invention was completed.

The enzyme agent for brewing of the present invention is α-amylase,
A combination of glucoamylase, acid protease and acid phosphatase is essential. It is most preferable to mix them all into one enzyme preparation, but to mix them at the time of use or at the time of preparation, mix them separately or by mixing two or more people as separate enzyme preparations for brewing of the present invention. It can also be an enzymatic agent.

As the enzyme preparation for brewing of the present invention, α-amylase 2 is added as an added amount per 1 g of polished rice, even if it takes the form of a combination of 4 persons, a separate type or a combination of 1 and 2 or more persons.
00 units or more, glucoamylase 40 units or more, acid protease 500 units or more, and acid phosphatase 10
It is preferable to use an enzyme preparation for brewing containing ~ 40 units.

The α-amylase used in the present invention may be any α-amylase, but biodiastase (originating from Aspergillus oryzae) of Amano Pharmaceutical Co., Ltd. can be easily obtained as a commercial product.

Any glucoamylase may be used as the glucoamylase, but the glucoamylase "Amano" (from Aspergillus niger) manufactured by Amano Pharmaceutical Co., Ltd.
Is preferred.

Any acidic protease may be used as the acidic protease, but Neurase (orisose origin) from Amano Pharmaceutical Co., Ltd. is effectively used.

Any acid phosphatase may be used as the acid phosphatase, and an easily available acid phosphatase is commercially available acid phosphatase (P-3627, wheat bran origin).
In addition, acid phosphatase purified by various chromatographies by culturing filamentous fungi such as Aspergillus oryzae, Assergillus niger, Aspergillus kawachi and yeasts such as Saccharomyces cerevisiae in a nutrient medium to produce acid phosphatase and purifying by various chromatography may be used. Furthermore, it was extracted with 0.5% saline solution from red rice bran (rice polishing rate 90 to 100%), and the 0.6 saturated ammonium sulfate precipitation fraction was treated with Biog.
The acid phosphatase having a high activity of acid phosphatase from which the low molecular weight fraction has been removed by gel chromatography using an el P2 column can also be used as the acid phosphatase of the enzyme agent for brewing of the present invention.

The amount of enzyme used in the present invention is 1
200 units or more of α-amylase as an addition amount per g,
Glucoamylase 40 units or more, acid protease 50
It is preferable to use a combination of 0 unit or more and acid phosphatase in an amount of 10 to 40 units, but in reality, the difference in the degree of milling of rice or the fermentation temperature may cause the amount of use to change depending on the region or factory. It is preferable that the combination unit of each enzyme is determined based on the experiment.

Next, experimental examples and examples of the present invention will be shown. The analytical methods and the like used in the present invention are as follows. (1) Analytical method etc. Alcohol, sake, acidity and amino acid content were determined by the National Tax Agency prescribed analysis method. Heavy metals were measured by an atomic absorption method, glucose was measured by a glucose-B-test kit (Wako Pure Chemical Industries, Ltd.), and total sugars were measured by a phenol-sulfuric acid method. The enzyme activity was measured by α-amylase, glucoamylase, and acidic protease by the National Tax Agency prescribed analysis method.
Acid phosphatase is obtained by the TORRIANI method (Bioc
hem Biophys Acta, 38, 460 (1
960)).

[0014]

[Experimental Example 1] Comparative brewing with a commercially available enzyme preparation Enzyme preparation commercially available as a substitute for the koji (Amano Pharmaceutical Co. Ltd.)
Gluc 100) was used as a substitute for all koji, and a comparative test with koji preparation was conducted. The glucoamylase activity of both was set to be the same as 40 units per 1 g of polished rice. As shown in Table 1, three-stage preparation of 100 g of total rice was carried out.
The temperature was kept constant at 15 ° C. and was kept on the 20th day. After completion of the fermentation, solid-liquid separation was performed by centrifugation to analyze the components of the sake liquor.

[0015]

[Table 1]

As shown in FIG. 1, the fermentation process is delayed in the preparation of the enzyme agent as compared with the preparation of koji. As for the alcohol content of the sake produced, 15.6% was produced by koji preparation.
The 0 preparation was as low as 9.5%, and the Gluc 100 enzyme preparation is a product in which the balance of α-amylase and glucoamylase is the same as that of koji, but from this result, the Gluc 100 enzyme preparation alone is used instead of koji. It turns out that it is impossible to do.

[0017]

[Experimental Example 2] Test brewing with addition of acidic protease Next, in addition to α-amylase and glucoamylase, an acidic protease was blended in the same manner as in the koji preparation, and comparative brewing was performed. An enzyme preparation containing 200 units of α-amylase, 40 units of glucoamylase, and 500 units of acidic protease per 1 g of white rice was prepared and compared with koji preparation. The charge composition and the temperature profile were the same as in Experimental Example 1.

As shown in FIG. 2, the fermenting rate in which the acidic protease was added was considerably improved as compared with the commercial enzyme preparation in Experimental Example 1, but the fermenting rate in the early stage of mashing was higher than that in the koji. Was also observed to be significantly delayed.

[0019]

[Experimental Example 3] Test brewing with addition of acid phosphatase Next, koji was charged and comparative brewing was carried out by adding acid phosphatase in addition to the addition of α-amylase, glucoamylase and acid protease. The acid phosphatase used was from Sigma. 200 units of α-amylase, 40 units of glucoamylase, 500 units of acid protease, and 15 units of acid phosphatase were added per 1 g of white rice, and the mixture was compared with koji preparation. The charge composition and the temperature profile were the same as in Experimental Example 1. As shown in FIG. 3, the acid phosphatase-containing preparation improved the fermentation rate delay in the early stage of mash, which was a drawback of the enzyme preparation preparation containing the acidic protease of Experimental Example 2, and improved fermentation throughout the mash. It was observed that the rate was significantly accelerated over koji charging. As is clear from FIG. 3, the koji preparation was 20 days mash, whereas the enzyme preparation was 15 days mash, which shortened the mash days by about 5 days. As shown in Table 2, the produced sake component produced a large amount of alcohol. Although the acidity was a little high, the effect of lowering the amino acidity, coloring degree, and iron content due to the addition of the enzyme agent was recognized.

[0020]

[Table 2]

From the above-mentioned experiments, as the enzyme agent for remedy,
It was found that by adding α-amylase, glucoamylase, acid protease, and acid phosphatase, a fermentation rate equal to or higher than that of koji-mixing can be obtained.

[0022]

[Experimental Example 4] Required amount of acid phosphatase The required amount of acid phosphatase charge activity was examined per 1 g of white rice. First, we tried to obtain acid phosphatase that does not contain other enzyme activities. Aspergillus oryzae RIB-40 was cultivated in liquid in Tchapek medium supplemented with 1% Triton X-100, and the broth was concentrated with an ultrafiltration membrane having a molecular weight cut off of 10,000, and then TSK-g.
Hydrophobic chromatography using an el Phenyl-5PW column was performed to collect the acid phosphatase active fraction, which was concentrated by the above-mentioned ultrafiltration membrane and then TSK-gel G30.
Gel chromatography using a 00SW column was performed to collect the acid phosphatase active fraction. As shown in Table 3, this fraction had almost no enzyme activity other than acid phosphatase, so it was decided to use this fraction.

[0023]

[Table 3]

Next, the required amount of acid phosphatase was examined by one-stage charging with 10 g of hot air dried α rice. White rice 1
Amylase is 250 units and glucoamylase is 4 per g
0 units and 500 units of acid protease were the same in each experimental system, and purified acid phosphatase was 0 and 1, respectively.
It was blended so as to be a series of 0, 20, 40, 80 units. The rate of pumping water was set to 180%, 0.2 ml of Kyokai No. 9 yeast pre-cultured with koji extract was added, and fermentation was performed at 15 ° C. After 20 days of mashing, solid-liquid separation was performed with a centrifuge and the components of the sake liquor were analyzed. As is clear from Table 4, the acid phosphatase in the section of 10 to 40 units has a small residual sugar and a high alcohol content, indicating that fermentation is proceeding smoothly. Particularly, acid phosphatase contains 20 units and 4
A remarkable effect was recognized in the 0 unit category. On the other hand, in the group to which acid phosphatase is not added, the residual sugar is large and the alcohol content is small, which indicates that fermentation is delayed.

[0025]

[Table 4]

[0026]

Example 1 A small preparation test was conducted using an enzyme preparation containing an acid phosphatase derived from Aspergillus oryzae. The charge composition is as shown in Table 1, and the composition of the enzyme agent for koji substitution is α-amylase 2 as the enzyme activity per 1 g of white rice.
The composition was 00 units, 40 units of glucoamylase, 500 units of acid protease, and 20 units of acid phosphatase. The charging method was the same as in Experimental Example 1. Fermentation temperature 15 ℃
A small preparation test was conducted on the 20th. The results are shown in Table 5.
In the same manner as in Experimental Example 3, the enzyme preparation for brewing of the brewing has a higher fermentation rate over the entire mash than the koji preparation, the mash days are shortened by about 5 days, and the steamed rice is dissolved in 54% of the koji preparation. In addition, the enzyme preparation was as high as 61%, the meal ratio was reduced, and the pure alcohol yield was also high. The ingredients of sake liquor were high in alcohol content, low in amino acid content, iron content, and other ingredients related to the quality of sake and in storage, and were as effective as or better than koji brewing.

[0027]

[Table 5]

[0028]

[Example 2] The mixing formulation and the amount of enzyme used were the same as those in Example 1, and the acid phosphatase was derived from Asukagillus oryzae instead of from red bran. The results are summarized in Table 6. As in Example 1, the fermentation rate was increased and the mashing days were shortened by 5 days as compared with the koji preparation.
As for the components of the sake brewed, the alcohol content was as good as that of the acid phosphatase of Aspergillus oryzae, and the baume was quickly broken. The degree of amino acid and the degree of coloring were low. In the use of red rice bran, the most problem is the mixing of iron into sake, but there is no significant difference in the iron content between koji and enzyme preparations, and the present invention is aimed at effective use of red rice bran, which is a by-product of sake brewing. Useful in.

[0029]

[Table 6]

[0030]

The present invention is aimed at the development of an enzyme agent that can be used in place of koji, and as is clear from the experimental examples and examples, the enzyme agent preparation according to the present invention is superior to koji preparation. As a result of the improvement of the dissolution rate of steamed rice, the rate of meal is reduced, the fermentation rate is increased, and the number of mashing days is shortened. Be expected.

[Brief description of drawings]

FIG. 1 Preparation test with a commercially available enzyme preparation (Gluc 100).
This enzyme preparation is a product in which the activity balance of α-amylase and glucoamylase is almost the same as that of koji. These are the results of a charging test in which both enzyme activities were the same as those for koji charging.

FIG. 2 shows the results of a comparative test between the preparation using an enzyme agent containing α-amylase, glucoamylase, and acid protease, and the koji preparation.

FIG. 3: α- using acid phosphatase from Sigma
It is the result of the comparison test of the preparation by the enzyme agent which mix | blended four members, amylase, glucoamylase, acidic protease, and acidic phosphatase, and koji preparation.

FIG. 4 is a result of comparison between the koji preparation and the preparation using an enzyme agent containing four kinds of α-amylase, glucoamylase, acid protease and acid phosphatase, using acid phosphatase prepared from red bran.

Claims (4)

[Claims] 1. An enzyme preparation for brewing comprising α-amylase, glucoamylase, acid protease and acid phosphatase. 2. An enzyme preparation for brewing, which comprises 200 units or more of α-amylase, 40 units or more of glucoamylase, 500 units or more of acid protease and 10 to 40 units of acid phosphatase as an added amount per 1 g of white rice. 3. The acid phosphatase is Aspergillus
Orize, Aspergillus Niger, Aspergillus
The enzyme agent for brewing according to claim 1 or 2, which is obtained from filamentous fungi such as kawachi, yeast such as Saccharomyces cerevisiae, or red bran. 4. Sake or shochu characterized by using the enzyme agent for brewing according to claim 1 or 2 in place of all or part of the rice malt in the main fermentation mash during the production of sake or shochu. Manufacturing method.