KR20220086129A - Fermenting agent for Yakju using Lichtheimia ramosa 83-1, preparation method and use thereof - Google Patents

Abstract

The present invention relates to a fermenting agent for medicinal liquor using Lichtemia ramosa 83-1, a preparation method and use thereof, and Lich. The ramosa 83-1 medicinal liquor strain has excellent starch decomposition ability and at the same time low protease activity. It has less concerns about problems such as off-flavor caused by amino acid components, so it is possible to manufacture high-quality medicinal liquor with superior sensory properties. Bar has a useful effect of securing the seed bacteria for the high quality of domestic medicinal liquor, and also has a more excellent fermenting agent for medicinal liquor by optimizing detailed conditions such as the culture characteristics, inoculum, raw material, imperial period, excipients, and drying method of the mold for medicinal liquor As it is provided, there is a useful effect of providing high-quality yakju by improving the quality of domestic yakju.

Description

Fermenting agent for medicinal liquor using Lichtheimia ramosa 83-1, manufacturing method and use thereof

The present invention relates to a fermenting agent for medicinal liquor using Lychtemia ramosa 83-1, a method for preparing the same, and a use thereof.

Traditional liquor, a representative fermented liquor in Korea, is a fermented beverage in which carbohydrates are decomposed by microorganisms to produce alcohol and other components, the most representative of which are yakju and takju. Yakju and takju as fermented liquors are traditionally manufactured by fermenting grains and yeast, and are fermented liquors in parallel with saccharification and alcoholic fermentation. After brewing, takju or makgeolli is takju or makgeolli that is cloudy in appearance by sieving sulphur, and yakju is obtained by filtering only the clear liquid from sulphur.

Yeast, which is used as a fermenting agent during the manufacturing process of yakju, is manufactured by grinding grains such as wheat, barley, and rice, adding about 25% of water to it, kneading it, molding it, and fermenting it naturally. Therefore, unlike the entry into Japan, which only plays the role of a saccharifying agent, fermented liquor is manufactured using koji grown by naturally epiphyte of various microorganisms derived from the environment. The characteristics of koji differ depending on the climate, climate, and manufacturing environment of the region where it is produced. The types and contents of sugars, organic acids and amino acids produced by mold and yeast contained in koji change, and various volatile fragrance components also appear in various ways. . After all, the diversity of yeast microorganisms shows differences in enzyme activity, alcohol fermentation power, organic acid and free amino acid content, etc., and this affects the quality characteristics such as taste, aroma, and color of traditional fermented wine.

In particular, various chemical reactions such as oxidation, hydrolysis, and dehydration merger are involved during the aging of alcoholic beverages. Some carbonyl compounds and pyrazines generated by the Mylard reaction between sugar and amino acids cause off-flavor and burnt smell that occurs when alcoholic beverages are stored for a long time, and change the color of alcoholic beverages. In addition, the indole compound and the Harman compound produced by photooxidation of amino acids change the color of the liquor. Polysulfide compounds made from amino acid changes may give alcoholic beverages an unpleasant odor.

In the case of Japanese sake made from rice and Korea, isovaleric acid, ethyl-isovaleric acid, or 1,1-diethoxy-3-butane, etc., have been identified as off-flavor components generated during long-term aging. Isoamyl alcohol, a higher alcohol of sake, is converted to isovaleraldehyde by enzymatic (alcohol dehydrogenase) oxidation derived from a fermenting agent. It is oxidized again to isovaleric acid, and an ethyl group is bound thereto to produce ethyl-isovaleraldehyde. Thereafter, when further oxidation proceeds, 1, 1-diethoxy-3-butane is produced. Therefore, in order to control this odor during the aging process of sake, in Japan, various methods such as milling rice to remove precursor materials, adsorbing off-flavor components with activated charcoal prior to aging, or developing yeast that produces less senile flavor Efforts are being made (Ko JS. Fun and easy to understand brewing Engineering. Yuhansa, Seoul, Korea. pp. 218-219 (2008)).

Various starchy raw materials such as rice and wheat are used in Korean yakju, and yeast contains various microorganisms and enzymes. For example, amino acids produced by peptidase present in mold are used as precursors of fusel oil during fermentation. If a large amount of amino acid components remain after fermentation, it acts as a flavor component that makes the taste too thick, and the remaining amino acid components react with the sugars of the fermentation broth during distribution, causing a mylard reaction. Coloring by Maillard reaction is a factor that lowers the sensory characteristics and quality of Yakju.

Therefore, in order to provide high-quality medicinal liquor, it is important to select a fungal strain with low protease activity so that it has excellent starch decomposition ability and at the same time reduces amino acid components that cause taste and odor. It is necessary to develop an excellent fermenting agent for medicinal liquor. By developing and using such a fermenting agent for yakju, the brewing quality of yakju is ultimately improved, so that high-quality yakju can be provided.

Under the above background, the present inventors, in order to solve the problem of quality improvement of domestic medicinal liquor and to provide high-quality medicinal liquor, have excellent starch decomposition ability and at the same time have low protease activity. , Lichtheimia ramosa 83-1 (hereinafter referred to as Lich. ramosa 83-1) of the present invention is an excellent strain with low protease activity that causes a problem of off-flavor while having excellent starch degradability among several domestic and foreign fungal strains. By optimizing detailed conditions such as inoculation amount, steamed rice raw material, imperial period, excipients, drying method, etc. suitable for manufacturing fermenting agent for medicinal liquor, an excellent fermenting agent for medicinal liquor was developed, and the present invention was completed.

An object of the present invention is to solve the problem of improving the quality of domestic medicinal liquor and to provide high-quality medicinal liquor, specifically, selecting an excellent yeast strain for yakju with high starch degradability and low protease activity, and an inoculum suitable for manufacturing fermenting agent for yakju It is to provide an excellent fermenting agent for medicinal liquor by optimizing detailed conditions such as raw materials for steamed rice, imperial period, excipients, and drying method.

In order to achieve the above object,

The present invention is Lich. ramosa 83-1 Provides a fungal strain for medicinal use.

In addition, the present invention is described in Lich. It provides a method for preparing a seed strain for medicinal use, comprising the step of culturing the ramosa 83-1 strain.

Furthermore, the present invention comprises the steps of: 1) inoculating the seed strain for medicinal liquor into steamed rice; and

2) step of imperializing steamed rice inoculated with seed bacteria; provides a method for producing a fermented agent for medicinal wine comprising.

In addition, the present invention provides a fermenting agent for yakju prepared by the method for producing the fermenting agent for yakju.

Furthermore, the present invention provides a method for manufacturing high-quality yakju using the fermenting agent for yakju.

Lich. The ramosa 83-1 medicinal liquor strain has excellent starch-decomposing ability and low protease activity. It is a mold for medicinal liquor that has little concern about problems such as off-flavor caused by amino acid components, and can produce high-quality medicinal liquor with superior sensory properties. Bar has a useful effect of securing the seed bacteria for the high quality of domestic medicinal liquor, and also has a more excellent fermenting agent for medicinal liquor by optimizing detailed conditions such as the culture characteristics, inoculum, raw material, imperial period, excipients, and drying method of the mold for medicinal liquor As it is provided, there is a useful effect of providing high-quality yakju by improving the quality of domestic yakju.

1 is a step-by-step flowchart of a method for manufacturing a fermentation agent for medicinal liquor of an example.
Figure 2 is a graph showing the change in moisture content according to the water immersion time for two types of rice in Samgwang-mi or Jeonju No. 615.
3 shows Asp. oryzae OF5-20 or Lich. It is a graph showing the change in the temperature of the solid seeds sown in steamed rice according to the imperial period in the production of fermented medicinal liquor using two types of mold of ramosa 83-1.
4 is a photograph of each device used in two types of drying methods: hot air drying and low temperature blowing drying used for drying solid seed bacteria.

Hereinafter, the present invention will be described in detail.

The present invention is Lich. ramosa 83-1 Provides a fungal strain for medicinal use.

Lich. The fungal strain ramosa 83-1 for medicinal liquor is a strain that has been patent deposited with accession number KACC 93350P, and is characterized by having enzyme activity suitable for the manufacture of medicinal liquor. The enzyme activity suitable for the preparation of the medicinal liquor can be understood to mean an excellent starch-decomposing activity, for example, as a strain with excellent glucoamylase activity, by finely decomposing the starch of grain raw materials such as rice to produce sugar It can be understood as a strain capable of smoothly supplying sugar to microorganisms that ferment alcohol, such as yeast.

See also Lich. The fungal strain ramosa 83-1 for medicinal liquor has low proteolytic enzyme (peptidase) activity and thus has low proteolytic power, and is suitable for the manufacture of medicinal liquor. If the proteolytic activity is high and the proteolytic power is high, problems such as an unwanted odor from the generated amino acids may be caused. For example, amino acids are used as precursors of fusel oil during alcoholic fermentation, giving off too strong a fragrance and making the taste worse. This can reduce the quality of the liquor. In addition, the residual amino acid components react with the saccharides of the fermentation broth during distribution, and are a factor that lowers the sensory characteristics and quality of yakju due to coloring by the Maillard reaction.

Therefore, for the production of high-quality medicinal liquor, it is preferable that the starch-degrading activity is excellent but the proteolytic activity is low, and the Lich. The fungal strain ramosa 83-1 is a strain with high glucoamylase activity showing starch-degrading activity and low peptidase activity showing proteolytic activity, and is suitable for manufacturing medicinal liquor.

In an embodiment of the present invention, glucoamylase, α-amylase, and peptidase enzyme activities were investigated using 4 types of media for the strains possessed by the Rural Development Administration and 14 strains of other domestic and foreign fungal strains. , Lich. It was confirmed that the ramosa 83-1 fungal strain had a high glucoamylase activity among 14 weeks, and almost no peptidase activity below the detection value (see Tables 2, 3 and 4).

In addition, the present invention is described in Lich. It provides a method for preparing a seed strain for medicinal use, comprising the step of culturing the ramosa 83-1 strain.

Lich. As described above, the ramosa 83-1 strain has excellent starch-degrading activity and low proteolytic activity, and is a suitable strain for medicinal use, and redundant descriptions will be omitted.

The culturing step is described in Lich. Any culture under conditions capable of growing the ramosa 83-1 strain is included in the present invention, for example, it may be prepared as a liquid seed by liquid culture.

The medium used in the culture is Lich. Any medium under conditions capable of growing the ramosa 83-1 strain is included in the present invention, and any commercially available medium or medium used for the purpose of conventional bacterial culture can be used, for example, REB, MEB, and It may be cultured with one or more media selected from the group consisting of PDB.

The culturing temperature is included as long as it is a temperature at which bacteria can grow, for example, it may be cultured at 20 to 30 ℃, 24 to 30 ℃, 25 to 30 ℃, 27 to 30 ℃, or about 28 ℃. The culture period may be, for example, culturing for 2 to 10 days, 3 to 10 days, 4 to 10 days, 4 to 9 days, 4 to 8 days, 5 to 7 days, or about 6 days. The culture method and apparatus may use all those commonly used for bacterial culture, for example, may be cultured with shaking, and may be cultured with shaking at 100 to 150 rpm or about 120 rpm with a shaking incubator.

Furthermore, the present invention

1) the step of inoculating the seed bacterium for medicinal liquor into Jeungjami; and

2) step of imperializing steamed rice inoculated with seed bacteria; provides a method for producing a fermentation agent for medicinal liquor comprising.

The seed for medicinal use is the Lich. As a seed strain obtained by culturing the ramosa 83-1 strain, the Lich. As described above, the ramosa 83-1 strain has excellent starch-degrading activity and low proteolytic activity, and is a strain suitable for medicinal use, and redundant description will be omitted.

The steamed rice is obtained by immersing rice in water, and preferably has an appropriate moisture content to be suitable for manufacturing a fermenting agent for yakju. The steamed rice has, for example, a moisture content of at least 29%, 29-40%, 29-38%, 29-35% or about 30%. When the moisture content is less than the lower limit range, Lich. Proliferation of ramosa 83-1 bacteria is difficult, and if the moisture content exceeds the upper limit, contamination of various bacteria such as bacteria may occur. In addition, the steamed rice may be to achieve the above-described moisture content by adjusting the soaking time for each rice variety.

The steamed rice may be, for example, steamed rice using three tailings or Jeonju No. 615 rice, and steamed rice using three tailings may be steamed rice prepared by immersion in water for 100 minutes or more, 120 minutes or more, or 150 minutes or more, preferably 120 It is steamed rice prepared by immersion in water for more than a minute (2 hours). Meanwhile, steamed rice using Jeonju No. 615 may be steamed rice prepared by immersion in water for 10 minutes or more, 15 minutes or more, 10 to 30 minutes or about 10 minutes, and preferably steamed rice prepared by immersion in water for 10 minutes or more. In the case of water immersion in the above-mentioned range, the moisture content of steamed rice can be achieved as above-mentioned 29% or more, 29 to 40%, 29 to 38%, 29 to 35%, or about 30%. If the moisture content exceeds the upper limit range, contamination of various germs such as bacteria may occur. In addition, the steamed rice may be to achieve the above-described moisture content by adjusting the soaking time for each rice variety.

In an embodiment of the present invention, as a result of preparing steamed rice by immersing three tailings in water, it was confirmed that it is desirable to soak for at least 120 minutes in order to achieve a moisture content of about 30%, and Jeonju No. 615 was immersed to prepare steamed rice. As a result, it was confirmed that in order to achieve a moisture content of 30%, it is preferable to immerse in water for 10 minutes or more (see Table 7).

The inoculation of step 1) is 1 to 10%, 1 to 9%, 2 to 10%, 2 to 8%, 2 to 7%, 3 to 8%, 3 to 6%, 2 to It may be a step of inoculating steamed rice at a concentration of 4% or about 2%. There is a problem in that it is difficult to achieve sufficient enzymatic activity when inoculated below the lower limit range, and even when inoculated beyond the upper limit range, sufficient enzymatic activity cannot be achieved or unnecessary protease hyperactivity may be caused.

In an embodiment of the present invention, as a result of examining the seed inoculum suitable for manufacturing a medicinal fermenting agent, when Samgwangmi and Jeonju No. 615 were used, the starch degrading activity was excellent at 2% inoculation, and at the same time the proteolytic activity could be kept low. , it was confirmed that it is most preferable to inoculate the seed with 2% (see Tables 8 and 9).

The imperial in step 2) may be to imperial the steamed rice inoculated with the seed at 30 to 40 ℃, 32 to 38 ℃, 34 to 36 ℃ or about 35 ℃. In addition, the empire of step 2 may be imperial for 2 to 10 days, 3 to 9 days, 3 to 6 days, 6 to 9 days, or about 6 days or about 9 days after seed inoculation. There is a problem in that it is difficult to achieve sufficient enzyme activity when it is impregnated below the lower limit, and when it is impregnated beyond the upper limit, there is a fear that unnecessary protease hyperactivity may be caused.

In an embodiment of the present invention, as a result of examining changes in enzyme activity according to the imperial period, when three tailings were used, the glucoamylase activity was the best on the 9th day of the empire, and the α-amylase activity was the best on the 6th day of the empire, and the protease It was confirmed that the activity was kept lowest at day 6 (see Table 10). On the other hand, when Jeonju No. 615 was used, the glucoamylase activity was the best on the 9th day, the α-amylase activity was the best on the 6th day, and it was confirmed that the protease activity was maintained at the lowest level on the 9th day, It was confirmed that it is preferable to incubate for 9 days in order to keep the protein-degrading activity low while having excellent starch-degrading activity (see Table 11).

In step 1), an excipient may be further added, and the excipient may be used for the purpose of protecting and maintaining the enzymatic activity of the spawn. The excipient includes all commercially available excipients, for example, at least one selected from the group consisting of skim milk, maltodextrin, cyclodextrin and lactomeal. can be used

In an embodiment of the present invention, as a result of examining changes in the enzymatic activity and spore count of bacteria according to the type of excipient, it was confirmed that skim milk or cyclodextrin is preferable, and cyclodextrin is more preferable. (see Tables 12 and 13).

The excipient may be added in an amount of 1 to 7%, 2 to 7%, 2 to 7%, 1 to 5%, 1 to 3%, or 5 to 7% by weight of steamed rice. When an excipient is used below the lower limit, there is a problem that it is insufficient to achieve the effect of protecting the desired enzyme activity, there is a concern that the enzyme activity may be hindered even when used in excess of the upper limit, and unnecessary protease hyperactivity There is a possibility that it may be caused.

The amount of the excipient added may be different depending on the type of excipient, for example, when the excipient is skim milk, 5 to 7% may be used in terms of starch degrading activity, and less than 5% in terms of protease activity , 1 to 5% or about 1% may be used. On the other hand, when the excipient is cyclodextrin, 3 to 7%, or about 5% may be used in terms of starch degrading activity, and 3 to 7%, or about 5% may be used in terms of protease activity. According to the above range, by using an excipient, it is possible to increase the starch decomposition activity and at the same time lower the glycoprotein decomposition activity, so it is suitable for manufacturing a fermentation agent for medicinal liquor.

In an embodiment of the present invention, as a result of examining the optimal amount of excipient added, 5 to 7% of cyclodextrin relative to the weight of steamed rice is preferable, and 5 to 7% is preferable in terms of starch degrading activity such as α-amylase and glucoamylase, It was confirmed that 5% is preferable in terms of low protease activity (see Table 14).

The manufacturing method may further include the step of drying the solid spawn obtained after the imperialization of step 2). The drying is included in the present invention as long as it is a drying method that does not inactivate enzyme activity, and may be a drying method with less microbial contamination such as bacteria. For example, the drying may be low-temperature blowing drying or hot-air drying, preferably low-temperature blowing drying. The low-temperature blow drying may be, for example, blow-drying at a temperature of 20 to 40 ℃, 30 to 40 ℃ or about 35 ℃, 10 to 40 hours, 12 to 36 hours, 16 to 30 hours, 20 to 28 hours Or it may be blow-drying for about 24 hours. On the other hand, the hot air drying may be, for example, hot air drying at a temperature of 40 to 60 ℃, 45 to 550 ℃ or about 50 ℃, 5 to 25 hours, 10 to 20 hours, 12 to 18 hours, 14 to 16 It may be hot air drying for an hour or about 15 hours.

In a specific example of the present invention, as a result of examining the enzyme activity of the fermenting agent for medicinal liquor according to the drying method, it was confirmed that the enzymatic activity inactivation range was smaller in the blowing drying method compared to the hot air drying method (see Table 15).

In an embodiment of the present invention, as a result of examining the degree of microbial contamination of the fermenting agent for medicinal liquor according to the drying method, the degree of contamination of ^both methods is maintained as low as 102 level, but preferably the method of blowing drying compared to hot air drying has a lower microbial contamination level was confirmed to be suitable (see Table 16).

Furthermore, the present invention provides a fermenting agent for yakju prepared by the method for producing the fermenting agent for yakju.

The fermenting agent for medicinal wine of the present invention is Lich. As a fermenting agent prepared by inoculating a seed strain obtained by culturing the ramosa 83-1 strain, the Lich. As described above, the ramosa 83-1 strain has excellent starch-degrading activity and low proteolytic activity, and is a suitable strain for medicinal use, and redundant descriptions will be omitted.

The fermenting agent for yakju has excellent starch decomposition activity, so that microorganisms that ferment alcohol such as yeast can supply sugar necessary for manufacturing yakju, as well as improve the taste of yakju, and above all, the fermenting agent for yakju is proteolytic It produces less amino acids due to low activity, less fusel oil and oily ingredients, less off-flavor, less concern about discoloration due to Maillard reaction, and improved sensory properties of higher quality A medicinal liquor may be provided.

In addition, the present invention provides a method for manufacturing high-quality yakju using the fermenting agent for yakju.

The fermenting agent for medicinal liquor has excellent starch-decomposing activity and low proteolytic activity, thereby improving the sensory characteristics of yakju to provide high-quality yakju, and redundant description will be omitted.

Hereinafter, the present invention will be described in detail by way of Examples and Experimental Examples.

However, the following Examples and Experimental Examples are merely illustrative of the present invention, and the content of the present invention is not limited to the following Examples and Experimental Examples.

<Experimental Example 1> Selection of fungal starters with excellent enzyme activity such as saccharification power

Fermented processed food and 14 weeks of mold stored in a cryogenic freezer (-80℃) were used (Table 1). The 14 weeks of the prepared mold were cultured in 4 types of mold media (REB, MEB, MERB (REB+MEB), PDB), and molds with excellent saccharification ability were selected.

Culture: △flask → medium 50 mL add → autoclave → spore counter (X10 ⁶ cells/mL) inoculated → shaking incubation (28℃, 120 rpm, 6 day) (Vision , Korea)

Crude enzyme solution : Broth → Centrifuge (3,000 rpm, 10 min) → sup. → 3 enzyme activity

α-amylase: Investigation of a liquefaction enzyme that hydrolyzes α-1,4 glycoside bonds in glucose-linked polysaccharides

No. Species/Strains No. Species/Strains One Lichtheimia ramosa / 83-1 8 Rhizopus delema r / 26-4 2 Lichtheimia ramosa / 69-1 9 Rhizopus delemar / 58-8 3 Lichtheimia ramosa / 78-1 10 Aspergillus terreus / 71-10 4 Lichtheimia ramosa / 81-9 11 Aspergillus luchuensis / 74-5 5 Lichtheimia ramosa / CN044 12 Aspergillus oryzae / OF5-20 6 Lichtheimia ramosa / 82-1 13 Aspergillus oryzae / SFO 7 Rhizopus oryzae / 71-9 14 Aspergillus oryzae / HJJ

<1-1> α-amylase activity

The activity of α-amylase, which hydrolyzes α-1,4 glycoside bonds in glucose-linked polysaccharides, was investigated. Table 2 shows the results of α-amylase activity of these fungi according to the type of medium after growth in 4 types of medium for mold culture.

No. Species/Strains REB
(U/mL) MEB
(U/mL) MERB
(U/mL) PDB
(U/mL) One Lichtheimia ramosa / 83-1 0.01 N.D. N.D. N.D. 2 Lichtheimia ramosa / 69-1 N.D. N.D. 0.03 N.D. 3 Lichtheimia ramosa / 78-1 0.04 1.06 0.01 N.D. 4 Lichtheimia ramosa / 81-9 0.03 1.16 N.D. N.D. 5 Lichtheimia ramosa / CN044 0.30 0.17 N.D. N.D. 6 Lichtheimia ramosa / 82-1 N.D. 0.02 0.08 N.D. 7 Rhizopus oryzae / 71-9 N.D. N.D. N.D. N.D. 8 Rhizopus delemar / 26-4 N.D. 0.10 0.29 N.D. 9 Rhizopus delemar / 58-8 N.D. 0.08 0.15 N.D. 10 Aspergillus terreus / 71-10 N.D. 5.21 2.01 N.D. 11 Aspergillus luchuensis / 74-5 0.46 0.25 1.38 N.D. 12 Aspergillus oryzae / OF5-20 N.D. 2.44 5.19 N.D. 13 Aspergillus oryzae / SFO N.D. 0.06 1.06 N.D. 14 Aspergillus oryzae / HJJ N.D. 29.51 49.67 N.D.

(N.D.: not detected)

As confirmed in Table 2, Lich. It was confirmed that ramosa 83-1 had low α-amylase activity on the medium.

<1-2> Glucoamylase activity

The glucoamylase activity of cleaving α-1, 4/α-1, 6 glycoside bonds from non-reducing ends to glucose was investigated. Table 3 shows the results of glucoamylase activity of these fungi according to the type of medium after growth in 4 types of medium for mold culture.

No. Species/Strains REB
(U/mL) MEB
(U/mL) MERB
(U/mL) PDB
(U/mL) One Lichtheimia ramosa / 83-1 0.32 17.81 5.30 0.10 2 Lichtheimia ramosa / 69-1 0.23 N.D. 6.89 0.25 3 Lichtheimia ramosa / 78-1 0.01 6.58 N.D. 0.17 4 Lichtheimia ramosa / 81-9 0.05 0.25 5.00 1.61 5 Lichtheimia ramosa / CN044 2.25 0.44 0.04 0.15 6 Lichtheimia ramosa / 82-1 0.46 0.03 0.10 N.D. 7 Rhizopus oryzae / 71-9 0.05 12.07 10.61 0.40 8 Rhizopus delemar / 26-4 0.01 15.88 13.65 4.67 9 Rhizopus delemar / 58-8 0.01 18.53 16.56 3.61 10 Aspergillus terreus / 71-10 0.36 N.D. 8.84 N.D. 11 Aspergillus luchuensis / 74-5 5.53 0.60 19.48 0.27 12 Aspergillus oryzae / OF5-20 0.85 N.D. 10.74 0.02 13 Aspergillus oryzae / SFO 0.64 2.16 2.51 0.10 14 Aspergillus oryzae / HJJ 0.23 2.57 2.55 0.16

(N.D.: not detected)

As can be seen in Table 3, glucoamylase is an enzyme that saccharifies starch and directly affects microorganisms that use monosaccharides such as glucose as a carbon source, most of which are described in Lich. It was confirmed that ramosa 83-1 had high glucoamylase activity on the medium.

<1-3> Protease activity

Table 4 shows the results of protease activity of these fungi according to the type of medium after growth in 4 types of medium for mold culture.

No. Species/Strains REB
(U/mL) MEB
(U/mL) MERB
(U/mL) PDB
(U/mL) One Lichtheimia ramosa / 83-1 N.D. N.D. N.D. N.D. 2 Lichtheimia ramosa / 69-1 9.18 N.D. N.D. 1.69 3 Lichtheimia ramosa / 78-1 33.35 N.D. 5.33 8.06 4 Lichtheimia ramosa / 81-9 0.96 N.D. N.D. 6.87 5 Lichtheimia ramosa / CN044 9.34 N.D. N.D. 0.33 6 Lichtheimia ramosa / 82-1 156.33 N.D. 30.20 17.28 7 Rhizopus oryzae / 71-9 0.65 N.D. N.D. 1.64 8 Rhizopus delemar / 26-4 0.65 N.D. N.D. 0.34 9 Rhizopus delemar / 58-8 0.33 N.D. N.D. 0.98 10 Aspergillus terreus / 71-10 13.87 9.74 23.10 7.87 11 Aspergillus luchuensis / 74-5 294.15 30.60 728.93 17.35 12 Aspergillus oryzae / OF5-20 27.35 53.59 124.48 14.31 13 Aspergillus oryzae / SFO 7.76 1223.46 1892.08 32.33 14 Aspergillus oryzae / HJJ 0.96 2184.95 1914.97 113.76

(N.D.: not detected)

As confirmed in Table 4, Lich. It was confirmed that ramosa 83-1 had no protease activity on the medium.

Accordingly, Lich. It was confirmed that ramosa 83-1 had excellent saccharification power, and at the same time, had low proteolytic power, which deteriorated the taste and aroma of yakju, or caused problems with off-flavor.

<Experimental Example 2> Measurement of dry cell mass by selection strain

After liquid culture (28°C, 5 days) of the selected 5 types of mold, filtration (Filter paper No. 2, Advantec Co., Japan) was performed to separate the cells and the culture medium. 8 g of the cells recovered for each type of mold were dried at 105° C. (Vision Scientific Co., Korea) for 8 hours, and the dry weight was measured to evaluate the cell productivity, and the results are shown in Table 5.

No. Species/Strains Cell mass (g) One Lichtheimia ramosa / 83-1 1.465 2 Aspergillus luchuensis / 74-5 1.277 3 Aspergillus oryzae / OF5-20 1.425 4 Aspergillus oryzae / SFO 1.324 5 Aspergillus oryzae / HJJ 1.323

As identified in Table 5, Lich. It was confirmed that ramosa 83-1 had excellent cell productivity.

<Experimental Example 3> Enzyme activity of fermentation agents by strain

As shown in Fig. 1, grain (rice, 200 g) was semi-refined, immersed, water-saving, and steamed for 5 types of mold selected to select a strain with high saccharification power, and then, 1% of the liquid broth of each mold was added. was inoculated with and inoculated at 35° C. for 3 days, and the enzyme activity of the fermentation agent for each fungal strain was investigated, and the results are shown in Table 6.

No. Species/Strains α-amylase
(Unit/g) glucoamylase
(Unit/g) protease
(Unit/g) One Lich. ramosa / 83-1 5.72 ±0.65 79.45 ± 3.05 279.93 ± 24.6 2 Asp. luchuensis / 74-5 59.31 ± 5.20 418.50 ± 0.87 4868.73 ± 11.4 3 Asp. oryzae / OF5-20 67.55 ± 12.93 44.51 ± 0.74 1,893.40 ± 6.97 4 Asp. oryzae / SFO 169.09 ± 8.15 9.81 ± 0.49 500.69 ± 8.14 5 Asp. oryzae / HJJ 144.05 ± 0.84 7.95 ± 0.31 1,047.41 ± 16.3

(N.D.: not detected)

As confirmed in Table 6, as a result of evaluating the enzyme activity with a fermenting agent for each fungal strain, Lich. It is confirmed that ramosa 83-1 has the best glucoamylase activity, which indicates the saccharification power of the fungus, and the lowest protease activity that makes the taste and aroma of yakju bad or causes the problem of off-flavor.

<Experimental Example 4> Development of solid spawn production technology using mold

<4-1> Optimal immersion time setting for raw materials for solid spawn production

Moisture content according to immersion time of raw rice was investigated using two types of rice (Samgwangmi, Jeonju 615).

Samgwangmi: Suwon 361 + Miryang 101 hybrid, mid-late, hard rice

Jeonju 615: Suwon 542 improved variety, early variety, powdery rice with coarsely arranged starch grains (garumi)

In order to set the optimum immersion time for each raw material, the water absorption rate was set as the optimum immersion time when the water content reached the saturated water content by immersion and the water content was 30% or more, and the results are shown in Table 7 and FIG. 2 .

hour
(minute) three tailings hour
(minute) Jeonju 615 Moisture absorption (%) Moisture content (%) Moisture absorption (%) Moisture content (%) 0 - 11.5 0 - 8.46 40 21.491 28.49 5 17.792 25.50 60 22.297 28.32 10 28.103 32.17 80 22.476 28.42 15 30.997 33.19 100 24.023 29.02 20 33.353 34.31 120 23.239 29.35 25 34.609 34.75 140 24.120 29.47 30 36.214 35.01 160 24.607 29.63

As can be seen in Table 7 and FIG. 2, since the moisture absorption rate for each variety is different, it is necessary to control the water immersion time by measuring the moisture absorption rate when the raw rice is different. If the moisture content is low, it is difficult for the fungal spawn to grow, and if the moisture content is high, contamination of various bacteria may occur. Therefore, the moisture content of steamed rice is preferably 30% or more.

Therefore, it is preferable to immerse the samgwangmi (light rice) used in the present invention for 2 hours or more, and for Jeonju 615 (bunjil rice/garumi), it is necessary to keep the immersion time of 10 minutes or less to obtain good steamed rice (godubap). have.

<4-2> Selection of inoculation amount of solid spawn production technology

Each of the selected fungi was inoculated in the bran medium with the same number of spores (10 ⁸ cells/mL) and cultured at 28°C and 120 rpm for 5 days. After filtration of the liquid culture medium to collect the sediment and the separated liquid, seed the godubap at each liquid spawn concentration (2, 4, 6%). Table 8 and Table 9 are shown.

Grain raw materials: Light rice (Samgwang, 2018, Nonsan) and Bunjil rice (Jeonju 615, 2019, Jeonbuk) are used.

Mold: used Lichtheimia ramosa 83-1, Aspergillus oryzae OF5-20

Activity (Unit/g) AO OF5-20
Inoculation (%) three tailings Jeonju 615 3 days 6 days 9 days 3 days 6 days 9 days α-amylase 2 572.23 594.08 565.27 566.27 591.10 634.81 4 575.21 662.63 626.87 582.16 601.04 601.04 6 604.02 687.47 627.86 568.25 575.21 652.70 Glucoamylase 2 845.65 949.50 1,137.42 798.67 947.03 1,117.64 4 823.40 1,251.17 1,592.39 773.94 838.23 1,174.51 6 986.59 1,036.04 1,275.89 882.74 942.08 1,105.28 Proteases 2 4,995.34 2,555.22 594.08 2,785.42 3,291.86 2,762.40 4 2,486.16 4,419.84 662.63 1,2684.02 3,936.42 2,04.78 6 3,153.74 3,798.30 687.47 2,716.36 2,555.22 6,307.48

As shown in Table 8, Asp. With respect to the solid spawn prepared with oryzae OF5-20, α-amylase activity was 6%>4%> 2% for samtail rice and 6%> 2% for Jeonju 615 when imperial for 9 days. > 4%. On the other hand, in the case of imperial for 9 days, glucoamylase activity was 4%>6%> 2% in Sam tailings and 4%>2%> 6% in Jeonju 615. On the other hand, when imperial for 9 days, protease activity was 2%<4%<6% in Samgwangmi and 4%<2%<6% in Jeonju 615.

Therefore, Asp. As a result of examining the inoculation amount according to the enzymatic activity of the solid spawn prepared with oryzae OF5-20, α-amylase and glucoamylase showed high activity at 4% inoculation at 3% tailings and Jeonju 615, and protease ( protease) is confirmed to have low activity at 2-4%.

Activity (Unit/g) LR 83-1
Inoculation (%) three tailings Jeonju 615 3 days 6 days 9 days 3 days 6 days 9 days α-amylase 2 6.40 8.68 8.96 15.34 20.47 16.69 4 3.38 7.19 8.38 12.74 15.28 14.98 6 5.29 9.02 19.73 7.37 11.05 15.82 Glucoamylase 2 114.11 125.73 151.33 179.89 196.90 307.60 4 71.71 114.61 140.94 164.56 178.16 258.64 6 101.38 130.19 148.48 110.28 141.15 222.04 Proteases 2 7.25 12.66 4.60 24.17 4.60 1.15 4 5.76 35.68 57.55 63.31 1.15 9.43 6 11.51 19.57 40.29 2.30 43.74 12.66

As shown in Table 9, Lich. For the solid spawn prepared with ramosa 83-1, α-amylase activity was 6%> 2%> 4% in the case of imperialization for 9 days, and 2%> 6% in Jeonju 615. > 4%. On the other hand, in the case of imperial for 9 days, glucoamylase activity was 2%> 6%> 4% in samtail rice and 2%> 4%> 6% in Jeonju 615. On the other hand, when imperial for 9 days, protease activity was 2%<6%<4% in Samgwangmi and 2%<4%<6% in Jeonju 615.

Thus, Lich. As a result of examining the amount of inoculation according to the enzymatic activity of the solid spawn prepared with ramosa 83-1, α-amylase and glucoamylase were highly active when inoculated with 6% or 2% in sambal and Jeonju 615. , and protease is confirmed to have the lowest activity at 2%.

From the above experimental results, it can be seen that there is a difference in enzyme activity depending on the mold used, the amount of inoculation and the taste of the raw material for the solid spores for medicinal liquor, and for the production of solid spores suitable for use in the manufacture of medicinal liquor, the above-mentioned mold and inoculum And it is necessary to set and control the conditions of raw rice.

<4-3> Changes in the temperature of solid spawn according to the period of the empire

Inoculate 2 types of steamed rice (10 kg each) with the optimal concentration (2, 4, 6%) of the selected fungal starter cultured in bran medium. Japan as Yaegaki). Changes in the temperature of the solid seed bacteria sown in steamed rice during the 9-day imperial period using the two selected types of mold were investigated, and the results are shown in FIG. 3 .

Mold: used Lichtheimia ramosa 83-1, Aspergillus oryzae OF5-20

Three tailings: Rice (10 kg) → Semi (5 times) → Water soaking (3 hours) → Water saving (1 hour) → Steam steaming (50 minutes) → Cooling (35℃) → Inoculation of each mold liquid → Empire (35℃, 9 days)

Jeonju No. 615 : Rice (10 kg) → Semi (3 times) → Water immersion (10 minutes) → Water saving (1 hour) → Steam steaming (50 minutes) → Cooling (35℃) → Inoculation of each mold liquid → Empire (35℃) , 9 days)

As can be seen in FIG. 3, microbial activity appears from the beginning of the empire (day 0), and the highest product temperature rose to 41° C. while exhibiting the most vigorous growth activity on the second day of the empire, and from the 3rd day, the product temperature change produced while the mold was growing was constant. It was confirmed that the product temperature dropped after 5 days, indicating that artificial adjustment was necessary. On the other hand, Lich. ramosa 83-1 is relatively Asp. Compared to oryzae OF5-20, the temperature showed a tendency to rise slightly later.

<4-4> Changes in Enzyme Activity according to the Imperial Period

The selected fungus was sown on steamed rice, and the enzymatic activity of the solid spawn according to these imperial periods was investigated, and the results are shown in Tables 10 and 11.

Mold: used Lichtheimia ramosa 83-1, Aspergillus oryzae OF5-20

Steamed rice: Use Samgwang rice or Jeonju No. 615

strain Empire period
(days) α-amylase (unit/g) Glucoamylase
(unit/g) Proteases
(unit/g) AO
OF5-20 3 572.23 845.65 4,995.34 6 594.08 949.50 2,555.22 9 565.27 1,137.42 3,867.36 LR
83-1 3 6.40 114.11 7.25 6 8.68 125.73 12.66 9 8.96 151.33 47.33

As can be seen in Table 10, when three tailings were used, the activity of α-amylase was Asp. oryzae OF5-20 is Empire 6th, Lich. The ramosa 83-1 was highest on the 9th day of the empire. Glucoamylase activity was determined by Asp. oryzae OF5-20 and Lich. Both ramosa 83-1 showed the highest activity on day 9 of imperial. On the other hand, when the protease activity is high, the protein is decomposed and converted into amino acids, and at this time, since the taste difference of the main substance is determined, it is advantageous that the protein decomposition power is low.

Therefore, the imperial period as a whole is α-amylase (α-amylase), glucoamylase (glucoamylase) activity is excellent, and the imperial 6 days is suitable for not too high proteolytic power.

strain Empire period
(days) α-amylase (unit/g) Glucoamylase
(unit/g) Proteases
(unit/g) AO
OF5-20 3 566.27 798.67 2,785.42 6 591.10 947.03 3,291.86 9 634.81 1,117.64 2,762.40 LR
83-1 3 15.34 179.89 24.17 6 16.79 199.54 4.60 9 16.69 307.60 1.15

As confirmed in Table 11, when Jeonju No. 615 was used, α-amylase activity was determined by Asp. oryzae OF5-20 was highest on Empire 9th, Lich. The activity of ramosa 83-1 increased with the passage of the empire period, and was highest on the 6th day of the empire, and then decreased again thereafter. As for the glucoamylase activity, the enzyme activity increased as the imperial period elapsed, and the highest activity was exhibited on the 9th day of the imperial period. Protease (protease) activity showed the lowest activity on the 9th day of imperial.

As a whole, 9 days of imperial empire with excellent α-amylase and glucoamylase activity and low proteolytic power are appropriate. It is confirmed that there is a difference in enzyme activity depending on the rice raw material used even when the same strain is used.

<4-5> Selection of excipients for solid spawns

In order to select a customized excipient that can maintain the activity without inactivating the enzyme of the solid spawn upon storage, 5% of one of the four types of excipients (skim milk, maltodextrin, cyclodextrin, lactomeal) was added to the solid spawn at 35 °C. It was dried for 24 hours, and the change in enzyme activity according to the type of excipient was investigated, and the results are shown in Table 12.

strain excipient α-amylase Glucoamylase Proteases AO
OF5-20 skim milk 828.54 969.28 7,665.66 maltodextrin 766.94 897.58 7251.3 cyclodextrin 709.32 904.99 5,662.92 lactomeal 793.77 949.50 5,478.76 LR
83-1 skim milk 1.69 21.64 26.00 maltodextrin 1.71 23.61 44.00 cyclodextrin 1.65 25.22 24.17 lactomeal 1.35 22.01 20.00

(Unit: Unit/g)

As confirmed in Table 12, α-amylase (α-amylase), glucoamylase (glucoamylase) activity is Asp. oryzae OF5-20 maintains the highest activity when skim milk is used as an excipient, and Lich. It was confirmed that ramosa 83-1 had the highest activity when cyclodextrin was used as an excipient.

<4-6> Change in the number of spores due to excipient treatment

Table 13 shows the results of measuring the change in the number of spores according to the type of excipient.

To measure mold spore count, add 2-3 drops of 1% methylene blue to 10 mL of 5% tween solution to 1 g of solid spawn prepared using each of 4 different excipients, dilute it, and measure the number of spores with a hemocytometer. was humble.

excipient Spore counts AO OF5-20 LR 83-1 skim milk 1.18 X 10 ⁸ 0.65 X 10 ⁸ maltodextrin 0.93 X 10 ⁸ 0.69 X 10 ⁸ cyclodextrin 1.86 X 10 ⁸ 0.71 X 10 ⁸ lactomeal 1.82 X 10 ⁸ 0.61 X 10 ⁸

As confirmed in Table 13, Asp. oryzae OF5-20 or Lich. It was confirmed that the highest number of spores was maintained when cyclodextrin was used as an excipient for all of the solid spawns using ramosa 83-1.

Therefore, Asp. oryzae OF5-20 or Lich. Cyclodextrin was found to be the most suitable excipient in terms of enzymatic activity and spore count of solid spawn using ramosa 83-1.

<4-7> Selection of optimal concentration of excipients for solid seed bacteria

Each enzyme activity was analyzed to select the appropriate concentration (0, 1, 3, 5, 7%) of the two selected excipients (cyclodextrin, skim milk) suitable for the storage capacity of the solid spawn, and the results are shown in Table 14. .

excipient strain density(%) α-amylase
(Unit/g) Glucoamylase (Unit/g) Proteases
(Unit/g) skim
milk AO OF5-20 0 565.27 949.50 3,867.36 One 608.98 872.85 6,169.36 3 692.43 1,045.94 7,596.60 5 828.54 969.28 7,665.66 7 714.29 1,053.35 7,596.60 cycleo
dextrin LR
83-1 0 0.08 15.58 47.33 One 4.09 28.06 43.74 3 1.35 27.82 59.85 5 4.17 32.64 11.51 7 2.03 29.67 46.04

As confirmed in Table 14, in terms of α-amylase activity, Asp. For solid spawn using oryzae OF5-20, it is most preferable to use skim milk at a concentration of 5% as an excipient, and Lich. For solid spawn using ramosa 83-1, it is most preferable to use cyclodextrin at a concentration of 5% as an excipient. In terms of glucoamylase activity, Asp. For solid spawn using oryzae OF5-20, it is most preferable to use skim milk at a concentration of 7% as an excipient, and Lich. For solid spawn using ramosa 83-1, it is most preferable to use cyclodextrin as an excipient at a concentration of 5%. On the other hand, in terms of protease activity that must be maintained low for the taste and aroma of yakju, Asp. For solid spawn using oryzae OF5-20, it is most preferable to use skim milk at a concentration of 1% as an excipient, and Lich. For solid spawn using ramosa 83-1, it is most preferable to use cyclodextrin as an excipient at a concentration of 5%.

<Experimental Example 5> Establishment of drying technology for solid seed bacteria

<5-1> Analysis of enzymatic activity of solid spawn according to drying method

The enzymatic activity of solid seed bacteria was compared according to the drying method (hot air drying or low temperature blowing drying).

Specifically, each solid spawn sample was dried with hot air (50° C., 15 hr) or low-temperature blow-dried (35° C., 24 hr) with the device of FIG. 4, and then the enzyme activity of these solid spawn samples was analyzed and the results are shown. 15 is shown.

strain drying method α-amylase Glucoamylase Proteases AO
OF5-20 blow 634.80 1,137.42 3,867.36 sirocco 626.87 907.47 6,169.36 LR 83-1 blow 2.41 23.61 4.61 sirocco 3.2 19.78 1.15

(Unit: Unit/g)

As confirmed in Table 15, Asp. In the case of solid spawn using oryzae OF5-20, the low-temperature blow-drying method maintains high activity of α-amylase and glucoamylase compared to the hot-air drying method, and the protease activity is maintained lower. , it was found that the low-temperature blow-drying method is preferable compared to the hot-air-drying method, and Lich. In the case of solid spawn using ramosa 83-1, the low-temperature blow-drying method maintains high activity of α-amylase and glucoamylase compared to the hot-air drying method. it can be seen that

Therefore, the low-temperature blow-drying method has a lower enzymatic activity inactivation than the hot-air drying, so it is suitable for drying solid seed bacteria.

<5-2> Analysis of microbial contamination of solid spawn according to drying method

The degree of microbial contamination of solid spores according to the drying method (hot air drying or low temperature blowing drying) was compared.

Specifically, after hot air drying (50°C, 15 hr) or low-temperature air drying (35°C, 24 hr) of each solid spawn sample with the device of FIG. The results are shown in Table 16.

strain CFU/g before drying Low temperature blow drying hot air dry AO OF5-20 0.6 X 10 ² 0.65 X 10 ² 0.72 X 10 ² LR 83-1 0.93 X 10 ² 0.85 X 10 ² 1.60 X 10 ²

As can be seen in Table 16, the contamination level of solid spores was 10 ² , which was confirmed to be low overall contamination. For reference, the above results are confirmed to be suitable for production and sale in that even seed producers in Japan, an advanced country, produce and sell products with a bacterial contamination level of 10 ² .

Therefore, low-temperature blowing drying is suitable for the drying method for industrial mass production of solid seed bacteria in the future.

Claims (17)

Lich deposited with accession number KACC 93350P. ramosa 83-1 fungal strain for medicinal use.
Lich. A method for preparing a seed strain for medicinal use, comprising culturing the ramosa 83-1 strain.
3. The method of claim 2,
The method for producing a seed for yakju, characterized in that the seed for yakju is a liquid starter.
3. The method of claim 2,
The culturing method for producing a seed strain for medicinal use, characterized in that the step of culturing in one or more media selected from the group consisting of REB, MEB, and PDB.
3. The method of claim 2,
The culture of claim 1 Lich. A method for producing a seed strain for medicinal use, comprising the step of culturing the ramosa 83-1 strain in a liquid at 20 to 30° C. for 2 to 10 days.
1) the step of inoculating the seed strain for medicinal liquor of claim 2 into the steamed rice; and
2) imperializing the steamed rice inoculated with the seed bacteria; a method for producing a fermentation agent for medicinal liquor comprising a.
7. The method of claim 6,
The inoculation of step 1) is a method for producing a fermentation agent for medicinal liquor, characterized in that the step of inoculating the steamed rice with the seed bacteria at a concentration of 1 to 10% based on the weight of the steamed rice.
7. The method of claim 6,
The imperial of step 2) is a method for producing a fermentation agent for medicinal liquor, characterized in that it is the step of imperializing the steamed rice inoculated with the seed culture at 30 to 40° C. for 2 to 10 days.
7. The method of claim 6,
The method for producing a fermentation agent for medicinal liquor, characterized in that the steamed rice has a moisture content of 30% or more.
7. The method of claim 6,
The steamed rice is a method for producing a fermenting agent for yakju, characterized in that it is steamed rice using Samgwangmi or Jeonju No. 615 rice.
7. The method of claim 6,
Method for producing a fermentation agent for medicinal liquor, characterized in that the excipient is added in an amount of 1 to 7% based on the weight of steamed rice in step 1).
12. The method of claim 11,
The excipient is skim milk (skim milk), maltodextrin (maltodextrin), cyclodextrin (cyclodextrin) and lactomeal (lactomeal) method for producing a fermentation agent for medicinal use, characterized in that at least one selected from the group consisting of.
7. The method of claim 6,
The method of manufacturing a fermentation agent for medicinal liquor, characterized in that it further comprises the step of drying the solid spawn obtained after the imperialization of step 2).
14. The method of claim 13,
The drying is a method of manufacturing a fermentation agent for medicinal liquor, characterized in that low-temperature blowing drying.
A fermenting agent for medicinal wine prepared by the method of claim 6 .
16. The method of claim 15,
The fermenting agent for yakju is yeast, characterized in that the fermenting agent for yakju.
A method for manufacturing high-quality yakju using the fermenting agent for yakju of claim 15.

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