KR102525960B1 - Fermenting agent for Yakju using Lichtheimia ramosa 3T-4, preparation method and use thereof - Google Patents

Abstract

The present invention relates to a fermentation agent for Yakju using Lichtamia ramosa 3T-4, a manufacturing method and use thereof, and Lich. The ramosa 3T-4 yakju mold strain has excellent starch decomposition ability and low protease activity. It is less concerned about problems such as off-flavor caused by amino acid components. It can produce high-quality yakju with excellent sensory characteristics. Bar has a useful effect of securing the starter for the high quality of domestic yakju, and also optimizes the detailed conditions such as the culture characteristics of the mold for yakju, inoculation amount, raw material, period of production, excipients, drying method, etc. Provided, there is a useful effect of providing high-quality Yakju by improving the quality of domestic Yakju.

Description

Fermenting agent for Yakju using Lichtheimia ramosa 3T-4, preparation method and use thereof {Fermenting agent for Yakju using Lichtheimia ramosa 3T-4, preparation method and use thereof}

The present invention relates to a fermenting agent for Yakju using Ricktamia ramosa 3T-4, a manufacturing method and use thereof.

Traditional liquor, a representative fermented liquor in Korea, is a fermented beverage in which various components including alcohol are produced as carbohydrates are decomposed by microorganisms. The most representative ones are yakju and takju. Yakju and takju as fermented liquors are traditionally prepared by fermenting grains and yeast, and are parallel fermented liquors in which saccharification and alcohol fermentation occur simultaneously. After brewing, mash is filtered through a sieve and the appearance is cloudy, which is Takju or Makgeolli, and Yakju is obtained by filtering only the clear liquid after receiving water in the mash.

Nuruk, which is used as a leavening agent during the manufacturing process of Yakju, is prepared by grinding grains such as wheat, barley, and rice, adding about 25% of water to them, kneading them, molding them, and naturally fermenting them. Therefore, unlike the entry of Japan, which only serves as a saccharification agent, fermented liquor is prepared using nuruk, in which various microorganisms derived from the environment are naturally epiphytic and grown. The characteristics of yeast vary depending on the climate, climate, and manufacturing environment of the region where it is produced. The types and contents of molds contained in yeast and sugars, organic acids, and amino acids produced by yeast vary, 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., which affects the quality characteristics such as taste, aroma, and color of traditional fermented liquor.

In particular, various chemical reactions such as oxidation, hydrolysis, and dehydration are involved during the aging of liquor. Some carbonyl compounds and pyrazines produced by the Maillard reaction between sugars and amino acids cause off-flavors and burnt smells that occur when the liquor is stored for a long time, and also changes the color of the liquor. In addition, indole compounds and Harman compounds produced by photo-oxidation of amino acids brown the color of the liquor. Polysulfide compounds made from changes in amino acids also impart unpleasant odors to alcoholic beverages.

In the case of Japanese rice wine made from rice and rice wine, isovaleric acid, ethyl-isovaleric acid, or 1, 1-diethoxy-3-butane have been identified as off-flavor components generated during long-term aging. Isoamyl alcohol, a higher alcohol of rice wine, is converted to isovaleraldehyde through enzymatic oxidation (alcohol dehydrogenase) derived from a fermentation agent. This is further oxidized to isovaleric acid, which is bonded with an ethyl group to form ethyl-isovaleraldehyde. Further oxidation thereafter produces 1,1-diethoxy-3-butane. Therefore, in order to control these off-flavors during the aging process of rice wine, in Japan, rice is milled to remove precursors, activated charcoal is used to absorb off-flavor components before aging, and yeasts that produce less aroma are developed. Efforts are being made (Ko JS. Fun and easy to understand brewing Engineering. Yuhansa, Seoul, Korea. pp. 218-219 (2008)).

Korean yakju also uses various starch ingredients such as rice and wheat, and there are various microorganisms and enzymes in nuruk. If a large amount of amino acid components remain after fermentation, they act as a flavor component that thickens the taste too much, and the remaining amino acid components react with sugars in the fermentation broth during distribution, resulting in a Maillard reaction. (Maillard reaction) is a factor that lowers the sensory characteristics and quality of Yakju.

Therefore, in order to provide high-quality Yakju, it is important to select fungal strains with low protease activity so that the amino acid components that cause taste and odor are reduced while having excellent starch decomposition ability. It is necessary to develop an excellent fermentation agent for Yakju. By developing and using such a fermenting agent for Yakju, the brewing quality of Yakju can ultimately be improved and high quality Yakju can be provided.

Under the above background, the present inventors, in order to solve the problem of improving the quality of domestic yakju and provide high-quality yakju, are striving to develop a mold for yakju and a fermentation agent for yakju, which has excellent starch decomposition ability and low protease activity. , Lichtheimia ramosa 3T-4 (hereinafter referred to as Lich. ramosa 3T-4) of the present invention has excellent starch decomposition ability among several domestic and foreign fungal strains and at the same time has a low protease activity that causes a problem of off-flavor. The present invention was completed by confirming that the fermentation agent for Yakju was most suitable, and by optimizing the detailed conditions such as inoculation amount, steamed rice raw material, imperial period, excipient, and drying method suitable for manufacturing the fermentation agent for Yakju.

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

In order to achieve the above purpose,

The present invention is deposited with accession number KACC 93337P Lich. Provides a fungal strain for ramosa 3T-4 yakju.

In addition, the present invention is the Lich. It provides a method for producing a spawn for medicinal purposes comprising the step of culturing the ramosa 3T-4 strain.

Furthermore, the present invention includes the steps of: 1) inoculating the seed for yakju into steamed rice; and

2) providing a method for producing a fermentation agent for yakju, including the step of repelling the rice inoculated with the spawn.

In addition, the present invention provides a fermentation agent for yakju prepared from the method for preparing a fermentation agent for yakju.

Furthermore, the present invention provides a method for producing high-quality Yakju using the fermentation agent for Yakju.

Lich of the present invention. The ramosa 3T-4 yakju mold strain has excellent starch decomposition ability and at the same time has low protease activity. As a yakju mold, there is little concern about problems such as off-flavors caused by amino acid components. It can produce high-quality medicinal liquor with excellent sensory characteristics. Bar has a useful effect of securing the starter for the high quality of domestic yakju, and also optimizes the detailed conditions such as the culture characteristics of the mold for yakju, inoculation amount, raw material, period of production, excipients, drying method, etc. Provided, there is a useful effect of providing high-quality Yakju by improving the quality of domestic Yakju.

1 is a step-by-step flow chart of a method for manufacturing a fermentation agent for yakju in one example.
Figure 2 is a graph showing the change in moisture content according to the soaking time for two types of rice, Samgwangmi or Jeonju No. 615.
Figure 3 is Asp selected for steam rice using two types of rice, Samgwangmi or Jeonju No. 615. oryzae OF5-20 or Lich. It is a graph showing the temperature change of the solid seed germs sown in steamed rice according to the imperial period in the manufacture of fermentation agent for Yakju using two types of fungi of ramosa 3T-4.
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 spawn.

Hereinafter, the present invention will be described in detail.

The present invention is deposited with accession number KACC 93337P Lich. Provides a fungal strain for ramosa 3T-4 yakju.

above Lich. The ramosa 3T-4 fungal strain for yakju is a strain for which a patent has been deposited under accession number KACC 93337P, and is characterized by having an enzyme activity suitable for manufacturing yakju. Enzyme activity suitable for preparing the yakju can be understood to mean excellent starch decomposition activity, for example, as a strain having excellent glucoamylase activity, by finely decomposing starch of cereal raw materials such as rice to produce sugar It can be understood as a strain that can smoothly supply sugar to microorganisms that perform alcohol fermentation, such as yeast.

Also, the above Lich. The ramosa 3T-4 fungal strain for yakju is suitable for manufacturing yakju as a strain with low proteolytic activity due to low peptidase activity. If the proteolytic activity is high and the proteolytic power is high, problems such as undesirable off-flavors may be caused from amino acids produced. For example, amino acids are used as precursors to fusel oil components during alcoholic fermentation, giving off too strong aroma components and adversely affecting the taste. It can degrade the quality of yakju. In addition, the remaining amino acid components react with the sugars of the fermentation broth during distribution, which is a factor in lowering the sensory characteristics and quality of Yakju due to coloring by Maillard reaction.

Therefore, for the production of high-quality yakju, it is preferable to have excellent starch decomposition activity but low proteolytic activity, and Lich of the present invention. The ramosa 3T-4 fungal strain has a high glucoamylase activity that decomposes starch and a low peptidase activity that decomposes proteolysis, and is suitable for preparing medicinal liquor.

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

In addition, the present invention is the Lich. It provides a method for producing a spawn for medicinal purposes comprising the step of culturing the ramosa 3T-4 strain.

above Lich. The ramosa 3T-4 strain, as described above, has excellent starch decomposing activity and low proteolytic activity, and is suitable for use as a yakju strain, and duplicate descriptions are omitted.

The culturing step is Lich. Any culture under conditions capable of growing the ramosa 3T-4 strain is included in the present invention, and, for example, liquid culture may be performed to prepare a liquid spawn.

The medium used in the culture is Lich. Any medium under conditions capable of growing the ramosa 3T-4 strain is included in the present invention, and any commercially available medium and conventional medium used for the purpose of culturing bacteria can be used, for example, REB, MEB, and It may be to culture in one or more types of media selected from the group consisting of PDB.

The temperature during the culture includes any temperature at which the bacteria can grow, but may be cultured at, for example, 20 to 30 ° C, 24 to 30 ° C, 25 to 30 ° C, 27 to 30 ° C, or about 28 ° C. The culturing 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 culturing method and apparatus may be all of those commonly used for bacterial culture, and may be, for example, shaking culture, and shaking culture at 100 to 150 rpm or about 120 rpm with a shaking cultivator.

Furthermore, the present invention

1) inoculating the starter for yakju into steamed rice; and

It provides a method for producing a fermentation agent for yakju, including; 2) imperializing the steamed rice inoculated with the spawn.

The spawn for medicinal liquor is the Lich of the present invention. As a spawn obtained by culturing the ramosa 3T-4 strain, the above Lich. The ramosa 3T-4 strain, as described above, has excellent starch decomposing activity and low proteolytic activity, and is suitable for use as a yakju strain, and duplicate descriptions are omitted.

The steamed rice is obtained by immersing rice in water, and preferably has an appropriate moisture content to be suitable for preparing a fermentation agent for yakju. The steamed rice has a moisture content of, for example, 29% or more, 29 to 40%, 29 to 38%, 29 to 35% or about 30%. Lich when the water content is less than the lower limit range. It is difficult to propagate ramosa 3T-4 bacteria, and when the moisture content exceeds the upper limit range, contamination with various germs such as bacteria may occur. In addition, the steamed rice may be to achieve the above moisture content by adjusting the soaking time for each rice variety.

The steamed rice may be, for example, steamed rice using Samgwangmi or Jeonju No. 615 rice, and steamed rice using Samkwangmi may be steamed rice prepared by soaking for 100 minutes or more, 120 minutes or more, or 150 minutes or more, preferably 120 Steamed rice prepared by soaking in water for more than 2 minutes (2 hours). On the other hand, steamed rice using Jeonju No. 615 may be steamed rice prepared by soaking for 10 minutes or more, 15 minutes or more, 10 to 30 minutes, or about 10 minutes, and preferably steamed rice prepared by soaking for 10 minutes or more. When immersed in water in the above-mentioned range, the water content of steamed rice can be achieved at 29% or more, 29 to 40%, 29 to 38%, 29 to 35% or about 30%. When the moisture content exceeds the upper limit range, contamination with germs and the like may occur. In addition, the steamed rice may be to achieve the above moisture content by adjusting the soaking time for each rice variety.

In a specific example of the present invention, as a result of preparing steamed rice by soaking Samgwangmi, it was confirmed that it is desirable to soak for 120 minutes or more to achieve a moisture content of about 30%, and to prepare steamed rice by soaking Jeonju No. 615. As a result, it was confirmed that it is preferable to soak in water for 10 minutes or more to achieve a water content of 30% (see Table 7).

The inoculation in step 1) is carried out at 1 to 10%, 1 to 9%, 2 to 10%, 2 to 8%, 2 to 7%, 3 to 8%, 3 to 6%, 2 to 10%, based on the weight of steamed rice. It may be a step of inoculating steamed rice at a concentration of 4% or about 4%. When inoculated below the lower limit, it is difficult to achieve sufficient enzyme activity, and even when inoculated above the upper limit, sufficient enzyme activity may not be achieved or unnecessary protease overactivity may occur.

In a specific embodiment of the present invention, as a result of examining the amount of spawn inoculum suitable for manufacturing fermenting agents for medicinal purposes, both Samgwangmi and Jeonju No. 615 showed excellent starch decomposition activity when inoculated at 4%, and at the same time, the proteolytic activity could be maintained low. , it was confirmed that it is most preferable to inoculate 4% of the spawn (see Tables 8 and 9).

The empire of step 2) may be to empire the steamed rice inoculated with the spawn at 30 to 40 ° C, 32 to 38 ° C, 34 to 36 ° C or about 35 ° C. In addition, the empire of step 2 may be 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. When the empire is less than the lower limit range, there is a problem that it is difficult to achieve sufficient enzyme activity, and when the empire exceeds the upper limit range, there is a concern that unnecessary protease activity may be caused.

In a specific embodiment of the present invention, as a result of examining the change in enzyme activity according to the imperial period, when Samgwangmi was 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, protease It was confirmed that the activity was maintained at the lowest level on day 6 (see Table 10). On the other hand, when Jeonju No. 615 was used, the glucoamylase activity was the highest on the 3rd day of the empire, the α-amylase activity was the highest on the 9th day of the empire, and the protease activity was the lowest on the 3rd day of the empire. In order to keep the proteolytic activity low at the same time as excellent, it was confirmed that it is preferable to ferment for 6 days (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 a specific embodiment of the present invention, as a result of examining the enzyme activity and spore count change of bacteria according to the type of excipient, it was confirmed that skim milk or cyclodextrin is preferred, and cyclodextrin is more preferred (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 using an excipient less than the lower limit, there is a problem of insufficient to achieve the desired effect of protecting enzymatic activity, and even when using an excipient exceeding the upper limit, there is a concern that enzyme activity may be hindered, and unnecessary protease overactivity There is a risk of causing

The amount of the excipient added may vary depending on the type of excipient, and for example, when the excipient is skim milk, it may be 5 to 7% in terms of starch decomposition 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, it may be used at 3 to 7%, or about 5% in terms of starch decomposition activity, and 3 to 7%, or about 5% in terms of protease activity. It is suitable for preparing a fermentation agent for yakju because it is possible to increase the starch decomposition activity and simultaneously lower the glycolysis activity by using an excipient according to the above range.

In an embodiment of the present invention, as a result of examining the optimal amount of excipients, 5 to 7% of cyclodextrin is preferably 5 to 7% based on the weight of steamed rice, and 5 to 7% is preferable in terms of starch decomposing 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 drying the solid spawn obtained after the empire in step 2). The drying is included in the present invention as long as the drying method does not deactivate the 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 air drying may be, for example, air drying at a temperature of 20 to 40 ° C, 30 to 40 ° C or about 35 ° C, 10 to 40 hours, 12 to 36 hours, 16 to 30 hours, 20 to 28 hours Alternatively, it may be air 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 ° C, 45 to 550 ° C or about 50 ° C, 5 to 25 hours, 10 to 20 hours, 12 to 18 hours, 14 to 16 hours It may be hot air drying for about 15 hours or about 15 hours.

In a specific example of the present invention, as a result of examining the enzyme activity of the fermentation agent for yakju according to the drying method, it was confirmed that the enzyme activity deactivation width was smaller in the air drying method compared to the hot air drying method (see Table 15).

In a specific embodiment of the present invention, as a result of examining the degree of microbial contamination of the fermentation agent for yakju according to the drying method, the degree of contamination in both methods is maintained at a low level of 10 ² , but preferably, the degree of microbial contamination is lower in the air drying method compared to the hot air drying method. It was confirmed that it was suitable for showing (see Table 16).

Furthermore, the present invention provides a fermentation agent for yakju prepared from the method for preparing a fermentation agent for yakju.

The fermentation agent for yakju of the present invention is Lich of the present invention. As a fermentation agent prepared by inoculating a spawn obtained by culturing the ramosa 3T-4 strain, the above Lich. The ramosa 3T-4 strain, as described above, has excellent starch decomposing activity and low proteolytic activity, and is suitable for use as a yakju strain, and duplicate descriptions are omitted.

The fermenter for yakju has excellent starch decomposition activity, so that microorganisms that perform alcohol fermentation, such as yeast, can not only supply sugar necessary for producing yakju, but also improve the taste of yakju, and above all, the fermenter for yakju decomposes protein. It has low activity and produces less amino acids, so there is less fusel oil and flavor components, less off-flavors, and less concern about discoloration due to the Maillard reaction, improving higher quality sensory characteristics. Yakju may be provided.

In addition, the present invention provides a method for producing high-quality Yakju using the fermentation agent for Yakju.

The fermenter for yakju has excellent starch decomposition activity and low proteolytic activity, thereby providing high-quality yakju by improving the sensory properties of yakju, and redundant descriptions are omitted.

Hereinafter, the present invention will be described in detail by examples and experimental examples.

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

<Experimental Example 1> Selection of fungal spawn with excellent enzyme activity such as glycosylation

Fermented processed foods and 14 molds stored in a cryogenic freezer (-80 ° C) were targeted (Table 1). Fungi with excellent saccharification ability were selected while culturing 14 prepared fungi in 4 fungal media (REB, MEB, MERB (REB+MEB), PDB).

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 activities

α-amylase: Investigation of liquefaction enzymes that hydrolyze α-1,4 glycoside bonds in glucose-linked polysaccharides

No. Species/Strains No. Species/Strains One Lichtheimia ramosa / 3T-4 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. After growing in four types of media for fungal culture, Table 2 shows the results of α-amylase activity of these fungi according to the type of media.

No. Species/Strains REB
(U/mL) MEB
(U/mL) MERB
(U/mL) PDB
(U/mL) One Lichtheimia ramosa / 3T-4 0.03 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. ramos a 3T-4 was confirmed to have low activity of α-amylase in the medium.

<1-2> Glucoamylase activity

The activity of glucoamylase, which cleaves α-1, 4/α-1, 6 glycoside bonds into glucose from the non-reducing end, was investigated. After growing in four types of media for fungal culture, the results of glucoamylase activity of these fungi according to the type of media are shown in Table 3.

No. Species/Strains REB
(U/mL) MEB
(U/mL) MERB
(U/mL) PDB
(U/mL) One Lichtheimia ramosa / 3T-4 0.74 20.32 22.23 19.18 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 confirmed in Table 3, glucoamylase is an enzyme that saccharifies starch and directly affects microorganisms using monosaccharides such as glucose as a carbon source, Lich. ramosa 3T-4 was confirmed to have high glucoamylase activity on the medium.

<1-3> Protease activity

After growing in four types of media for fungal culture, the protease activity results of these fungi according to the type of media are shown in Table 4.

No. Species/Strains REB
(U/mL) MEB
(U/mL) MERB
(U/mL) PDB
(U/mL) One Lichtheimia ramosa / 3T-4 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. ramosa 3T-4 was confirmed to have no protease activity on the medium.

Therefore, Lich of the present invention. It was confirmed that ramosa 3T-4 had excellent saccharification ability, and at the same time, it had low protein decomposition ability that deteriorated the taste and aroma of Yakju or caused the problem of off-flavor generation.

<Experimental Example 2> Measurement of dry cell mass for each selected strain

After liquid culture (28 ℃, 5 days) for the selected 5 species of fungi, filtration (Filter paper No. 2, Advantec Co., Japan) was performed to separate the cells and the culture medium. Cell productivity was evaluated by measuring the dry weight by drying 8 g of cells recovered for each mold type at 105 ° C. (Vision Scientific Co., Korea) for 8 hours, and the results are shown in Table 5.

No. Species/Strains Cell weight (g) One Lichtheimia ramosa / 3T-4 1.367 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 confirmed in Table 5, Lich. ramosa 3T-4 is confirmed to have excellent cell productivity.

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

After grains (rice, 200 g) were semi-, immersed, water-saving and steamed as shown in FIG. 1 for the 5 types of fungi selected to select strains with high saccharification power, 1% broth of each fungus was added thereto. Inoculated with, and imperial for 3 days at 35 ° C., 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 / 3T-4 5.06 ±0.72 51.06 ± 1.94 458.87 ± 23.8 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 as a fermentation agent for each fungal strain, Lich of the present invention. It is confirmed that ramosa 3T-4 has the best glucoamylase activity, which indicates the saccharification power of mold, and the lowest protease activity, which makes the taste and aroma of Yakju bad or causes a problem of off-flavor generation.

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

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

Using two types of rice varieties (Samgwangmi, Jeonju 615), the moisture content of raw rice was investigated according to the soaking time.

Samgwangmi: Suwon 361 + Miryang No. 101 hybridization, mid-late maturation, hard rice

Jeonju 615 : Suwon 542 Improved, early-ripening, powdered rice with coarsely arranged starch grains (Karumi)

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

hour
(minute) Samkwangmi hour
(minute) Jeonju 615 Water absorption rate (%) Moisture content (%) Water absorption rate (%) 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 confirmed in Table 7 and FIG. 2, since the moisture absorption rate for each variety is different, it is necessary to adjust the soaking time by measuring the moisture absorption rate when the raw material rice is different. If the moisture content is low, it is difficult for mold spawn to proliferate, and if it is high, contamination with various germs may occur. Therefore, the moisture content of steamed rice is preferably 30% or more.

Therefore, Samgwangmi (hard rice) used in the present invention is preferably soaked for 2 hours or more, and Jeonju 615 (floured rice/garumi) requires a soaking time of 10 minutes or less to obtain good steamed rice (hard rice). there is.

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

Each of the selected fungi was inoculated with the same number of spores (10 ⁸ cells/mL) in a bran medium and cultured for 5 days at 28°C and 120 rpm. After filtering the liquid culture medium and recovering the liquid separated from the sediment, it was seeded in godubap by liquid spawn concentration (2, 4, 6%) to select the inoculation amount by analyzing the enzyme activity of the solid spawn by period, and the results Table 8 and Table 9 show.

Grain raw materials: 2 types of hard rice (Samkwang, 2018, Nonsan) and powdered rice (Jeonju 615, 2019, Jeonbuk) are used

Mold: Lichtheimia ramosa 3T-4, Aspergillus oryzae OF5-20 used

Activity (unit/g) AO OF5-20
Inoculation amount (%) Samkwangmi 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 Protease 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 2,048.78 6 3,153.74 3,798.30 687.47 2,716.36 2,555.22 6,307.48

As shown in Table 8, Asp. For the solid spawn prepared with oryzae OF5-20, the α-amylase activity was 6% > 4% > 2% in Samgwangmi and 6% > 2% in Jeonju 615 when it was incubated for 9 days. > 4%. On the other hand, in the case of empire for 9 days, glucoamylase activity was 4%>6%> 2% for Samkwangmi and 4%>2%> 6% for Jeonju 615. On the other hand, in the case of imperial fermentation for 9 days, protease activity was 2% < 4% < 6% in Samgwangmi and 4% < 2% < 6% in Jeonju 615.

Thus, Asp. As a result of examining the inoculation amount according to the enzyme activity of the solid spawn prepared by oryzae OF5-20, α-amylase and glucoamylase showed high activity when inoculated at 4% in Samgwangmi and Jeonju 615, and protease ( protease) is confirmed to have a low activity at 2-4%.

Activity (unit/g) LR 3T-4
Inoculation amount (%) Samkwangmi Jeonju 615 3 days 6 days 9 days 3 days 6 days 9 days α-amylase 2 4.39 6.44 3.79 12.84 18.97 8.62 4 3.70 3.85 4.67 10.69 14.56 13.75 6 5.40 6.34 3.87 9.86 11.83 6.87 Glucoamylase 2 109.17 135.87 174.82 174.82 208.80 325.90 4 99.90 134.51 179.89 177.17 213.32 385.24 6 105.34 132.91 170.98 186.69 202.36 345.18 Protease 2 21.87 29.93 65.61 43.74 215.24 227.90 4 29.93 132.37 359.11 12.66 382.13 195.67 6 18.42 462.70 521.40 24.17 370.98 250.92

As shown in Table 9, Lich. Regarding the solid spawn prepared with ramosa 3T-4, the α-amylase activity was 4% > 6% > 2% in Samgwangmi and 4% > 2% in Jeonju 615 for 9 days. > 6%. On the other hand, in the case of empire for 9 days, glucoamylase activity was 4%>2%> 6% for Samgwangmi and 4%>6%> 2% for Jeonju 615. On the other hand, in the case of imperial fermentation for 9 days, protease activity was 2% < 4% < 6% in Samgwangmi and 4% < 2% < 6% in Jeonju 615.

Thus, Lich. As a result of investigating the inoculation amount according to the enzymatic activity of the solid spawn prepared with ramosa 3T-4, α-amylase and glucoamylase showed high activity when inoculated at 4% in Samgwangmi and Jeonju 615, The protease was found to have the lowest activity at 4%.

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 raw material rice of the mold solid spawn for yakju. And it is necessary to set and adjust the conditions of the raw material rice.

<4-3> Changes in the quality of solid spawn according to the imperial period

The selected fungal spawn was inoculated into 2 types of steamed rice (10 kg each) with the optimal concentration (2, 4, 6%) of the liquid spawn cultured in the bran medium, and was inoculated at 35 ° C for 9 days. Japanese Yaegaki) Empire. The temperature change of the solid spawn cultures sown in steamed rice was investigated during the 9-day imperial period using the selected two types of fungi, and the results are shown in FIG. 3.

Mold: Lichtheimia ramosa 3T-4, Aspergillus oryzae OF5-20 used

Samgwangmi: Rice (10 kg) → Semi-prepared rice (5 times) → Soaking (3 hours) → Water saving (1 hour) → Increased rice (50 minutes) → Cooling (35℃) → Inoculation of each mold liquid soup → Empire (35℃, 9 days)

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

As confirmed in Figure 3, microbial activity appears from the start of the empire (day 0), and the highest growth activity was exhibited on the 2nd day of the empire, with the highest food temperature rising to 41 ° C. It was maintained, and it was confirmed that the product temperature fell after 5 days, indicating that artificial control was necessary. Meanwhile, Lich. ramosa 3T-4 is relatively Asp. oryzae OF5-20 showed a tendency to increase the food temperature a little later.

<4-4> Changes in enzyme activity according to the imperial period

The selected fungi were sown on Jeungjami to investigate the enzyme activity of the solid spawn according to these imperial periods, and the results are shown in Tables 10 and 11.

Mold: Lichtheimia ramosa 3T-4, Aspergillus oryzae OF5-20 used

Jeungjami: Use Samgwangmi or Jeonju No. 615

strain empire period
(days) α-amylase (unit/g) Glucoamylase
(unit/g) Protease
(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
3T-4 3 4.39 109.17 21.87 6 6.44 135.87 29.93 9 3.79 174.82 62.15

As confirmed in Table 10, when Samkwangmi was used, α-amylase (α-amylase) activity was Asp. oryzae OF5-20, Lich. In all spawns of ramosa 3T-4, it was the highest on the 6th day of the empire, and the enzyme activity decreased as the empire period passed. Glucoamylase activity showed the highest activity on the 9th day of the empire. On the other hand, if the protease activity is high, the protein is decomposed and converted into amino acids, and at this time, it is advantageous that the protein decomposition power is low because the difference in taste of the liquor is determined.

Therefore, the Imperial period is suitable for the Imperial period of 6 days, which has excellent α-amylase and glucoamylase activity as a whole and does not have too high protein dissolving power.

strain empire period
(days) α-amylase (unit/g) Glucoamylase
(unit/g) Protease
(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
3T-4 3 12.84 174.82 43.74 6 8.84 205.23 215.24 9 8.62 325.90 227.90

As confirmed in Table 11, when Jeonju No. 615 was used, α-amylase activity was Asp. When oryzae OF5-20 was used, it was highest on the 9th of the Empire, and Lich. The activity of ramosa 3T-4 showed a tendency to increase and then decrease with the passage of the imperial period. As for the glucoamylase activity, the enzymatic activity increased as the imperial period passed, and showed the highest activity on the 9th imperial day.

As for the imperial period, the six-day imperial period is suitable because it has excellent α-amylase and glucoamylase activities and does not have high protein dissolving power. Even when the same strain is used, it is confirmed that there is a difference in enzyme activity depending on the rice raw material used.

<4-5> Selection of excipients for solid spawn

In order to select a customized excipient that can maintain activity without inactivating the enzyme of the solid spawn during storage, 5% of one of the four excipients (skim milk, maltodextrin, cyclodextrin, lactomeal) was added to the solid spawn and 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 Protease 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
3T-4 skim milk 6.32 30.78 75.00 maltodextrin 9.02 30.66 230.20 cyclodextrin 9.42 37.34 151.93 lactomeal 6.04 29.05 29.93

(Unit: Unit/g)

As confirmed in Table 12, α-amylase (α-amylase) and glucoamylase (glucoamylase) activities of Asp. oryzae OF5-20 maintains the highest activity when skim milk is used as an excipient, and Lich. It was confirmed that the activity of ramosa 3T-4 was the highest when cyclodextrin was used as an excipient.

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

The change in the number of spores according to the type of excipient was measured and the results are shown in Table 13.

The method of measuring the number of fungal spores is to add 2 to 3 drops of 1% methylene blue to 10 mL of 5% tween solution for 1 g of the solid spawn prepared using four different excipients, dilute it, and count the number of spores with a hemocytometer. I was humbled.

excipient Spore counts AO OF5-20 LR 3T-4 skim milk 1.18 X 10 ⁸ 0.72 X 10 ⁸ maltodextrin 0.93 X 10 ⁸ 0.85 X 10 ⁸ cyclodextrin 1.86 X 10 ⁸ 0.89 X 10 ⁸ lactomeal 1.82 X 10 ⁸ 0.83 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 solid spawns using ramosa 3T-4.

Thus, Asp. oryzae OF5-20 or Lich. Cyclodextrin was found to be the most suitable excipient in terms of enzymatic activity and spore count of all solid spawns using ramosa 3T-4.

<4-7> Selection of optimal concentration of excipient for solid spawn

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

excipient strain density(%) α-amylase
(Unit/g) Glucoamylase (Unit/g) Protease
(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
3T-4 0 1.03 24.60 62.15 One 0.68 27.32 89.78 3 0.72 27.20 59.85 5 1.07 28.93 29.93 7 0.44 29.43 52.95

As confirmed in Table 14, in terms of α-amylase activity , Asp. For the 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 3T-4, it is most preferable to use cyclodextrin at a concentration of 5% as an excipient. In terms of glucoamylase activity , Asp. For the 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 3T-4, it is most preferable to use cyclodextrin at a concentration of 7% as an excipient. On the other hand, Asp. For the 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 3T-4, it is most preferable to use cyclodextrin at a concentration of 5% as an excipient.

<Experimental Example 5> Establishment of drying technology for solid spawn

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

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

Specifically, after hot air drying (50 ° C, 15 hr) or low temperature blowing drying (35 ° C, 24 hr) for each solid spawn sample with the device shown in FIG. 15.

strain drying method α-amylase Glucoamylase Protease AO
OF5-20 blowing 634.80 1,137.42 3,867.36 sirocco 626.87 907.47 6,169.36 LR 3T-4 blowing 0.95 56.01 62.15 sirocco 0.89 19.78 11.51

(Unit: Unit/g)

As confirmed in Table 15, Asp. In the case of solid spawn using oryzae OF5-20, the low-temperature air drying method maintains higher α-amylase and glucoamylase activities and lower protease activity compared to the hot air drying method. , it was found that the low-temperature blow drying method was preferable to the hot air drying method, and Lich. In the case of solid spawn using ramosa 3T-4, the low-temperature blow-drying method maintains higher activity of α-amylase and glucoamylase compared to the hot-air drying method, so the low-temperature blow-drying method is more preferable than the hot air drying method. can know that

Therefore, the low-temperature blow drying method has a lower enzyme activity deactivation range than hot air drying, so it is suitable for drying solid spawn.

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

Microbial contamination of the solid spawn 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 shown in FIG. 4, and then the degree of microbial contamination (bacterial contamination) of the solid spawn was analyzed. The results are shown in Table 16.

strain CFU/g before drying low temperature air drying hot air dry AO OF5-20 0.6 X 10 ² 0.65 X 10 ² 0.72 X 10 ² LR 3T-4 0.3 X 10 ² 0.71 X 10 ² 0.89 X 10 ²

As confirmed in Table 16, the contamination level of the solid spawn 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 spawn producers in Japan, an advanced country, produce and sell products with a bacterial contamination of about 10 ² .

Therefore, as a drying method for industrial mass production of solid spawn in the future, low-temperature blow drying is suitable.

Claims (17)

1) Lich deposited with accession number KACC 93337P. ramosa 3T-4 liquid culture for 2 to 10 days at 20 to 30 ° C. to obtain a spawn for medicinal liquor;
2) inoculating the spawn for yakju obtained in step 1) into steamed rice containing 30% or more moisture at a concentration of 2 to 4% based on the weight of steamed rice; and
3) A method for producing a fermentation agent for yakju comprising the step of imperializing the steamed rice inoculated with the spawn at 30 to 40 ° C. for 6 to 9 days.
delete According to claim 1,
The method for producing a fermentation agent for yakju, characterized in that the spawn for yakju is a liquid spawn.
According to claim 1,
The culturing is a method for producing a fermentation agent for yakju, characterized in that the step of culturing in at least one medium selected from the group consisting of REB, MEB, and PDB.
delete delete delete delete delete delete According to claim 1,
The manufacturing method is a method for producing a fermentation agent for yakju, characterized in that in step 2), the excipient is added in an amount of 1 to 7% based on the weight of steamed rice.
According to claim 11,
Wherein the excipient is at least one selected from the group consisting of skim milk, maltodextrin, cyclodextrin and lactomeal.
According to claim 1,
The manufacturing method of the fermentation agent for yakju, characterized in that it further comprises the step of drying the solid spawn obtained after the empire of step 3).
According to claim 13,
The drying is a method for producing a fermentation agent for yakju, characterized in that the low-temperature blow drying.
A fermentation agent for Yakju produced by the method of manufacturing the fermentation agent for Yakju of claim 1.

delete delete

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