KR20170053927A - Middle temperature fermenting nuruk and manufacturing method thereof - Google Patents

Abstract

The present invention relates to a mesophilic fermentation yeast and a preparing method thereof. More specifically, the mesophilic fermentation yeast is prepared by grinding wheat, mixing water and the wheat, and infiltrating the mixture to form the yeast. The yeast is then fermented for two days and at 36C while being flipped to the other side there-between and dried for seven days at 45C. The mesophilic fermentation yeast prepared by using a conventional method has a low acidity rate to effectively prevent micro-organism contamination and has a low amino acid level to lower the greasy taste of alcohol prepared therewith. The mesophilic fermentation yeast has excellent -amylase and glucoamylase enzyme activities as well as glycation abilities and is determined to have excellent taste and flavors based on the analysis of the organic acids, free amino acids, and flavor components while having excellent storability due to the excellent residual capacity of the enzymes in accordance with the storage period. Accordingly, the present invention provides a method for preparing high-quality yeast by using a conventional method as well as the yeast being beneficial to the preparation and production of yeast.

Description

TECHNICAL FIELD The present invention relates to a middle temperature fermentation nuruk and a manufacturing method thereof,

The present invention relates to a medium-temperature fermented yeast and a method for producing the same, and more particularly, to a method capable of producing yeast having improved quality and improved storage stability.

Each country in the world has been handed out unique liquor, which is local alcoholic beverages, such as traditional alcoholic beverages, traditional alcoholic beverages or regional beverages, depending on the climate, climate and customs of the area. In Korea, traditional wine called "takju" (makgeolli), yakju and distilled liquor has been passed down for a long time. Takju (makgeolli) is a type of fermented soybean fermented by using yeast made by cultivating fungi or yeast in cereals. It has higher nutritional value because it contains more dietary fiber and physiologically active substances than yakju and distilled liquor.

As mentioned above, despite the high nutritional value of Korean traditional liquor, Takju is not a typical mainstream. Recently, consumers' desire for liquor has diversified and the tendency to find high quality liquor has become stronger. Accordingly, there is a need for manufacturing a liquor having a high quality and a standardized process.

Nuruk is fermented by various microorganisms at low temperature by molding wheat as a main raw material. Through such fermentation process, various enzymes are activated and glycosylation proceeds, resulting in a characteristic taste and smell of Korean traditionalism (Kim MJ, 2002. The study about traditional Nuruk. Korean J Food Sci Technol 9: 324-329).

Because of the complicated manufacturing process and diverse fermentation microorganisms, the production of standardized products and the standardization of quality are urgently needed. In Korean traditional yeast companies, the natural fermentation and experience-dependent production of yeast, the microbial growth, the scientific control of the metabolic system, and the establishment of the standardized brewing process are not properly performed.

Nuruk is classified into traditional yeast and modified yeast according to the manufacturing method. Traditional nuruk is a method in which various microorganisms present in natural environment are produced by proliferation. The taste and aroma of sake produced by saccharification and alcohol fermentation by these strains have a unique advantage. The modified yeast is prepared by inoculating fermented seeds such as Aspergillus and Rhizopus into a sterilized starch raw material in order to prevent contamination with germs and to produce a uniform quality wine.

As described above, the traditional nuruk manufacturing process has a disadvantage in that it is difficult to produce a standardized and uniform quality wine because the flavor of the wine varies according to the season. Because the quality of the commercial nuruk is inferior, the competitiveness of our liquor is poor as well. In order to overcome this, we have studied the preliminary study on the technology of making yeast according to our sake by using fermenting microorganisms such as yeast and lactic acid bacteria which affect the flavor of our sake through scientific improvement researches such as improvement of quality of yeast, More than necessary.

Accordingly, the present invention has been accomplished on the basis of the quality characteristics of the yeast according to the fermentation temperature in the conventional process for producing the yeast, and the present invention has been completed based on this finding, and a technique for producing yeast having an excellent enzyme activity and a unique taste and aroma has been established .

It is an object of the present invention to provide a warm fermented yeast and a method for producing the same.

In order to achieve the above object,

1) crushing the wheat;

2) adding water to the milled mill of step 1) and then infiltrating;

3) shaping the infiltrated mill of step 2);

4) fermenting the shaped mill of step 3) at a temperature of 34 to 38 占 폚; And

5) the fermentation of step 4), and drying the fermented yeast.

The present invention also provides a yeast produced by the method for producing a middle-temperature fermented leaven of the present invention.

The present invention also provides a Takju prepared with the koji of the present invention.

More specifically, the present invention relates to a medium-temperature fermented yeast and a method for producing the same. More specifically, the wheat is pulverized, mixed with water, infiltrated to form yeast, fermented at an interval of 2 days at a temperature of 36 ° C, The fermented yeast has a low acidity and is effective in preventing microbial contamination and has a low amino acid content. Thus, it is believed that the flavor produced during the production of takju is offset by the conventional method, and the α-amylase, glucoamylase enzyme activity and glycation ability are excellent , Organic acids, free amino acids and fragrance components, it was judged that the quality of the flavor and aroma would be excellent. By confirming that the storage ability was excellent due to the excellent residual capacity of the enzyme according to the storage period, the fermentation yeast of the present invention and its preparation A method of manufacturing a high quality yeast using a conventional method is provided, and a yeast produced by the method is provided, It may be used for that.

FIG. 1 is a view showing a manufacturing process of a conventional yeast.
FIG. 2 is a graph showing acidic yeast and Songyun leu, which are commercially available yeast, as comparative sphere.
FIG. 3 is a graph showing a change in middle temperature fermented yeast according to a fermentation period. FIG.
4 is a graph showing changes in temperature of middle-temperature fermentation yeast and natural fermentation yeast.
FIG. 5 is a graph showing changes in physicochemical properties of the middle-temperature fermented yeast according to fermentation period. FIG.
FIG. 5A is a graph showing pH change of middle temperature fermented yeast according to fermentation period. FIG.
FIG. 5B is a graph showing changes in acidity of middle-temperature fermented yeast according to fermentation period. FIG.
FIG. 5c is a graph showing the change in amino acid level of the middle-temperature fermented yeast according to fermentation period. FIG.
FIG. 6 is a graph showing changes in enzyme activity of mesophilic fermented yeast according to fermentation period. FIG.
FIG. 6A is a graph showing the change in α-amylase activity of mesophilic fermented yeast according to fermentation period. FIG.
6B is a graph showing the glucoamylase activity of mesophilic fermented yeast according to fermentation period.
FIG. 6c is a graph showing changes in acidic protease activity of mesophilic fermented yeast according to fermentation period. FIG.
FIG. 6 (d) is a graph showing the change in sugar strength of mesophilic fermented yeast according to fermentation period. FIG.
Fig. 7 is a graph showing a GC-MS chromatogram of the volatile perfume compounds identified in the yeast. Fig.
7A is a graph showing a GC-MS chromatogram of an acidic nuruk volatile perfume compound.
7B is a graph showing a GC-MS chromatogram of a volatile flavor compound of Song-Nyurikun.
7C is a graph showing a GC-MS chromatogram of volatile flavor compounds of mesophilic fermented yeast.
8 is a graph showing changes in enzyme activity of mesophilic fermented yeast according to storage periods.
8A is a graph showing the change of? -Amylase activity of mesophilic fermented yeast according to the storage period.
FIG. 8B is a graph showing the glucoamylase activity of mesophilic fermented yeast according to the storage period. FIG.
FIG. 8C is a graph showing changes in acidic protease activity of mesophilic fermented yeast according to storage periods. FIG.
FIG. 8D is a graph showing the change in sugar strength of mesophilic fermented yeast according to the storage period. FIG.

Hereinafter, the present invention will be described in detail.

According to the present invention,

1) crushing the wheat;

2) adding water to the milled mill of step 1) and then infiltrating;

3) shaping the infiltrated mill of step 2);

4) fermenting the shaped mill of step 3) at a temperature of 34 to 38 占 폚; And

5) the fermentation of step 4), and drying the fermented yeast.

In the above method, the amount of water added in step 2) is preferably 20 to 30%, more preferably 24 to 28%, and most preferably 26 to 28%.

In this method, the molding of step 3) may be performed using a square, a circle, or an ellipse. In a specific embodiment of the present invention, molding is performed using a yeast molding mold.

In the above method, the fermentation of step 4) is preferably fermented at a temperature of 34 to 38 캜, more preferably at a temperature of 36 to 37 캜. In addition, it is preferable to invert and ferment at intervals of 2 to 4 days in the fermentation period.

In the above method, the fermentation of step 4) is preferably 1 to 30 days.

In the above method, drying in step 5) is preferably performed at a temperature of 40 to 50 ° C, more preferably 42 to 48 ° C, and most preferably 45 to 47 ° C. The drying is preferably carried out for 5 to 10 days, more preferably for 6 to 9 days, most preferably for 7 to 8 days,

In a specific embodiment of the present invention, the present inventors have found that wheat is crushed, mixed with water, infiltrated into yeast, molded into yeast, flipped at a temperature of 36 ° C every two days, fermented, and dried at 45 ° C for 7 days The medium-temperature fermented yeast prepared by the conventional method is effective in preventing microbial contamination and has a low amino acid level due to its low acidity (see Table 2 and FIG. 5) Analysis of organic acids, free amino acids and fragrance components showed excellent taste and flavor quality (see Table 6, Table 8 and Table 10) (See Table 11 and FIG. 8), the medium-temperature fermented yeast of the present invention and its preparation method are useful as a method for producing yeast of excellent quality by using the conventional method It can be used.

The present invention also provides a yeast produced by the method for producing a middle-temperature fermented leaven of the present invention.

The present invention also provides a Takju prepared with the koji of the present invention.

The nuruk is characterized by excellent enzyme activity and storage stability.

In a specific embodiment of the present invention, the koji produced by the medium temperature yeast production method of the present invention is effective in preventing microbial contamination (see Table 2 and FIG. 5), has excellent enzyme activity and glycation ability (see Table 3 and FIG. 6) (See Table 6, Table 8, and Table 10), and confirming that the shelf life is excellent (see Table 11 and FIG. 8) Can be usefully used for manufacturing excellent yeast and takju.

Hereinafter, the present invention will be described in detail with reference to Examples, Comparative Examples and Experimental Examples.

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

< Example  1> Middle temperature  Manufacture of fermented yeast

The fermented yeast was prepared by using the conventional method and the quality change of the yeast was confirmed during the manufacturing process.

Specifically, Korean millet (gangmul wheat) cultivated in Gwangju, Jeonnam Province was purchased and crushed just before making yeast. As shown in Fig. 1, the wheat was pulverized with a roller mill and then repeated three times at 7-mesh (2.8 mm), water-mixed at a water content of 26%, and infiltrated at room temperature for 1 hour. The infiltrated wheat was mixed, weighed 340 g, placed in a yeast molding mold, molded with yeast in the same size and weight using a molding machine, and fermented at a temperature of 36 ° C for 2 days. Dried at 45 ° C for 7 days, and then used as an assay sample for quality characteristics according to fermentation periods 0, 3, 6, 10, 20 and 30 days (Fig. 1).

< Comparative Example  1> Manufacture of natural fermented yeast

Using the same wheat as the above <Example 1>, yeast was naturally fermented and used as a control.

Specifically, just before making the same milnu leukocyte as in Example 1, the mixture was pulverized with a roller mill and then repeated three times with a 7-mesh (2.8 mm) length. The mixture was water-mixed at a water content of 26% Infiltrate. The infiltrated wheat was mixed and weighed 340 g each. Weighing the yeast using a molding machine, and then fermenting the mixture at 20 to 25 ° C for 2 days.

< Comparative Example  2> Commercial yeast

In order to compare the quality with the medium-temperature fermented yeast produced in Example 1, the commercially available fermented acidic yeast of Busan and the yeast ginseng of Gwangju were purchased and the same quality analysis experiment was performed (FIG. 2). Specifically, the acidic yeast was purchased from Kimgeongsan makkolli, and Songhaknulk was purchased from Songjak.

< Experimental Example  1> Middle temperature  Characterization of fermented yeast

The outline changes of the commercially available yeast (acidic yeast and yeast gruel) of the medium-temperature fermented yeast produced in Example 1 and that of Comparative Example 2 were compared.

Specifically, the weight of the yeast was measured with a micro weighing scale (MW-Ⅱ 6000N, CAS Co.) and the size was measured with a digimatic caliper (CD-20CPX, Mitutoyo Co.) 50, AND Co.).

As a result, as shown in Fig. 3, there was no significant difference in appearance between the medium-temperature fermented yeast and commercial fermented yeast at the 0th day of fermentation. In the early fermentation period, Litheia spp. (Fig. 3).

As a result of analyzing the weight, size and moisture content of the yeast, the weight and size of the yeast tended to decrease with the fermentation period as shown in Table 1. As the fermentation period became longer, . As a result of moisture content analysis, the acidic yeast and sesame yeast of control were 11.9% and 8.66%, respectively, and the moisture content of middle temperature fermented yeast was 1 to 2% (Table 1). In case of commercially available yeast, the moisture content is 8 to 10%. However, since the mesophilic yeast of the present invention has been dried at 45 ° C for 7 days, the moisture content as a whole is as low as 1 to 2%. The reason why the drying process of the yeast is artificially roasted in the present invention is to artificially lower the water content so as to inhibit the growth of other microorganisms and to enhance the storage stability of the yeast.

yeast Fermentation period (days) Weight (g) size
(Diameter x height mm) Water content (%) Control (commercially available yeast) acid - - 11.9 ± 0.1 ¹⁾ Control (Commercial Nuruk) Songhak - - 8.7 ± 0.2 Middle temperature
Fermentation
yeast 0 247.7 118.1 x 31.7 1.8 ± 0.1 3 241.4 118.4 x 31.9 1.5 ± 0.1 6 241.5 118.8 x 30.5 1.6 ± 0.0 10 242.8 118.6 x 31.6 1.9 ± 0.2 20 242.1 118.6 x 31.5 1.3 ± 0.1 30 241.3 117.5 x 30.0 2.0 ± 0.2

¹⁾ values are mean ± SD (N = 3).

< Experimental Example  2> Middle temperature  The fermentation period of fermented yeast Product temperature  Confirm change

The change in temperature of the neutral fermented yeast prepared in Example 1 and the natural fermented yeast in Comparative Example 1 were measured.

Specifically, the fermentation of pumon change by using a data logger U12-013's HOBO ^ⓡ was measured pumon changes in yeast according to the fermentation time (0, 3, 6, 10, 20, 30) every six hours.

As a result, as shown in FIG. 4, in the case of the mesophilic fermented yeast, the fermentation proceeded at 36 ° C., the koji microorganism started to operate from day 0, the highest temperature reached 45 ° C. during the most vigorous propagation on day 2, The fermentation was stagnant and the temperature of the fermented yeast remained constant at 36 DEG C for 30 days (Fig. 4). The fermentation was carried out at 20 to 25 ° C in the case of the yeast which had been subjected to the natural fermentation without any temperature and temperature control. From day 0, the yeast microorganism started its activity, 27 ℃, and after 3 days, the temperature remained constant at 24 ± 1 ℃. Although the fermentation temperature was different, overall growth and growth of microorganisms were similar to those of middle temperature fermented yeast.

< Experimental Example  4> Middle temperature  Identification of Physicochemical Properties of Fermented Yeast

The medium-temperature fermented yeast prepared in Example 1 Changes in physicochemical properties according to fermentation period were confirmed by measuring pH, total acidity and amino acidity.

Specifically, first of all, 20 ml of distilled water was added to the test sample, and the filtrate was prepared by leaching at room temperature for 3 hours. Samples for enzyme activity analysis were prepared by adding 50 ml of sodium chloride solution to 10 g of the sample and leaching it at low temperature (4 ° C) overnight or at room temperature (15 to 20 ° C) for 3 hours with occasional shaking and filtration. Respectively.

 The pH was measured at room temperature with a pH meter (orion 3 star, Thermo scientific Co.). Total acidity was determined by taking a certain amount of sample and filtering 10 ml of the sample through a filter paper into a 100 ml Erlenmeyer flask. 2 ~ 3 drops of 0.5% phenolphthalein indicator were added and neutralized with 0.1 N NaOH until red color was obtained. Was converted into lactic acid (%).

The amino acid level was reduced by 2 to 3 drops of 0.5% phenolphthalein indicator in 10 ml of the filtered sample, neutralized with 0.1 N NaOH until it became pale red, and 5 ml of neutral formalin solution was added thereto. The solution was titrated with 0.1 N NaOH and expressed as the number of ml of 0.1 N NaOH required to become pale pink.

As a result, as shown in the following Table 2 and FIG. 5, the acidic koji extract in commercial koji of Comparative Example 2 as a control had a pH of 6.64, and the pH of the koji-koji extract was 6.29. In the case of the mesophilic yeast of the present invention, the pH decreased from 6.18 to 5.84 according to the fermentation period (Table 2 and Fig. 5). The pH of the extracts of the commercially available nuruk extract was weakly neutral and the change between them was not significant.

In the case of acidity, acidity of acidic yeast and sesame yeast was 0.19% and 0.15%, respectively. The acidity of mesophilic yeast increased from 0.046% to 0.078% according to fermentation period (Table 2 and Fig. 5). The acidity of middle - temperature fermented yeast prepared from commercial nuruk was 2 to 5 times lower than that of commercial nuruk, which was expected to be effective in preventing the contamination of harmful bacteria such as harmful microorganisms in the early stage of koji fermentation.

In the case of amino acid, the acidic yeast and Songjun leu were the same value as 1.28 ㎖, and the middle temperature fermented yeast increased amino acid degree according to fermentation period. From 10 days of fermentation, it decreased to 0.48 ㎖ according to fermentation period ). Therefore, the prepared amino - acid fermented yeast was about 3 times lower than the commercial yeast of the control, so that when the Takju or Yakju was prepared, the taste of the tail was much canceled. This is thought to affect the taste change.

yeast Fermentation period (days) pH Acidity
(%, lactic acid) Amino acid degree
(0.1 N NaOH / 10 mL) Control (commercially available yeast) acid 6.64 ± 0.03 ¹⁾ 0.1881 ± 0.0068 1.28 + - 0.10 Control (Commercial Nuruk) Songhak 6.29 + 0.02 0.1518 0.0143 1.28 ± 0.01 Middle temperature
Fermentation
yeast 0 6.18 ± 0.05 0.0462 ± 0.0019 0.36 ± 0.01 3 5.90 + - 0.01 0.0708 0.0027 0.68 ± 0.02 6 5.88 ± 0.01 0.0786 0.0018 0.70 + 0.02 10 5.86 ± 0.02 0.0735 ± 0.0014 0.59 + 0.02 20 5.85 ± 0.01 0.0699 + - 0.0014 0.48 + 0.03 30 5.84 ± 0.01 0.0783 + - 0.0009 0.48 + 0.03

¹⁾ values are mean ± SD (N = 3).

< Experimental Example  3> Middle temperature  Identification of Enzymatic Properties of Fermented Yeast

The medium-temperature fermented yeast prepared in Example 1 Changes in enzymatic properties during fermentation period were confirmed by measuring α-amylase, glucoamylase, acidic protease activity and saccharifying power (SP).

Specifically, α-amylase activity was obtained by adding 50 ml of sodium chloride solution to 10 g of yeast, leaching at low temperature overnight or at room temperature for 3 hours, and then diluting the filtered solution to prepare a crude enzyme solution. 2 ml of 1% starch solution is taken in a test tube and preheated at 40 ° C for 5 minutes. Then, 0.1 ml of the enzyme solution is added to initiate the reaction, and 10 ml of the iodine solution is preliminarily pipetted at 0.1 minute intervals The mixture was put in a test tube, and the resulting color was maintained at 25 ° C., passed through a thick 10 mm, and the transmittance T% was measured by comparing the color at 670 nm. The enzyme activity (unit) was calculated by the following equation (1) according to Wohlgemuth value.

[Equation 1]

T _{30 min} : Transmittance after enzymatic reaction for _{30 min}

T _{0 min} : Transmittance before enzyme reaction

 Acidic protease activity was measured by adding 50 ml of sodium chloride solution to 10 g of the nuruk sample and leaking at low temperature (4 ° C) overnight or at room temperature (15 to 20 ° C) for 3 hours. 10 ml of the filtrate was put into a dialysis membrane and dialyzed overnight at 4 ° C in 10 to 2 M acetic acid buffer, and then the enzyme solution was diluted to prepare a crude enzyme solution. 1.0 ml of pH 3.0 McBain buffer and 0.5 ml of crude enzyme solution were added to 1.5 ml of casein solution, and reacted at 40 ° C for 60 minutes. Then, 3 ml of TCA solution was added to stop the reaction and remove the precipitate. 5 ml of a sodium carbonate solution and 1 ml of a phenol reagent were added to 1 ml of the above solution, and the mixture was developed at 40 ° C for 30 minutes. Absorbance was measured at 660 nm. An enzyme solution was added to the control immediately before the addition of the TCA solution. The enzyme activity was calculated by the following equation (2).

&Quot; (2) &quot;

Glucoamylase activity was obtained by adding 0.2 ml of 0.2 M acetic acid buffer to 1 ml of a 2% starch solution, preheating the mixture at 40 ° C for 5 minutes, reacting the mixture with 0.1 ml of the above koji enzyme solution at 40 ° C for 20 minutes, adding 0.1 ml of 1 N NaOH solution After the reaction was stopped, it was left to stand for 30 minutes, and 0.1 N hydrochloric acid solution was neutralized. The control used in the experiment was 0.2 ml of 0.2 M acetic acid buffer in 1 ml of the starch solution, and the mixture was preheated at 40 ° C for 5 minutes. The control was neutralized by adding 0.1 ml of 1 N hydrochloric acid solution as in the case of the sample, and the enzyme activity was determined by measuring the reducing sugar by the DNS method. Glucoamylase activity was determined as 1 unit of 1 mg of glucose production activity from soluble starch at 40 ° C for 60 minutes, and the glucoamylase activity of 1 g of the sample was calculated by the following equation (3).

&Quot; (3) &quot;

 In order to measure the saccharifying power (SP), the above-mentioned Nuruk enzyme solution was diluted with two dilutions of diluted water at least 10 ml, and then used as a crude enzyme solution. 50 ml of a 2% starch solution and 30 ml of a vinegar acid buffer solution were taken in a 100 ml volumetric flask and then preheated in a constant temperature water bath preheated to 55 캜 for 10 minutes and 10 ml of the enzyme solution was sacrificed for 60 minutes. In the control, 10 ml of distilled water was added and saccharified in the same manner for 60 minutes. The enzyme reaction was stopped by adding 10 ml of 0.5 N NaOH, quenched, and adjusted to 100 ml with distilled water.

5 ml of Felling A reagent, 5 ml of Felling B reagent and 40 ml of distilled water were added to a 250 ml Erlenmeyer flask, and 10 ml of distilled water was added to the Erlenmeyer flask containing the Felling reagent. The Erlenmeyer flask was boiled, (About 22 ㎖ of boiling water was consumed in the beginning, consuming about 24 ~ 26 ㎖). When the blue color of the copper sulfate in the Pehling reagent gradually disappears, 4 drops of the methylene blue solution are dropped. While the solution is being boiled, the standard glucose solution is dropped, and when the blue color disappears, the end point is defined as the number of consumed ml of the standard glucose solution. Next, add 10 ml of the enzyme solution into the Erlenmeyer flask containing the Pelling reagent, and boil and titrate with standard glucose solution. Add 4 drops of methylene blue solution to the solution until no blue color disappears. Respectively. Assuming that the number of consumed ml of the standard glucose solution until the completion of the titration is M, the saccharification rate is calculated by the following equations (4) to (6).

&Quot; (4) &quot;

&Quot; (5) &quot;

&Quot; (6) &quot;

As a result, as shown in Table 3 and FIG. 6, the α-amylase activity was 175.1 and 280.5 units / g in the case of commercial koji of Comparative Example 2 as a control, respectively. The medium-temperature fermented nuruk (TN-A) increased its activity by fermentation period, and its α-amylase activity was 310.3 units / g, which was 1.2 times higher than that of commercial nuruk (Table 3 and FIG. 6).

Glucoamylase activities were 908 and 3,372 units / g in acidic yeast and sesame yeast, and the activity of yeast in medium temperature fermented yeast decreased with increasing fermentation period. The activity of 5,592 units / g was higher than that of control yeast 2 to 3-fold higher activity (Table 3 and Figure 6). In conclusion, the fact that the saccharifying power of the medium-temperature fermented yeast prepared in Example 1 is higher than that of commercial yeast indicates that the effect of decomposing the starch of rice with the saccharide is large due to the enzyme activity of the yeast, which is the raw material of the yeast.

 The activity of acidic koji and sesame yeast were 4,779.8 and 4,212.5 units / g, respectively. The fermented yeast showed a tendency to increase and decrease with fermentation period. On the 30th day of fermentation, enzyme activity was 1,928.5 units / g, (Table 3 and Fig. 6). &Lt; tb &gt; &lt; TABLE &gt; Especially, when the protein decomposition ability is excessively high and the protein is decomposed and converted into an amino acid, it is advantageous that the protein decomposition ability is low because the taste difference of the protein is determined.

As a result of the sugar strength (SP) measurement, acidic koji and sesame koji were 467.3 and 915.5 SP, and medium temperature fermented yeast was maintained constant after the increase of sugar strength after fermentation on the third day. The saccharifying power of mesophilic fermented yeast of fermentation 30 days was 1055.6 SP, which was 1.5 times superior to that of control fermented yeast (Table 3 and FIG. 6). In the case of saccharification power, the value of 719.33 SP was also observed at 0 day of fermentation, which was thought to be the fermentation of the yeast in the course of drying for 7 days at 45 ° C.

As a whole, it was found that the α-amylase, glucoamylase activity and glycation ability of the mesophilic fermented yeast of the present invention were superior to commercially available nuruk, and that the commercially available nuruk had excellent acid-decomposing ability.

yeast Fermentation period
(Work) Enzyme activity (units / g) Saccharogenic power (SP) α-amylase Glucoamylase Acid protease Control (commercially available yeast) acid 175.07 ± 7.63 908.00 ± 147.50 4779.76 + 325.73 467.35 Control (Commercial Nuruk) Songhak 280.51 ± 17.90 3372.00 ± 39.34 4212.48 占 77.12 915.52 Middle temperature
Fermentation
yeast 0 186.50 ± 6.91 4428.00 ± 78.17 288.02 + - 57.43 719.33 3 267.44 ± 9.37 4007.50 ± 411.17 1340.28 ± 20.08 952.42 6 245.32 + - 41.96 4713.75 ± 23.42 2467.20 ± 29.43 978.02 10 256.29 + - 13.17 3895.50 ± 130.91 1500.00 ± 37.58 961.74 20 293.22 + - 11.82 5580.75 ± 173.49 1684.96 ± 30.64 1041.68 30 310.28 + 14.37 5392.00 ± 90.07 1928.52 + 89.81 1055.64

< Experimental Example  5> Middle temperature  Fermented yeast Viable cell count  Confirm change

The changes in the viable cell counts of the commercially available yeast (acidic yeast and yeast gruel) of the medium-temperature fermented yeast produced in Example 1 and that of Comparative Example 2 were measured.

Specifically, 10 g of the yeast was sampled and placed in a sterile bag, and 90 ml of sterile physiological saline was added thereto. The resulting solution was dissolved in a homogenizer and diluted with sterilized physiological saline in a stepwise manner. The microbial count on the PCA medium was expressed as CFU / g Respectively.

As a result, as shown in Table 4, the viable cell counts of commercially available yeast and control yeast were in the range of 10 ⁹ to 10 ^10, and the viable cell count of the yeast fermented by fermentation during the fermentation period was high at the early stage of fermentation And the fermentation period was decreased (Table 4). And Lichethemia sp., Rhizopus sp. Mucor sp. Etc.) were excluded from this experiment.

yeast Fermentation period (days) Number of living cells (cfu / g) Control (commercially available yeast) acid 1.8 占 0.2 占¹⁰¹⁾ Control (Commercial Nuruk) Songhak 3.7 ± 0.3 × 10 ⁹ Middle temperature
Fermentation
yeast 0 7.2 ± 1.7 × 10 ⁹ 3 3.6 ± 0.2 × 10 ¹⁰ 6 1.2 ± 0.6 × 10 ¹⁰ 10 1.8 ± 0.5 × 10 ¹⁰ 20 1.3 ± 0.3 × 10 ¹⁰ 30 8.4 ± 1.1 × 10 ⁹

¹⁾ values are mean ± SD (N = 3).

< Experimental Example  6> Middle temperature  Analysis of organic acids in fermented yeast

The organic acid of the medium-temperature fermented yeast prepared in Example 1 and the commercially available yeast (acidic yeast and yeast gruel) of Comparative Example 2 were analyzed.

 Specifically, 5 ml of 20% ethanol was added to 1 g of the nuruk sample, and the mixture was sonicated at 30 ° C. for 20 minutes under sonicator. The mixture was centrifuged at 3,000 rpm at 4 ° C. for 10 minutes, 5 ml of ethanol was added and the mixture was repeated three times in the same manner as above to give a total volume of 20 ml. The supernatant was taken and filtered through a 0.2 ㎛ membrane filter (Mullepore Cp., Ireland) and analyzed. The organic acid was analyzed using HPLC (LC-20A Prominence, Shimadzu Co., Japan) under the conditions shown in Table 5 below. Columns were KC-811 (7.8 x 300 mm, Shodex Co.) The flow rate was 0.6 ml / min, the injection volume was 10 μl, and the PDA (210 nm) was used for the detector (Table 5).

Configuration Condition  HPLC  LC-20A Prominence, Shimadzu Co., Japan  Column  KC-811 (300 mm, Shodex Co.)  Mobile phase  4 mM sulfuric acid  유율  0.6 ml / min  Injection volume  10 μl  Detector  PDA (210 nm)

As a result, as shown in Table 6, citric acid (citric acid) was detected as a major organic acid in the case of commercially available yeast (Comparative Example 2), and citric acid, malic acid (malic acid) and lactic acid acid (lactic acid) were analyzed as major organic acids (Table 6). Citric acid, malic acid and oxalic acid were analyzed in the case of medium temperature fermented yeast prepared in Example 1 of the present invention. Citric acid (citric acid) as a main organic acid was increased in the early stage of fermentation, (Table 6). The amount and type of organic acid were different according to the leek, which was thought to be the difference depending on the raw materials, the related fermenting microorganisms and the fermentation conditions.

Organic acid Composition (mg%) Control Control Medium temperature fermented yeast (days) Acidic yeast Songhak-yeol 0 3 6 10 20 30 Oxalic nd ²⁾ n.d 23.43 + 5.09 n.d n.d n.d 2.63 ± 1.05 10.49 + - 0.99 Citric 298.52 ± 45.89 ¹⁾ 186.65 + - 71.53 145.89 + - 22.03 205.95 ± 29.96 189.35 ± 29.23 174.83 + - 4.32 164.66 ± 2.66 155.56 ± 76.64 Tartaric n.d n.d n.d 2.03 ± 1.05 n.d n.d 2.57 + - 0.23 n.d Malic n.d 57.23 + - 19.74 41.04 + - 4.21 36.19 ± 15.26 35.09 + - 1.05 29.65 + - 6.40 28.78 ± 12.72 22.20 ± 1.05 Succinic n.d n.d n.d n.d n.d n.d n.d n.d Fumaric n.d n.d n.d n.d n.d n.d n.d n.d Lactic n.d 33.28 + - 11.37 n.d n.d n.d n.d n.d n.d Formic n.d n.d n.d n.d n.d n.d n.d n.d Acetic n.d n.d n.d n.d n.d n.d n.d n.d Pyroglutamic n.d n.d n.d n.d n.d n.d n.d n.d Total 298.52 277.16 210.36 244.17 224.44 204.49 198.65 188.25

¹⁾ values are mean ± SD (N = 3).

²⁾ nd was not detected.

< Experimental Example  7> Middle temperature  The fermentation period of fermented yeast Wheat  Free amino acid analysis

The free amino acids of the commercial yeast (acidic yeast and yeast gruel) of the medium-temperature fermented yeast produced in Example 1 and that of Comparative Example 2 were analyzed.

25 ml of 80% ethanol was added to 1 g of the sample, homogenized with a homogenizer, and extracted at 30 ° C for 15 minutes. The extract was centrifuged at 3,000 rpm for 10 min at 4 ° C, and 1 ml or 2 ml of the supernatant was taken out, evaporated to dryness using a concentrator or dry oven, and dissolved in 1 ml or 2 ml of 0.02 N HCl. (L-8900, Hitachi Co., Japan) under the conditions shown in Table 7 below.

 Configuration  Condition   Amino acid analysis   L-8900   Column Cation separation Lithum. 4.6mm
Cation Lithum filter. 4.6mm   Detector   UV-Vis (440 nm ~ 570 nm)   Buffer flow rate   0.45 ml / min   Ninhydrin flow rate   0.25 ml / min   Injection volume   100 μl   Mobile phase A (pH 2.85): Lithium citrate 1.41% + citric acid 0.7% + methanol 5% + HCl 0.9%
B (pH 4.20): Lithium citrate 1.41% + citric acid 0.7% + HCl 0.6%
C (pH 3.30): Lithium citrate 1.88% + lithium chloride 5.07% + HCl 1%

As shown in Table 8, the major amino acids of commercially available acidic yeasts of Comparative Example 2 are proline (sweet / bitter), alanine (sweet), valine (weak bitter), glutamic acid and aminobutyric acid. Total amino acid content was 602.92 ㎍ / ㎖, and the content of essential amino acid was 21.14 ㎍ / ㎖. The major free amino acids of Songhaknuruk consisted of 24 free amino acids such as proline (sweet / bitter), alanine (sweet), valine (weak bitter), urea and α-aminobutyric acid and total amino acid content was 929.98 ㎍ / ㎖ , And the content of essential amino acid was 103.63 占 퐂 / ml (Table 8).

Fermented yeast of the present invention was composed of eight free amino acids such as proline (sweet / bitter taste), valine (weak bitter taste), taurine, alanine and γ-aminobutyric acid and the total amino acid content was 107.23 Mu] g / ml, and the content of essential amino acid was 12.27 [mu] g / ml (Table 8).

From free amino acid analysis, it was found that the content of free amino acid in commercial nuruk was higher than that of middle temperature fermented yeast, and it was composed of various amino acids. It was thought that the difference in the raw materials, climate environment and fermentation conditions of the manufacturing area and the fermentation efficiency of takju as a fermentation agent were obvious.

Glass
amino acid Control Middle temperature fermented yeast (㎍ / ㎖, day) Acidic yeast Songhak-yeol 0 3 6 10 20 30 P-Ser 8.71 ± 0.65 ¹⁾ 16.49 + 1.05 n.d n.d n.d n.d n.d n.d Tau nd ²⁾ n.d 10.47 ± 1.55 6.67 ± 0.38 6.25 ± 1.68 5.04 ± 2.26 9.78 ± 1.48 6.17 ± 2.65 Urea 18.49 + - 3.84 34.41 + - 4.77 n.d n.d n.d n.d n.d n.d Asp 10.03 + - 1.72 7.67 ± 4.05 n.d n.d n.d n.d n.d n.d Thr ^c 1.58 + - 0.12 8.11 ± 4.91 n.d n.d n.d n.d n.d n.d Ser 2.53 + - 0.73 9.29 ± 5.89 n.d n.d n.d n.d n.d n.d Glu 20.43 + - 3.64 15.78 ± 1.13 n.d n.d n.d n.d n.d n.d Gly 13.38 + - 2.52 9.30 0.74 3.00 0.40 2.26 + 1.01 2.50 + - 0.84 3.81 ± 1.05 2.04 ± 0.53 1.44 + - 0.10 Ala 36.57 8.47 44.02 + - 2.61 2.47 ± 0.66 2.68 ± 0.51 2.98 ± 1.57 4.25 ± 0.87 2.57 + - 0.80 3.35 0.32 Cit n.d 1.70 + 1.05 n.d n.d n.d 6.77 n.d n.d a-ABA n.d 28.84 + - 4.85 15.91 + - 3.52 n.d n.d n.d n.d n.d Val ^c 22.56 ± 0.81 38.66 ± 2.15 12.38 + - 0.58 13.80 + - 4.74 12.14 + - 2.35 8.74 + - 4.32 13.35 + - 4.75 12.27 ± 7.89 Cys n.d 4.09 ± 1.05 7.67 ± 0.40 10.01 + - 1.05 10.58 ± 0.92 8.94 ± 1.05 n.d n.d Ile ^c n.d 22.47 ± 5.65 n.d n.d n.d n.d n.d n.d Leu ^c n.d 19.09 ± 2.95 n.d n.d n.d n.d n.d n.d Phe n.d 21.64 ± 0.39 n.d n.d n.d n.d n.d n.d b-Ala 17.17 + - 3.04 4.89 ± 0.55 n.d n.d n.d n.d n.d n.d b-AiBA n.d 1.64 0.15 n.d n.d n.d n.d n.d n.d g-ABA 19.80 + - 4.32 16.45 ± 9.16 4.00 0.93 1.53 + - 0.20 1.38 + - 0.54 1.72 ± 0.28 3.07 + - 0.72 2.01 ± 1.05 NH3 68.89 ± 3.53 62.20 + - 4.16 71.02 + - 5.93 42.14 + - 6.76 21.65 ± 3.90 31.29 ± 2.17 19.70 ± 1.59 36.36 ± 3.08 Lys ^c n.d 2.13 ± 0.15 n.d n.d n.d n.d n.d n.d His ^c n.d 5.63 ± 0.15 n.d n.d n.d n.d n.d n.d Arg ^c n.d 6.36 ± 0.67 n.d n.d n.d n.d n.d n.d Hypro 2.10 ± 1.05 2.04 ± 1.26 n.d n.d 2.40 ± 1.05 1.10 ± 0.15 4.17 ± 1.28 1.54 + 1.05 Pro 360.69 ± 9.49 547.07 + - 4.83 1.18 ± 0.78 39.72 ± 1.05 43.54 + - 5.23 49.53 + 1.58 17.15 ± 0.91 44.08 + - 8.16 Totals 602.92 929.98 128.10 118.82 103.41 127.15 71.83 107.23 Essential amino acids ^{3 )} 24.14 103.63 12.38 13.80 12.14 8.74 13.35 12.27

¹⁾ values are mean ± SD (N = 3).

²⁾ nd was not detected.

³⁾ Essential amino acids

< Experimental Example  8> Middle temperature  Analysis of aroma components of fermented yeast

The aroma components of the commercial yeast (acidic yeast and yeast gruel) of the medium-temperature fermented yeast produced in Example 1 and the comparative example 2 were analyzed.

 Specifically, 100 ml of an ether-pentane mixed solution (1: 1) was added to 10 g of the nuiru sample, and the mixture was extracted at 150 rpm at 20 ° C. overnight. The mixture was filtered and then 50 ml of an ether- And re-extracted at 150 rpm and 20 ° C for 1 hour. The resulting solution was filtered and concentrated. The filtrate was filtered through a 0.2 μm syringe filter (Whatman Co., USA) and analyzed by GCMS (GCMS-QP2010plus ) Were analyzed under the conditions shown in Table 9 below (Table 9).

Content Condition GC-MS QP2010plus Column HP-INNOWAX (60 m x 0.25 mm i, d x 0.25 m film thikness) Injection temp. 250 ℃ Column flow 1.0 cm / sec (linear velocity, N ₂ ) Split ration 20 Column temp.

Detection FID 250 ° C Injection volume 1 μl

As a result, as shown in Table 10, a total of 48 fragrance components were detected, 32 of hydrocarbone series, 10 of ester series, 4 of acid series and 2 of alcohols. The common fragrance components of the three kinds of yeast (middle-temperature fermented yeast, acidic yeast, and Songjung yeast) analyzed were octane, formic acid ethyl ester, (E) -6-methyl-3-undecene, decane, methyl benzene, undecane, metyhllaurate, 1-ethyl-3-methyl-benzene, acidic acid and n-hexadecanoin acid (Table 10).

The major constituents of Nuruk are acidic Nuruk (22Z, 12Z) -9,12-octadecadienoate ethyl, ethyl palmitate, n-hexadecanoic acid, 8-heptadecene and 9-octadecenoic acid. N-hexadecanoic acid, octadec-9-enoic acid, methyl octadeca-9,12-dienoate and 1-tricosene, and the middle temperature fermented yeast TN- 4-ethoxy-benzoic acid ethyl ester, 1,3-dimethyl-benzene and 2-methyl-octane (Table 10). Especially. There is no significant difference in the constituents of the commercial nuruk and the aroma of the medium-temperature fermented yeast of the present invention.

peak RT (min) ingredient yeast Acidic yeast Songhak-yeol Middle temperature
Fermented yeast One 5.467 Octane 0.72 0.81 1.03 2 5.861 3-Methyl-octane 0.29 3 5.982 Formic acid ethyl ester 0.48 0.6 0.66 4 6.445 Dodecane 1.12 5 6.448 2-Methyl-octane 1.45 6 6.574 (E) -6-Methyl-3-undecene 0.57 0.63 0.75 7 6.793 Tetrahydro-2-methyl-furan 0.25 8 7.344 Nonane 0.6 0.86 9 9.151 2-Methyl-nonane 0.35 10 10.76 Decane 0.34 0.4 0.69 11 11.322 3,3-Dimethyl-octane 0.28 12 12.574 3,7-Dimethyl decane 0.39 13 12.805 Methyl benzene 0.93 0.98 1.35 14 15.876 Undecane 0.17 0.2 0.33 15 17.57 Methyl laurate 0.35 0.39 16 17.593 Ethyl-benzene 0.51 17 18.467 1,3-Dimethyl-benzene 1.07 1.17 1.65 18 21.236 1,2-Dimethyl-benzene 0.48 19 21.249 Xylene 0.5 0.69 20 23.861 1-Ethyl-3-methyl-benzene 0.52 0.59 0.86 21 25.12 1,3,5-Trimethyl-benzene 0.21 22 27.637 1,2,4-Trimethyl-benzene 0.78 23 27.649 1,3,5-Trimethyl-benzene 0.85 1.28 24 38.34 3-Hexadecene 0.6 25 39.584 Acetic acid 0.43 0.69 1.07 26 44.157 Acetic acid ethyl ester 23.55 33.8 27 45.258 2,3-Butanediol 0.61 28 47.947 n-Pentadecane 0.46 0.31 29 50.651 1-Hexadecene 0.53 2.45 30 50.671 9-Octadecene 0.95 31 61.891 9-Eicosene 1.04 32 61.909 1-Octadecene 2.97 33 66.855 8-Pentadecanone 0.52 34 72.137 3-Eicosene 0.84 35 72.14 1-Nonadecene 36 72.314 Ethyl tetradecanoate 0.56 37 78.364 4-Ethoxy-benzoic acid ethyl ester 1.7 38 81.908 Ethyl palmitate 7.7 39 90.247 1-Tricosene 4.3 40 90.694 Methyl octadeca-9,12-dienoate 7.99 41 90.745 Ethyl stearate 1.03 42 91.598 9-Octadecenoic acid ethyl ester 5.37 43 93.605 (9Z, 12Z) -9,12-Octadecadienoate ethyl 8.05 44 98.297 1-Eicosanol 0.5 45 100.562 1,2-Benzenedicarboxylicacid, dibutylester 0.18 46 110.209 n-Hexadecanoic acid 7.36 10.4 7.67 47 128.877 8-Heptadecene 6.43 48 128.935 Octadec-9-enoic acid 8.29

Acetic acid ethyl ester and n-hexadecanoic acid accounted for more than 30 ~ 40% of the fragrance components of the leek. Acetic acid ethyl ester was the most abundant in the fruits such as pineapple fruit It is a component of the fragrance that is present in the wine. Octadec-9-enoic acid is a fragrant ingredient with a sour flavor, which is characterized as a characteristic fragrance of Songhaknuruk, and 4-Ethoxy-benzoic acid The ethyl ester was a fragrant component of soy sauce and was analyzed as a characteristic aroma component of middle - temperature fermented yeast.

< Experimental Example  9> Middle temperature  Identification of storage stability of fermented yeast

The residual capacity of the koji enzyme was analyzed according to the storage period of the medium-temperature fermented yeast prepared in Example 1 above.

Specifically, the activity of α-amylase, glucoamylase, acidic protease and the saccharifying power (SP) were measured, and the same procedure as in Example 3 was carried out. During the storage period of 0, 8 and 12 weeks, The residual capacity of koji enzyme was analyzed at.

As a result, as shown in the following Table 11 and FIG. 8, the α-amylase activities of the yeast were maintained at 4 ° C and 310 units / g, respectively. 99.9% was retained, and 0.1% enzyme was inactivated. At 15 ℃ storage condition, 271 units / g was maintained at 87.3%, 12.7% enzyme was inactivated and at 30 ℃ storage condition, 83% was maintained at 257.4 units / g and 17% enzyme was inactivated 8).

Glucoamylase activity was maintained at 95.2% at 5,136 units / g at 4 ℃, but the enzyme was inactivated at 4.8%. At 15 ℃ storage condition, 5,038 units / g was maintained at 93.4%. In fact, 6.6% enzyme was inactivated and at 30 ℃ storage condition, it was 88.6% at 4,776 units / g and 11.4% of enzyme was inactivated And Fig. 8).

Acidic protease activity showed that the 12 - week enzyme activity was maintained at 94.8% at 2,776.3 units / g at 4 ℃ and 5.2% enzyme was inactivated. At 15 ℃ storage condition, 2,708.8 units / g was maintained at 92.5% and the actual 7.5% enzyme was inactivated. At 30 ℃ storage condition, 2,629.9 units / g was maintained at 89.8% and 10.2% of enzyme was inactivated 8).

As a result of analyzing saccharogenic power, which is widely used in Korea, by SP saccharogenic power unit, the 12 - week enzyme activity of medium - temperature fermented yeast was maintained at 97.1% at 1,025.3 SP at 4 ℃ storage condition. Analysis of the yeast at that point in time showed that 2.9% was inactivated. At 15 ℃ storage condition, 95.9.2 units / g was maintained at 90.9%. In fact, 9.1% enzyme was inactivated and at 30 ℃ storage condition, 85.5% was maintained at 902.9 units / g and 14.5% of enzyme was inactivated 8).

As a result of analyzing the residual capacity of these enzymes, α-amylase, glucoamylase, acidic protease, and saccharogenic power were the best surviving enzymes at 4 ℃ storage condition for 12 weeks (90 days) The residual capacity of the enzyme was excellent. It is believed that the inhibition of microbial proliferation and enzymatic degradation at 4 ° C is maintained so that the enzymatic activity is maintained over 90% even during the storage period of 12 weeks.

Storage period (week) Temperature (℃) 4 15 30 α-amylase (units / g, weeks) 0 310.28 ± 14.37 ¹⁾ 310.28 + 14.37 310.28 + 14.37 8 309.17 + - 11.08 290.30 ± 14.90 278.69 + - 11.03 12 310.03 + - 3.11 271.00 ± 9.06 257.44 + - 6.58 Glucoamylase (units / g, weeks) 0 5392 ± 90.07 5392 ± 90.07 5392 ± 90.07 8 5230 + - 81.02 5258 ± 86.56 5160 ± 32.79 12 5136 + 76.91 5038 ± 77.78 4776 ± 10.49 Acidic protease (units / g, weeks) 0 2928.52 + 89.81 2928.52 + 89.81 2928.52 + 89.81 8 2921.48 + - 8.63 2859.58 + - 71.97 2779.12 + 27.01 12 2776.32 + - 21.70 2708.76 + - 6.85 2629.92 + - 63.79 Saccharogenic power
(SP, weeks) 0 1055.64 1055.64 1055.64 8 1045.27 1009.25 1002.34 12 1025.26 959.15 902.94

¹⁾ values are mean ± SD (N = 3).

Claims (7)

1) crushing the wheat;
2) adding water to the milled mill of step 1) and then infiltrating;
3) shaping the infiltrated mill of step 2);
4) fermenting the shaped mill of step 3) at a temperature of 34 to 38 占 폚; And
5) the fermentation of step 4), followed by drying.
The method according to claim 1, wherein the fermentation in step (4) is performed at a temperature of 36 to 37 ° C.
[3] The method according to claim 1, wherein the fermentation of step 4) is performed for 1 to 30 days.
The method according to claim 1, wherein the drying in step 5) is performed at a temperature of 40 to 50 ° C for 5 to 10 days.
The yeast produced by the method of manufacturing the mesophilic fermented leaven of claim 1.
[6] The koji according to claim 5, wherein the koji is excellent in enzyme activity and storage stability.
A takju prepared by the koji of claim 5.

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