KR102184735B1 - Manufacturing method of barley vinegar having high GABA content using farm scale surface fermentation and the barley vinegar manufactured thereby - Google Patents

Abstract

The present invention relates to a method for producing barley vinegar having a high GABA content through farmhouse-type surface fermentation and barley vinegar prepared thereby. Specifically, barley vinegar prepared by the farmhouse-type barley vinegar production method using the native fermentation seeds of the present invention has high acidity, high DPPH and ABTS radical scavenging ability, so it has excellent antioxidant activity, high GABA content, color, taste, Since overall preference such as fragrance is excellent, the barley vinegar manufacturing method can be usefully used in the field of manufacturing functional fermented vinegar including barley vinegar.

Description

Manufacturing method of barley vinegar having high GABA content using farm scale surface fermentation and the barley vinegar manufactured thereby}

The present invention relates to a method for producing barley vinegar having a high GABA content through farmhouse-type surface fermentation and barley vinegar prepared thereby.

Vinegar, a fermented food that has been used for a long time, is not only used as a seasoning during cooking, but is also widely used as a food preservative and medicine. Vinegar is manufactured by fermenting with microorganisms using grains, fruits, and liquor rich in sugar or starch as the main ingredients. Brewed vinegar made by fermentation has the aroma of the raw material and the aroma generated during the fermentation process. It contains various organic acids, sugars, amino acids, esters, etc. Vinegar has a very strong sterilizing power, so it is widely used in processed foods, and has a stress relief effect, and the unique acidity of vinegar stimulates appetite and promotes digestive juice secretion, which is beneficial for health. In recent years, a new functional fermented vinegar market such as healthy fermented vinegar using local agricultural products has been formed, and it has been developed in the form of beverages for purposes such as diet, skin care, adult disease prevention, and fatigue recovery.

Barley, one of the grains that can be used as a raw material for grain vinegar, contains a large amount of tocotrienol, which inhibits cholesterol synthesis, and contains much more calcium, fiber, and B vitamins than rice, making the skin elastic, and tocopherol. Since it contains an ingredient, it can act as an antioxidant (Per Aman et al. J. Agric. Food Chem., 35(5): 704-709, 1987). In oriental medicine, germinated barley is called malt and is used as a medicinal material, and it is mainly applied to stomach weakness and indigestion because it has an action to promote digestion and function of geonwi (健胃). Especially, it is used when the stomach is full due to food, pain, digestion, and tastelessness (Young-eun Lee et al., Herbal Food Materials, Mun Undang, pp36-39, 2013).

On the other hand, GABA (γ-amino butyric acid) is a kind of amino acid that is widely distributed in general cereals and animals and plants. It is a neurotransmission inhibitory substance present in the mammalian cerebellum. It activates brain cell metabolism to prevent stroke and mental concentration. Effects on strengthening, memory enhancement, and insomnia have been confirmed, and efficacy has been reported to relieve hangover, relieve stress, lower hypertension, activate liver function and promote alcohol metabolism, relieve obesity, promote kidney function, and promote growth and development.

An object of the present invention is to provide a method for producing barley vinegar having a high GABA content through farmhouse-type surface fermentation and barley vinegar prepared thereby.

In order to achieve the above object, the present invention is a step of 1) saving water after immersing rice in water; 2) increasing the rice of step 1) to prepare godubap; 3) inoculating and culturing the seed of Aspergillus luchuensis 74-5 (KACC93235P) to the godubap of step 2); 4) drying the cultured godubap of step 3) to prepare rice malt; 5) mixing the rice malt, fresh water, and Saccharomyces cerevisiae Y204 (KACC93237P) yeast seed of step 4), and fermenting it to prepare a base liquor; 6) preparing barley makgeolli by fermenting godubap made of barley and marinated water in the mash of the base liquor of step 5); And 7) Gluconacetobacter saccharivorans ( Gluconacetobacter saccharivorans ) CV1 (KACC17058) acetic acid bacteria or Acetobacter pasteurianus ( Acetobacter pasteurianus ) CV3 (KACC17057) by mixing seeds prepared with acetic acid bacteria in the barley rice wine of step 6) It provides a method for producing barley vinegar comprising the step of fermenting.

In addition, the present invention provides barley vinegar prepared by the above method.

Barley vinegar prepared by the farmhouse-type barley vinegar production method using the native fermentation seeds of the present invention has high acidity, high DPPH and ABTS radical scavenging ability, so it has excellent antioxidant activity, high GABA content, and overall color, taste, fragrance, etc. Since the preference is excellent, the barley vinegar manufacturing method can be usefully used in the field of functional fermented vinegar including barley vinegar.

1 is a graph showing a result of measuring the moisture absorption rate and moisture content according to the soaking time of Ohdae rice.
Figure 2 is a schematic diagram showing a manufacturing method of rice koji (white soup ) using Aspergillus luchuensis 74-5 seeds.
3 is a graph (A), and a photograph (B) of a photograph of the prepared rice malt, as a result of measuring the change in temperature of rice malt according to imperial time in the process of manufacturing rice malt using Aspergillus luchuensis 74-5 seed.
Figure 4 is a schematic diagram showing a manufacturing method of rice malt (Hwangguk) using Aspergillus duck material ( A. oryzae ) 75-2 seed.
FIG. 5 is a graph (A) of a result of measuring the change in temperature of rice malt according to imperial time in the process of manufacturing rice malt using Aspergillus duck material 75-2 seed, and a photograph (B) of the prepared rice malt.
Figure 6 is a total acidity according to the initial pH of barley vinegar prepared using Gluconacetobacter saccharivorans CV1 acetic acid bacteria (A) or Acetobacter pasteurianus CV3 acetic acid bacteria (B) It is a graph of the measurement result.
7 is a graph showing the results of measuring the total acidity according to the fermentation temperature of barley vinegar prepared using gluconacetobacter sachariborans CV1 acetic acid bacteria (A) or Acetobacter pasteurianus CV3 acetic acid bacteria (B).
FIG. 8 is a graph showing results of measuring DPPH radical scavenging activity (A), ABTS radical scavenging activity (B), and GABA content (C) of barley vinegar prepared using CV1 or CV3.
9 is a graph showing results of analysis of multiple fragrances of barley vinegar prepared using CV1 or CV3.
10 is a graph showing the results of measuring the preference (A) by age and preference (B) by sex of barley vinegar prepared using CV1 or CV3.

Hereinafter, the present invention will be described in detail.

The present invention comprises the steps of 1) saving water after immersing rice in water; 2) increasing the rice of step 1) to prepare godubap; 3) inoculating and culturing the seed of Aspergillus luchuensis 74-5 (KACC93235P) to the godubap of step 2); 4) drying the cultured godubap of step 3) to prepare rice malt; 5) mixing the rice malt, fresh water, and Saccharomyces cerevisiae Y204 (KACC93237P) yeast seed of step 4), and fermenting it to prepare a base liquor; 6) preparing barley makgeolli by fermenting godubap made of barley and marinated water in the mash of the base liquor of step 5); And 7) Gluconacetobacter saccharivorans ( Gluconacetobacter saccharivorans ) CV1 (KACC17058) acetic acid bacteria or Acetobacter pasteurianus ( Acetobacter pasteurianus ) CV3 (KACC17057) by mixing seeds prepared with acetic acid bacteria in the barley rice wine of step 6) It provides a method for producing barley vinegar comprising the step of fermenting.

The rice of step 1) may be Odae rice or Hwaseonchal rice, but is not limited thereto, and all varieties of rice that are commercially available may be used.

The immersion in step 1) may be made for 40 to 100 minutes, 50 to 100 minutes, 40 to 90 minutes, 50 to 90 minutes, 40 to 80 minutes, 50 to 80 minutes, 40 to 70 minutes, or 50 to 70 minutes. have.

Aspergillus luchuensis 74-5 seed of step 3) is 0.01 to 10%, 0.01 to 5%, 0.01 to 1%, 0.01 to 0.5%, 0.05 to 10%, 0.05 to 5%, 0.05 to 1 %, or 0.05 to 0.5%.

Step 3) after inversion of the mixture, the temperature is 25 to 40 ℃, 30 to 40 ℃, 33 to 40 ℃, 25 to 38 ℃, 30 to 38 ℃, 33 to 38 ℃, 25 to 36 ℃, 30 to 36 ℃ Or it may include a step of lowering to 33 to 36 ℃. By lowering the temperature after turning over, it is possible to induce the production of citric acid, which has a sour taste.

The drying in step 4) is 45 to 60°C, 48 to 60°C, 50 to 60°C, 45 to 57°C, 48 to 57°C, 50 to 57°C, 45 to 55°C, 48 to 55°C, or 50 to It can be done at 55°C.

Saccharomyces serviziae Y204 yeast seed in step 5) is 0.001 to 1.0%, 0.001 to 0.5%, 0.001 to 0.1%, 0.005 to 1.0%, 0.005 to 0.5%, or 0.005 to It can be mixed at a concentration of 0.1%.

The barley makgeolli of step 7) has an alcohol content of 3 to 10%, 4 to 10%, 5 to 10%, 3 to 8%, 4 to 8%, 5 to 8%, 3 to 7%, 4 to 7% , Or it may be prepared in 5 to 7%.

The barley makgeolli of step 7) was adjusted to pH 2.5 to 4.5, 2.8 to 4.5, 3.0 to 4.5, 2.5 to 4.2, 2.8 to 4.2, 3.0 to 4.2, 2.5 to 4.0, 2.8 to 4.0, or 3.0 to 4.0 I can.

Seeds prepared with gluconacetobacter sachariborance CV1 (KACC17058) acetic acid bacteria or Acetobacter pasturianus CV3 (KACC17057) acetic acid bacteria of step 7) are 0.1 to 30%, 1 to 30%, 5 to 30 in barley rice wine %, 0.1 to 20%, 1 to 20%, 5 to 20%, 0.1 to 15%, 1 to 15%, or 5 to 15%.

The fermentation of step 7) is 10 to 35°C, 12 to 35°C, 14 to 35°C, 20 to 35°C, 10 to 33°C, 12 to 33°C, 14 to 33°C, 20 to 33°C, 10 to 20 It may be made at ℃, 12 to 20 ℃, or 14 to 20 ℃.

The fermentation of step 7) may be performed for 5 to 50 days, 5 to 40 days, 5 to 30 days, or 5 to 25 days.

In addition, the present invention provides barley vinegar prepared by the barley vinegar production method.

The barley vinegar manufacturing method may have the characteristics as described above. In one example, the method comprises the steps of 1) immersing rice in water and then saving water; 2) increasing the rice of step 1) to prepare godubap; 3) inoculating and culturing the seeds of Aspergillus luchuensis 74-5 (KACC93235P) to the godubap of step 2); 4) drying the cultured godubap of step 3) to prepare rice malt; 5) mixing the rice malt, fresh water, and Saccharomyces serviziae Y204 (KACC93237P) yeast seed of step 4), and fermenting it to prepare a base liquor; 6) preparing barley makgeolli by fermenting godubap made of barley and marinated water in the mash of the base liquor of step 5); And 7) barley vinegar comprising the step of fermenting the barley makgeolli of step 6) by mixing and fermenting seeds prepared with gluconacetobacter saccharibolans CV1 (KACC17058) acetic acid bacteria or Acetobacter pasteurianus CV3 (KACC17057) acetic acid bacteria It may be a manufacturing method of.

The barley vinegar may have an enhanced GABA content by acetic acid fermentation by the acetic acid bacteria.

In a specific embodiment of the present invention, the present inventors derive the optimum soaking time of raw rice in the process of manufacturing rice malt, and prepared using Aspergillus luchuensis 74-5 seed or Aspergillus duck material 75-2 seed. Aspergillus luchuensis 74-5 seeds suitable for preparing barley vinegar were derived by confirming the change in temperature of rice malt and comparing the enzyme activity (see Tables 1 to 3 and FIGS. 1 to 5).

In addition, the present inventors derived the optimal yeast spawn and temperature conditions in the process of preparing barley makgeolli using the rice malt and yeast spawn Saccharomyces serviziae Y204 spawn or Saccharomyces serviziae Y283 spawn. (See Tables 4 and 5).

In addition, the present inventors of the present invention in the process of producing barley vinegar using the barley makgeolli and fermentation seed, gluconacetobacter sachariborance CV1 (KACC17058) acetic acid bacteria or Acetobacter pasteurianus CV3 (KACC17057) acetic acid bacteria, and The fermentation temperature conditions were derived, and the quality characteristics of barley vinegar prepared under optimal conditions were checked, and sensory characteristics were evaluated. The acidity of barley vinegar prepared by the production method of the present invention was high, and DPPH and ABTS radical scavenging ability was high, so that the antioxidant activity was high. It was excellent, it was confirmed that the GABA content was high, and the overall acceptability such as color, taste, and fragrance was excellent (see Tables 6 to 12 and FIGS. 6 to 10).

Therefore, the method for producing barley vinegar may be usefully used in the field of producing functional fermented vinegar including barley vinegar.

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

However, the following examples are merely illustrative of the present invention, and the contents of the present invention are not limited by the following examples.

Experimental Example 1. Establishment of Optimal Conditions for Manufacturing Method of Rice Nuruk

In order to establish the optimum conditions for the first step of the barley vinegar production process, the rice malt (fermentation agent) manufacturing method, the following experiment was performed.

1-1. Establishment of soaking time conditions for raw rice

Since it is important to set the soaking time of raw rice in the manufacturing process of rice malt, Odae rice (Cheorwon-gun, Gangwon-do, Republic of Korea, 2016) and Hwaseonchal rice (Ganghwa-gun, Incheon Metropolitan City, Republic of Korea, 2016) were purchased as grain raw materials for making rice malt , These rice were immersed for 100 minutes at intervals of 10 minutes, and the water absorption rate of the raw rice was calculated by the following calculation formula 1.

[Calculation 1]

Moisture absorption rate (%) = (weight after moisture absorption-weight before moisture absorption) / (weight before moisture absorption) × 100

In addition, the moisture content of each sample was measured using a moisture measurement scale (MX-50, And Co., Ltd., Japan).

As a result, the moisture content before immersion of Ohdae and Hwaseonchal rice was 10.53% and 9.14%, respectively, and after 1 hour of soaking, the moisture absorption rate reached 28.01% and 39.22%, respectively, reaching saturated moisture content (30.36% and 36.50%, respectively). It was confirmed that (Table 1 and Fig. 1). In addition, the moisture absorption rate of Hwaseonchal rice is about 1.4 times higher than that of Odae rice, and the saturated moisture content is about 1.2 times higher, so that the raw material rice was selected as Hwaseonchal rice.

1-2. Comparison of the quality of rice malt according to the fungal spawn

In order to select the optimal seed by comparing the quality of rice malt according to the fungal spawn, as the fungal spawn, Aspergillus luchuensis 74-5 strain and Baekguk bacteria Aspergillus duck material ( A. oryzae ) 75 -2 strains were distributed from the Korean Agricultural Culture Collection (KACC) from the National Academy of Agricultural Sciences (Table 2), and rice malt was prepared using these strains, and the quality was compared and analyzed.

Strain name The scientific name of the strain KACC accession number 74-5 Aspergillus luchuensis 93235P 75-2 A. oryzae 93236P

1-2-1. Preparation of rice malt (white soup) using Aspergillus luchuensis 74-5 seed

According to the result of Experimental Example 1-1, after washing Hwaseonchal rice, soaking for 1 hour, removing water for 50 minutes, and then steaming for 1 hour using a steaming machine to prepare godubap, and the moisture content of the godubap Was about 33%. After that, cool the godubap to 40℃ with a cooler, inoculate Aspergillus luchuensis 74-5 seeds with 0.1%, and then use an imperial fermentor (MINI-15, Yaegaki Food, Japan) for 20 hours. And the empire process for a total of 28 hours with a second empire 8 hours, and dried at 50 ℃ for 17 hours. In order to induce the production of citric acid that gives off a sour taste in this process, after the second inversion, lower the temperature to 35°C and incubate for 16 to 17 hours, then leave the country and go through the drying process to produce sour rice koji (white soup). Was prepared (Fig. 2). In particular, this white soup is a rice malt that is widely used in makgeolli production.

1-2-2. When manufacturing rice malt using Aspergillus luchuensis 74-5 seed, check the change in quality of rice malt according to imperial time

In the process of manufacturing the rice malt of Experimental Example 1-2-1, the change in the temperature of the rice malt was measured from the time of seed inoculation until departure. Pumon of ssalnuruk was measured using a sensor with the data logger ^{(Data logger, U12-013, HOBO ®} , USA).

As a result, the temperature of rice koji dropped and then increased in the stage before the first turnover, which is interpreted as a result of the adaptation of the inoculated Aspergillus luchuensis 74-5 fungi to the environment. After the first and second reversal, the temperature of the product rose rapidly, and then maintained constant, which is interpreted as the smooth growth of rice malt due to smooth circulation of air introduced from the outside (FIG. 3).

1-2-3. Preparation of Rice Nuruk (Hwangguk) Using Aspergillus Duck Material 75-2 Seed

After preparing and cooling godubap from Hwaseonchal rice in the same manner as described in Experimental Example 1-2-1, the Aspergillus duck material 75-2 seed was inoculated with 0.1%, and then imperial fermentation (MINI-15, Yaegaki Food) was used to go through the empire process. In order to increase the starch resolution in this process, after the second flip, the heater was stopped to promote the production of sweetness by allowing the rice koji to grow spontaneously.After cultivation for 16 hours, the rice koji that tastes sweet after leaving and drying (Hwangguk) was prepared (Fig. 4). In particular, these yellow soups are rice malt that is widely used in the production of takju, yakju and cheongju.

1-2-4. When manufacturing rice malt using Aspergillus duck ash 75-2 seed, the change in quality of rice malt according to imperial time was confirmed

In the process of manufacturing the rice malt of Experimental Example 1-2-3, the change in product temperature of the rice malt was measured from the time of seed inoculation until departure. The temperature of rice malt was measured using a sensor-equipped data logger (Data logger, U12-013, HOBO ^® ).

As a result, the temperature of rice koji dropped by about 1℃ before the first turnover, and then increased. This is interpreted as a result of adaptation of the inoculated Aspergillus duckweed 75-2 fungi to the environment. After the first flip, the pum temperature fell and then rose again to the set heater temperature (38°C), and after the 2 nd flip, the pum temperature dropped and then rapidly increased, and then maintained constant. It is interpreted that the circulation of air introduced from the outside is smooth and the growth of rice malt is made vigorously (FIG. 5).

1-2-5. Comparison of Enzyme Activity of Each Rice Nuruk Made with Two Fungal Sprouts

The activities of alpha-amylase, glucoamylase, and acidic carboxypeptidase were measured and compared in the white and yellow soups prepared in Experimental Examples 1-2-1 and 1-2-3, respectively. .

1-2-5-1. Alpha-amylase activity measurement method

Baekkuk prepared in Experimental Examples 1-2-1 and 1-2-3, respectively, in 10 ml of a buffer solution in which a 0.1 N acetic acid solution and a 0.1 N sodium acetate solution were mixed at a volume ratio of 1:2 1 g of yellow soup was suspended to leach the enzyme. Thereafter, 2 ml of a 1% starch solution was put in a test tube and preheated at 40° C. for 5 minutes, and then 0.1 ml of the enzyme solution was added to initiate the reaction, and 0.1 ml of the reaction solution was taken out at intervals of 1 minute to prepare 10 iodine solution in advance. It was mixed by putting it in the test tube put in each ml. By measuring the absorbance of 670 nm through which the iodine mixture was transmitted through a 10 mm quartz cell, the transmittance (T, %) was calculated, and the enzyme activity was calculated by the following calculation formula 2 according to the Wohlgemuth value.

[Calculation 2]

Enzyme activity (Units/g) = {12.75 × (T _{30 min} -T _{0 min} ) / 30 (min)} × dilution factor

(T _{30 minutes} : permeability 30 minutes after the start of the enzyme reaction; And

T _{0 min} : Permeability before the start of the enzyme reaction)

1-2-5-2. Method for measuring glucoamylase activity

To 1 ml of 2% starch solution, 0.2 ml of 0.2 M acetic acid buffer was added, preheated at 40°C for 5 minutes, and 0.1 ml of the enzyme solution prepared in the same manner as described in Experimental Example 1-2-5-2 was added to 40°C. After reacting for 20 minutes, 0.1 ml of 1N sodium hydroxide (NaOH) solution was added to stop the reaction. Thereafter, the reaction solution was allowed to stand for 30 minutes, and then 0.1 ml of 1 N hydrochloric acid solution was added to neutralize. The control was used as a sample mixed with reagents in the same manner as above without an enzyme solution, and the enzyme activity was calculated as the amount of glucose produced by measuring the reducing sugar by DNS method. One unit, which is a unit of glucoamylase activity, was defined as producing 1 mg of glucose from soluble starch at 40°C for 60 minutes, and the glucoamylase activity of 1 g of the sample was calculated by the following calculation formula 3.

[Calculation 3]

Enzyme activity (Units/g) = Produced glucose weight (mg) × 60/20 (reaction time) × 1/0.1 (enzyme amount) × dilution factor

1-2-5-3. Method for measuring acidic carboxypeptidase activity

To 1.5 ml of the casein solution, 1.0 ml of a pH 3.0 Mcllvaine buffer and 0.5 ml of a crude enzyme solution were added and reacted at 40° C. for 60 minutes, and then 3 ml of a TCA (trichloroacetic acid) solution was added to stop the reaction and the precipitate was removed. After adding 5 ml of sodium carbonate solution and 1 ml of phenol reagent to 1 ml of the reaction solution, color development was performed at 40°C for 30 minutes, and the absorbance was measured at 660 nm. As a control, enzyme solution was added immediately before adding the TCA solution. The absorbance was measured using the value. The enzyme activity was calculated by the following calculation formula 4.

[Equation 4]

Enzyme activity (Units/g) = y × 6/1 (reaction liquid amount) × 1/0.5 (enzyme liquid amount) × dilution factor

1-2-5-4. Enzyme activity comparison

The enzyme activity of each rice malt measured by the method described in Experimental Examples 1-2-5-1 to 1-2-5-3 is shown in Table 3 below.

Fungal spawn used Enzyme activity (Units/g) Alpha-amylase
(α-amylase) Glucoamylase
(glucoamylase) Acidic carboxypeptidase
(acidic carboxypeptidase) Aspergillus luchuensis 74-5 spawn 38.74 ± 1.21 1,209.38 ± 34.36 2,199.42 ± 57.88 Aspergillus duckweed 75-2 spawn 2,427.99 ± 57.9 691.36 ± 23.67 1,193.92 ± 42.79

The enzymatic activity of white guk made with Aspergillus luchuensis 74-5 seed was strong in the order of acidic carboxypeptidase, glucoamylase and alpha-amylase activity, showing 31.2 times higher saccharification power than liquefaction power, and Aspergillus duckweed 75-2 seed The enzyme activity of Hwangguk prepared with was strong in the order of alpha-amylase, acidic carboxypeptidase, and glucoamylase, and the liquefaction power was 3.5 times higher than that of glycation (Table 3). This suggests that the enzyme activity may vary depending on the fungal spawn used, and it is predicted that the taste of the prepared rice malt will also be affected.

Experimental Example 2. Establishment of Optimal Conditions for the Method of Manufacturing Barley Makgeolli according to the Type of Yeast Seed

In order to establish the optimum conditions for the method of manufacturing barley makgeolli according to the types of yeast seeds, white sticky barley is used as a raw material, imported from Europe and widely used in the brewing industry, Saccharomyces cerevisiae Fermivin (SCF). The strain was used as a control yeast seed, and barley makgeolli was prepared using two types of yeast seed in Table 4 below.

Strain name The scientific name of the strain KACC accession number Fermivin Saccharomyces cerevisiae - Y204 Saccharomyces cerevisiae 93237P Y283 Saccharomyces cerevisiae 93238P

Specifically, barley makgeolli was prepared by combining white and yellow soup prepared in Experimental Example 1 with the three types of yeast seeds in a total of six combinations (AL74-5 + SCF; AO75-2 + SCF; AL74-) 5 + Y204; AO75-2 + Y204; AL74-5 + Y283; and AO75-2 + Y283). To each 300 g of rice malt, 600 ml of immersion water was added, and 0.01% of each yeast seed was added and fermented for 2 days to prepare a base liquor. At this time, the fermentation temperature was 15, 20 or 25°C. Barley makgeolli was prepared by adding 300 g of godubap (white sticky barley) and 600 ml of immersion water to the entire amount of the mash of the base liquor. The alcohol production ability, acidity, amino acid level, pH, sugar content, organic acid change, and the pattern of fragrance components of each prepared barley makgeolli were compared and evaluated.

Grain raw material Fermenting agent (sprouting) Yeast spawn Fermentation temperature (℃) White sticky barley Aspergillus luchuensis 74-5 Saccharomyces cervicale Y204 25

As a result, aspergillus luchuensis 74-5 seed was used as a fermenting agent, Saccharomyces serviziae Y204 seed was used as yeast, and barley makgeolli with a fermentation temperature set at 25°C had 15% alcohol production ability and It showed a 1.6% change in acidity, showing the best quality. Therefore, the barley makgeolli production conditions in Table 5 were selected as optimal conditions.

Experimental Example 3. Establishment of Optimal Conditions for Fermented Vinegar Manufacturing Method According to the Type of Fermentation Seed

3-1. Preparation of barley makgeolli according to optimal mixing conditions

Rice koji (white soup) was prepared in the same manner as described in Experimental Example 1-2-1 under the conditions of Table 5 derived from Experimental Examples 1 and 2, and Cycaromyces servi in the same manner as described in Experimental Example 2. Barley makgeolli was prepared using Jiae Y204 yeast seed.

3-2. Establishment of initial pH conditions for manufacturing barley vinegar

In preparing barley vinegar using the barley makgeolli and acetic acid bacteria, which are indigenous fermentation seeds in Table 6 below, the acidity according to the initial pH was measured.

Specifically, the barley makgeolli prepared in Experimental Example 3-1 was prepared to have an alcohol content of 6%, and then the pH was adjusted to 2, 3, and 4 with a saturated aqueous solution of citric acid and sodium carbonate. Each of the seeds produced with 200 ml of the prepared barley makgeolli with two types of acetic acid bacteria ( Gluconacetobacter saccharivorans ) CV1 strain and Acetobacter pasteurianus CV3 strain) of Table 6 were each 10% Fermentation was started at 30° C. by inoculating each. Immediately after the start of fermentation, a certain amount of barley vinegar sample after 20, 40, 60, or 80 hours was taken and filtered through a filter paper, and 10 ml of each of the filtrate was placed in a 100 ml Erlenmeyer flask, and 0.5% phenolphthalein indicator was added from 2 to 3 drops dropped. Thereafter, neutralization titration was performed with 0.1 N sodium hydroxide (NaOH) until it turned red, and the total acidity was calculated by converting the amount of the consumed solution to lactic acid (%) by the following calculation formula 5.

[Equation 5]

Acidity (lactic acid, %) = sodium hydroxide appropriate amount × 0.009 (conversion coefficient) / 10 ml (sample amount) × 100

Strain name The scientific name of the strain KACC accession number CV1 Gluconacetobacter saccharivorans 17058 CV3 Acetobacter pasteurianus 17057

As a result, it was found that there was no change in the acidity of barley vinegar at pH 2, which is interpreted as a result of the failure of each acetic acid bacteria to grow properly because they did not adapt to the environment. On the other hand, at pH 3 and 4, it was found that the total acidity of barley vinegar increased from 20 hours after fermentation of each acetic acid bacteria, suggesting that the acetic acid fermentation proceeded well as each acetic acid bacteria were well adapted to the environment. In particular, the acidity was higher when the CV1 strain was used compared to the CV3 strain, and the acidity was higher when the initial pH was 3 than when the initial pH was 4 (FIG. 6).

3-3. Establishment of fermentation temperature conditions for manufacturing barley vinegar

Barley vinegar was prepared using the barley makgeolli as a raw material and two kinds of acetic acid bacteria, and the total acidity of barley vinegar according to the fermentation temperature was measured.

Specifically, the barley makgeolli prepared in Experimental Example 3-1 was prepared so that the alcohol content was 6%, and the pH was adjusted to 3 using a saturated aqueous solution of citric acid and sodium carbonate, and then 10 seed seeds made with CV1 and CV3 acetic acid bacteria were each prepared. Percent inoculation and fermentation was started at 15, 30 or 40°C. Barley vinegar in the same manner as described in Experimental Example 3-2, except that samples from the 3rd, 6th, 9th, 12th, 15th, 18th or 20th day were measured immediately after the start of fermentation. The total acidity of was measured.

As a result, in the case of acetic acid fermentation at 40°C, neither of the acetic acid bacteria CV1 and CV3 were able to grow, whereas in the case of acetic acid fermentation at 15°C, each of the acetic acid bacteria adjusted to a fermentation temperature lower than 3 days, and then the acidity continued to increase and fermented 18 The acidity increased to 6.0 or more from the first order. In addition, in the case of acetic acid fermentation at 30°C, it reached a stationary phase on the 6th day of fermentation, the acidity increased to 5.5, and then decreased again (Fig. 7). In the case of using acetic acid bacteria CV1 and CV3, both showed similar patterns, and the above results suggest that both seeds are suitable for barley vinegar fermentation, and high-quality barley vinegar with high acidity when fermented for 20 days or longer under low-temperature fermentation conditions at 15℃ Will be produced.

Experimental Example 4. Quality characteristics of barley vinegar prepared according to optimal initial pH and fermentation temperature conditions

According to the optimum conditions derived in Experimental Example 3, the barley makgeolli prepared in Experimental Example 3-1 was prepared so that the alcohol content was 6%, and then the pH was adjusted to 3 with citric acid and a saturated aqueous sodium carbonate solution, and the prepared barley. Fermentation was started at 30°C by inoculating 10% of seeds made with acetic acid bacteria CV1 or CV3 in 200 ml of makgeolli. Immediately after the start of fermentation, the quality characteristics of barley vinegar on the 6th, 12th, or 20th day were analyzed as follows.

4-1. Check the acidity and pH of barley vinegar according to fermentation period

The pH of barley vinegar was measured at room temperature with a pH meter (orion 3 star, Thermo Scientific Co. USA), and the acidity was measured in the same manner as described in Experimental Example 3-2.

Effective period (days) Acidity (%) pH CV1 CV3 CV1 CV3 0 0.6 ± 0.0 ^d 0.5 ± 0.0 ^d 3.5 ± 0.0 ^a 3.6 ± 0.0 ^a 6 1.4 ± 0.1 ^c 1.2 ± 0.0 ^c 3.3 ± 0.1 ^b 3.4 ± 0.1 ^b 12 2.9 ± 0.1 ^b 2.3 ± 0.2 ^b 3.0 ± 0.0 ^c 3.2 ± 0.0 ^c 20 5.2 ± 0.0 ^a 4.1 ± 0.2 ^a 2.8 ± 0.0 ^d 2.9 ± 0.0 ^d

^a to ^d : Different alphabets in the same column indicate a statistically significant difference in Duncan's multiple range test (p <0.001).

As a result, the acidity of both barley vinegar using CV1 and CV3 increased according to the fermentation period and increased to 5.2% and 4.1%, respectively, on the 20th day of fermentation, and barley vinegar using CV1 was about 21% higher than that of barley vinegar using CV3. This suggests that the acid production rate of CV1 is faster than that of CV3. The pH value of barley vinegar using CV1 and CV3 on the 20th day of fermentation was 2.8 and 2.9, respectively, as the pH value decreased with the fermentation period in the same way as the acidity change (Table 7).

4-2. Confirmation of organic acid content of barley vinegar according to fermentation period

10 ml of each barley vinegar sample was taken and filtered through a 0.2 μm membrane filter (Millipore Co., Ireland), and the organic acid content was measured by high performance liquid chromatography (HPLC) analysis. The c column is an ODS column (TSK gel ODS-100V, 5 µm, 4.6 × 25 cm, TOSOH Co., Japan), the mobile phase is 8 mM perchloric acid, the flow rate is 1.0 ml/min, the sample injection volume Silver 10 μL, UV detection was analyzed using an HPLC instrument (LC-20A Prominence, Shimadzu Co., Japan) under 440 nm conditions.

As a result, the main organic acid of barley vinegar using CV1 or CV3 was acetic acid, which was more produced depending on the fermentation period. In barley vinegar using CV1, malic acid, lactic acid, fumaric acid, and propionic acid were detected in trace amounts. acid) and the like were detected in trace amounts. In both types of barley vinegar, tartaric acid was not detected, and succinic acid was not detected as fermentation proceeded (Table 8).

4-3. Confirmation of free amino acid content of barley vinegar according to fermentation period

Add 5 ml of 5% trichloroacetic acid (Junsei Chemical Co., Ltd., Japan) to 5 ml of each barley vinegar sample and centrifuge (4℃, 12,000 rpm, 15 minutes) to add 1 or 2 ml of the supernatant It was taken, evaporated to dryness using an oven, and dissolved by adding 1 or 2 ml of a 0.02 N hydrochloric acid (HCl) solution. After filtering with a 0.2 μm syringe filter (Syringe filter, Millipore Co.), the content of free amino acids was analyzed under the conditions of Table 9 using an amino acid analyzer (L-8900, Hitachi Co., Japan).

As a result, the total free amino acid content of barley vinegar using CV1 or CV3 increased with time, and the main free amino acids of barley vinegar using CV1 were proline, alanine, and leucine ( leucine), etc., the main free amino acids of barley vinegar using CV3 were proline, alanine, valine, leucine, etc., and the others were found in trace amounts (Tables 10 and 11).

4-4. Confirmation of antioxidant activity of barley vinegar according to fermentation period

4-4-1. DPPH radical scavenging activity confirmation

An appropriate amount of ethanol was added to 0.8 ml of a 0.4 mM DPPH (2,2-diphenyl-1-picrylhydrazyl) solution so that the absorbance at 525 nm was 0.95-0.99, and the amount of ethanol to be added was set. After adding the set amount of ethanol to 0.8 ml of 0.4 mM DPPH solution, shake it vigorously for 10 seconds, add 0.8 ml of shaken DPPH solution every 30 seconds to 0.2 ml of each barley vinegar sample, mix well, and react for 10 minutes in each dark cow. And the absorbance was measured at 525 nm. As a control, a sample without barley vinegar was used, and DPPH radical scavenging ability was calculated by the following calculation formula 6 using the measured absorbance value.

[Equation 6]

DPPH radical scavenging ability (%) = 100-{(absorbance of sample/absorbance of control) × 100}

As a result, DPPH radical scavenging activity immediately after the start of acetic acid fermentation of barley vinegar using CV1 or CV3 was 41.42% and 43.18%, respectively, and the highest scavenging activity was shown at 43.58% and 47.9%, respectively, on the 6th day of fermentation, and thereafter, The activity decreased again as the fermentation period elapsed. In addition, the activity of barley vinegar using CV3 was slightly higher than that of CV1 (FIG. 8A).

4-4-2, ABTS radical scavenging activity confirmation

A 7.4 mM ABTS (2,2'-Azino-bis (3-ethylbenzothiazoline-6-sulphonic acid)) solution was diluted with PBS (phosphate buffered saline, pH 7.4) so that the absorbance at 732 nm was 0.700 ± 0.030. To 50 µl of each barley vinegar sample, 950 µl of the ABTS solution was added every 30 seconds, and each mixture was reacted for 10 minutes in a dark place, and the absorbance was measured at 732 nm. A sample without barley vinegar was used as a control, and the ABTS radical scavenging ability was calculated by the following calculation formula 7 using the measured absorbance value.

[Equation 7]

ABTS radical scavenging ability (%) = 100-{(absorbance of sample/absorbance of control) × 100}

As a result, the ABTS radical scavenging activity immediately after the start of acetic acid fermentation of barley vinegar using CV1 or CV3 showed the highest activity at 53.4% and 39.30%, respectively, and after the fermentation period elapsed, the activity rapidly decreased. Both types of barley vinegar showed little activity, and ABTS radical scavenging activity was slightly higher in barley vinegar using CV1 than in CV3 (FIG. 8B). This suggests that it would be desirable to make the fermentation period within 20 days in order to obtain ABTS radical scavenging activity.

4-5. Confirmation of GABA content in barley vinegar according to fermentation period

After taking 5 ml of each barley vinegar sample into a conical tube, it was filtered through a membrane filter (0.45 μm). Amino acid analyzer (S7130 amino acid organizer, Sykam, Germany), sample injector (S5200 sample injector, Sykam), solvent delivery system (S2100 solvent delivery system, Sykam) and cation separation column (LCA K06/NA cation separation column, 250 mm × 4.6 mm) was used to measure the GABA content in the sample, and the flow rate of the mobile phase was 0.45 ml/min.

As a result, the GABA content of barley vinegar using CV1 or CV3 continuously increased with the passage of the fermentation period, and GABA contents of 2,164 µg/ml and 5,339 µg/ml, respectively, on the 20th day of fermentation were shown. Overall, CV3 was higher than CV1. In the barley vinegar used, the GABA content was about 2 to 3 times higher (FIG. 8C).

4-6. Analysis of multiple flavor components of barley vinegar according to fermentation period

0.5 ml of each barley vinegar sample is placed in a 10 ml vial (vial, La-Pha-Pack GmbH, Germany), stirred at 40° C. for 30 minutes at 500 rpm, and then electronic nose (Fast GC based HERACLES flash Electronic nose. Alpha Mos, France) Multiple fragrance components were measured using. The electron nose was used with two columns (DB5 apolar and DB1701 Slightly polar) attached, and a FID (Flame Ionization Detecter) was used as the detector. The sample was injected with a syringe type (5.0 ml-HS) at a column head pressure of 1.0 psi while the column temperature was maintained at 25°C. During the analysis, the temperature of the injector and the detector was set to 200°C and the pressure of the detector was set to 39.0 psi. Depending on the detected peak, a sensor with a discrimination power of 0.900 or more and a relative standard deviation (RSD) of less than 20% is selected, and discriminant function analysis and SIMCA (discriminant function analysis) and SIMCA ( Soft Independent Modeling of Class Analogy). Before sample analysis, a peak value of C6-C16 was obtained using Kovats (Custom Alkanes Blend Standard) and used as a standard.

As a result of the analysis, the scent pattern was different according to the seed and fermentation period, and barley vinegar using CV1 or CV3 did not show an intensive scent pattern on the 0th, 6th, and 20th days of fermentation, but the intensive scent pattern was observed on the 12th day of fermentation. Showed. The difference according to the spawn was not large in general, but the greatest difference was shown on the 20th day of fermentation, and it seemed that the difference in the fragrance pattern according to the spawn occurred as the fermentation period elapsed (FIG. 9).

Experimental Example 5. Confirmation of sensory properties of barley vinegar prepared according to optimum initial pH and fermentation temperature conditions

In the same manner as described in Experimental Example 4, barley makgeolli was fermented with acetic acid, and sensory evaluation was performed using barley vinegar on the 20th day of fermentation.

Specifically, 10 expert members evaluated the color, aroma, taste and overall acceptability of each fermented vinegar on a 9-point scale, and rice vinegar fermented for 20 days by CV1 or CV3 as a control group (CV1 Glu-rice and CV3 -Glu-rice) was used.

Item Barley vinegar CV1 CV3 color 6.9 ± 2.0 6.6 ± 2.4 incense 5.0 ± 2.6 4.1 ± 2.8 flavor 5.7 ± 2.7 4.6 ± 2.4 Overall acceptability 6.0 ± 2.8 4.8 ± 2.1

As a result, barley vinegar using CV1 showed better color, flavor, taste, and overall preference than barley vinegar using CV3 (Table 12). As a result of analyzing the preference for each age, barley vinegar using CV1 in their 40s, and barley vinegar using CV3 in their 50s more preferred, and as a result of analysis according to sex, both men and women preferred barley vinegar using CV1 more (Fig. 10). This suggests that barley vinegar using CV1 can be developed as customized barley vinegar, although the preference of barley vinegar using CV1 is better overall, but targeting middle-aged people.

Claims (11)

1) water saving after immersing rice in water;
2) increasing the rice of step 1) to prepare godubap;
3) inoculating and culturing the seed of Aspergillus luchuensis 74-5 (KACC93235P) to the godubap of step 2);
4) drying the cultured godubap of step 3) to prepare rice malt;
5) mixing the rice malt, fresh water, and Saccharomyces cerevisiae Y204 (KACC93237P) yeast seed of step 4), and fermenting it to prepare a base liquor;
6) preparing barley makgeolli by fermenting godubap made of barley and marinated water in the mash of the base liquor of step 5); And
7) A method for producing barley vinegar with enhanced GABA content comprising the step of fermenting and fermenting seeds prepared with Acetobacter pasteurianus CV3 (KACC17057) acetic acid bacteria in the barley makgeolli of step 6).
The method of claim 1, wherein the immersion in step 1) is performed for 40 to 100 minutes.
The method of claim 1, wherein the Aspergillus luchuensis 74-5 seed in step 3) is inoculated with a godubap at a concentration of 0.01 to 10%.
The method of claim 1, wherein the step 3) comprises lowering the temperature to 25 to 40°C after the mixture is turned over.
The method of claim 1, wherein the drying in step 4) is performed at 45 to 60°C.
The method for producing barley vinegar according to claim 1, wherein the Saccharomyces serviziae Y204 yeast seed in step 5) is mixed in a mixture of rice malt and fresh water at a concentration of 0.001 to 1.0%.
The method according to claim 1, wherein the barley makgeolli of step 7) is prepared with an alcohol content of 3 to 10%.
The method of claim 1, wherein the barley makgeolli of step 7) is adjusted to a pH of 2.5 to 4.5.
The method of claim 1, wherein the fermentation of step 7) is performed at 10 to 35°C.
The method of claim 1, wherein the fermentation of step 7) takes place for 5 to 50 days.
Barley vinegar with improved GABA content prepared by the method of claim 1.

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