KR20220076708A - Manufacturing method of complex fermented vinegar using black barley and barries - Google Patents

Abstract

The present invention relates to a method for producing a complex fermented vinegar using black barley and berries, and more particularly, to a black barley fermented liquid production step for preparing a black barley fermented broth, a berry fermented liquid production step to prepare a berry fermented broth, and the black barley fermented liquid production A raw material mixing step of mixing the berry fermentation broth prepared through the berry fermentation broth production step with the black barley fermented broth prepared through the step, a seed seed mixing step of mixing the seed seed into the mixture prepared through the raw material mixing step, the seed seed mixing step It consists of an acidity control step of adjusting the acidity by mixing glacial acetic acid with the mixture in which the seeds are mixed through the fermentation step, and a fermentation step of fermenting the mixture whose acidity is adjusted through the acidity control step.
The complex fermented vinegar produced through the above process promotes the production of acetic acid to increase acidity, and contains berries, which increases the content of phenolic compounds and organic acids, thereby exhibiting excellent antioxidant effect.

Description

Manufacturing method of complex fermented vinegar using black barley and berries {MANUFACTURING METHOD OF COMPLEX FERMENTED VINEGAR USING BLACK BARLEY AND BARRIES}

The present invention relates to a method for producing a complex fermented vinegar using black barley and berries, and more particularly, the production of acetic acid is promoted to increase the acidity, and the content of phenolic compounds and organic acids is increased due to the berry component being contained, thereby providing excellent antioxidant It relates to a method for producing a complex fermented vinegar using black barley and berries, which provides a complex fermented vinegar showing an effect.

The origin of vinegar is related to the discovery of wine, and when wine comes into contact with air during storage, acid is naturally generated and turned into vinegar. Vinegar has been industrialized since the 1800s as the metabolic mechanism of the fermentation process was revealed, and vinegar manufacturing methods such as Sokyang method and deep fermentation method were diversified.

Barley has been used as a representative traditional vinegar as one of the raw materials of the staple food. It contains physiologically active substances such as β-glucan and polyphenols, and as the effect of improving immunity and anti-allergic effect of barley polyphenol extract is known, consumers' interest Although it is used to make soy sauce, barley tea, and noodles, the types of prepared products are very limited. Recently, research on colored barley with high antioxidant activity and processing into raw materials such as rice cakes and confectionery is being conducted.

On the other hand, aronia and bokbunja are representative berries with high antioxidant activity. Anthocyanin and polyphenol compounds, the main ingredients, have high antioxidant activity and reduce cholesterol, and aronia contains useful ingredients that reduce lifestyle diseases such as cardiovascular disease. Bokbunja is known to have an immune-boosting effect. Because bokbunja is limited as a raw material for processing due to the high price of its fruit, cost reduction and functional supplementation can be expected by mixing it with colored barley with high antioxidant activity.

In addition, grain vinegar uses grains such as rice, barley, and brown rice as raw materials and undergoes saccharification and alcohol fermentation through yeast or entry into the country and then enters acetic acid fermentation. Vinegar can be obtained by proceeding.

However, since the alcohol fermentation process is different for berries and grains, improvement in the fermentation process is needed, and a study on the characteristics of complex fermentation is needed.

Accordingly, the developer of the present invention uses black barley, which is a raw material with a high anthocyanin content among grains, bokbunja, and aronia among berries to improve production efficiency and produce nutritionally excellent vinegar by using two methods for the characteristics of raw materials. A manufacturing method was invented.

Korean Patent Registration No. 10-0911108 (2009.07.30) Korean Patent Registration No. 10-1256352 (2013.04.15)

It is an object of the present invention to provide a complex fermented vinegar using black barley and berries that promotes the production of acetic acid, increases acidity, and provides a complex fermented vinegar that contains berries and exhibits excellent antioxidant effects by increasing the content of phenolic compounds and organic acids To provide a manufacturing method of

An object of the present invention is to prepare a black barley fermented liquid production step for producing a black barley fermented broth, a berry fermented liquid production step for preparing a berry fermented broth, and a black barley fermented broth prepared through the black barley fermented liquid production step. A raw material mixing step of mixing the berry fermentation broth, a seed seed mixing step of mixing a seed seed into the mixture prepared through the raw material mixing step, an acidity control step of adjusting the acidity by mixing glacial acetic acid with the seed seed mixed mixture through the seed seed mixing step and a fermentation step of fermenting a mixture whose acidity has been adjusted through the acidity control step.

According to a preferred feature of the present invention, the raw material mixing step is made by mixing 30 to 400 parts by weight of the fermented berry solution prepared through the fermented berry solution manufacturing step with 100 parts by weight of the black barley fermented solution prepared through the black barley fermented solution manufacturing step. do.

According to a more preferred feature of the present invention, in the step of preparing the black barley fermented liquid, the black barley is soaked in water for 5 to 7 hours and then steamed for 30 to 90 minutes, and 2 to 3 parts by weight of yeast to 100 parts by weight of the steamed black barley , to prepare a mixture by mixing 1.5 to 2.5 parts by weight of yeast and 100 to 120 parts by weight of water, and 200 to 250 parts by weight of black barley, 4 to 5 parts by weight of yeast and water, which are heated to 100 parts by weight of the mixture after 20 to 30 hours It is supposed to be prepared by mixing 250 to 300 parts by weight and fermenting and filtering for 6 to 8 days.

According to a more preferred feature of the present invention, the fermented berries are shredded berries, then the sugar content is adjusted to 25 to 30 Brix by mixing high-fructose sugar with the crushed berries, and yeast is mixed with the shredded berries whose sugar content is controlled 24 It is prepared by fermentation and filtration for 7 to 9 days at a temperature of 26 ° C., and the berries are made of one or more selected from the group consisting of aronia and bokbunja.

According to an even more preferred feature of the present invention, the fermentation step is to be performed for 20 to 22 days at a temperature of 28 to 32 ℃.

The method for producing complex fermented vinegar using black barley and berries according to the present invention promotes the production of acetic acid to increase acidity, and contains berry components to increase the content of phenolic compounds and organic acids to show excellent antioxidant effect. It exhibits an excellent effect providing

1 is a flowchart showing a method for manufacturing a complex fermented vinegar using black barley and berries according to the present invention.
Figure 2 is a graph showing the analysis of the physicochemical properties of the black barley fermented broth, the aronia fermented broth and the fermented bokbunja fermented broth.
3 is a graph showing the physicochemical properties of acetic acid fermented broth according to mixing of black barley fermented broth and aronia fermented broth.
Figure 4 is a graph showing the physicochemical properties of the acetic acid fermentation broth according to the mixing of the fermented black barley and bokbunja fermented broth.
5 is a photograph showing the appearance of vinegar, which was fermented with acetic acid by mixing fermented black barley and fermented berries.
6 is a graph showing the measurement of pH, sugar content, acidity and alcohol content of a fermented broth mixed with black barley and bokbunja.
7 is a graph showing the measurement of pH, sugar content, acidity and alcohol content of acetic acid fermentation broth mixed with black barley and bokbunja.
8 is a photograph showing the separation process of acetic acid bacteria in SM medium.
9 is a graph showing the growth curve of the selected acetic acid bacteria.

Hereinafter, a preferred embodiment of the present invention and the physical properties of each component will be described in detail, which is intended to describe in detail enough that a person of ordinary skill in the art to which the present invention pertains can easily carry out the invention, This does not mean that the technical spirit and scope of the present invention is limited.

The method for producing a complex fermented vinegar using black barley and berries according to the present invention includes a black barley fermented liquid production step (S101) for preparing a black barley fermented broth, a berry fermented liquid production step (S101-1) for preparing a berry fermented broth (S101-1), the black barley The raw material mixing step (S103) of mixing the berry fermentation broth prepared through the berry fermentation broth production step (S101-1) with the black barley fermented broth prepared through the fermentation broth production step (S101), and the raw material mixing step (S103) Manufactured through An acidity control step (S107) and the acidity control step (S107) of mixing glacial acetic acid to the mixture in which the seedlings are mixed through the seedling mixing step (S105), the seedling mixing step (S105) ) consists of a fermentation step (S109) of fermenting a mixture whose acidity has been adjusted.

The black barley fermented liquid manufacturing step (S101) is a step of preparing a black barley fermented broth, and the black barley is soaked in water for 5 to 7 hours, then steamed for 30 to 90 minutes, and yeast 2 to 100 parts by weight of the steamed black barley 3 parts by weight, 1.5 to 2.5 parts by weight of yeast, and 100 to 120 parts by weight of water to prepare a mixture, and after 20 to 30 hours, 200 to 250 parts by weight of black barley, 4 to 5 parts by weight of yeast, which are heated to 100 parts by weight of the mixture And 250 to 300 parts by weight of water is mixed and prepared through a process of fermentation and filtration for 6 to 8 days.

The fermented berries manufacturing step (S101-1) is a step of preparing a fermented berry broth. After crushing the berries, high-fructose sugar is mixed with the crushed berries to adjust the sugar content to 25 to 30 Brix, and berries whose sugar content is controlled. It is prepared by mixing yeast and fermenting and filtering at a temperature of 24 to 26° C. for 7 to 9 days, and the berries are made of one or more selected from the group consisting of aronia and bokbunja.

At this time, the yeast is not particularly limited, and any normal yeast capable of fermenting berries can be used, but it is preferable to use permivin.

The raw material mixing step (S103) is a step of mixing the fermented berries prepared through the fermented berries manufacturing step (S101-1) with the fermented black barley prepared through the black barley fermented liquid manufacturing step (S101), the black barley It is preferable to mix 30 to 400 parts by weight of the fermented berry broth prepared through the fermented berry broth production step (S101-1) with 100 parts by weight of the black barley fermented broth prepared through the fermentation broth production step (S101).

In this case, it is preferable to use the black barley fermented broth and the fermented berries diluted to 5 to 7%, more preferably 6% by mass concentration of alcohol.

The seedling mixing step (S105) is a step of mixing the seedlings with the mixture prepared through the raw material mixing step (S103), and the seedlings 8 to 100 parts by weight of the mixture prepared through the raw material mixing step (S103) This is a step of mixing 12 parts by weight.

In this case, the seed seed may use one or more selected from the group consisting of grain seed seed, aronia seed seed, and bokbunja seed seed.

In order to separate acetic acid bacteria from the acetic acid fermentation broth, acetic acid strains were isolated from grain seeds, blueberry vinegar, aronia seeds, and bokbunja seeds as samples.

Each sample was inoculated into GYE medium (yeast extract 1%, glucose 5%, ethanol 3%) as a pre-culture and incubated at 30° C. for 48 hours. The pre-cultured sample was inoculated into SM medium (Acetobacter selective medium, yeast extract 0.5%, glucose 3.0%, CaCO ₃ 1.0%, agar 2.0%, ethanol 5.0%) and cultured at 30° C. for 48 hours, and then the strain was isolated.

Each of the isolated strains was tooth picked on SM medium, and the size of a clear ring around the strain was measured to separate acetic acid bacteria. Acetic acid peroxidation (glucose 0.5%, glycerol 0.5%, polypeptone 0.5%, yeast extract 0.5%, bromocresol purple 0.003%, acetic acid 1 %, ethanol 1%, agar 1.5%), acetic acid (glucose 0.5%, glycerol 0.5%, polypeptone 0.5%, yeast extract 0.5%, acetic acid 3%, ethanol 2%, agar 1.5%) and pH tolerance (glucose 0.5%) , glycerol 0.5%, polypeptone 0.5%, yeast extract 0.5%, ethanol 2%, agar 1.5%, pH 3.0-3.4), alcohol resistance (yeast extract 1%, glucose 5%, CaCO ₃ 3%, agar 2.5%, ethanol 10 to 20%) was tested to select excellent strains, and then microorganisms were identified.

Microorganisms were identified by 16s ribosomal RNA sequencing, and the analysis was commissioned by Solgent. For PCR and sequencer, ABI 9700 PCR (Applied Biosystems, Foster City, CA, USA) and ABI 3730 XL DNA analyzer (Applied Biosystem) were used, respectively. The 16s rRNA base sequence of the analyzed microorganism was identified using the Basic Local Alignment Search Tool of the National Center for Biotechnology Information (NCBI).

To select the final excellent strain among the selected strains, the viability of the strain was evaluated using YPM medium (yeast extract 0.5%, peptone 0.3%, mannitol 2.5%, agar 1.5%). Fermentation suitability was evaluated through vinegar production.

The acidity control step (S107) is a step of adjusting the acidity by mixing glacial acetic acid with the mixture in which the seedlings are mixed through the seedling mixing step (S105), and glacial acetic acid in the seedlings mixed mixture through the seedlings mixing step (S105). It is a step to adjust the acidity of the mixture to 2% by mixing.

The fermentation step (S109) is a step of fermenting the mixture whose acidity has been adjusted through the acidity control step (S107), and the mixture whose acidity has been adjusted to 2% through the acidity control step (S107) is heated at 28 to 32°C. It consists of a process of fermentation at temperature for 20 to 22 days.

After the above fermentation step (S109), the production of complex fermented vinegar using black barley and berries according to the present invention is completed.

Hereinafter, the manufacturing process of the complex fermented vinegar using black barley and berries according to the present invention will be described by way of example.

In the description below, since fermented black barley is manufactured through a method similar to the method for manufacturing makgeolli, the expression “black barley makgeolli” is used interchangeably in addition to the expression “fermented black barley”.

Since the fermented berry juice is manufactured through a method similar to the method of manufacturing wine, the expression berry wine was used in addition to the expression fermented berry.

1. Vinegar is manufactured through mixed acetic acid fermentation after alcohol fermentation of barley and berries, respectively.

-Experiment method

After each alcoholic fermentation of black barley and berries, alcohol was mixed in a certain ratio, followed by acetic acid fermentation to prepare vinegar.

(1) Production of fermented black barley broth (black barley makgeolli)

After washing 900 g of black rice and soaking it for 6 hours to reach moisture saturation, steaming for 1 hour and cooling, 24 g of yeast, 18 g of yeast, and 1,120 mL of water were mixed evenly. After boiling and steaming, 42 g of yeast and 2,630 mL of water were mixed, fermented for 7 days, and filtered through a juicer to be used as a sample for analysis and as a raw material for acetic acid fermentation.

(2) Production of berry fermentation broth (berry wine)

After crushing the aronia and bokbunja fruits respectively, they were sieved to 28°bx using high fructose, fermented at 25°C for 8 days with commercial yeast, and the filtered filtrate was used as a sample for analysis and as a raw material for acetic acid fermentation.

(3) Preparation of complex fermented vinegar

For acetic acid fermentation of black barley and berries, each fermented broth was diluted to an alcohol concentration of 6% and mixed in the same ratio as in Table 1 below, followed by acetic acid fermentation. After adding 10% of seed vinegar to the fermented broth of black barley and berries, adjusting the initial acidity to 2% with glacial acetic acid, and fermenting at 30°C for 14 days, complex fermented vinegar (BA) mixed with black barley and aronia ingredients and black barley And bokbunja components were mixed to prepare a complex fermented vinegar (BB). Mixed fermented vinegar was named BA0~BA100 and BB0~BB100 based on the berry mixing ratio.

<Table 1> Mixing ratio of black barley makgeolli and berry wine for acetic acid fermentation

(4) Fermentation characteristics of black barley and berries

The characteristics of each fermented broth prepared with black barley, aronia and bokbunja as raw materials were investigated. Depending on the fermentation period, sugar content, pH, and color were measured with a saccharometer, pH meter, and spectrophotometer, and acidity was converted into acetic acid content after measuring the amount required for neutralization titration using 0.1 N NaOH. The alcohol content of the sample was measured using an alcoholizer.

(5) Characteristics of mixed acetic acid fermentation broth

After mixing black barley and berry alcohol in the ratio shown in Table 1, the characteristics of vinegar were investigated while acetic acid fermentation was carried out. Acetic acid fermentation characteristics were measured by measuring sugar content, acidity, pH, color, and alcohol content in the same manner as in the fermentation broth.

(6) organic acid content analysis

HPLC was used to determine the organic acid content of the complex fermented vinegar mixed with black barley and berries. Distilled water was added to the complex fermented vinegar fermented for 14 days, followed by ultrasonic extraction for 1 hour, and 10 μL of the sample extracted for 1 hour with a stirrer was injected into the column and detected using a detector. The mobile phase was analyzed by using a 0.01NH ₂ SO ₄ solution at a flow rate of 0.5 mL/min. As standards, citric acid, oxalic acid, and fumaric acid were manufactured by Showa Denko, lactic acid, succinic acid, and shikimic acid were produced by Sigma-Aldrich, and malic acid by Kanto Chemical. A standard curve was drawn and each sample was analyzed and expressed as mg/kg.

(7) Total polyphenol and total anthocyanin content

The total polyphenol content was measured by applying the National Health Functional Foods Method. That is, 7.5 mL of distilled water, 0.5 mL of folin-ciocalteu's phenol reagent, and 1 mL of 35% sodium carbonate were added to 1 mL of a solution prepared with a 10 mg/10 mL concentration of a complex fermented vinegar sample mixed with black barley and berries. It was left still and colorimetric quantification was carried out at 760 nm using a UV/VIS spectrophotometer. At this time, a calibration curve was prepared using tannic acid as a standard material, and the total polyphenol content was obtained from this.

For the total anthocyanin content, 1 g of each sample was extracted with 40 mL of an 80% methanol solution containing 0.1% HCl for 24 hours, and then absorbance was measured at 528 nm using a UV/VIS spectrophotometer. At this time, a calibration curve was prepared using cyanidin as the standard material, and the total anthocyanin content was obtained from this.

(8) Measurement of DPPH radical scavenging ability

To measure the antioxidant activity of black barley and berry mixed fermented vinegar, DPPH radical scavenging ability was measured. This method is a method of measuring antioxidant activity by using the characteristic that the purple color is discolored as the DPPH radical reacts with a compound having antioxidant activity and receives electrons or hydrogen to reduce it. The measurement method was as follows. 0.2 mL of ethanol was added to 0.1 mL of each sample prepared at a concentration of 50 ~ 500 μg/mL, and 0.3 mL of 2x10 ^-4 M DPPH solution was added, followed by stirring. After reacting for 30 minutes, absorbance was measured at 517 nm using a microplate reader, and the control was tested by adding ethanol instead of the sample. The radical scavenging ability was calculated by substituting the measured absorbance into the formula below.

Inhibition(%) = {1-(A _Experiment - A _Blank )/A _Control }×100

(9) Measurement of ABTS radical scavenging ability

ABTS radical scavenging ability was measured as follows by applying the method of Arts MJTJ et al. (2004). To measure the ABTS radical scavenging ability of 50% ethanol extracts of Aronia fruit of different maturity levels, samples were prepared at a concentration of 50 to 500 μg/mL. After adding 0.195 mL of ABTS radical solution to 0.005 mL of sample and reacting for 7 minutes, absorbance was measured at 734 nm using a microplate reader (Biotec), and ethanol was used as a control instead of the sample.

result

(1) Characteristics of fermented black barley and berries

In order to prepare a complex fermented vinegar mixed with black barley and berries, alcohol content, sugar content, pH, and acidity were investigated while alcohol fermentation was performed with each raw material. The results are shown in FIG. 1 below. In makgeolli, monosaccharides are produced by saccharification of starch contained in raw grains, and alcohol is produced from monosaccharides. Therefore, starch must be saccharified prior to alcohol fermentation. As for the black barley, the fermentation rate increased from the 2nd day as the fermentation proceeded by adding extra sake on the 1st day, and the rate of increase was slowed from the 4th day. The sugar content of black barley alcohol increased from the 2nd day onwards as saccharification by yeast proceeded in the early stage of fermentation, and then it became mild after the 4th day. The acidity increased from 0.02% to 0.64% until the 4th day, and then slightly increased.

After the 1st day, the alcohol fermentation proceeded rapidly, and the fermentation continued until the 8th day, and the alcohol content reached 12.94%, and the sugar content decreased rapidly until the 4th day as the fermentation progressed, and then decreased gently after the 4th day, and the pH and acidity was maintained in the range of 3.74~4.01 and 0.51~0.68, respectively, without significant change.

In bokbunja, alcohol fermentation occurred rapidly from the beginning of fermentation and gradually increased after 4 days, and the alcohol production amount was the highest at 14.77% on the 8th day. The change in sugar content decreased rapidly at the beginning of fermentation in response to alcohol production and decreased gently after 4 days, acidity slightly increased from 1.01% to 1.30% until the 2nd day of fermentation, and then remained similar, and pH was maintained without change during fermentation. .

Also, as shown in Table 2 below, the results of measuring the chromaticity of the fermented broth showed that the brightness decreased and the redness and yellowness increased in the case of black barley, and in the case of aronia, the brightness, redness, and yellowness all decreased. There were hardly any.

<Table 2> Alcohol fermentation characteristics of black barley makgeolli, aronia wine, and bokbunja wine

(2) Characteristics of acetic acid fermentation broth mixed with black barley and berries

After diluting the alcohol content of black barley, aronia, and fermented bokbunja alcohol to 6% each, black barley-aronia alcohol (BA) and black barley-bokbunja alcohol (BB) were mixed in different ratios as shown in Table 1 to 14 Daily acetic acid fermentation was carried out. In both black barley-aronia mixed fermented vinegar (BA) and black barley-bokbunja mixed fermented vinegar (BB), alcohol content decreased and acidity increased as the acetic acid fermentation proceeded, pH decreased, and sugar content was maintained constant. Factors that affect acetic acid production are alcohol concentration, initial acetic acid concentration, and acetic acid bacteria inoculation, which are fermentation substrates. Fermentation characteristics similar to those of this study were reported as acetic acid fermentation progressed.

Black barley-aronia mixed fermented vinegar (BA) reached an alcohol content of 0.67~3.77%, sugar content of 6.85~7.98, pH 3.05~3.32, and acidity of 3.55~6.90% depending on the ratio of aronia after 14 days of fermentation. As the mixing ratio of aronia increased, the alcohol content, pH decrease, and acidity increase were small. In particular, BA100 fermented with 100% aronia alcohol in acetic acid after 14 days had a high alcohol residual and a low acidity of 3.55%, so fermentation took place slowly . was found to appear (Fig. 3 below).

In addition, as shown in FIG. 4 below, the mixed fermented black barley-bokbunja vinegar (BB) had an alcohol content of 0.67 to 0.73%, a sugar content of 5.15 to 7.98, a pH of 3.03 to 3.09, and an acidity of 6.90 to 7.08% after fermentation. The black barley-bokbunja mixed fermented vinegar also showed an effect according to the bokbunja mixing ratio, but it was not as great as the black barley-aronia mixed fermented vinegar (BA), and the acetic acid fermentation promotion effect by the addition of black barley alcohol hardly appeared.

In addition, the chromaticity of the mixed fermented vinegar of black barley and berries was shown in Tables 3 to 4. In black barley-aronia mixed fermented vinegar (BA), when the aronia content was high, the L and a values tended to decrease as the fermentation progressed, and the b values increased. As the content of aronia alcohol increased, the brightness and yellowness decreased and the degree of redness increased, and as the ratio of black barley alcohol increased, the red color gradually became lighter (Fig. In the black barley-bokbunja mixed fermented vinegar (BB), there was almost no change in color depending on the fermentation period, and as shown in FIG. It was difficult to tell the difference. The higher the alcohol content of bokbunja, the lower the brightness and the higher the yellowness and redness.

<Table 3> Chromaticity of acetic acid fermentation according to black barley makgeolli-Aronia wine mixture

<Table 4> Chromaticity of acetic acid fermentation solution by mixing black barley makgeolli-bokbunja wine

(3) Organic acid content of black barley and berry mixed fermented vinegar

Table 5 below shows the results of measuring the organic acid content after the black barley-aronia mixed fermented vinegar and black barley-bokbunja mixed fermented vinegar were fermented for 14 days. In all samples, acetic acid was produced the most, and fumaric acid, shikimic acid, and quinic acid were detected in addition to acetic acid in the case of black barley-aronia mixed fermented vinegar, and when the aronia alcohol content was 75% or more (BA100, BA75) Only citric acid and malic acid were detected. Citric acid, fumaric acid, and shikimic acid were also detected in black barley-bokbunja mixed fermented vinegar. In both Aronia and Bokbunja, when vinegar was prepared by mixing 25% black barley makgeolli (BA75, BB75), it was determined that mixing black barley makgeolli with the highest acetic acid content could promote acetic acid fermentation.

Ye XJ et al. (2004) reported that acetic acid and citric acid were detected in barley vinegar, but in this study, it was confirmed that berries contain various organic acids beneficial to health according to the addition of alcohol.

<Table 5> Organic acid content (mg/kg) of vinegar fermented with acetic acid by mixing black barley makgeolli and berry wine

(4) Total polyphenol and anthocyanin content

Polyphenols and anthocyanins are known as substances that protect cells exposed to reactive oxygen species (Bidlack W 1999). In the present invention, total polyphenol and anthocyanin contents were measured to investigate the effect of the addition of black barley on antioxidant activity in berries with high antioxidant activity. As shown in Table 6, total polyphenol and anthocyanin in 100% aronia alcohol vinegar (BA100) were 18.21 mg/mL and 2.19 mg/mL, respectively, and the content of total polyphenol and anthocyanin increased as the aronia ratio increased. In the case of bokbunja, 100% alcohol vinegar (BB100) contained more antioxidants than aronia vinegar with total polyphenols and anthocyanins of 44.24 mg/mL and 6.69 mg/mL, respectively. The content increased, and in most samples, the total polyphenol content and total anthocyanin content were higher than that of vinegar prepared only with black barley makgeolli.

<Table 6> Total polyphenol and anthocyanin content of vinegar fermented with acetic acid by mixing black barley makgeolli and berry wine

(5) DPPH and ABTS radical scavenging activity of black barley and berry mixed fermented vinegar

Table 7 shows the results of measuring the antioxidant activity of black barley-berry mixed fermented vinegar with DPPH and ABTS radical scavenging ability. As a result of calculating the IC ₅₀ value by measuring the DPPH radical scavenging ability and ABTS radical scavenging ability by concentration of black barley-aronia mixed fermented vinegar, the results were 22.26 mg/mL and 35.57 mg/mL in the case of 100% aronia alcohol (BA100). As the barley alcohol content increased, the IC ₅₀ value increased, confirming that the antioxidant activity was increased by the addition of aronia. The IC ₅₀ values calculated by the measured DPPH and ABTS radical scavenging activity of 100% fermented bokbunja alcohol vinegar (BB100) were 9.68 mg/mL and 13.76 mg/mL. showed

The high antioxidant activity of aronia vinegar and bokbunja vinegar is thought to be influenced by antioxidants such as polyphenols and anthocyanins derived from aronia and bokbunja fruit and alcoholic fermentation broth . It was found that the antioxidant activity was increased.

<Table 7> DPPH and ABTS radical scavenging activity of vinegar fermented with acetic acid by mixing black barley makgeolli and berry wine

(6) Analysis of Phenolic Compounds in Black Barley and Berries Mixed Fermented Vinegar

The results of analyzing the phenolic compounds of black barley, aronia and bokbunja mixed fermented vinegar are shown in Table 7 below. Galic acid, Chlorogenic acid, Caffeic acid and Quercetin were detected in Black Barley-Aronia mixed fermented vinegar (BA). Chlorogenic acid, Caffeic acid and Quercetin were higher as the aronia content was higher. Galic acid, Caffeic acid, Ellagic acid, and Quercetin were detected in the black barley-bokbunja mixed fermented vinegar (BA). The higher the bokbunja content, the higher the phenolic compound content. Only galic acid was detected in black barley vinegar. Various phenolic compounds of mixed fermented vinegar depend on the berries used as raw materials, and various phenolic compounds were detected according to the addition of berries.

<Table 8> Phenolic compound content (mg/L) of vinegar fermented with acetic acid by mixing black barley makgeolli and berry wine

2. Vinegar is manufactured through acetic acid fermentation after mixed alcohol fermentation of barley and berries.

Experimental method

(1) Black barley pretreatment

Black barley powder was prepared by grinding black barley and passing it through a 100 mesh sieve.

The gelatinized black barley powder was prepared by pouring enough water into the black barley powder, immersing it in water for 1 hour, and heating it at 121°C.

Black barley granulated powder was used as a granulated powder sample by grinding black barley by puffing machine and passing it through a 100 mesh sieve.

(2) Preparation of saccharification solution by enzyme treatment

To the gelatinized liquid gelatinized by adding 360 mL of water to 40 g of barley powder, and the gelatinized liquid mixed by adding 360 mL of water to 40 g of barley powder, add purified enzyme for table wine (1% of the weight of the powder) and heat at 25℃ waterbath for 6 After shaking for an hour, the saccharification solution was prepared by centrifugation at 8,000 rpm for 10 minutes. As a control, an enzyme treatment was performed in the same way using a powder that was not pretreated with barley.

(3) Wort production

After grinding the malt and passing it through a 100 mesh sieve, 4 times the amount of water was poured and shaking in a water bath at 40° C. for 2 hours. After centrifuging the malt solution at 5,000 rpm for 10 minutes, the supernatant was used as wort.

(4) Preparation of saccharification solution by wort

Add 260 g of wort to the gelatinized liquid gelatinized by adding 100 mL of water to 40 g of barley powder and 100 mL of water to 40 g of gelatinized barley powder. A saccharification solution was prepared by centrifugation for 10 minutes. As a control, wort was treated in the same way using a powder that was not pretreated with barley.

(5) Investigation of characteristics of saccharification solution

For the characteristics of barley saccharification solution, sugar content, pH, and acidity were measured in the same way as in Chapter 1, and reducing sugar was measured by the DNS method.

(6) Manufacture of mixed fermented alcohol with black barley and bokbunja

After crushing 3 kg of bokbunja, add 3 kg of black barley saccharified solution to 100% (DW0), 75% (DW1), 50% (DW2), and 0% (DW3) concentrations and dilute it with white sugar. It was propagated at 28°bx. Herein, permibin, a commercially available yeast, was added and fermented at 25° C. for 9 days, followed by filtration to prepare an alcoholic fermentation broth.

(7) Manufacture of mixed fermented vinegar with black barley and bokbunja

To prepare black barley and bokbunja mixed fermented vinegar, the mixed alcoholic fermentation broth prepared above was diluted to an alcohol concentration of 6% and acetic acid fermentation was performed. Black barley and bokbunja mixed fermented vinegar was prepared by adding 10% of seed vinegar to the diluted alcoholic fermented black barley and bokbunja mixed alcohol, adjusting the initial acidity to 2% with glacial acetic acid, and fermenting it at 30°C for 14 days.

(8) Black barley and bokbunja mixed alcohol fermentation characteristics

The characteristics of alcoholic fermentation broth prepared by mixing black barley and bokbunja were investigated. According to the fermentation period, sugar content, pH, acidity, color, and alcohol content were measured in the same manner as those used in vinegar production through mixed acetic acid fermentation after alcohol fermentation of barley and berries, respectively.

(9) Characteristics of acetic acid fermentation broth

The characteristics of vinegar were investigated while carrying out acetic acid fermentation by diluting black barley and bokbunja mixed fermentation alcohol. Acetic acid fermentation characteristics were measured as in alcoholic fermentation broth, sugar content, acidity, pH, color, and alcohol content.

(10) organic acid content analysis

To find out the organic acid content of black barley and bokbunja mixed fermented vinegar, 1. Barley and berries were respectively alcohol-fermented and then analyzed using HPLC as in vinegar production through mixed acetic acid fermentation.

(11) Total polyphenol and total anthocyanin content

Total polyphenol and anthocyanin contents were measured in the same manner as in vinegar production through alcohol fermentation of barley and berries, respectively, and then mixed acetic acid fermentation.

(12) Measurement of DPPH and ABTS radical scavenging activity

The DPPH and ABTS radical scavenging ability of black barley and bokbunja mixed fermented vinegar was measured in the same manner as in vinegar production through alcohol fermentation of barley and berries, respectively, and then mixed acetic acid fermentation.

result

(1) Characteristics of saccharification solution by enzyme treatment

Black barley saccharified solution to be mixed with berry juice was prepared in order to proceed with alcoholic fermentation of wine manufacturing method by mixing black barley and berries. After pre-treating black barley with gelatinization or gelatinization treatment, a saccharification solution was prepared through enzyme treatment for 6 hours, and sugar content, pH, acidity, and reducing sugar were measured. As time increased, reducing sugar increased, and it was found that reducing sugar was most produced when water was added to the gelatinized powder and enzymatic treatment was performed.

<Table 9> Sugar content, pH, acidity, reducing sugar of black barley saccharification solution prepared by enzyme treatment

(2) Characteristics of black barley saccharified solution prepared from wort

Black barley was pre-treated in the same way as above, and a saccharification solution was prepared using wort instead of enzyme treatment. The results of measuring the sugar content, pH, acidity, and reducing sugar of the saccharification solution are shown in the table below. As the reaction time increased, the reducing sugar increased. When the wort was treated with the gelatinized powder, the reducing sugar was higher, but the amount of reducing sugar production was lower than that of the enzyme treatment .

<Table 10> Sugar content, pH, acidity, reducing sugar of black barley saccharified solution prepared from wort

(3) Characteristics of mixed alcohol fermentation of black barley and bokbunja

Black barley saccharification liquid and bokbunja were mixed and alcohol fermentation was carried out according to the wine manufacturing method. Black barley saccharified solution was 100% (DW0), 75% (DW1), 50% (DW2), and 0% (DW3) in the same amount as bokbunja. After alcohol fermentation at 25℃ for 9 days, pH, sugar content , acidity, and alcohol content were measured. As shown in FIG. 6 below, as the fermentation proceeded, the pH of the alcoholic fermentation broth hardly changed, and as the sugar content used as food for yeast decreased during the fermentation period, the sugar content decreased, the acidity slightly increased, and the alcohol content increased significantly.

As a result of analyzing the characteristics of the alcoholic fermentation broth according to the concentration of saccharified black barley , the concentration of the saccharified solution did not affect pH and acidity. It was confirmed that the addition of the liquid promotes alcoholic fermentation.

Table 11 shows the results of measuring the chromaticity of black barley and bokbunja mixed alcohol fermented broth. As the fermentation progressed, the brightness slightly decreased in all samples, the redness did not change, and only when the saccharification solution was added, the yellowness increased.

<Table 11> Color of black barley bokbunja mixed alcoholic fermentation broth

(4) Black barley and bokbunja mixed acetic acid fermentation characteristics

After mixing black barley saccharification liquid and bokbunja, alcohol fermentation was carried out in a wine method, acetic acid fermentation was performed. After acetic acid fermentation at 30° C. for 11 days, the results of pH, sugar content, acidity, alcohol content, and color were measured as shown in FIGS. 7 and 12 below.

The pH and sugar content of the acetic acid fermentation broth were maintained almost constant during the fermentation period in all samples, but as the fermentation progressed, the acidity increased and the alcohol content used as a substrate for the acetic acid fermentation decreased. As a result of measuring the chromaticity of the acetic acid fermentation broth, the brightness slightly decreased as the fermentation progressed, but the redness and yellowness were almost unchanged.

<Table 12> Color of black barley bokbunja mixed acetic acid fermentation broth

(5) Antioxidant activity of black barley and bokbunja mixed vinegar

① Total polyphenol and anthocyanin content

To analyze the antioxidant activity of a mixed alcoholic fermented black barley-bokbunja mixed alcohol fermented solution by mixing black barley saccharified solution and bokbunja, and the vinegar prepared therefrom, the contents of total polyphenols and anthocyanins, which are antioxidant active substances, were measured. As shown in Table 13, the total polyphenol and anthocyanin content increased to the maximum after alcohol fermentation and then decomposed again in the acetic acid fermentation process. It was confirmed that the polyphenol and anthocyanin content was higher.

<Table 13> Total polyphenol and anthocyanin content of black barley-bokbunja mixed alcoholic fermentation broth and vinegar

② DPPH and ABTS radical scavenging ability

To analyze the antioxidant activity of black barley-bokbunja mixed alcoholic fermentation broth and vinegar prepared from alcohol fermentation by mixing black barley saccharified solution and bokbunja, the DPPH and ABTS radical scavenging ability was measured by concentration, and then the IC ₅₀ value was calculated. (Table 14).

As a result of measuring the antioxidant activity by DPPH radical scavenging ability, it was found that the IC ₅₀ value of the black barley-bokbunja mixed solution increased through alcohol fermentation and acetic acid fermentation, and thus the antioxidant activity decreased. However, it was judged that bokbunja vinegar (DW0) containing 100% saccharification solution had a lower IC ₅₀ value than vinegar without saccharification solution (DW3), so it was judged to have superior antioxidant activity.

Antioxidant activity measured by ABTS radical scavenging activity also showed the same trend as DPPH radical scavenging activity.

<Table 14> Antioxidant activity of black barley-bokbunja mixed alcohol fermented broth and vinegar

3. Selection of excellent acetic acid fermented bacteria

Experimental method

(1) Isolation and identification of acetic acid fermentation strains from barley vinegar

In order to separate acetic acid bacteria from acetic acid fermentation broth, acetic acid strains were isolated from grain seeds, aronia seeds and bokbunja seeds as samples.

Each sample was inoculated into GYE medium (yeast extract 1%, glucose 5%, ethanol 3%) as a pre-culture and incubated at 30° C. for 48 hours. The pre-cultured sample was inoculated into SM medium (Acetobacter selective medium, yeast extract 0.5%, glucose 3.0%, CaCO ₃ 1.0%, agar 2.0%, ethanol 5.0%) and cultured at 30° C. for 48 hours, and then the strain was isolated. Each of the isolated strains was tooth picked on SM medium, and the size of a clear ring around the strain was measured to separate acetic acid bacteria.

Acetic acid peroxidation (glucose 0.5%, glycerol 0.5%, polypeptone 0.5%, yeast extract 0.5%, bromocresol purple 0.003%, acetic acid 1 %, ethanol 1%, agar 1.5%), acetic acid (glucose 0.5%, glycerol 0.5%, polypeptone 0.5%, yeast extract 0.5%, acetic acid 3%, ethanol 2%, agar 1.5%) and pH tolerance (glucose 0.5%) , glycerol 0.5%, polypeptone 0.5%, yeast extract 0.5%, ethanol 2%, agar 1.5%, pH 3.0~3.4), alcohol resistance (yeast extract 1%, glucose 5%, CaCO ₃ 3%, agar 2.5%, ethanol 10-20%) was tested to select excellent strains, and then the microorganisms were identified. Microorganisms were identified by 16s ribosomal RNA sequencing, and the analysis was commissioned by Solgent. For PCR and sequencer, ABI 9700 PCR (Applied Biosystems, Foster City, CA, USA) and ABI 3730 XL DNA analyzer (Applied Biosystem) were used, respectively. The 16s rRNA base sequence of the analyzed microorganism was identified using the Basic Local Alignment Search Tool of the National Center for Biotechnology Information (NCBI).

To select the final excellent strain among the selected strains, the viability of the strain was evaluated using YPM medium (yeast extract 0.5%, peptone 0.3%, mannitol 2.5%, agar 1.5%). Fermentation suitability was evaluated through vinegar production.

(2) Evaluation of fermentability suitability of barley and berries using selected strains

Using the finally selected strain through the medium, the selected strain was inoculated into a sample fermented with barley makgeolli and aronia alcohol, and pH, sugar content, acidity, color, alcohol content and number of acetic acid bacteria were checked for 14 days. The number of acetic acid bacteria was confirmed using YPM medium, which is an Acetobacter culture medium.

→ Separation and selection of strains with excellent acetic acid peroxidation, acetic acid and pH resistance, alcohol resistance and growth through the medium

result

(1) Separation of acetic acid bacteria from acetic acid fermentation broth

For the separation of acetic acid strains, the acetic acid strains were isolated using the Acetobacter selective medium (SM medium) after primary culture in GYE medium with pre-culture of grain seeds, blueberry vinegar, aronia seeds and bokbunja seeds. A total of 117 species of acetic acid bacteria with transparent rings isolated from each sample using SM medium were identified, including 20 species in grain seeds, 20 species in aronia seeds, 8 species in blueberry vinegar, and 69 species in bokbunsa seeds. These strains were isolated from pure water to select the optimal excellent strain through the medium.

(2) Primary selection of strains isolated from acetic acid fermentation broth

In order to select excellent strains of strains purely isolated from seedlings of grains and berries, after culturing with tooth pick on SM medium, strains with a size of 1 cm or more were first selected by measuring the size of the clear ring. (FIG. 8)

A total of 62 strains were first selected, and as a result of confirming the characteristics of these strains through acetic acid peroxidation medium, it was confirmed that there were almost none. Acetic acid was confirmed that most of the strains produced acetic acid as a result of checking whether or not culture was performed after strain inoculation using an acetic acid producing medium. As a result of checking the presence or absence of culture after strain inoculation using a medium prepared by adjusting pH to 3, most of the strains had pH resistance. Alcohol resistance was analyzed by the presence or absence of transparent rings through media prepared with alcohol content of 10, 15, and 20% in SM medium. As the alcohol content increased, the active strain decreased, and the activity of the most strains at 10% it was confirmed that there is Therefore, 52 strains with a transparent ring size of 1 cm or more in SM medium and high alcohol resistance according to pH, acetic acid resistance, and alcohol content were selected and identified through 16s ribosomal RNA sequencing analysis, and the analysis was requested by Solgent. .

(3) Identification of strains isolated from acetic acid fermentation broth

As a result of identification using the 16S rRNA nucleotide sequence for the strains selected as excellent strains among a total of 20 strains isolated from grain seedlings, it was confirmed as shown in Table 15.

All 11 excellent strains isolated from grain seedlings were identified as Acetobacter pasterianus .

<Table 15> Identification results of strains isolated from grain seeds

The strains selected as excellent strains among a total of 20 strains isolated from Aronia spp. were identified using the 16S rRNA nucleotide sequence, and as a result, it was confirmed as shown in Table 16. Among the superior strains isolated from Aronia spp., 6 were identified as Acetobacter pasterianus , and 4 as Komagataeibacter saccharivorans .

<Table 16> Identification results of strains isolated from aronia spp.

As a result of identification using the 16S rRNA nucleotide sequence for the strains selected as excellent strains out of a total of 69 strains isolated from bokbunja seed, it was confirmed as shown in Table 17.

All of the excellent strains isolated from bokbunja seedlings were identified as Acetobacter pasterianus .

<Table 17> Identification results of strains isolated from bokbunja seed

Therefore, as shown in Table 18 below, as a result of measuring the acetic acid production transparent ring size, pH resistance, acetic acid production, and alcohol resistance, 6 excellent strains were finally selected, and the strains were G7, G10, G15, A7, BJ20, BJ62. All of them were identified as Acetobacter pasteurianus .

Therefore, we want to select an acetic acid fermentation starter strain that can be used for final vinegar fermentation by selecting strains with excellent growth rates for these strains.

<Table 18> Selection of excellent acetic acid strains using medium

(4) Selection of excellent strains for growth of the selected strains

In order to select strains with excellent viability among the selected strains, the strains were inoculated in LM medium and absorbance (O.D. 600 nm) was measured at 12-hour intervals for 4 days at 30 degrees to confirm the growth of the strains. The selected strains were selected as excellent strains of acetic acid through the medium, and a total of 6 strains, G7, G10, G15, A7, BJ20, and BJ62, were used to confirm excellent growth.

As a result of measuring the absorbance according to the culture time of the selected strains and confirming the growth, the BJ20 strain showed the highest absorbance as shown in FIG. 9 below. In addition, most of the strains showed the highest absorbance after 48 hours of incubation, confirming that the number of bacteria was high. In the case of strain BJ20, the absorbance value was confirmed to be 1.383 at 60 hours, and in the case of strain G10, the absorbance value was confirmed to be 1.349 at 72 hours. Therefore, the BJ20 strain, which is a strain with high growth rate, was selected to prepare barley and berry vinegar, and the fermentability suitability was evaluated.

(5) Evaluation of fermentability suitability of barley and berries using selected strains

Acetobacter pasteurianus BJ20, a strain with excellent acetic acid and pH resistance, alcohol resistance, and growth, was selected and barley and berry vinegars were manufactured to evaluate fermentability suitability. Black barley makgeolli, aronia and bokbunja liquor were inoculated with Acetobacter pasteurianus BJ20, an excellent strain, and fermented with acetic acid at 30°C for 14 days.

As shown in Table 19 below, the pH of black barley vinegar showed a similar level of 3.51 to 3.52, and it was confirmed that the pH of aronia vinegar decreased slightly to 3.66 to 3.58 and that of bokbunja vinegar was 3.72 to 3.25. In terms of sugar content, 5.4-4.5 brix for black barley vinegar, 6.4-6.8 brix for aronia vinegar, and 7.7-6.8 brix for bokbunja vinegar.

In the case of acidity, the acidity of black barley vinegar increased from 1.41% at the initial stage to 4.15% after 2 weeks after fermentation, and in the case of aronia vinegar, the initial acidity increased from 1.13% to 4.62% after 2 weeks. Also, in the case of bokbunja vinegar, the initial acidity increased from 1.44% to 6.75% after 2 weeks.

The alcohol content of the prepared vinegar decreased from 11.05% in the initial stage to 1.05% after 2 weeks for black barley vinegar, and from 7.79% at the beginning to 1.25% for aronia vinegar after 2 weeks.

In addition, the alcohol content of bokbunja vinegar decreased from 10.68% initially to 1.13% after 2 weeks.

<Table 19> Analysis of physicochemical properties of barley and berry vinegar using excellent strains

Using the excellent strain Acetobacter pasteurianus BJ20, the color and number of acetic acid bacteria were measured for each fermentation period according to the production of barley and berry vinegar (Table 20). As for the chromaticity of black barley vinegar, the L value decreased as the fermentation progressed, and the a and b values increased. It was confirmed that the color of bokbunja vinegar decreased as the fermentation progressed, the L value, the a value, and the b value.

As a result of the acetic acid strain analysis of vinegar according to the fermentation period, black barley vinegar increased from 1.23±0.03 Log CFU/ml at the initial stage to 6.57±0.10 Log CFU/ml after 2 weeks, and in the case of aronia vinegar, the initial 1.12±0.04 Log CFU/ml increased to 6.83±0.02 Log CFU/ml after 2 weeks. Also, in the case of bokbunja vinegar, it increased from 1.19±0.06 Log CFU/ml at the beginning to 5.92±0.04 Log CFU/ml after 2 weeks. As for the number of acetic acid bacteria in vinegar according to fermentation period, the highest number of bacteria was confirmed in the first week of fermentation for black barley, aronia and bokbunja vinegar.

<Table 20> Analysis of color and strain of barley and berry vinegar using excellent strains

Therefore, in the method for producing complex fermented vinegar using black barley and berries according to the present invention, the production of acetic acid is promoted to increase acidity, and berry components are included to increase the content of phenolic compounds and organic acids, thereby exhibiting excellent antioxidant effect. Fermented vinegar is provided.

S101; Black barley fermented liquid manufacturing step, S101-1; Berry fermented liquid manufacturing step
S103; Raw material mixing stage
S105 ; Seedling mixing stage
S107; acidity control stage
S109; Fermentation stage

Claims (5)

Black barley fermented liquid production step of preparing the black barley fermented broth;
A berry fermented liquid preparation step of preparing a berry fermented broth;
a raw material mixing step of mixing the fermented berries prepared through the fermented berries manufacturing step with the fermented black barley prepared through the black barley fermented liquid manufacturing step;
Seedling mixing step of mixing the seedling with the mixture prepared through the raw material mixing step;
an acidity control step of adjusting the acidity by mixing glacial acetic acid with the mixture in which the seedlings are mixed through the seedling mixing step; and
A method for producing a complex fermented vinegar using black barley and berries, characterized in that it comprises; a fermentation step of fermenting a mixture whose acidity has been adjusted through the acidity control step.
The method according to claim 1,
Black barley and berries, characterized in that the raw material mixing step is made by mixing 30 to 400 parts by weight of the fermented berry solution prepared through the fermented berry solution manufacturing step with 100 parts by weight of the black barley fermented solution prepared through the black barley fermented solution manufacturing step Method for manufacturing complex fermented vinegar using.
The method according to claim 1 or 2,
The black barley fermented solution manufacturing step involves soaking black barley in water for 5 to 7 hours, then steaming it for 30 to 90 minutes, and adding 2 to 3 parts by weight of yeast, 1.5 to 2.5 parts by weight of yeast and water to 100 parts by weight of the steamed black barley. A mixture is prepared by mixing 100 to 120 parts by weight, and after 20 to 30 hours, 200 to 250 parts by weight of black barley, 4 to 5 parts by weight of yeast and 250 to 300 parts by weight of water are mixed with 100 parts by weight of the mixture and 6 to A method for producing a complex fermented vinegar using black barley and berries, characterized in that it is produced by fermentation and filtration for 8 days.
The method according to claim 1 or 2,
After crushing the berries, the fermented berry juice is mixed with crushed berries with high fructose sugar to adjust the sugar content to 25 to 30 Brix, and yeast is mixed with the berries for which the sugar content is controlled, and 7 to 9 at a temperature of 24 to 26 ° C. It is prepared by fermentation and filtration for one day,
The method for producing a complex fermented vinegar using black barley and berries, characterized in that the berries are made of one or more selected from the group consisting of aronia and bokbunja.
The method according to claim 1,
The fermentation step is a method for producing a complex fermented vinegar using black barley and berries, characterized in that it is performed for 20 to 22 days at a temperature of 28 to 32 ℃.

Images