KR102334548B1 - Manufacturing method of low-temperature fermented jujube vinegar using Acetobacter oryzifermentans - Google Patents

Abstract

The present invention relates to a method for producing fermented jujube vinegar using Acetobacter originantance. Specifically, Acetobacter oryzifermentans ( Acetobacter oryzifermentans ) As a result of preparing jujube fermented vinegar using KACC 19301 strain, when fermented for 14 days at pH 4, 20 ° C. It was confirmed that high-quality fermented jujube vinegar with a high content of functional ingredients could be manufactured because it increased in a short time. Therefore, through the present invention, it is possible to prepare fermented jujube vinegar with excellent acetic acid production ability even under low temperature conditions.

Description

The manufacturing method of low-temperature fermented jujube vinegar using Acetobacter oryzifermentans

The present invention relates to a method for producing a low-temperature fermented jujube fermented vinegar using Acetobacter orifermentance, and to a low-temperature fermented jujube fermented vinegar prepared by the manufacturing method.

Vinegar is one of the fermented foods that have been manufactured and used for a long time in both East and West, and is a representative seasoning with a sour taste that is easily used in daily life. Vinegar is divided into fermented vinegar and diluted acetic acid according to the manufacturing method. Fermented vinegar is vinegar made by fermenting grains, fruits, and alcoholic beverages as main ingredients, and diluted acetic acid is vinegar made by diluting glacial acetic acid or acetic acid with drinking water. Fermented vinegar is further divided into natural fermented vinegar and alcoholic fermented vinegar according to the production method. Natural fermented vinegar is made by fermenting alcohol to make alcohol and then fermenting it again with acetic acid. Compared to alcoholic fermented vinegar that ferments acetic acid, it has richer functional ingredients and is beneficial to health. In addition, fermented vinegar is largely divided into two types depending on the raw material, and grain vinegar fermented as a raw material and fruit vinegar fermented as a raw material are representative. Various organic acids and amino acids present in vinegar are involved in energy metabolism in the body, thereby inhibiting the accumulation and production of lactic acid, which is a fatigue substance, and lipid peroxide that causes arteriosclerosis, thrombosis, and the like. In addition, vinegar prevents obesity and is good for skin health and beauty, as well as removing toxins and hangovers in the human body. In addition, it has been reported that various physiological activities such as increase in calcium absorption rate, relief of constipation, alleviation of eye fatigue, and increase in resistance are reported.

Jujube, a deciduous broad-leaved arboreal fruit of the genus zizyphus in the Rhamnaceae family, has long been known as a food that comforts the stomach and protects the five intestines (Cheng N, Du B, Wang Y, Gao H, Cao). W, Zheng J, Feng J. Antioxidant properties of jujube honey and its protective effects against chronic alcohol-induced liver damage in mice. Food Funct. 5: 900-908 (2014)). In addition, there is a record that if you eat for a long time, your complexion will improve and your body will become lighter and you will not get old.

It has been widely used in oriental medicine as a pain reliever (Gao QH, Wu CS, Wang M. The jujube (Ziziphus jujuba Mill.) fruit: A review of current knowledge of fruit composition and health benefits J. Agr. Food Chem. 61: 3351 -3363 (2013)). Jujube fruit is composed of various sugars, organic acids, vitamins and trace inorganic elements along with nutrients such as protein and fat. In addition, various sterols, alkaloids, saponins, vitamins, organic acids, amino acids known to have pharmacological effects such as laxatives, diuretics, tonics, cholestasis, sedatives, physical recovery, expectorants, and anti-inflammatory drugs. It is known that a large amount of medicinal ingredients such as rye are present (Kwon SH, Cho KY, Kim SY, Kim MJ. Application of Ziziphus jujube fruit for dietary life. Korean J. Food Sci. Technol. 5: 1-14 (1993) ), phenolic compounds contained in jujube are known to have antioxidant and anticancer activity. In particular, c-AMP (cyclic adenosine monophosphate, adenosine, 3'-, 5'-cyclic monophosphate), it has been reported that only those contained in jujubes have pharmacological and physiological activities (Choi KS. Changes in physiological and chemical characteristics of jujube fruits (Zizyphus jujuba Miller) var. bokjo during maturity and postharvest ripening. J. Resour. Dev. 9: 47-53 (1990)).

Various physiologically active substances and antioxidants contained in these jujubes are unstable and toxic to the human body after extraction. As a part of it, a biological transformation method using microorganisms or enzymes is being developed. Fermentation is a representative method of bioconversion. Among them, vinegar produced through acetic acid fermentation makes nutrients such as organic acids and amino acids such as acetic acid, citric acid, and lactic acid that give a sour taste, and has a lactic acid decomposition function, so it has a fatigue recovery effect. It has various health and beauty effects, such as lowering the viscosity of blood and removing cholesterol from the inner walls of blood vessels.

Dates have a very high concentration of sugar. Jujube fruit juice generally contains 84.4-76.4% of saccharides and is mainly composed of fructose, glucose, oligosaccharides, and the like. However, it has been reported that there is a physiologically active effect that promotes the growth of lactic acid bacteria even at high sugar concentration. Therefore, in the case of making jujube vinegar, it is expected that the health functional beverage market that adds the nutritional functions of fermented vinegar to the excellent nutritional properties and physiological activity of jujube will expand.

In general, it is known that during acetic acid fermentation for fermented vinegar production, if the culture temperature is too low, the fermentation rate is slowed, and if the temperature is too high, ethanol and acetic acid are lost, resulting in loss of flavor (Kwon SH, Cho KY, Kim SY, Kim) MJ. Application of Ziziphus jujube fruit for dietary life. Korean J. Food Sci. Technol. 5: 1-14 (1993)). Therefore, it is important to establish optimal conditions for a method for producing jujube vinegar that has a fast fermentation rate without loss of active ingredients.

As a prior art related to jujube vinegar, Korean Patent No. 10-1285738 discloses a jujube vinegar drink, and Korean Patent No. 10-1790414 discloses a method for producing jujube vinegar or jujube vinegar manufactured by the above method. , There is no disclosure of the method for producing fermented jujube vinegar according to the pH, fermentation temperature and fermentation period established in the present invention.

Accordingly, the present inventors used yeast with excellent alcohol-producing ability and aroma-producing ability and acetic acid bacteria with excellent acid-producing ability, and produced jujube fermented vinegar using only jujube extract according to specific conditions of pH, fermentation temperature and fermentation period. The present invention was completed by succeeding in selecting the conditions for increasing the acidity within a period of time.

An object of the present invention is to provide a method for producing fermented jujube vinegar having excellent acid-producing ability at pH 4 and 20° C. and fermented jujube vinegar prepared by using the method.

In order to achieve the above object, the present invention

1) preparing a jujube extract;

2) alcohol fermentation of the jujube extract of step 1) to prepare a fermented jujube wine;

3) preparing the fermented jujube vinegar by inoculating the Acetobacter oryzifermentans strain deposited with the accession number KACC 19301 into the fermented jujube strain of step 2); It provides a method for producing fermented jujube vinegar, comprising a.

In addition, the present invention provides a low-temperature fermented jujube fermented vinegar prepared by the above method.

The present invention relates to a method for producing fermented jujube vinegar using Acetobacter originantance. Specifically, Acetobacter oryzifermentans ( Acetobacter oryzifermentans ) As a result of preparing jujube fermented vinegar using KACC 19301 strain, when fermented for 14 days at pH 4, 20 ° C. It was confirmed that high-quality fermented jujube vinegar with a high content of functional ingredients could be manufactured because it increased in a short time. Therefore, through the present invention, it is possible to prepare fermented jujube vinegar with excellent acetic acid production ability even under low temperature conditions.

1 is a view showing that the CO ₂ production ability was measured to determine the alcohol production ability of Saccharomyces servisiae yeast of 22 weeks, confirming that YM32, YM22, and YM25 have excellent CO 2 production capability.
Figure 2 is a qualitative evaluation of the acid-producing ability of acetic acid bacteria of 8 weeks, and is a diagram showing the excellent acid-producing ability of SLV-7.
Figure 3 is a diagram showing a phylogenetic diagram of the isolated and identified Acetobacter origin Fermentance SLV-7 acetic acid bacteria.
Figure 4 shows the sugar content and alcohol concentration when the jujube extract was fermented with alcohol according to various temperature conditions (18, 25 and 30 ° C) and fermentation period using Saccharomyces servisiae YM22 and Saccharomyces servisiae YM27 yeast. is the diagram shown.
Figure 5a is a diagram showing the analysis of free sugar content when the jujube extract is fermented with alcohol according to various temperature conditions (18, 25 and 30 ℃) and fermentation period using Saccharomyces servisiae YM22 yeast.
Figure 5b is a diagram illustrating the analysis of free sugar content when the jujube extract is fermented with alcohol according to various temperature conditions (18, 25 and 30 ℃) and fermentation period using Saccharomyces servisiae YM27 yeast.
6a is a diagram showing the organic acid content when the jujube extract is fermented with alcohol according to various temperature conditions (18, 25 and 30° C.) and fermentation period using Saccharomyces servisiae YM22 yeast.
Figure 6b is a diagram showing the organic acid content when the jujube extract is alcohol-fermented according to various temperature conditions (18, 25 and 30 ° C) and fermentation period using Saccharomyces servisiae YM27 yeast.
Figure 7a is a diagram showing the alcohol content and acid production ability when acetic acid fermentation of jujube fermented wine of pH 2 according to various temperature conditions (20, 30 and 35 ° C) and fermentation period using Acetobacter orifermentance acetic acid bacteria.
Figure 7b is a diagram showing the alcohol content and acid production ability when acetic acid fermentation of jujube fermented wine of pH 3 according to various temperature conditions (20, 30 and 35 ° C) and fermentation period using Acetobacter origin acetic acid bacteria.
Figure 7c is a diagram showing the alcohol content and acid production ability when acetic acid fermentation of jujube fermented liquor of pH 4 according to various temperature conditions (20, 30 and 35 ℃) and fermentation period using Acetobacter orifermentance acetic acid bacteria.
Figure 7d is a diagram showing the alcohol content and acid production ability when acetic acid fermentation of jujube fermented wine of pH 5 according to various temperature conditions (20, 30 and 35 ℃) and fermentation period using Acetobacter orifermentance acetic acid bacteria.

Hereinafter, the present invention will be described in detail.

the present invention

1) preparing a jujube extract;

2) alcohol fermentation of the jujube extract of step 1) to prepare a fermented jujube wine;

3) preparing fermented jujube vinegar by inoculating the Acetobacter oryzifermentans strain deposited under the accession number KACC 19301 into the fermented jujube strain of step 2); It provides a method for producing fermented jujube vinegar, comprising a.

The jujube extract of step 1) can be prepared by washing, watering and extracting jujubes.

The extraction solvent of the extraction may be water, alcohol, or a mixed solvent thereof, specifically water.

The extraction method of the extraction may be shaking extraction, Soxhlet extraction, or reflux extraction. At this time, the extraction temperature may be 40 to 95 ℃, specifically 50 to 90 ℃, more specifically 60 to 85 ℃, but may be, but is not limited thereto. In addition, the extraction time may be 1 to 20 hours, specifically 2 to 10 hours, more specifically 4 to 8 hours, but is not limited thereto. In a range outside the conditions of the extraction temperature or extraction time, the extraction is not sufficiently performed, so the content of the active ingredient in the jujube extract may be insignificant. Alternatively, the extraction yield may no longer increase and the efficiency of the extraction operation may decrease.

The jujube extract can be used after being concentrated to 15 to 30 ° Brix, specifically 18 to 25 ° Brix, more specifically 18 to 22 ° Brix, concentrated to a sugar content. If the sugar content of the jujube extract is lower than 18 ° Brix, the sugar content of vinegar may be lowered, and overall preferences such as taste and aroma may also be lowered. .

Alcohol fermentation in step 2) may be performed by inoculating Saccharomyces cerevisiae YM22 or Saccharomyces cerevisiae YM27 (Saccharomyces cerevisiae) deposited with accession number KACC 48337 and fermenting, preferably Saccharomyces cerevisiae YM22 (Saccharomyces cerevisiae) is inoculated and fermented.

The alcoholic fermentation of step 2) may be fermentation at 15 to 35 °C, specifically 17 to 31 °C, more specifically 20 to 27 °C, more specifically 22 to 26 °C. have.

The alcoholic fermentation of step 2) may be carried out for 3 to 20 days, specifically 3 to 18 days, 3 to 17 days, 3 to 16 days, 4 to 16 days, 5 to 16 days, or 6 to 16 days, More specifically, it may be carried out for 6 to 15 days, 7 to 15 days, 8 to 15 days, 9 to 15 days, or 10 to 14 days.

The alcoholic fermentation in step 2) may be stationary fermentation.

The fermented jujube vinegar of step 3) is prepared by inoculating the seeds prepared using the Acetobacter oryzifermentans SLV-7 strain deposited under the accession number KACC 19301.

The seed seed may be inoculated at a concentration of 7 to 15% (v/v), specifically, may be inoculated at a concentration of 9 to 12% (v/v).

The acetic acid fermentation of step 3) is carried out by inoculating Acetobacter oryzifermentans strains in jujube fermented strains of pH 2 to 6, specifically pH 3 to 5, and more specifically pH 3.5 to 4.5. can

The acetic acid fermentation of step 3) may be performed at 15 to 35 °C, specifically 18 to 30 °C, more specifically 18 to 23 °C.

The acetic acid fermentation of step 3) may be fermented for 3 to 25 days, specifically 5 to 25 days, more specifically, stationary fermentation for 10 to 22 days.

In step 3), acetic acid fermentation takes place, and the acetic acid fermentation may be carried out by the method of farmhouse-type stationary fermentation (surface fermentation) or enterprise-type rapid fermentation (deep fermentation), and specifically, it may be performed by the method of stationary fermentation. Stationary fermentation is a method of acetic acid fermentation in a stationary state after inoculation of acetic acid bacteria in the medium. The fermentation facility is simple, so the initial investment cost can be minimized and a mild sour taste can be produced. Fermentation conditions such as skill level must be controlled, fermentation must be carried out for a long time, and there are problems in that the yield is low and the vinegar already has an off-flavor. On the other hand, deep fermentation can control fermentation conditions such as fermentation temperature and aeration, so the fermentation period is shorter than that of stationary fermentation (surface fermentation), and the yield and acidity of vinegar can be increased. There is a problem that the sensibility is reduced.

In addition, the present invention provides a low-temperature fermented jujube fermented vinegar prepared by the above manufacturing method.

In a specific embodiment of the present invention, Saccharomyces serbiae YM22 and YM27 having excellent alcohol-producing ability and fragrance-producing ability were selected (FIGS. 1 and Table 1), and Acetobacter oryzifermentans having excellent acid-producing ability (Acetobacter oryzifermentans) SLV -7 strains were isolated and identified (see Tables 2 to 4 and FIGS. 2 to 3), and as a result of preparing jujube fermented vinegar using the YM22 and SLV-7 acetic acid bacteria, the result was fermented for 14 days at pH 4 and 20 ° C. It was confirmed that high-quality fermented jujube vinegar with a high content of functional ingredients could be prepared because the acidity was quickly increased in spite of the acetic acid fermentation at a low temperature ( FIGS. 7a to 7d ).

Therefore, the present invention can produce fermented jujube vinegar excellent in acetic acid production ability even under low temperature conditions.

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

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

<Example 1> Selection of yeast and acetic acid bacteria for production of fermented jujube vinegar

<1-1> Yeast selection for production of fermented jujube wine

In order to manufacture fermented jujube vinegar, the manufacturing process of fermented jujube wine must be preceded. In order to manufacture high-quality fermented jujube wine, it is necessary to select yeast that produces alcohol well and has excellent aroma-generating ability. Therefore, yeast having excellent alcohol-producing ability and aroma-producing ability was selected by the following method.

Specifically, yeast breaks down glucose to produce alcohol and CO ₂ . Therefore, _{it can be judged that the more CO 2} is produced, the better the alcohol production ability. 22 kinds of Saccharomyces servisiae yeast were inoculated into YPD broth and cultured (30° C., 72 hours) while the weight (g) of the culture medium was measured every 24 hours to measure quantitative CO ₂ production.

In addition, in order to produce fruit vinegar with excellent flavor and flavor, it is necessary to use yeast excellent in aroma production ability to produce fruit flavor. After inoculating 100 μl of yeast in 5 mL of YPD broth and culturing with shaking for 2 days, linearly spread it on YPD Agar medium and compare the strain cultured at 30°C for 4 days with the uncultivated YPD Agar medium (control) for sniffing test. carried out. A total of three sessions were conducted with an interval of 30 minutes, and items in which no unusual odor was found were marked with ‘-’.

As a result, as shown in FIG. 1 , _{most of the strains produced a large amount of CO 2 for} 0 to 48 hours of fermentation, and a small amount was produced for 48 to 72 hours. YM32 produced the largest amount with a total of 0.16 g, followed by YM22, YM25, YM27, and YM31 produced 0.14 g.

In addition, as shown in Table 1 below, YM22 had a sweet scent, YM27 had a grape scent, YM31 and YM35 had a savory scent, YM38 had a banana scent, YM39, YM40 had a fruity scent, and YM46 and YM47 had a floral scent. classified. On the other hand, YM24 and YM26 had an ethanol smell, YM36 and YM37 had an unpleasant odor, and YM44, which had a gaseous smell, was classified as a bad smell.

Combining the above results, YM22 and YM27 yeasts having excellent aroma production ability were selected as yeasts to be used in fruit vinegar production for the production of fragrant fruit vinegar while having excellent alcohol production ability.

yeast strain 1 round after 0 minutes of fermentation 2nd round after 30 minutes of fermentation 3 times after 1 hour of fermentation YM22 sweet scent - - YM24 ethanol ethanol - YM25 - - - YM26 ethanol - - YM27 grape flavor - - YM28 - - - YM29 - - - YM31 fragrant scent - - YM32 - - - YM34 - - - YM35 fragrant scent - - YM36 unpleasant odor - - YM37 unpleasant odor - - YM38 banana flavor - - YM39 fruit flavor ethanol - YM40 fruit flavor - - YM42 - - - YM46 - - - YM44 smell of gas - - YM45 - - - YM46 floral scent - - YM47 floral scent - -

<1-2> Selection of acetic acid bacteria

In order to select acetic acid bacteria with excellent acid-producing ability, the acidity of acetic acid produced by the acetic acid bacteria was measured qualitatively and quantitatively.

Specifically, the color of the acetic acid bacteria solid medium is opaque due to the added calcium carbonate. When acetic acid bacteria are cultured in this medium, calcium carbonate is decomposed by acetic acid produced by the acetic acid bacteria and a clear zone is formed. Based on this principle, in order to confirm the qualitative production performance of acetic acid bacteria, it was attempted to measure the size of the transparent ring formed after culturing in a solid medium containing calcium carbonate. First, 10 ml of acetic acid bacteria liquid medium (yeast extract 0.5%, glucose 0.5%, glycerin 1%, MgSO4·7H ₂ O 0.02%, EtOH 6%, acetic acid 1%) is dispensed into each test tube, and each acetic acid bacteria is inoculated with 1 spot. Then, the culture was shaken at 30 ℃ for 72 hours at 160 rpm. Then, put a paper disk on a solid medium for acetic acid bacteria selection (yeast extract 0.5%, glucose 3%, CaCO3 1%, EtOH 3%, agar 2%), dispense 50 μl of acetic acid bacteria culture medium, and incubate at 30°C for 72 hours, then carbonic acid By measuring the size of the transparent ring formed by decomposition of calcium (CaCO ₃ , 1.0% v/v) using a digital caliper, the acid-producing ability of each acetic acid bacteria was confirmed. The diameter of the transparent ring was measured three times to the second decimal place, and when the size of the transparent ring is 30 mm or more, “+++” ; “++” for more than 20 mm ; “+” for more than 10 mm ; When it is less than 10 mm, it is marked with “-”. In addition, in order to quantitatively measure the production performance of acetic acid bacteria, an acetic acid bacteria liquid medium containing ethanol (6% v / v) and acetic acid (1% v / v) was prepared, and each acetic acid bacteria was inoculated to prepare a culture solution. Then, seeds were prepared using each acetic acid bacteria culture medium, and the acidity was measured by converting the acidity into acetic acid (%). First, 50 ml of acetic acid bacteria liquid medium (yeast extract 0.5%, glucose 0.5%, glycerin 1%, MgSO4 7H2O 0.02%, EtOH 6%, acetic acid 1%) was dispensed into each test tube as a pre-culture, and then each acetic acid bacteria was incubated. It was inoculated and incubated at 30° C. for 72 hours at 160 rpm. Then, 500 ml of makgeolli prepared at an alcohol concentration of 6.24% was dispensed into an Erlenmeyer flask for seed preparation, and 50 ml of the pre-culture solution was inoculated, followed by stationary culture at 30°C. For acidity measurement, 1 ㎖ of sample was collected every 3 days, diluted 10 times with 9 ㎖ of distilled water, 2~3 drops of 1% phenolphthalein solution was added, and the appropriate consumption of 0.1N NaOH solution was calculated using the equation below.

Acidity (%) =V×F×A×D×1/S×100

V: Appropriate consumption of 0.1N-NaOH solution (ml)

F: titer of 0.1N-NaOH solution

A: Amount of organic acid equivalent to 1 ml of 0.1N-NaOH solution (acetic acid = 0.006)

D: dilution factor

S: Sample amount (ml)

As a result, as shown in Tables 2 and 3, there were 3 types (No. 3, 5, 7) of acetic acid bacteria with a transparent ring measuring 30 mm or more among acetic acid bacteria of 8 weeks, and 3 types (No. 3, 5, 7) of 20 mm or more ( No. 2, 4, 8), and 10 mm or more was shown as 1 type (No. 1). Strains measuring 10 mm or less were No. It was one of six. Among these, No. 5 strain was measured the highest with 36.71 mm, followed by No. 7 and No. 3 was measured to be 32.62 mm and 31.92 mm, confirming the excellent production performance qualitatively. These results were measured to be less than 10 mm, indicating the lowest production performance of No. It could be judged as a qualitative result with about 4 times higher activity compared to the 8.64 mm transparent ring of 4.

In addition, as shown in Table 4 and FIG. 2 , as a result of measuring the change in acidity by preparing a seed seed using makgeolli, it was measured on the 9th day of fermentation, which is the time of fermentation, based on the measurement of the acidity as 0.66% on the 0th day of fermentation. The results were compared to confirm the production performance of each acetic acid bacterium. The strain Acetobacter originating from the highest acid-producing ability was measured to be 7.18% at the end of fermentation (9 days), followed by SLV-7 as 6.00%. At this time, Acetobacter origin and SLV-7 produced similar levels of acid at 1.90% and 2.00%, respectively, on the 3rd day of fermentation, and 4.18% on the 6th day of fermentation, respectively, while showing the difference in production performance on the 9th day of fermentation. could be seen to indicate These results were significant as strains that were confirmed as strains showing high production performance as the size of the transparent ring was measured to be 30 mm or more in the qualitative experimental results of both acetobacillus acetobacter origin and SLV-7, which were measured to have an acidity of 6.0% or higher. results could be judged.

Therefore, as a result of comparing the qualitative and quantitative production performance of 8 strains of acetic acid bacteria, strain SLV-7 was selected as the acetate bacteria to be used in the second year of reproduction vinegar.

No. strain acid production No. strain acid production One Gluconobacter oxydans 16.78±0.16 + 5 Acetobacter oryzifermentans 36.71±0.60 +++ 2 Gluconobacter thailandicus 26.14±0.12 ++ 6 Acetobacter pasteurianus A6 8.64±0.14 - 3 Acetobacter pasteurianus 31.92±0.52 +++ 7 SLV-7 32.62±0.18 +++ 4 Komagataeibacter europaeus JY49 25.35±0.18 ++ 8 Y2019-1 24.56±0.01 ++

Fermentation period/strain 0 3 6 9 Gluconobacter oxydans 0.66±0.12 0.74±0.09 1.28±0.03 3.26±0.03 Gluconobacter thailandicus 0.66±0.12 0.70±0.03 1.74±0.06 3.02±0.03 Acetobacter pasteurianus 0.66±0.12 0.68±0.03 1.46±0.03 3.12±0.00 Komagataeibacter europaeus JY49 0.66±0.12 0.76±0.03 1.28±0.03 3.02±0.03 Acetobacter oryzifermentans 0.66±0.12 1.90±0.03 4.18±0.09 7.18±0.07 Acetobacter pasteurianus A6 0.66±0.12 0.74±0.03 1.48±0.03 2.80±0.03 SLV-7 0.66±0.12 2.00±0.09 4.18±0.03 6.00±0.06 Y2019-1 0.66±0.12 0.68±0.03 1.10±0.03 3.44±0.03

<Example 2> Taxonomic identification of acetic acid bacteria for production of fermented jujube vinegar

For the identification of the acetic acid bacteria selected in Example 1, Acetobacter aceti KCTC 12290 (=NCIMB 8621), Acetobacter strain KACC 13994 (= LMG 1262), LMG 1590 and LMG 1591 (each conventionally A. pasteurianus subsp. pasteurianus) , A.pasteurianus subsp.ascendens, and A.pasteurianus subsp. paradoxus), Acetobacter pomorum KCTC 22319 (=LHT 2458) and Acetobacter pomorum DM001 strains were used as reference strains for 16s rRNA analysis.

The 16S rRNA gene was sequenced as described in the following literature using SEQ ID NO: 1 and SEQ ID NO: 2 primers (Kim, JM, Le, NT, Chung, BS, Park, JH, Bae, JW, Madsen, EL, Jeon) , CO (2008) Influence of soil components on the biodegradation of benzene, toluene, ethylbenzene, and o-, m-, and p-xylenes by the newly isolated bacterium Pseu-doxanthomonas spadix BD-a59. Appl. Environ. Microbiol. 74 , 7313-7320.). The obtained sequence was compared with the 16S rRNA gene sequence of the reference strain available from GenBank using the BLAST program (http://www.ncbi.nlm.nih.gov/blast/). The 16S rRNA gene sequence of SLV-7 and the 16s rRNA sequence in the reference bacteria were aligned using an Infernal secondary-structure aware aligner available from the Ribosomal Database Project. Phylogenetic trees were constructed using the Neighbor-Joining (NJ), Maxim-Parsimony (MP) and Maximum-Lowhood (ML) algorithms of the PHYLIP software (version 3.695). The resulting schematic topology was evaluated using bootstrap analysis based on a dataset resampled 1000 times from the PHYLIP package. MLSA was performed using the nucleotide sequence (7728 bp) of three housekeeping genes [dnaK (1911 bp), groEL (1644 bp) and rpoB (4173 bp)] of the Acetobacter strain derived from the whole genome sequence. Linked gene sequences were aligned using a web-based tool (CLUSTAL Omega, http://www.ebi.ac.uk/Tools/msa/clustalo/) and phylogenetic trees with bootstrap values were calculated using the NJ algorithm of MEGA7 software. was constructed using

SEQ ID NO: sequence (5'-3') SEQ ID NO: 1 AGAGTTTGATCMTGGCTCAG SEQ ID NO: 2 TACGGYTACCTTGTTACGACTT

As a result, as shown in FIG. 3, the isolated and identified SLV-7 acetic acid bacteria were found to have 100% nucleotide sequence homology with Acetobacter origin DM001. Accordingly, the new acetic acid bacteria isolated and identified was named Acetobacter origin Fermentance SLV-7 strain, and deposited at the Korea Agricultural Microorganisms Conservation Center (KACC) under the accession number KACC19301.

<Example 3> Preparation of jujube extract for production of fermented jujube vinegar

After washing 42 kg of jujubes in running water and draining water, the total raw materials were hydrolyzed (D.W. 420L) to 200% (w/w) and extracted in hot water at 80° C. for 5 to 9 hours to prepare a hot water extract of jujubes. Then, after filtration with a 20㎛ line filter, it was concentrated to 18 to 22 ° Brix to prepare a jujube extract concentrate for fermented jujube vinegar.

<Example 4> Preparation of fermented jujube wine for production of fermented jujube vinegar

<4-1> Manufacture of fermented jujube wine

Saccharomyces servisiae YM22 (KACC 48337) and Saccharomyces servisiae YM27 were used as yeast for the production of fermented jujube. Yeast 2 weeks (YM22, YM27) were pre-cultured in YPD medium (Yeast extract 0.5%, peptone 0.5%, dextrose 0.5%) by stirring at 30° C. and 160 rpm for 48 hours. After that, 0.1% (v/v) of the yeast pre-culture was inoculated into the jujube extract prepared in Example 3, and left still at 25° C. and cultured for 72 hours to prepare a main hair.

Inoculate two kinds of jujube extract with 0.1% (v/v) each of the prepared jujube extract, and leave the fermentation period (0, 3, 6, 9, 14 days) while stationary fermentation at each temperature (18℃, 25℃, 30℃). ), and then analyzed the sugar content, alcohol concentration, free sugar and organic acid content of the fermented jujube as follows to explore the optimal conditions for the production of fermented jujube.

<4-2> Analysis of sugar content and alcohol concentration of the prepared fermented jujube wine

If a lot of sugar is consumed and a lot of alcohol is produced, it is judged that the fermentation power is excellent. Accordingly, in order to evaluate the fermentation power according to the fermentation period of the prepared fermented jujube, the sugar content and alcohol concentration of the fermented jujube prepared in Example 4-1 were analyzed as follows.

Specifically, 100 ml of the collected sample and 100 ml of distilled water are put in a test tube, 2 to 3 drops of an antifoaming agent are added, distilled with a distiller, and 80 ml of the distilled water is defined as 100 ml of distilled water, and a density meter (AT/DMA500M, Anton paar, USA) to measure the alcohol content of the fermented jujube wine. Sugar content was measured using a refractometer (B626544, Atago, Japan) of 2 ml of the sample.

As a result, as shown in FIG. 4 , it was confirmed that sugar content and alcohol were in inverse proportion to an increase in alcohol as sugar was consumed. When the fermentation temperature was 18°C, the alcohol concentration reached 8% on the 6th to 9th days of fermentation, whereas at 25°C and 30°C, the alcohol concentration reached 8% within 3 days of fermentation, maintaining the alcohol concentration until 14 days of fermentation. At 18 ℃, on the 3rd day, it was confirmed that the YM22 strain was more than twice as fast as the YM27.

<4-3> Analysis of free sugar content of the prepared fermented jujube wine

In order to evaluate the free sugar content according to the fermentation period of the prepared fermented jujube, the free sugar content of the fermented jujube prepared in Example 4-1 was analyzed as follows.

Specifically, free sugar was analyzed using HPLC (Waters 2695, Waters Co., Miliford, MA, USA). The sample was filtered through a PVDF membrane filter having a pore size of 0.45 μm as the test solution, and the prevail carbohydrate ES (5 μm, 4.6×250 mm) was used for the column, and the mobile phase was an acetonitrile:water mixture (70:30, v/ v), the flow rate was 1.0 ml/min, and the detection was analyzed using an ELSD sigmal (Waters 2424, Waters Co., Miliford, MA, USA).

As a result, as shown in FIGS. 5A and 5B , glycerol as a sugar alcohol, fructose as a monosaccharide, glucose, and sucrose as a disaccharide were detected. It showed a similar trend to the change in sugar content according to the fermentation period, and at 18 °C, almost all of fructose, glucose, and sucrose were consumed after 6 days of fermentation, and at 25 °C and 30 °C, almost all of them were consumed in 3 days. The glycerol content showed a similar trend to the alcohol change, showing the highest content on days 9 to 14 of fermentation at 18 °C, and the highest on days 3 to 6 at 25 °C and 30 °C. At 18°C, on the 3rd day, the YM22 strain consumed about 2.3 times more glucose than the YM27.

<4-4> Analysis of organic acid content of the prepared fermented jujube wine

The organic acid of the fermented jujube prepared in Example 4-1 was analyzed as follows.

Specifically, organic acids were analyzed by post column method using HPLC (LC-20A, Shimadzu Co., Kyoto, Japan). The column for organic acid analysis was used by connecting two RSpak KC-811 (8.0 mm×300 mm, Showa Denko, Tokyo, Japan) to a Shodex RSpack KC-G (6.0 mm×50.0 mm) guard column. As the mobile phase, 3 mM perchloric acid (Kanto chemical, Tokyo, Japan) was used, the flow rate was 0.8 ml/min, and the temperature of the column oven was 63°C. The separation solution was prepared with 0.2 mM bromothymol blue (Sigma Chemical Co., St. Louis, MO, USA), 15 mM sodium hydrogen phosphate (Na2HPO4) (Sigma Chemical Co.), 2 mM sodium hydroxide and After the reaction, it was detected at UV 440 nm. At this time, the flow rate of the reaction solution was 1.0 ml/min, and the reaction temperature was 30°C. 10 ml of the sample was taken and filtered (0.2 μm, Millipore Co.) for use.

As a result, as shown in FIGS. 6A and 6B , 9 types of organic acids were detected. Formic acid, citric acid, acetic acid, and malic acid were found to be the main organic acids, which is thought to be due to the properties of the raw materials. It is known that sugar is decomposed during fermentation and contributes to the production of organic acids. In the YM27 strain, 18℃ condition, the total amount of organic acid was the highest on the 6th day of fermentation, but the total amount of organic acid was the highest on the 3rd day of fermentation in the remaining experimental groups. By strain, the organic acid content of YM22 strain was generally lower than that of YM27, and the organic acid content was lower at 30℃ than at 18℃ and 25℃ by temperature. In particular, the organic acid content was significantly high at YM27, 18℃ and YM27, 25℃ conditions, which is thought to be mainly used for organic acid production without conversion of sugar to alcohol.

Combining the above results, fermentation with YM22, which is well converted to alcohol and has excellent energy efficiency, at 25° C. for 14 days was set as the optimal condition for the production of fermented jujube wine.

<Example 5> Preparation of fermented jujube vinegar

<5-1> Seed production

Seed vinegar was prepared using acetic acid bacteria to prepare fermented jujube vinegar.

Specifically, ALM medium (Acetic acid bacteria liquid medium; yeast extract 0.5%, glucose 0.5%, glycerin 1.0%, MgSO4·7H ₂ O 0.02%, acetic acid 1.0%, EtOH 6.0% ) at 30 ° C. at 160 rpm and stirring for 48 hours, and then inoculated with 10% (v/v) of the lactic acid bacteria culture solution into the jujube fermented stock prepared in 4-1 above, and the acidity was 3 to 3.5 at 30 ° C. for 7 days. % was prepared by stationary culture until the seed.

<5-2> Manufacture of fermented jujube vinegar

After crude filtration of the fermented jujube wine prepared based on the alcoholic fermentation conditions of the optimal fermented jujube wine established in Example 4-4, and the preparation to an alcohol concentration of 8%, the acidity was adjusted to pH 2, 3, It was adjusted to 4 and 5. After that, 10% (v/v) of the seedling prepared in Example 5-1 was inoculated, and the fermentation period (0, 3, 6, 9, 14 , 21 days), fermented malt was collected and the characteristics and functional ingredients were analyzed as follows.

<5-3> Measurement of changes in alcohol concentration and acidity of fermented jujube vinegar

For acidity measurement, 10 ml of the sample is collected on the 0th, 3rd, 6th, 9th, 14th and 21st days of fermentation, put in a 30 ml beaker, 2 to 3 drops of 1% phenolphthalein solution is added, and then 0.1N The optimal consumption of NaOH solution was calculated using the following equation. Alcohol concentration was measured in the same manner as in Example 4-2.

Acidity (%) =V×F×A×D×1/S×100

V: Appropriate consumption of 0.1N-NaOH solution (ml)

F: titer of 0.1N-NaOH solution

A: Amount of organic acid equivalent to 1 ml of 0.1N-NaOH solution (acetic acid = 0.006)

D: dilution factor

S: Sample amount (ml)

As a result, as shown in FIGS. 7A to 7D , when the pH of the fermented jujube wine was set to 2, the acidity was measured to be 20.2%. It is thought that the edible citric acid used to control the pH had a major influence, and it is estimated that the acidity was high due to citric acid rather than acetic acid, which is not suitable for vinegar fermentation. When the pH was set to 3, the acidity was 3.3%, but the acidity did not rise significantly as the fermentation progressed. Only jujube vinegar fermented at 35℃ had the highest acidity of 5.3% on the 14th day of fermentation. When the pH was set to 4, the acidity was measured to be 0.7%. Jujube vinegar fermented at 20°C ideally increased the acidity. Alcohol was consumed on the 9th day of fermentation, and the acidity increased to 10.4% on the 14th day of fermentation. The acidity did not rise significantly as the fermentation progressed under the conditions of 30°C and 35°C. In addition, even when the pH was set to 5, the rate of increase in acidity was slow according to the fermentation period under the conditions of 20°C and 25°C, and the acidity did not increase significantly as the fermentation proceeded at the condition of 35°C.

That is, the conditions for producing jujube fermented vinegar by acetic acid fermentation for 14 days under conditions of 20°C and pH 4, which are remarkably excellent in acid production, were identified as optimal conditions.

<110> REPUBLIC OF KOREA (MANAGEMENT : RURAL DEVELOPMENT ADMINISTRATION) <120> Manufacturing method of low-temperature fermented jujube vinegar using Acetobacter oryzifermentans <130> 2019P-11-047 <160> 2 <170> KoPatentIn 3.0 <210> 1 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> forward primer <400> 1 agagtttgat cmtggctcag 20 <210> 2 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> reverse primer <400> 2 tacggytacc ttgttacgac tt 22

Claims (10)

1) preparing a jujube extract;
2) alcohol fermentation of the jujube extract of step 1) to prepare a fermented jujube wine;
3) preparing the fermented jujube vinegar by inoculating the Acetobacter oryzifermentans SLV-7 strain deposited under the accession number KACC 19301 into the fermented jujube strain of step 2); A method for producing fermented jujube vinegar, comprising a.
The method of claim 1, wherein the alcoholic fermentation of step 2) is performed by inoculating and fermenting Saccharomyces cerevisiae YM22 deposited with accession number KACC 48337.
The method according to claim 1, wherein the alcoholic fermentation in step 2) is subjected to stationary fermentation at 20 to 27°C.
The method according to claim 1, wherein the alcoholic fermentation in step 2) is a stationary fermentation for 3 to 16 days.
According to claim 1, wherein the production of fermented jujube vinegar in step 3) Acetobacter oryzifermentans deposited under the accession number KACC 19301 ( Acetobacter oryzifermentans ) It is prepared by inoculating the seed prepared using the SLV-7 strain. , Manufacturing method of fermented jujube vinegar.
The method of claim 1, wherein the fermented jujube wine in step 3) has a pH of 3 to 5, the fermented jujube vinegar.
The method of claim 1, wherein the fermented jujube vinegar preparation in step 3) is prepared by stationary fermentation at 18 to 23°C.
The method of claim 1, wherein the fermented jujube vinegar preparation in step 3) is prepared by stationary fermentation for 5 to 25 days.
The method of claim 1, wherein the fermented jujube vinegar has an acidity of 6 to 12%.
The fermented jujube vinegar according to any one of claims 1 to 9, prepared by the method.

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