KR20130015581A - Jujube vinegar - Google Patents

Abstract

PURPOSE: A jujube vinegar beverage is provided to use Acetobacter for an alcohol dilute fermentation or two stages fermentation. CONSTITUTION: A jujube vinegar beverage comprises the following steps: The jujube vinegar beverage is made by injecting Acetobacter aceti(KCCM 12654) to a dried jujube juice containing 7.5-8.0% of ethanol and shake culturing it at 21-23°C. The dried jujube juice is 20-25% dried jujube extract obtained by pulverizing dried jujubes with the third distilled water. 3-5%(v/v) of the Acetobacter aceti(1KCCM 12654) is injected. The jujube vinegar beverage is obtained by injecting Saccharomyces cerevisiae to the dried jujube juice, fermenting in alcohol, centrifugating and taking the supernatant liquid, injecting Acetobacter aceti(KCCM 12654), and shake culturing at 21-23°C.

Description

Jujube vinegar drink {JUJUBE VINEGAR}

The present invention relates to a jujube vinegar beverage and a method for manufacturing the same, and more particularly, to a jujube vinegar beverage with enhanced taste and functionality and a method for manufacturing the same.

Vinegar is a traditional food that has been used for a long time in both East and West. It is a representative fermented food with a unique aroma and sour taste that contains a small amount of volatile and nonvolatile organic acids, sugars, amino acids, and esters. By promoting the secretion of digestive fluids to help digestion and refreshing appetite is known to give.

In addition, vinegar is not only used as a seasoning to give a sour taste when cooking, but also softens the taste of foods such as salty and sweet flavors, adds a distinctive flavor, reduces fishy smell, and makes meat lighter. It is also used as a raw material for sauces, mayonnaise, dressings, ketchups, and flavoring agents.

In addition, vinegar contains a large amount of acetic acid and other organic acids involved in the TCA cycle, so that the TCA cycle proceeds smoothly without accumulating lactic acid in the body, which is good for fatigue recovery due to intense exercise or overwork, and makes the blood weakly alkaline. do.

In addition, vinegar is known to be effective in diet, break down lipid peroxide to prevent atherosclerosis, promote the secretion of corticosteroids to manage stress, promote digestion and appetite, reduce blood pressure, recover fatigue, It has several physiological functions, such as promoting calcium absorption.

Representative strains of such vinegar are Acetobacter aceti , Acetobacter pasteurianus , Gluconobacter oxydans These strains are gram negative and do not form spores with motility aerobic bacilli or cocci, and Gluconobacter sp. Prefers sugars to alcohol and lacks the ability to re-acetate acetic acid, while Acetobacter sp. It has a TCA circuit that can make acetic acid a property.

However, until now, only basic research on the chemical composition of vinegar has been conducted, and there are no studies related to the preference related to commodity. In particular, there is no study comparing the preference between two-stage fermentation and alcoholic distilled acetic acid fermentation. There is a need to decide.

Recently, as the mortality rate due to circulatory diseases such as hypertension and arteriosclerosis increases, and the incidence of adult diseases such as diabetes and obesity, interest in health functional foods based on natural products has been increasing. The situation is rising.

Jujube is a deciduous broad-leaved arborescent fruit of the genus Rhamnaceae zizyphus, which is distributed throughout Korea, Europe and Asia. Jujube has traditionally been used in food and medicine, and is rich in nutrients, vitamins, and dietary fiber.

Although jujube is nutritious and contains various physiologically active substances, the main consumption is dry, mainly for dehydration, herbal medicine, health food, etc. A small amount is added. In addition, in the development of processed food using jujube, the sugar concentration of jujube shows an excessive sweetness, and thus has a disadvantage of falling in preference.

Accordingly, the inventors of the present invention have attempted to develop and provide vinegar having a high degree of preference while utilizing various sugars of jujube using acetic acid fermentation and having various physiological functions such as blood pressure drop, fatigue recovery, and calcium absorption promotion due to various fermented products.

An object of the present invention is to provide a jujube vinegar beverage prepared using acetic acid bacteria.

In addition, the present invention aims to provide the optimum conditions of jujube acetic acid fermentation through the reaction surface analysis.

In addition, an object of the present invention is to provide a jujube vinegar beverage prepared using alcohol dilution fermentation or two-stage fermentation.

In addition, an object of the present invention is to provide a jujube vinegar beverage with enhanced palatability and bioactive function.

In order to achieve the above object, the present invention is obtained by inoculating acetobacter aceti ( Acetobacter aceti (KCCM 12654)) in a dry jujube containing 7.5 ~ 8.0% ethanol shaking culture at 21 ~ 23 ℃ To provide a jujube vinegar drink.

In addition, the present invention is inoculated with Saccharomyces cerevisiae in dried jujube juice alcohol fermentation and centrifugation to take only the supernatant, Acetobacter Aceti ( Acetobacter was inoculated aceti (KCCM 12654)) provides a jujube vinegar drink, characterized in that which is obtained by shaking culture at 21 ~ 23 ℃.

Hereinafter, the jujube vinegar beverage of the present invention will be described in detail.

The present invention relates to a jujube vinegar beverage fermented by inoculating acetic acid bacteria in dried jujube juice, it is preferable to use Acetobacter aceti (KCCM 12654) as acetic acid bacteria. Acetobacter When inoculated and fermented with aceti (KCCM 12654), the production of jujube vinegar with excellent taste and flavor is excellent due to the high production of acetic acid.

Here, the dried jujube juice contains ethanol for acetic acid fermentation, preferably 7.5 to 8.0% ethanol, more preferably 7.69% ethanol. Further, in cases jujube juice acetonitrile bakteo Oh Shetty (Acetobacter aceti (KCCM 12654)) for immunization preferably at 21 ~ 23 ℃, more preferably in good to shaking culture at 21.75 ℃. In order to optimize the production of acetic acid, the reaction surface analysis was carried out using the initial ethanol content and the fermentation temperature as the factor variables for 19 days according to the central synthesis planning method. When the initial ethanol content was 7.69% and the fermentation temperature was 21.75 ℃, the acetic acid production was excellent. This is because it is possible to produce jujube vinegar, which has high taste and flavor.

In addition, the dried jujube juice in the present invention is not particularly limited, for example, 20 ~ 25% dry juice extract obtained by grinding the dry jujube with the third distilled water can be used. This is because the amount of acetic acid produced is exceptionally high.

In addition, the inoculation amount of Acetobacter aceti (KCCM 12654) in the present invention is not particularly limited, for example, 3 to 5% (v / v) can be inoculated.

In addition, the jujube vinegar beverage of the present invention may be prepared by distilled fermentation as described above, but may also be prepared through two-stage fermentation. Preferably, inoculated with Saccharomyces cerevisiae in dried jujube juice alcohol fermentation and centrifugation to take only the supernatant, Acetobacter was inoculated aceti (KCCM 12654)) can be produced jujube vinegar drink by shaking culture at 21 ~ 23 ℃. The jujube vinegar beverage produced through the two-stage fermentation has an advantage that the flavor and preference is excellent.

According to the present invention can be prepared jujube vinegar drink using acetic acid bacteria.

In addition, the present invention can provide the optimum conditions (initial ethanol content and fermentation temperature) of jujube acetic acid fermentation through the reaction surface analysis.

In addition, according to the present invention can be produced jujube vinegar beverage using alcohol dilution fermentation or two-stage fermentation.

In addition, according to the present invention can be produced not only excellent taste, flavor, preference, but also jujube vinegar beverages with enhanced physiological activity.

1 shows Acetobacter in modified YPD medium aceti (A), Acetobacter pasteurianus (B), and Gluconoacetobacter xylinus subsp. xylinus It is a photograph showing the transparent ring in the case of culturing (C).
Figure 2 shows A. aceti , A. pasteurianus and G. xylinus in jujube juice subsp. Using xylinus is a graph showing the concentration of bacteria when acetic acid was fermented at an initial ethanol content of 3% and a temperature of 30 ° C.
Figure 3 shows A. aceti , A. pasteurianus and G. xylinus subsp. Using xylinus is a graph showing the pH change when acetic acid was fermented at an initial ethanol content of 3% and a temperature of 30 ° C.
Figure 4 shows A. aceti , A. pasteurianus and G. xylinus subsp. Using xylinus is a graph showing the acetic acid content when acetic acid fermentation was performed at an initial ethanol content of 3% and a temperature of 30 ° C.
5 shows A. aceti , A. pasteurianus and G. xylinus in jujube subsp. It is the HPLC chromatogram which shows the acid product in the case of acetic acid fermentation using xylinus .
Figure 6 is a graph showing the bacteria concentration and the ethanol content (A), and acetic acid content and the reducing sugar content (B) at the time when the acetic acid fermentation for 72 hours by using a different inoculum size chosangyun in jujube juice and A. aceti.
Figure 7 is a graph showing the bacteria concentration and the ethanol content (A), and acetic acid content and the reducing sugar content (B) of the time using a different inoculum size chosangyun in jujube juice and A. aceti hayeoteul the acetic acid fermentation for 16 days.
8 is a graph showing the bacterial concentration and ethanol content (A), and the acetic acid content and reducing sugar content (B) when the acetic acid fermentation for 72 hours using A. aceti different in the initial sugar concentration in jujube juice.
FIG. 9 is a graph showing the bacterial concentration and ethanol content (A), and the acetic acid content and reducing sugar content (B) when the acetic acid was fermented for 16 days using A. aceti with different initial sugar concentrations in jujube juice.
FIG. 10 is a graph showing the bacterial concentration and ethanol content (A), and the acetic acid content and reducing sugar content (B) when acetic acid was fermented for 72 hours using A. aceti .
FIG. 11 is a graph showing the bacterial concentration and ethanol content (A), and the acetic acid content and the reducing sugar content (B) when the initial ethanol content of jujube juice is different and the fermentation of acetic acid for 16 days using A. aceti .
12 is a graph showing the bacterial concentration and ethanol content (A), and the acetic acid content and reducing sugar content (B) when the fermentation temperature in jujube juice and fermentation of acetic acid for 72 hours using A. aceti .
Figure 13 is a graph showing the bacterial concentration and ethanol content (A), and the acetic acid content and reducing sugar content (B) when the fermentation temperature in jujube juice and the fermentation of acetic acid for 16 days using A. aceti .
14 is a reaction surface (A) and a ridge (B) showing the effect of the initial ethanol content and fermentation temperature on the amount of acetic acid produced in the fermentation of jujube using A. aceti of jujube juice.
15 is a reaction surface (A) and ridge line (B) showing the effect of the initial ethanol content and fermentation temperature on the acidity in acetic acid fermentation using A. aceti of jujube juice.
Figure 16 shows the bacterial concentration (A), acetic acid content (B), reducing sugar content (C) when jujube fermentation of jujube juice for 19 days at optimum conditions (21.75 ℃, 7.69% initial ethanol content) using A. aceti And ethanol content (D).
Figure 17 Saccharomyces 5 days in jujube This graph shows the ethanol content of ethanol fermentation using cerevisiae .
Fig. 18 is an HPLC chromatogram showing the acid product of alcohol distillation (A) and two-stage fermentation (B) in jujube juice.
Figure 19 is a graph comparing the pH and sugar content when jujube dilution fermentation (left) and two-stage fermentation (right) in jujube juice.
20 is a graph comparing the acidity and sugar content of jujube vinegar and the conventional vinegar beverage.

Hereinafter, the content of the present invention will be described in more detail in the following examples, but the scope of the present invention is not limited only to the following examples, and includes modifications of equivalent technical ideas.

Experimental Materials and Strains Used

In the following experiment, dried jujube was used for bovine jujube purchased from Boeun. In addition, the acetic acid bacteria used in the following experiment was purchased from the Korea Microbiological Preservation Center affiliated with the Korea Spawn Association, and used the bacteria having the acetic acid performance, the types of bacteria are shown in Table 1. At this time, the medium composition for the culture of acetic acid bacteria is shown in Table 2.

bacteria KCCM ATCC IFO DSM NRRL Acetobacter liquefaciens 11834 14835 12388 5603 Acetobacter liquefaciens 11346 14835 12388 5603 Acetobacter pasteurianus 40201 33445 3509 Acetobacter pasteurianus 11882 12875 13753 4491 Acetobacter pasteurianus 32413 12876 13752 46223 Acetobacter pasteurianus 32831 23753 13751 4493 Acetobacter pasteurianus 40011 6438 3170 2324 B-468 Acetobacter pasteurianus 40052 9325 3225 B-570 Acetobacter pasteurianus 40117 12879 Acetobacter pasteurianus 40169 3299 Acetobacter pasteurianus 40021 12874 13755 2347 Acetobacter pasteurianus 40163 3259 Acetobacter aceti 40229 15973 14818 3508 Acetobacter aceti 12655 3284 Acetobacter aceti 12654 3283 Acetobacter sp. 40085 3297 Acetobacter xylinum 40407 3288 Gluconoacetobacter xylinus subsp. xylinus 40123 23767 15237 6513 Gluconoacetobacter xylinus subsp. xylinus 41431 11142 2325 Gluconoacetobacter xylinus subsp. xylinus 40198 23768 14814 2004 Gluconoacetobacter xylinus subsp. xylinus 40216 10245 3173 46604 Gluconoacetobacter xylinus subsp. xylinus 40231 23767 15237 6513 Gluconoacetobacter hansenii 40230 35959 14820 5602

KCCM: Korean Culture Center of Microorganisms

ATCC: American Type Culture Collection

IFO: Institute for Fermentation in Osaka

DSM: Deutsche Sammlung von Microorganismund Zellkulturen Gmb H

NRRL: The Northern Regional Reserch Laboratory

YPD Badge modified YPD badge Yeast extract 10 g Yeast extract 10 g Eptone 20 g Eptone 20 g Dextrose 20 g Dextrose 20 g Ethanol 30 mL Agar 15 g Distilled water 1 L Ethanol 30 mL CaCO ₃ 10 g Distilled water 1 L

<Measurement and Analysis Method>

-Growth of isolated acetic acid strains

The bacterial growth rate during shaking culture was measured using a spectrophotometer (Optizen 2120UV, Mecasys, Daejeon, Korea) to measure the absorbance of the culture (660 nm).

pH and acidity measurements

The pH of the culture was measured using a pH meter (Orion star series, Thermo electron, Beverly, MA, USA), and the acidity was neutralized and titrated with 0.1 N NaOH solution using 10% of the culture solution as an indicator and 0.1% phenolphthalein. Converted to.

-Determination of sugar and quantification of reducing sugar

The supernatant was collected by centrifugation of the fermentation broth, and the sugar content was measured using a sugar meter (PR-101α, ATAGO, Tokyo, Japan). Reducing sugar quantification of the culture was quantified by the DNS method. In other words, the reaction solution was taken and diluted, 0.5 mL of the reaction solution was reacted with 0.5 mL of DNS solution, heated for 5 minutes, cooled, and the absorbance was measured at 575 nm using a spectrophotometer (Mecasys). The absorbance measured was obtained by reducing the equivalent weight from the glucose standard curve.

-Acetic acid and organic acid analysis

Analysis of the organic acid was analyzed by HPLC (600 controller, Waters, Milford, MA, USA) by passing the culture supernatant through a 0.2 μm membrane filter. The organic acid analysis conditions were SUPELCOGEL column (C-610H, SUPELCO, Bellefonte, PA, USA), and 0.1% phosphoric acid as mobile phase (0.5 mL / min) to 10 μL injection volume to UV detector (210 nm). Detection was quantified.

-Ethanol content analysis

In ethanol content analysis, the culture supernatant was analyzed by HPLC (Waters) through a 0.2 μm membrane filter. The organic acid analysis conditions were SUPELCOGEL column (C-610H, SUPELCO), 0.1% phosphoric acid as a mobile phase (0.5 mL / min) to 10 μL injection volume was detected by UV detector (203 nm) and quantified.

Experimental Example  1: high acidity Spawn  Selection

In the present experimental example, the single colony obtained by smearing and culturing was cultured by shaking, and plated at 30 ° C for 3 days using a disk method in modified YPD medium, and then the formation of a transparent ring was observed. The size of the resulting transparent ring is shown in Table 3 below.

bacteria KCCM Clear zone size (mm) Acetobacter liquefaciens 11834 18 ± 1.73 Acetobacter liquefaciens 11346 - Acetobacter pasteurianus 40201 12.67 ± 0.58 Acetobacter pasteurianus 11882 24.33 ± 8.08 Acetobacter pasteurianus 32413 22.00 ± 0.00 Acetobacter pasteurianus 32831 19.33 ± 3.06 Acetobacter pasteurianus 40011 16.33 ± 3.51 Acetobacter pasteurianus 40052 19.67 ± 4.93 Acetobacter pasteurianus 40117 19.67 ± 3.21 Acetobacter pasteurianus 40169 21.67 ± 3.06 Acetobacter pasteurianus 40021 - Acetobacter pasteurianus 40163 - Acetobacter aceti 40229 13.33 ± 1.15 Acetobacter aceti 12655 16.5 ± 4.00 Acetobacter aceti 12654 25.00 ± 1.00 Acetobacter sp. 40085 14.17 ± 0.29 Acetobacter xylinum 40407 - Gluconoacetobacter xylinus subsp. xylinus 40123 - Gluconoacetobacter xylinus subsp. xylinus 41431 - Gluconoacetobacter xylinus subsp. xylinus 40198 - Gluconoacetobacter xylinus subsp. xylinus 40216 23.33 ± 3.21 Gluconoacetobacter xylinus subsp. xylinus 40231 - Gluconoacetobacter hansenii 40230 -

As shown in Table 3, the strain showing the largest transparent ring of the experimental strain is Acetobacter aceti (KCCM 12654), Acetobacter pasteurianus (KCCM11882) and Gluconoacetobacter xylinus subsp. xylinus (KCCM 40216) showed clear rings of 25.00 ± 1.00, 24.33 ± 8.08, and 23.33 ± 3.21 cm, respectively. Transparent rings of these three strains are shown in FIG. In the following experiments, the three strains were used by culturing at 200 ° C and 200 rpm in YPD medium.

Experimental Example  2: acetic acid fermentation results by acetic acid bacteria

In this experimental example, three strains Acetobacter showing high acid generating ability through the selection medium in Experimental Example 1 aceti (KCCM 12654, less than AA), Acetobacter pasteurianus (KCCM11882, hereinafter AP) and Gluconoacetobacter xylinus subsp. Acetic acid fermentation was carried out using xylinus (KCCM 40216, hereinafter GX).

After adding 5% (v / v) of directly cultured bacteria to jujube juice, the concentration, pH, and acetic acid content of the bacteria were measured every 24 hours with acetic acid fermentation at 30 ° C and 200 rpm for 10 days.

Figure 2 shows the results of measuring the concentration of bacteria. According to this, in the case of AA, the bacteria grew rapidly until day 5 with little lag time, but the other bacteria had a long induction period and the maximum bacterial concentration was not higher than that of AA. As a result, AA showed more than twice the bacterial concentration when compared to AP.

Figure 3 shows the pH change of each bacteria for 10 days. AA decreased in pH up to 4 days after fermentation and showed no significant difference thereafter, while GX had a similar pH level as AA, but slowed down.

4 shows the acetic acid content results. In the case of AA, the lag time was short and increased rapidly until the 5th day, but the acetic acid content gradually increased after 5 days, and in the case of GX, the acetic acid content began to increase after 3 days and the content was less than half of AA. Crazy On the other hand, AP showed high pH and low acetic acid content, and jujube juice was found to have little acetic acid production.

As a result of comparing the bacteria concentration, pH, acetic acid content, it was found that AA is more suitable for jujube juice acetic acid fermentation than GX, AP.

In addition, AA, AP and GX were fermented for 10 days to compare the acid composition. Figure 5 shows the result of comparing the acid composition by fermenting AA, AP and GX for 10 days.

According to FIG. 5, three acetic acid bacteria showed various acid compositions, and AA had the highest acetic acid content than other acids, and the composition of other acids had almost no change and only the amount of acetic acid produced increased for 10 days. AP showed little increase in acetic acid content and oxalic acid content was the highest. However, the acid content did not change for 10 days and the amount of levulinic acid increased. GX showed a steady increase in acetic acid content and, unlike other fungi, increased tartaric acid content.

In the vinegar, the higher the acetic acid content, the better the flavor and the higher the taste preference. Therefore, AA was the most suitable for the fermentation of dried jujube.

Experimental Example  3: acetic acid fermentation by conditions

Based on Experimental Examples 1 and 2, A. aceti (AA) having excellent bacterial growth and acetic acid production ability was selected as an acetic acid fermentation strain. In this experimental example, the effects on initial bacterial inoculum, sugar concentration, ethanol concentration, and fermentation temperature were examined first to optimize acetic acid fermentation conditions. The fermentation periods of 72 hours and 16 days where most logarithmic proliferation of AA occurred were selected.

Basically, to produce dried jujube vinegar, centrifuged dried jujube juice (jujube: distilled water = 1: 3) with 3rd distilled water at 11,590 × g, and then inoculated with precultured acetic acid bacteria into a 500 mL Erlenmeyer flask. Shake culture at 200 rpm in a volume of 100 mL.

Experimental Example 3-1: Influence by Acetic Acid Inoculation

In acetic acid fermentation, acetic acid inoculum is an important factor in the final bacterial concentration. In order to investigate the effect of inoculation of acetic acid bacteria on the growth and production of acetic acid bacteria, 1, 3, 5, 10% (v / v) of acetic acid precultured in 25% dry extract juice containing 5% alcohol The growth and acidity, reducing sugar, ethanol concentration was measured while shaking and incubating at 30 ° C. at 200 rpm. 6 and 7 show the results of the fungi, ethanol, acetic acid and reducing sugar concentrations after 72 hours and 16 days of incubation.

According to FIG. 6 (A), generally, the higher the inoculation amount, the shorter the induction period, but there was a separate inoculation amount suitable for growth of bacteria. The highest bacterial concentration was obtained when the inoculation amount was 3% in 72 hours when the growth of the bacteria occurred, and the ethanol content was less than 1% after only 72 hours to satisfy the ethanol content required by the vinegar. In the case of 1% inoculation, ethanol consumption was relatively low due to the low bacterial concentration.

According to FIG. 6 (B), the acetic acid concentration was not significantly affected by the inoculation amount, and the reducing equivalent was as low as 127.4 mg / mL when specifically 5%.

According to FIG. 7 (A), after 16 days, the ethanol content was very low at about 0.5% or less for all inoculation amounts. This is because it was used as a substrate for acetic acid fermentation. In addition, the bacterial concentration showed a constant level for all doses. This is because even if there is a difference in induction period, the bacteria grow to almost the same concentration after sufficient time in the limited substrate of the bacteria.

According to FIG. 7 (B), even after 16 days, the acetic acid content did not show a large change compared to 72 hours, and the effect by the inoculation amount could not be found.

Experimental Example 3-2: Effect by Initial Sugar Concentration

In order to investigate the effect of initial sugar concentration on the growth and production of acetic acid bacteria, 5% (v / v) of acetic acid bacteria pre-cultured in dry extracts containing 5% ethanol was inoculated and shaken at 200 rpm at 30 ° C. , At this time, using 5, 10, 15, 20, 25% dry extract juice, growth and acidity, reducing equivalents, ethanol concentration was measured.

8 and 9 show the results of the bacterial, ethanol, acetic acid and reducing sugar concentrations after 72 hours and 16 days of incubation with different initial sugar concentrations.

According to FIG. 8 (A), the bacterial concentration also increased as the sugar concentration increased during the initial 72 hours, but did not show a significant difference at 20% or more, and the ethanol concentration was maintained at 1% or less without significant difference.

According to FIG. 8 (B), acetic acid concentration also showed no significant difference at about 20 mg / mL level for all sugar concentrations. This is because a high concentration of glucose may decrease the acidity, which may inhibit acetic acid production. On the other hand, sugars that are not converted to alcohol are involved in the growth of harmful bacteria and off-flavor, so an appropriate concentration of sugar is required.

According to FIG. 9 (B), even after 16 days, the amount of acetic acid produced was not significantly different depending on the juice concentration. Changes in reducing equivalents were significantly different due to different concentrations of juice. After 16 days, the reducing equivalent was lower than that at 72 hours. As a result, it was found that reducing sugar was consumed for the growth of bacteria even after 72 hours.

Experimental Example 3-3: Effect by Initial Ethanol Content

Acetic acid bacteria are microorganisms that produce acetic acid by oxidizing ethanol. The initial ethanol concentration is very important for acetic acid production. In order to examine the effect of the initial ethanol concentration inoculated with 5% (v / v) of acetic acid bacteria pre-cultured in 25% dry extract juice containing ethanol and shaking culture at 200 rpm at 30 ° C, the ethanol content 3 , 4, 5, 6, 7% of the experiment was adjusted to the growth, acidity, reducing equivalent, ethanol concentration was measured.

10 and 11 show the results of the bacterial, ethanol, acetic acid and reducing sugar concentrations after 72 hours and 16 days of incubation with different initial ethanol contents.

According to FIGS. 10 (A) and 11 (A), the ethanol content of the bacterial concentration was found to decrease rather in the case of 7% ethanol at 72 hours involved in bacterial growth. The higher the concentration of ethanol, the longer the induction period, the slower the growth of bacteria and acid production capacity was judged to be lowered. After 16 days, the initial ethanol content was found to be highly involved in acetic acid production. 6% ethanol content showed high bacteria production.

10 (B) and 11 (B), the change of acetic acid content according to the ethanol content does not have a significant effect in the initial 72 hours, but after acetic acid fermentation for 16 days showed a high acetic acid content at 6% ethanol content. As a result, acetic acid is a fermentation product which appears in the normal phase after the growth of acetic acid bacteria, but it does not affect the initial stage, but the effect is large after fermentation.

Regarding the sugar content, the reducing sugar content was high when the ethanol content was 3% at the first 72 hours, but there was no significant difference in other ethanol concentrations. After 16 days, reducing sugars were reduced to similar levels for all ethanol concentrations.

Experimental Example 3-4: Influence by Fermentation Temperature

In general, when the fermentation temperature is too low at the time of acetic acid fermentation rate is slow, too high ethanol and acetic acid loss occurs to lose the flavor. In this Experimental Example, in order to investigate the effect of fermentation temperature of acetic acid bacteria on the growth and production of acetic acid bacteria, 25% dry extracts containing 5% alcohol were inoculated so that 5% (v / v) of acetic acid bacteria were precultured. Growth, acidity, reducing equivalents, and ethanol concentrations were measured while shaking culture at 200 rpm at 30, 33 and 40 ° C.

12 and 13 show the results of the bacterial, ethanol, acetic acid and reducing sugar concentrations after 72 hours and 16 days of incubation at different fermentation temperatures.

According to FIG. 12, the initial 72 hours showed a significant effect on the growth of bacteria according to the fermentation temperature. Increasing the temperature to the peak of 30 ℃ significantly inhibited the growth of bacteria. However, according to FIG. 13, the longer the fermentation time was, the less the influence of temperature was observed. On the 16th, the bacteria maintained a constant level even at 30 ° C. or higher. This was not the optimum temperature, but the growth of bacteria occurred steadily and the concentration was determined to be uniform due to the limited mass used in the experiment.

On the other hand, the bacterial concentration was greatly affected by temperature, especially at the beginning of growth, but did not significantly affect ethanol concentration, acetic acid concentration and reducing equivalent.

In general, in case of AA selected as jujube acetic acid bacterium, 30 ° C is known as the optimum temperature. However, since the optimum growth and the optimum acetic acid production temperature of the bacteria may be different, it was determined that it is necessary to obtain the optimum temperature according to the intended use.

Experimental Example  4: Optimization of Acetic Acid Production by Response Surface Methodology

Experimental Example 3 it can be seen that the effect of the initial ethanol content and the fermentation temperature is large in acetic acid fermentation of jujube among many factors. In order to optimize the production of acetic acid, the reaction surface analysis was performed using the initial ethanol content and the fermentation temperature as factor variables for 19 days according to the central synthesis planning method, and the results are shown in Tables 4 to 8, FIGS. 14 and 15.

The experiment was conducted according to the face centered design (FCD) for the optimum culture of jujube vinegar and SAS program was used for the response surface analysis. Fermentation temperature (X ₁ ) was set as a factor variable by injecting 100% of 20% dried jujube juice (13.25 ± 0.21 ° Bx) into 500 mL Erlenmeyer flask and inoculating 3% (v / v) of precultured A. aceti. The experiments were performed under 11 set conditions by encoding three levels (-1,0,1) with the initial ethanol content (X ₂ ). At this time, the reaction variables related to the characteristics of acetic acid culture medium were set to acetic acid content (Y ₁ ) and acidity (Y ₂ ).

Table 4 shows the results of the variance analysis for the regression equation. As a result of the reaction surface formed by the quadratic regression equation, the R ² value was 0.71 and 0.78 for acetic acid content and acidity, respectively.

In addition, as shown in Table 5, the effect of the factor variable on the acetic acid content and acidity, the reaction variable, was significantly affected by the fermentation temperature among the factor variables ( p <0.05). Table 6 shows the reaction surface polynomials for acetic acid production and acidity.

14 and 15 showed a similar pattern of acetic acid content and acidity reaction surface, and temperature and initial ethanol concentration affected. The peak point of acetic acid content was the highest point, and the peak point was the saddle point in case of acidity. Acidity was determined to have the highest production of acetic acid through ridge analysis (Table 7). As a result, an initial ethanol concentration of 7.69% can be expected to give 52.76 mg / mL of acetic acid when fermented at 21.75 ° C for 19 days, and an initial ethanol concentration of 7.86% can be expected to give an acidity of 5.53% when fermented at 22.16 ° C for 19 days. (Table 8).

Experimental Example  5: jujube vinegar culture at the optimum point

In this experimental example, we examined acetic acid fermentation according to the culture period under the optimum conditions determined by reaction surface analysis, that is, the initial ethanol concentration of 7.69% and the fermentation temperature of 21.75 ° C.

Figure 16 shows the acetic acid bacteria, acetic acid, reducing sugar, ethanol content when incubated for 19 days at the condition of the initial ethanol concentration of 7.69%, fermentation temperature 21.75 ℃. According to this, the growth of acetic acid bacteria was continuously increased without induction period in the early stage, the highest at 5 days, and then gradually decreased. However, even when acetic acid bacteria decreased, the amount of acetic acid was found to increase steadily. Reduction equivalents were almost unchanged and ethanol decreased rapidly from the first day of fermentation and was very low.

Experimental Example  6: Comparison of Alcohol Dilution Fermentation and Two-stage Fermentation of Jujube Vinegar

Since the initial ethanol concentration is an important factor for acetic acid fermentation, in this experimental example, the validity of the two-stage fermentation of acetic acid fermentation after obtaining proper ethanol using yeast was verified.

Experimental Example 6-1: Ethanol Fermentation

Saccharomyces pre-cultured in dried jujube, ground with tertiary distilled water The cerevisiae was inoculated with 5% of jujube juice and shaken at 30 ° C. and 200 rpm with a volume of 100 mL in a 500 mL flask. Figure 17 shows the change in ethanol content during 5 days ethanol fermentation using yeast Saccharomyces cerevisiae in jujube juice.

According to this, the ethanol concentration fermented with yeast gradually increased with the increase of fermentation time, and the ethanol content increased up to 6% which is suitable for acetic acid fermentation for 5 days. On the other hand, it was determined that ethanol can be obtained in a short time by optimizing the initial sugar concentration or temperature which affects ethanol fermentation.

Experimental Example 6-2: Comparison between alcohol dilution fermentation and two-stage fermentation

Saccharomyces pre-cultured in dried jujube, ground with tertiary distilled water The cerevisiae was inoculated with 5% of jujube juice and shaken at 30 ° C. and 200 rpm with a volume of 100 mL in a 500 mL flask. During the ethanol fermentation, the ethanol content was measured to make the optimum ethanol content. The alcoholic fermented jujube juice was centrifuged at 11,590 × g for 15 minutes to extract only the supernatant and passed through a 40 μm filter to completely remove the residue. Acetobacter precultured here Inoculation of aceti 3% by volume shaking culture was performed at 21.8 ° C, 200 rpm.

In order to compare the organic acid compositions of alcohol distillation and two-stage fermentation, Fig. 18 shows HPLC chromatograms of acid products of alcohol distillation (A) and two-stage fermentation (B) in jujube juice.

According to this, it can be seen that the composition of the organic acid is different when the two-stage fermentation is different from the distillation fermentation. In the case of alcohol distillation fermentation, the amount of acetic acid was the highest, but in the case of two-stage fermentation, the content of tartaric acid was high and the acetic acid content was very low. The peak of oxalic acid did not appear clearly, and the peak of levulunic acid appeared and it was composed of organic acid of different composition. In ethanol fermentation, it was found that the tartaric acid content increased like the ethanol content. Also, after ethanol fermentation, the lowered sugar concentration could be affected.

In addition, FIG. 19 shows the pH and the sugar content of alcohol distillation and two-stage fermentation. According to this, the pH and sugar content of the two-stage fermentation were relatively lower than those of the distilled fermentation. This is because, as shown in the organic acid composition results above, the overall pH of the organic acid produced during the two-stage fermentation was relatively low, and it was determined that more sugar was consumed for alcohol fermentation using yeast.

Most of the main acids of jujube are levulunic acid and the rest are oxalic acid and malic acid. However, it was found that the composition is changed a lot through acetic acid fermentation. In addition, acetic acid and citric acid are very important factors affecting acidity, but the amount of both acids did not increase in two-stage fermentation. As a result, it was determined that it is necessary to adjust the ethanol fermentation conditions in order to use two-stage fermentation to control the initial ethanol concentration.

Experimental Example  7: jujube vinegar drink

Acetobacter precultured in 25% dried jujube juice containing 7.69% ethanol and by shaking culture at 200rpm to 21.75 ℃ inoculated aceti (KCCM 12654) 5% ( v / v) was prepared jujube vinegar drink.

Experimental Example 7-1: Sugar and Acidity Test

The sugar and acidity of the jujube vinegar beverage prepared above were compared with those of three commercially available vinegar beverages. Sugar and acidity were measured by applying the above method as shown in Figure 20.

According to FIG. 20, in the case of jujube vinegar, the sugar content was 15.0 ° Bx, which is higher than that of 9.7-11.9 ° Bx of commercial vinegar drinks, and the acidity was relatively high, at 5.8%. Therefore, it was judged that it is necessary to determine the appropriate dilution drainage in order to prepare a commercially available beverage, and therefore, an appropriate amount of dilution after dilution was necessary to adjust the acidity.

Experimental Example 7-2: Preference Test

In this experimental example, the preference was compared by juicing dilution fermentation and two-stage fermentation of jujube vinegar as a sample through sensory evaluation. Here, two-stage fermentation was performed by the method of Experimental Example 6. The taste, aroma, and overall acceptability of jujube vinegar were described for 23 experimental subjects of food science and biotechnology in Seoul National University, and the objectives and evaluation items were evaluated. Nine points were very good and 0 points were very dislike. The statistical analysis of the results was analyzed by one-way ANOVA at 5% confidence level using SAS.

In addition, the preference of fermented jujube distilled jujube distilled beverage with different dilution rate of 50, 75, 100% was also evaluated using the same method.

Table 9 shows the evaluation results of liquor dilution fermentation and two-stage fermentation.

According to Table 9, the taste (6.3 ± 0.6) of the distilled fermented samples was significantly better than that of the secondary fermented samples (5.3 ± 0.1), but the flavor and overall acceptability were inferior.

Table 10 shows the evaluation results of the preference of alcoholic distilled jujube distilled beverage of distilled distillation.

According to Table 10, no significant difference was found in taste, aroma and preference even with different dilution factors.

All the experiments were repeated three or more times. In case of significant difference by variance analysis, SAS was analyzed and Duncan's test was used to verify the significance at the significance level p = 0.05. In response surface analysis, the dependent variable Y (acidity, acetic acid content) for the independent variables X ₁ and X ₂ is represented by the following quadratic regression where β ₀ is a constant and β _i , β _ii , and β _ij are regression Coefficient. Response surface regression (RSREG) was performed using statistical software SAS (9.1 SAS Institute Inc., Cary, NC, USA), and the response surface graphs were shown using Mathematica (Mathematica 7.1, Champaign, IL, USA).

Claims (4)

Jujube vinegar drink, characterized in that obtained by inoculating acetobacter aceti ( Acetobacter aceti (KCCM 12654)) in a dry jujube containing 7.5 ~ 8.0% ethanol shaking culture at 21 ~ 23 ℃. The method of claim 1,
The jujube jujube drink jujube vinegar, characterized in that 20 ~ 25% dried jujube juice obtained by grinding the dry jujube with the third distilled water. The method of claim 1,
Acetobacter aceti ( Acetobacter aceti (KCCM 12654)) is a jujube vinegar drink, characterized in that 3 to 5% (v / v) inoculation. Saccharomyces cerevisiae was inoculated into dried jujube juice, alcohol fermented, and centrifuged to take only the supernatant, followed by Acetobacter. jujube vinegar drink characterized in that the inoculated aceti (KCCM 12654)) obtained by shaking culture at 21 ~ 23 ℃.

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