Novel acetobacter pasterianus and quick producing method of marigold vinegar using the same strain

Abstract

The present invention relates to a novel strain of Acetobacter pasterianus and a method for producing rapid fermented vinegar using the same. More specifically, the invention relates to an Acetobacter pasterianus S25 strain (accession number: KFCC11970P) having 16S ribosomal DNA containing the nucleotide sequence indicated by SEQ ID NO. 1, capable of rapid acetic acid fermentation, thereby shortening the acetic acid fermentation period while exhibiting excellent flavor, and a method for producing rapid vinegar with excellent flavor using the same.

Description

Technology Field

[0001] The present invention relates to a novel Acetobacter Pasteurianus strain and a method for producing rapid fermented vinegar using the same. More specifically, the invention relates to a novel Acetobacter Pasteurianus S25 strain isolated from Makgeolli and a method for producing vinegar capable of rapid production of fermented vinegar by performing acetic acid fermentation using the same. Background Technology

[0003] Vinegar is a type of fermented food that has long been used as an important seasoning to enhance the flavor of food with its distinctive sour taste. Vinegar is classified into fermented vinegar (brewed vinegar), which is produced by fermenting grains, alcoholic beverages, or fruit juices, and synthetic vinegar, which is made by diluting glacial acetic acid or acetic acid with water and adding amino acids or sugars; traditionally, food service establishments have widely used synthetic vinegar. However, with the improvement of dietary habits and the growing interest in well-being culture, and the resulting shift in consumer perception that prioritizes the functionality of food, interest and demand for brewed vinegar, which is produced using natural ingredients that are harmless to the human body and rich in nutrients, are increasing.

[0004] The acidity of vinegar is determined by the content of organic acids, which are produced through acetic acid fermentation using acetic acid bacteria. Typically, the standard acidity for classifying vinegar is 4% (w / v). Vinegar is classified according to acidity into low acidity (4 to 5% w / v), general acidity (6 to 7% w / v), and high acidity vinegar containing twice, three times, etc., of these values. Regarding the acidity of general acetic acid fermented products, it is known that the acetic acid production characteristics of the starter cultures involved in fermentation have a relatively greater influence on the increase or decrease in acidity and the fermentation rate than the fermentation method. Therefore, in order to produce high-quality fermented vinegar, it is necessary to discover and improve superior acetic acid bacteria.

[0005] Meanwhile, 16S rRNA (ribosomal RNA) is a component of the 30S subunit of prokaryotic ribosomes and binds to the Shine-Dalgarno sequence. The gene region encoding it is widely used in phylogenetic studies of fungi because it is very well conserved among different species of bacteria or archaea due to its slow evolutionary rate.

[0006] Marigold Calendula arvensis Field marigold is an annual plant belonging to the Asteraceae family, native to Southern Europe, and is also known as the marigold. It grows to a height of 30–50 cm, branches out, and is covered in fine hairs that emit a distinctive scent, which is known to be effective in repelling insects. It is widely planted in flowerbeds and pots as an ornamental plant in the spring, and is also known to possess medicinal properties effective in treating dermatitis and otitis media. Furthermore, it is known to be effective in improving eyesight due to its high content of lutein and zeaxanthin, which are widely known as effective ingredients for eye protection and antioxidant effects; therefore, it is consumed in the form of flower tea or vinegar. Prior art literature

[0008] (Patent Document 0001) KR 101815389 B1 The problem to be solved

[0009] The present invention aims to provide a novel strain of Acetobacter pasteurianus that can produce vinegar with excellent acidity while reducing production time by shortening the fermentation period during vinegar production, and a method for producing vinegar by acetic acid fermentation using the same. means of solving the problem

[0011] According to one aspect of the present invention, Acetobacter pasteurianus having a 16S rRNA gene comprising the nucleotide sequence represented by SEQ ID NO. 1 and capable of rapid acetic acid fermentation ( Acetobacter pasterianus ) S25 strain (accession number: KFCC11970P) may be provided.

[0012] In addition, the above strain can produce a fermented product with an acetic acid concentration of 4% (w / v) within 10 days of the start of fermentation.

[0013] According to another aspect of the present invention, a first step of preparing raw materials; a second step of alcohol fermenting the raw materials to produce an alcohol fermentation liquid; and Acetobacter Pasteurianus ( Acetobacter pasterianus A method for producing vinegar may be provided, comprising: a third step of inoculating with the S25 strain (accession number: KFCC11970P) to ferment acetic acid.

[0014] In addition, the above raw material is marigold ( Calendula arvensis It may include ).

[0015] In addition, the above acetic acid fermentation can be carried out by fermenting for 5 to 90 days at a temperature of 25 to 35°C.

[0016] According to another aspect of the present invention, vinegar produced by the above-described manufacturing method may be provided. Effects of the invention

[0018] Novel Acetobacter Pasteurianus according to one aspect of the present invention ( Acetobacter pasterianus The S25 strain has excellent alcohol tolerance and acid production ability, so it can produce high concentrations of acetic acid even at high alcohol concentrations. Through this, blackening caused by the proliferation of unwanted bacteria is reduced, and fermented vinegar with excellent preservation and flavor can be produced.

[0019] In addition, since the acetic acid fermentation rate is fast and the acid production capacity is excellent, when used in vinegar production, the fermentation period can be shortened and acetic acid fermentation can be carried out with rapid efficiency. Through this, there is an advantage in being able to produce fermented vinegar quickly. In addition, vinegar with excellent flavor can be produced.

[0020] In addition, when using marigold raw materials containing a high concentration of active ingredients with excellent anti-aging and vision-improving effects, it is possible to produce vinegar with excellent functionality and sensory properties and high consumer preference.

[0021] A method for producing vinegar according to another aspect of the present invention can improve the yield and reduce contamination caused by unwanted bacteria during fermentation by separating alcohol fermentation and acetic acid fermentation. Brief explanation of the drawing

[0023] Figure 1 illustrates the results of confirming the total acidity (%) according to the fermentation period of nine types of acetic acid bacteria isolated according to the first manufacturing example of the present invention. Fig. 2 shows isolated Acetobacter Pasteurianus ( Acetobacter pasteurianus This is a phylogenetic tree of the S25 strain, and Figure 3 illustrates the results of comparing the total acidity (%) according to the fermentation period of the S25 strain and the control strain according to one aspect of the present invention. Specific details for implementing the invention

[0024] The present invention relates to a novel Acetobacter Pasteurianus isolated from Makgeolli ( Acetobacter pasterianus This invention relates to the S25 (KFCC11970P) strain and a method for producing vinegar using the same.

[0025] The present invention will be described in more detail below.

[0026] In describing the present invention, detailed descriptions of known technologies related to the invention are omitted if it is determined that such descriptions would unnecessarily obscure the essence of the invention. Furthermore, the terms described below are defined considering their functions in the present invention, and these definitions may vary depending on the intentions or practices of the user or operator. Therefore, their definitions should be based on the content throughout this specification.

[0027] Unless otherwise defined, technical and scientific terms used in this specification may be interpreted in the sense commonly understood by those skilled in the art to which this invention pertains. Additionally, singular expressions include plural expressions unless the context clearly indicates otherwise. Furthermore, terms such as 'comprising' or 'having' are intended to specify the existence of the features, numbers, steps, components, or combinations thereof described in the specification, and should not be understood as excluding the existence or addition of one or more other features, numbers, steps, components, or combinations thereof.

[0029] According to one aspect of the present invention, a novel Acetobacter Pasteurianus ( Acetobacter pasterianus ) S25 (KFCC11970P) strain may be provided.

[0030] The above strain was isolated and identified from Makgeolli and was deposited with the Korean Culture Collection of Microorganisms on August 9, 2023 (Deposit No. KFCC11970P). As a result of analyzing the genetic information of the above strain, it was confirmed to be a novel strain belonging to *Acetobacter pasteurianus* having a gene encoding 16S rRNA containing the nucleotide sequence indicated by SEQ No. 1.

[0031] The above strain has excellent alcohol tolerance and acid production ability, and can exhibit excellent acetic acid fermentation efficacy even at high alcohol concentrations. In addition, it can achieve an acetic acid concentration of 4% (w / v) within 10 days of the start of acetic acid fermentation, allowing for rapid acetic acid fermentation, making it suitable for use in producing rapid fermented vinegar. Here, the above acetic acid concentration may refer to total acidity.

[0032] More specifically, the above acetic acid concentration may refer to the total acidity value obtained by taking 10 mL of the acetic acid fermentation product, diluting it to 100 mL using distilled water, taking 20 mL from the result, adding 2 to 3 drops of phenolphthalein solution to the sample, titrating with 0.1 N NaOH solution, and calculating the amount consumed according to the following mathematical formula 1.

[0034]

[0035] (In the above Equation 1, V is the consumption of 0.1N NaOH solution (ml), F is the titer of 0.1N NaOH solution, A is the amount of organic acid equivalent to 1 ml of 0.1N NaOH solution (g) (acetic acid: 0.006), D is the dilution factor, and S is the sample amount (g).)

[0037] According to another aspect of the present invention, Acetobacter Pasteurianus according to one aspect of the present invention ( Acetobacter pasterianus A method for producing vinegar using the S25 (KFCC11970P) strain may be provided. The vinegar production method comprises: a first step (S1) of preparing raw materials; a second step (S2) of producing an alcohol fermentation liquid by alcohol fermenting the raw materials; and Acetobacter pasteurianus ( Acetobacter pasterianus ) It may be performed by including the third step (S3) of acetic acid fermentation by inoculating the S25 strain (accession number: KFCC11970P).

[0038] First, a step of preparing the raw material is performed (S1). The raw material can be prepared by crushing or juicing the plant raw material to be used to produce vinegar using a conventional method. Plant flowers may be used as the raw material. In a preferred embodiment of the present invention, marigold ( Calendula arvensis It is explained by using ). As the above raw material, at least one selected from the group consisting of leaves, stems, flowers, and roots of marigold may be used, and preferably, leaves are used.

[0039] If necessary, water or sugar may be added to the above raw materials in addition to the plant raw materials. When sugar is added, it is preferable to prepare the raw materials by adding sugar such that the sugar content is 15 to 30 Brix in order to optimize fermentation efficiency during alcohol fermentation. In order to optimize alcohol fermentation, the plant raw materials, water, and sugar may be mixed in a weight ratio of 1:20 to 50:50 to 200 to prepare the raw materials for alcohol fermentation, but are not limited thereto.

[0040] Next, an alcohol fermentation liquid can be prepared by inoculating the above raw material with a microorganism for alcohol fermentation and performing alcohol fermentation (S2). The microorganism for alcohol fermentation may be selected from among the yeasts typically used for alcohol fermentation. Preferably, a yeast species typically used for wine production is selected to further enhance the flavor and aroma of the vinegar produced and to promote secondary lactic acid fermentation. When inoculating the above raw material with the microorganism for alcohol fermentation, the raw material and the microorganism may be mixed in a weight ratio of 1:0.0001 to 0.001, but are not limited thereto. The alcohol fermentation conditions may be adjusted by considering the temperature conditions suitable for the alcohol fermentation of the selected microorganism.

[0041] The above alcohol fermentation may be performed in two stages. Specifically, it may be divided into a first alcohol fermentation stage in which yeast is added to the prepared raw material and first fermented at 20 to 35°C, preferably 25 to 30°C, for 7 to 10 days, and a second alcohol fermentation stage in which the first alcohol fermentation liquid obtained from the first alcohol fermentation stage is filtered and further fermented at 20 to 35°C for 2 weeks to 2 months. Through this, the taste and aroma of the fermented product produced can be improved and the alcohol fermentation yield can be further enhanced. In the second alcohol fermentation stage, the method of filtering the first alcohol fermentation liquid may be performed using a conventional method, for example, by filtering the first fermentation liquid using a filter screen to remove solid matter, but is not limited thereto. The alcohol fermentation liquid produced in this step (S2) may have an alcohol concentration of 5 to 15% (w / v) and a sugar content of 5 to 10% (w / w), but is not limited thereto.

[0042] Next, Acetobacter Pasteurianus ( Acetobacter pasterianus Acetic acid fermentation can be performed by inoculating the S25 strain (accession number: KFCC11970P) (S3).

[0043] An acetic acid bacteria culture solution containing the strain Acetobacter Pasteurianus S25 (Accession No.: KFCC11970P) can be added to the alcohol fermentation liquid and inoculated. The acetic acid bacteria culture solution may be obtained by inoculating a strain according to one aspect of the present invention into a culture medium for culturing microorganisms and culturing it using a microorganism culture method known in the art. Known culture methods may include, for example, static culture or agitation culture. In one embodiment according to the present invention, it is described as being prepared by inoculating a single colony of the strain Acetobacter Pasteurianus S25 (Accession No.: KFCC11970P) grown on a solid medium into 300 to 1,000 ml of liquid medium and culturing it at 25 to 35°C for 1 to 5 days, but is not limited thereto. The solid medium may include a yeast extract, a carbon source, ethanol, and agar. Preferably, the medium may contain 0.1 to 1% yeast extract, 1 to 5% carbon source, 1 to 5% agar, and 1 to 5% ethanol, with the remainder being water. The liquid medium may contain yeast extract, a carbon source, and ethanol. Preferably, the medium may contain 0.1 to 1% yeast extract, 1 to 5% carbon source, and 1 to 5% ethanol, with the remainder being water. The % refers to weight relative to volume (w / v). Sucrose or glucose may be selected as the carbon source, but is not limited thereto. If necessary, the solid medium and liquid medium may further include additives to promote the growth of the culture strain. In terms of improving acetic acid fermentation efficiency, this step (S3) can be carried out by fermenting for 5 to 90 days, preferably 5 to 50 days, at a temperature of 25 to 35°C, which is the optimal temperature for the growth and acetic acid production of the Acetobacter Pasteurianus S25 (KFCC11970P) strain.

[0044] A method for producing vinegar using a strain according to one aspect of the present invention has the advantage of rapidly producing vinegar with excellent preservation properties and flavor by using a novel Acetobacter pasteurianus strain that possesses excellent alcohol tolerance and acetic acid production capabilities and can perform acetic acid fermentation quickly. Additionally, by separating alcohol fermentation and acetic acid fermentation, the yield of the produced vinegar can be improved, and contamination caused by unwanted microorganisms during long-term fermentation can be reduced.

[0046] Hereinafter, to aid in understanding the present invention, the present invention will be described in more detail based on the following examples. However, these examples are merely illustrative of the present invention and do not limit the appended claims. It is obvious to those skilled in the art that various changes and modifications to the examples are possible within the scope and spirit of the present invention, and that such variations and modifications fall within the scope of the appended claims.

[0048] Examples

[0049] Isolation of Acetic Acid Bacteria from Fermented Foods

[0050] To isolate acetic acid bacteria, a total of 11 samples were collected and used, including 6 types of makgeolli and 5 types of vinegar from Suncheon-si. 1 mL of each sample was taken and diluted stepwise in 9 mL of sterile 0.85% NaCl solution. 100 μ of the diluted solution was spread onto a GYCE agar (3% glucose, 0.5% yeast extract, 0.5% soy extract, 1% calcium carbonate, 3% ethanol, 1% calcium carbonate, 2% agar) solid medium plate and incubated at 30°C for 48 hours. After incubation, acetic acid bacteria that formed a clear zone around the colony due to the decomposition of CaCO3 were selected. Six types of collected makgeolli were stored at room temperature for 8 weeks and used as isolation samples. Five types of vinegar samples were activated in GYE broth (0.5% yeast extract, 0.5% soy extract, 3% glucose, 3% ethanol) and plated onto GYCE agar (3% glucose, 0.5% yeast extract, 0.5% soy extract, 1% calcium carbonate, 3% ethanol, 1% calcium carbonate, 2% agar), an agar medium for isolating acetic acid-producing bacteria. These were incubated at 30°C for 3 days, and a total of 9 single colonies were isolated by first selecting based on the presence or absence of a clear zone formed as calcium carbonate (CaCO3) dissolves around the acetic acid bacteria colonies, followed by multiple subcultures.

[0051] The isolated strains were preserved by suspending them in a 10% skim milk solution (BD Difco, Sparks, MD, USA) and storing them at -80°C. To activate the acetic acid bacteria, GYE (3.0% glucose, 0.5% yeast extract, 0.5% soy extract, 3% ethanol) liquid medium was used and pre-cultured.

[0053] Selection of Strains with Superior Acetic Acid Production Ability

[0054] To select acetic acid bacteria exhibiting high total acidity over a short period through rapid fermentation from 9 isolated acetic acid bacteria species, acetic acid bacterial colonies were inoculated into 5 mL of acetic acid bacterial culture medium GYE (0.5% yeast extract, 0.5% soy extract, 3% glucose, 3% ethanol) and cultured with shaking at 30°C for 48 hours, after which this was used as the pre-culture medium. For the main culture, 500 mL of acetic acid bacterial culture medium GYE was prepared, inoculated with the 1% acetic acid bacterial pre-culture medium, and cultured with shaking at 30°C for 48 hours. Total acidity was measured at 7-day intervals, and the results are shown in Table 1 and Figure 1 below.

[0056] No strain name Total acidity (%) Separation source 0 days 7 days 14th 21st 1 S25 0.46 4.38 5.73 5.73 Makgeolli 2 SC09 0.47 1.90 1.71 1.86 wine vinegar 3 S24 0.47 3.03 2.93 3.00 Makgeolli 4 SC02 0.46 1.33 3.67 3.93 mulberry vinegar 5 SC30-2 0.47 1.35 1.34 1.33 Fresh Makgeolli 6 8 0.47 1.18 1.54 1.41 Makgeolli 7 SC06 0.47 1.35 1.31 1.24 Makgeolli 8 SC08 0.46 2.19 2.24 2.28 Makgeolli 9 GO08-1 0.46 2.72 2.72 2.73 Makgeolli

[0058] Referring to Table 1 above, it can be seen that among the nine acetic acid bacteria, strain S25 exhibited a significantly higher acidity of 4.38% after 7 days. The acidity of strain S25 was 5.73% after 14 days, and it maintained the same acidity even after 21 days, thus ending the acetic acid fermentation. As shown in Figure 1, among the total of nine isolated acetic acid bacteria, it was confirmed that S25 produced the most acetic acid during fermentation compared to other isolated acetic acid bacteria. Accordingly, strain S25 was selected as the acetic acid bacterium with superior acetic acid production performance among the isolated acetic acid bacteria.

[0060] <Identification of Selected Acetic Acid Bacteria>

[0061] To perform molecular identification and construct a phylogenetic tree of the selected strains, the strains were inoculated into GYE brophre and cultured at 30°C for 48 hours. Cells were recovered by centrifugation, and DNA was extracted using the ZR Fungal / Bacterial DNA Miniprep kit (Zymo Research Corp., CA., USA). The extracted DNA was synthesized using universal primers 785F (SEQ No. 2, GGATTAGATACCCTGGTA) and 907R (SEQ No. 3, CCGTCAATTCMTTTRAGTTT) to amplify the 16S rRNA gene fragment. The PCR conditions consisted of an initial denaturation of 5 minutes, followed by 30 cycles of denaturation at 95°C for 30 seconds, annealing at 55°C for 2 minutes, and extension at 68°C for 90 seconds. The amplified PCR products were purified using the QIAquick PCR Purification kit (QIAGEN) and then sent to Macrogen for DNA sequencing analysis. The analysis results were submitted to the National Center for Biotechnology Information (NCBI, http: / / www.ncbi.nlm.nih.gov / After analyzing phylogenetic relationships by comparing the sequence homology with standard strains registered in GeneBank using nucleotide BLAST, the sequences of the standard strains were obtained, and a phylogenetic tree was constructed using the MEGA X program after comparing the sequences, which is shown in Figure 2. The phylogenetic tree was constructed using a neighbor-joining algorithm, and the robustness of the constructed phylogenetic tree was confirmed by bootstrapping through 1,000 iterations.

[0062] As a result of performing a BLAST search based on the 16S rRNA gene sequence analysis results of the finally selected S25 Acetobacter pasteurianus It was identified as. S25(1341bp) is the standard strain Acetobacter pasteurianusIt showed 100% homology with LMD 22.1 (NR026107.1), and the maximum composite Likehood method was used as a model for inferring evolutionary distances to construct a phylogenetic tree using the nucleotide sequence.

[0063] The finally selected strain Acetobacter pasteurianus It was named S25, and the above strain was deposited with the Korean Culture Center of Microorganisms (KCCM) and assigned accession number KFCC 11970P. In subsequent experiments, the above deposited strain was used as an acetic acid fermentation bacterium to produce marigold vinegar.

[0065] Marigold Wine Production

[0066] Marigold leaves were purchased from the tea shop Mohusil Co., Ltd. in Suncheon. Marigold raw materials were prepared by mixing 150g of dried marigold leaves, 6kg of sugar, and 22L of water. Based on the 22L of prepared marigold raw materials, 5g of wine yeast (EC1118, lallemand, Canada) was added to carry out alcoholic fermentation. Additionally, 20g of phosphate, a yeast nutrient, and 2g of sulfite were mixed to prevent bacterial growth, and alcoholic fermentation was carried out.

[0067] Alcohol fermentation through the production of marigold wine was carried out in two stages. Yeast was inoculated into the marigold raw material, and primary alcohol fermentation was performed at a temperature of 25 to 30°C for 7 to 10 days. The filtered fermented liquid was then placed in a 23L carboy tank, and secondary alcohol fermentation was carried out at 20°C for 3 weeks. After 2 months, the yeast was allowed to settle, and then removed by racking using a siphon to prepare the marigold alcohol fermented liquid (marigold wine) for acetic acid fermentation, which was used as the raw material. The alcohol concentration of the prepared marigold alcohol fermented liquid was measured at 11%, and the sugar content at 8%. To produce vinegar using the marigold wine, it was diluted to an alcohol concentration of 7 to 7.5% and used as the fermented liquid for vinegar production.

[0069] <Making Marigold Vinegar>

[0070] To the marigold alcohol fermentation liquid prepared in the previous step Acetobacter pasteurianus Marigold vinegar was produced through acetic acid fermentation by inoculating with the S25 (KFCC11970P) strain. As a control, the patented strain owned by Suncheon City is A. pasteurianus The SC08 (KACC92126P) strain was used. Inoculation of each strain was performed by adding the acetic acid bacteria culture solution to the prepared alcohol fermentation solution.

[0071] First, colonies of the strain grown on GYE solid agar plates were picked with a platinum loop and inoculated into 5 ml of GYE liquid medium. A pre-culture solution was prepared by shaking culture at 30°C for 48 hours. For the main culture solution, 1% of the pre-culture solution was inoculated into 500 ml of GYE liquid medium and shaken at 30°C for 48 hours to be used as the acetic acid bacteria culture solution for the production of marigold vinegar. Using a total of 5 L jars, 0.5 L of water was added to 1.5 L of the marigold wine prepared in the previous step, and 0.5 L of the acetic acid bacteria culture solution was added to each jar to inoculate the acetic acid bacteria. Acetic acid fermentation was carried out under static culture conditions at 30°C, and the total acidity was measured.

[0073] Measurement of Total Acidity in Marigold Vinegar

[0074] The total acidity of the prepared marigold vinegar was measured at 7-day intervals. On each day after inoculation, 10 mL of vinegar was taken, placed in a volumetric flask, and diluted to 100 mL using distilled water. 20 mL of this was taken into a 250 mL Erlenmeyer flask, 2-3 drops of phenolphthalein solution were added to the sample, and the amount consumed was measured by titrating with 0.1 N NaOH solution. The total acidity content (%) was calculated based on the amount of NaOH solution consumed using the formula in Equation 1 above, and the results are shown in Table 2 and Figure 3 below.

[0076] 0 days 7 days 14th 21st 28th 35 days 42 days 49 days SC08 0.7 2.2 4.3 4.9 5.1 5.5 5.8 6.0 S25 0.7 4.0 6.6 6.9 7.0 7.3 7.3 6.9 56 days 63 days 70 days 77 days 84 days SC08 7.1 7.1 7.1 7.0 6.8 S25 6.5 6.6 6.9 7.0 6.9

[0078] Referring to Table 2 and Figure 3 above, the results of acetic acid fermentation using two strains with marigold fermentation liquid show that the total acidity concentration in acetic acid fermentation using strain S25 was superior to that in acetic acid fermentation using the existing patented strain SC08 starting from the 7th day of fermentation. S25 achieved an acetic acid concentration of 4.0%, which is the standard acidity for vinegar, within 7 days of fermentation, confirming the possibility of rapid acetic acid fermentation in vinegar production. Therefore, it was confirmed that there is a possibility to reduce production time by shortening the fermentation period during marigold vinegar production, increase the acidity of the produced marigold vinegar, and reduce contamination caused by the proliferation of unwanted bacteria.

[0080] <Survey on Marigold Vinegar Preference>

[0081] A preference survey of marigold vinegars prepared by inoculating the control group SC08 and the experimental group S25 acetic acid bacteria strains, respectively, was conducted using a panel of 14 trained researchers. The preference characteristics of the vinegar—color, sourness, aroma, and overall preference—were evaluated using a 5-point scale (1: very weak, very poor; 5: very strong, very good). Each marigold vinegar was centrifuged (3,000 rpm, 10 min, 4°C) to remove impurities, and 20 ml portions were served in clear plastic cups for the panelists to evaluate. They were instructed to rinse their mouths with water between servings of different samples. The evaluation results are shown in Table 3 below.

[0083] A. pasteurianus SC08 A. pasteurianus S25 Color 3.45 ±0.34 1) 3.89 ±0.66 Flavor 3.21 ±0.74 4.50 ±0.74 Sour Tate 3.75 ±0.57 4.18 ±0.57 Overall preference 3.82 ±0.63 4.50 ±0.22 5 point Likert scale: 1-extremely dislike, 5-extremely like, Each value represents the mean ±SD

[0085] Referring to Table 3 above, A. pasteurianus As a result of conducting a sensory evaluation after producing vinegar using two types, there was no difference between the two strains in color, and in terms of aroma and overall preference, the strain according to one aspect of the present invention A. pasteurianus S25 strain showed a higher preference. In terms of sourness as well. A. pasteruianus The S25 strain was evaluated as having a stronger sour taste.

[0086] It was confirmed that when vinegar is produced by acetic acid fermentation of marigold alcohol using the novel acetic acid fermentation strain Acetobacter pasteurianus S25 (KFCC11970P) according to one aspect of the present invention, an acidity of 4% or more is achieved within 7 days of inoculation of the strain, and vinegar with an excellent acidity of 5% or more can be produced within 14 days. This is the result of static culture; however, in the case of shaking culture, the acetic acid fermentation period can be shortened to one-half to one-third of the above period, thereby reducing the time required for vinegar production and further improving productivity. It was confirmed that the novel acetic acid fermentation strain according to one aspect of the present invention has excellent alcohol tolerance and can perform acetic acid fermentation at a high alcohol concentration of 7% or more. Since it exhibits excellent flavor and aroma while shortening the acetic acid fermentation period required for fermenting vinegar, it can be usefully utilized for the rapid production of fermented vinegar. In addition, when using marigold as a raw material and a novel acetic acid fermentation strain according to one aspect of the present invention, there is an advantage in being able to produce marigold fermented vinegar with high antioxidant capacity and functional component content.

[0088] Name of depositing institution: Korean Culture Collection of Microorganisms Trustee Number: KFCC11970P Date of Deposit: 2023-08-09

Claims

Claim 1 Acetobacter pasteurianus for the production of marigold vinegar, deposited under accession number KFCC11970P, which is isolated from Makgeolli, possesses a 16S rRNA gene containing the nucleotide sequence indicated by SEQ No. 1, is capable of rapid acetic acid fermentation, and can produce a fermented product with an acetic acid concentration of 4% (w / v) within 7 days of the start of fermentation in a GYE medium containing 0.5% yeast extract, 0.5% soy extract, 3% glucose, and 3% ethanol based on a total of 100% (w / v). Acetobacter pasterianus ) S25 strain. Claim 2 delete Claim 3 Marigold Calendula arvensis ) A first step of preparing raw materials by mixing leaves, water, and sugar; a second step of producing marigold wine by adding wine yeast to the raw materials and performing alcoholic fermentation; and Acetobacter pasteurianus deposited under accession number KFCC11970P ( Acetobacter pasterianus A method for producing marigold vinegar comprising a third step of inoculating with an S25 strain and fermenting with acetic acid. Claim 4 A method for producing marigold vinegar according to claim 3, wherein in the second step, the alcohol fermentation comprises: a first alcohol fermentation step in which wine yeast is added to the raw material and first fermented at 20 to 35°C for 7 to 10 days; and a second alcohol fermentation step in which the first alcohol fermentation liquid obtained in the first alcohol fermentation step is filtered and further fermented at 20 to 35°C for 2 weeks to 2 months. Claim 5 A method for producing marigold vinegar according to claim 3, wherein the acetic acid fermentation is performed by fermenting for 5 to 90 days at a temperature of 25 to 35°C. Claim 6 Marigold vinegar produced by a manufacturing method according to any one of paragraphs 3 to 5.

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