KR101778436B1 - Acetobacter sp. SLV-7 as a novel strain with high acetic acid producing ability and use thereof - Google Patents

Abstract

The present invention relates to an Acetobacter sp. SLV-7 strain (accession No. KACC 92002P). The strain of the present invention has ethanol resistance, can grow in a wide temperature range, and has excellent acetic acid producing ability, and thus, is useful for vinegar production.

Description

A novel acetobacterium strain SLV-7 having excellent acetic acid production ability and its use {Acetobacter sp. SLV-7 as a novel strain with high acetic acid producing ability and use thereof.

The present invention relates to a strain of the genus Acetobacter isolated from a traditional fermented vinegar makgeolli vinegar. More particularly, the present invention relates to a strain of Acetobacter ( Acetobacter ) having excellent acetic acid production activity sp . ) SLV-7 strain (Accession No. KACC 92002P).

Vinegar is one of the oldest fermented foods. Currently, the vinegar is used as a health food and is reported to have therapeutic effect as a medicine. A variety of physiological activities such as antibiotic effect, antioxidant function, lowering of blood sugar, prevention of obesity, lowering of cholesterol, prevention of arteriosclerosis and hypertension have been reported.

Previous studies have been carried out to increase the yield of acetic acid production. However, nowadays, Frings of Germany has developed a submerged culture to produce more than 17% of high acidity vinegar. . Traditional fermented vinegar is fermented in two stages. First, alcohol is produced by the yeast in the raw material containing sugar, followed by acetic acid to produce acetic acid. Traditional fermented vinegar generally has a long fermentation period of about one month. Start culture using natural vinegar.

The bacteria used for vinegar production belong to Acetobacteraceae and are absolutely aerobic, with an optimum growth temperature of 25 to 30 ° C and an optimum pH of 5.0 to 6.5. Acetic acid bacteria are known to have resistance even under acidic conditions below pH 5.0, and the main acetic acid bacteria used for vinegar production are usually two genera of Acetobacter and Gluconobacter .

The domestic vinegar industry is mainly composed of large enterprises and low-priced products, and there is a lack of efforts to develop traditional vinegar. In the domestic fermentation industry, most of the accumulation of experience-oriented know-how based on the contents of technology is based on the field. Since the professional development process of the technology has not been systemized, rapid industrialization of the bio-industry material, There are many cases of difficulties.

In order to overcome these problems and take a leap forward in the biotechnology industry, we will concentrate on core technologies such as database construction of various microbial characteristics, fermentation culture process and strain development technology, and scale-up technology related to practical application, It is necessary to establish a highly efficient technology development system by efficiently integrating the element technologies based on the function / characteristics of the target microorganisms. The traditional fermented food manufacturing process in Korea utilizes the natural fermentation process by natural microorganisms introduced from raw materials or air by adding and mixing the raw materials to the main raw materials without adding the seeds (starter). Therefore, since the microorganisms participating in the fermentation are not uniform, it is difficult to standardize the manufacturing process, and thus the quality and taste of the fermented food produced are variously formed.

One aspect of the present invention is to provide a method of producing an Acetobacter sp . ) Strain SLV-7 (Accession No. KACC 92002P).

Another aspect of the present invention is a culture medium containing at least one selected from the group consisting of the acetobacterium strain SLV-7 of the present invention (accession number KACC 92002P), the culture of the strain, the concentrate of the strain or the culture and the dried product thereof And to provide a composition for the fermentation of acetic acid.

Another aspect of the present invention is a method for producing a plant of the present invention comprising culturing the acetobacterium strain SLV-7 (Accession No. KACC 92002P) of the present invention to produce a herbaceous plant; Fermenting the prepared herbicide in a medium containing ethanol; And a step of filtering the fermented medium, and a vinegar produced by the method.

One aspect of the present invention is to provide a method of producing an Acetobacter sp . ) Strain SLV-7 (Accession No. KACC 92002P).

The term " Acetobacter " of the present invention is an absolute aerobic bacterium belonging to the gram-negative alpha- Proteobacteria river and belonging to Acetobacteraceae , and fermentation of ethanol produces acetic acid . It is present in a single or an oval or single-stranded form, does not form spores, has no motility, and has an optimal pH of 3.5 to 6.5.

According to one embodiment of the present invention, the isolate SLV-7 in the genus Acetobacter was identified by isolating it from the conventional fermented vinegar makgeolli vinegar, and the 16S rRNA gene sequence (SEQ ID NO: 4) of the strain was analyzed. As a result, Acetobacter pasteurianus subsp . pasteurianus ) LMG 1262T. However, as a result of analyzing the average nucleotide identity (ANI) of the whole genome, it was found to be 90.52%, which is a novel acetic acid strain. Therefore, the above strain was deposited with the National Institute of Agricultural Science and Technology, National Institute of Agricultural Science, and received the accession number of KACC 92002P.

According to one embodiment, the strain of the genus Acetobacter SLV-7 grows in a temperature range of 10 to 45 ° C, preferably 15 to 40 ° C, more preferably 20 to 37 ° C.

According to one embodiment, the strain of the genus SLV-7 in the genus Acetobacter is grown in a medium having an ethanol concentration of 0 to 15%, preferably 0 to 10%, more preferably 0 to 8% have.

According to one embodiment, the strain of the genus SLV-7 in the genus Acetobacter is grown in a medium having a nitric acid concentration of 0 to 8%, preferably 0 to 6%, more preferably 0 to 4% have.

Further, another aspect of the present invention includes at least one selected from the group consisting of the strain of the genus acetobacterium SLV-7 (Accession No. KACC 92002P) of the present invention, the culture of the strain, the concentrate of the strain or the culture and the dried product thereof The composition for fermentation with acetic acid.

The term "culture product" of the present invention is interpreted as a concept including a culture medium obtained by culturing a medium or a strain containing the strain, its metabolites, extra nutrients, etc. obtained by culturing the medium for a certain period of time, and then removing the strain.

According to one embodiment, the culture of the strain of the genus Acetobacterium SLV-7 of the present invention can be used in a culture medium used for culturing microorganisms, a medium easily selected by a person skilled in the art depending on the purpose, More preferably, the YPGDE medium described in Example 1 below can be selected and used.

According to one embodiment, the culture of the strain of the genus Acetobacter SLV-7 of the present invention can be prepared by inoculating the strain of the present invention into the microorganism culture medium and culturing the microorganism in a microorganism culture method (for example, stationary culture, stirring Culturing, preferably in a cultivation method of acetic acid, more preferably 10% by weight (based on the total weight of the culture medium) and culturing at 25 to 37 for 2 to 10 days. More preferably, the strain of the present invention can be prepared by inoculating the culture medium with 10% by weight (based on the total weight of the culture medium) and culturing at 30 캜.

The strain of the genus Escherichia coli SLV-7 or the concentrate of the culture and the dried product thereof can be easily prepared according to the method of concentrating or drying the microorganism or the culture medium known in the art, and the strain of the genus SLV-7 of Acetobacter, The culture, the strain or the concentrate of the culture solution and the dried product thereof are not limited thereto, but they may be contained in the composition for producing acetic acid of the present invention in an amount of 1 to 15% by weight.

In another aspect of the present invention, there is provided a method for producing a plant, comprising culturing an acetobacterium strain SLV-7 (Accession No. KACC 92002P) to produce a herbaceous plant; Fermenting the prepared herbicide in a medium containing ethanol; And filtering the resultant, and a vinegar produced by the method.

The term "cultivation" of the present invention means that microorganisms are grown under moderately artificially controlled environmental conditions. Specifically, examples of the culture method include, but are not limited to, a batch culture, a continuous culture, and a fed-batch culture. These various methods are disclosed, for example, in "Biochemical Engineering" (James M. Lee, Prentice-Hall International Editions, pp. 138-176, 1991).

The term " vinegar starter "of the present invention means a candle which becomes a species used for soaking vinegar newly, and means a candle in which acetic acid bacteria actively produce acetic acid. Generally, a portion of the fermentation broth which has been partially fermented, or part of the fermentation broth which is normally fermenting, is used. The acetic acid bacteria contained in the herbaceous plants rapidly grow the bacterial membrane to slow the aging of the bacteria membrane and preserve the membrane until the end of fermentation. Thus, the rate of acid production is fast, and the acid production is continued until the end, It is preferable that the fermentation liquid is not turbid.

According to one embodiment, the step of preparing the herbaceous plant may be carried out in a culture medium containing acetic acid, and the acetic acid may be contained at a concentration of 0.5 to 5%, more preferably 1 to 4%.

According to one embodiment, the ethanol may be contained in the medium at a concentration of 2 to 10%, preferably 4 to 8%, more preferably 5 to 6%.

According to one embodiment, the herbaceous plant may be contained in a medium containing ethanol in an amount of 1 to 15%, preferably 10%. The fermentation step may be performed at a temperature of 10 to 40 ° C, Deg.] C to 40 [deg.] C, and more preferably 30 to 37 [deg.] C.

The Acetobacter of the present invention sp . ) SLV-7 strain (Accession No. KACC 92002P) has ethanol resistance, can grow in a wide temperature range, and has excellent acetic acid producing ability, so it can be useful for vinegar production.

FIG. 1 shows the rep-PCR result of the strain SLV-7 in Acetobacter.
FIG. 2 is a graph showing the results of culturing the strain of the genus SLV-7 in Acetobacter in a medium containing ethanol at different concentrations. FIG.
Fig. 3 is a graph showing the results of incubation of the strain SLV-7 in Acetobacter in a medium containing acetic acid at different concentrations.
FIG. 4 shows the results of culturing the strain of the genus Acetobacter SLV-7 in various temperature ranges.

Hereinafter, one or more embodiments will be described in more detail by way of examples. However, these embodiments are intended to illustrate one or more embodiments, and the scope of the present invention is not limited to these embodiments.

Example  1: Isolation of acetic acid bacteria

Acetic acid bacteria were isolated from makgeolli vinegar and 10 kinds of herbaceous plants which were prepared by fermenting 38 kinds of rice wine from 30 to 4 to 6 weeks.

Specifically, the decimal dilution method and the rice wine vinegar jongcho (serial dilution) to calcium carbonate (CaCO ₃₎ is 2% contained YPGDE agar medium (0.5% yeast extract is diluted, 0.5% peptone, 0.5% glycerol , 0.5% D- glucose, 1.5% agar and 4% ethanol). The agar plate was incubated at 30 ℃ for 5 days, and 38 isolates were selected.

Only repressive extragenic palindromic PCR (PCR) was performed using GTG5 primers. Specifically, only the selected colonies were taken to obtain the template DNA, and the cells were boiled at 100 for 10 minutes in a Chelex-100 solution (Bio-Rad, USA). (1.5 g), GTG5 (5'-GTGGTGGTGGTGGTTG-3 '') primer (SEQ ID NO: 1), 1.25 U Taq polymerase (solgent, Korea), dNTP mixute (solgent, Korea), 1X Taq buffer Total volume was adjusted to 50 μl using pure water and amplified using a C1000 thermal cycler (Bio-Rad, USA). PCR conditions were 94 ° C for 5 minutes (1 cycle); 94 ° C for 30 seconds, 85 ° C for 1 minute, and 65 ° C for 8 minutes (30 cycles); 65 ° C for 16 minutes (1 cycle) was set.

After the PCR was completed, the product was electrophoresed at 100 V for 45 minutes using 1.5% agarose gel (0.5% TBE buffer), stained with ethidium bromide (EtBr) for 15 minutes, Was confirmed. Nineteen strains were selected by re-selecting only the sample showing a specific pattern.

The selected 19 strains were inoculated into a YPGDE agar medium containing 2% of calcium carbonate, and the strain SLV-7 having a high growth rate was selected based on the size of the transparent ring formed in a single colony .

Example  2: Identification of selected acetic acid bacteria

The homology between the previously reported microorganisms and the SLV-7 strain isolated in Example 1 was compared through the 16S rRNA gene.

First, colony polymerase chain reaction (PCR) was performed to confirm the base sequence of the 16S rRNA gene. Selected colonies were extracted from the colonies in a Chelex-100 solution (Bio-rad, USA) and boiled for 10 minutes at 100 ° C to decompose the cell wall. Primer (SEQ ID NO: 2), F1 (5'-AGA GTT TGA TCM TGG CTC AG-3 ') primer (SEQ ID NO: ), 1.25 U Taq polymerase, dNTP mixute, 1X Taq buffer, and purified water, and then amplified using a C1000 thermal cycler. PCR conditions were 94 5 min (1 cycle); 94 DEG C for 45 seconds, 60 DEG C for 45 seconds, and 72 DEG C for 1 minute (30 cycles); 72 ° C for 10 minutes (one cycle) was set. Amplified PCR products were purified using PCR product purification kit (Solgent, Korea) and analyzed for the base sequence of 16S rRNA gene through Macrogen (Korea).

The 16S rRNA sequence (SEQ ID NO: 4) of the SLV-7 strain was amplified using EzTaxon (Chun et al., 2007) of the Korea Bioinformation Center (KOBIC) The nucleotide sequence was compared with similarity.

As a result, the present inventive Acetobacter sp.) The strain SLV-7 is an Acetobacter pacificia subsp. pasteurianus subsp . pasteurianus ) LMG 1262T strain and 99.9%, Acetobacter pasteurianus subsp . ascendens ) with LMG 1590T strain, 99.8%, Acetobacter pacificianus paradoxus ( Acetobacter pasteurianus subsp . paradoxus LMG 1591T strain was 99.7% homologous.

However, as shown in Fig. 1, the GTG5-PCR pattern was compared. As a result, the pattern of Acetobacter SLV-7 strain was Acetobacter pasteurianus subsp . ascendens LMG 1590T strain and A. pasteurianus subsp . paradoxus LMG 1591T strain, and A. pasteurianus subsp . pasteurianus LMG 1262T ( A. pasteurianus subsp . Pasteurianus KACC 13994T), but the average nucleotide identity (ANI) of the whole genome was analyzed to be 90.52%, which was a novel acetic acid strain (FIG. 1).

Example  3: Acetobacter SLV -7 Characterization of strains

Two previously known isolates, Acetobacter pomorum) KCTC 22319T (wine, separating bacteria in vinegar) and acetonitrile bakteo Oh Shetty (Acetobacter aceti) KCTC 12290T (= Acetobacter ATCC 15978T, by selecting the strains) used for vinegar production characteristics of the strains with the candidate strain Respectively.

3-1: Identification of ethanol resistance

Ethanol was added to the YPGD medium at each concentration (0, 2, 4, 6, 8, 10, 12 and 14%) to prepare plates. In the YPGDE medium, until an OD ₆₀₀ value of 1 was reached, Acetobacter sp. The SLV-7 strain was cultured, and the culture was diluted to 10 < ^-3 > and 5 [mu] L of the strain was inoculated on the plate and cultured at 30 DEG C for 48 hours.

As a result, Acetobacter sp. SLV-7 strain showed high ethanol resistance (Fig. 2).

3-2: Acid resistance confirmation

Plates were prepared by adding acetic acid to the YPGDE medium at various concentrations (0, 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4 and 4.5%). In the YPGDE medium, until the OD ₆₀₀ value reaches 1, the Acetobacter sp. The SLV-7 strain was cultured, and the culture was diluted to 10 < ^-3 > and 5 [mu] L of the strain was inoculated on the plate and cultured at 30 DEG C for 48 hours.

As a result, Acetobacter sp. SLV-7 strains were found to grow relatively rapidly at higher acetic acid concentrations than other strains (FIG. 3).

3-3: Check the growth temperature range

In the YPGDE medium, until an OD ₆₀₀ value of 1 was reached, Acetobacter sp. SLV-7 strain was cultured, and the culture solution was diluted to 10 < ^-3 > and 5 [mu] l was inoculated on a YPGDE medium plate. After inoculation, the cells were cultured at 10, 15, 20, 25, 30, 37, 40 and 45 캜 for 48 hours.

As a result, it was confirmed that the optimal culture temperature of all the acetic acid bacteria was 30 ℃. Especially, Acetobacter sp. The strain SLV-7 was found to grow in a wide temperature range from 20 to 40 DEG C (FIG. 4).

3-4: Acetic acid Generation  evaluation

The acetic acid bacteria were cultured using a fermenter, and the growth curve and yield of acetic acid production were confirmed. The operating conditions of the fermenter are shown in Table 1 below.

Fermenter operating conditions badge YPDE (Yeast extract 0.5%, Peptone 0.5%, D-glucose 0.5%, Ethanol 5%) Acetic acid concentration One% Medium capacity 700 ml Incubation temperature 30 ℃ Rotation speed 250 rpm Amount of strain inoculated 10% Air injection amount 2 vvm

When the residual ethanol concentration decreased to 1% or less, 5% ethanol was added. Acetic acid concentration (%), minimum pH and yield of acetic acid production were determined at the maximum fermentation value, and the yield of acetic acid production was calculated using the following equation (1).

The amount of acetic acid produced and the amount of ethanol consumed during the fermentation period were detected by HPLC quantitative analysis using a RI (Refractive Index) detector (mobile phase: 1 mM sulfuric acid, flow rate: 0.5 ml / min, detector: RI detector, Column: Repromer H, column temperature: 50 < 0 > C).

As a result, the initial pH and pH were similar for all three strains, but the lowest pH was lowest for A. aceti KCTC 12290T strain, Acetobacter sp. SLV-7, and A. pomorum KCTC 22319T strains. Acetic acid concentration at the highest fermentation was Acetobacter sp. SLV-7 strains were the highest, and acetic acid yield was also higher in Acetobacter sp. SLV-7 strains were the most excellent (Table 2).

Acetobacter
sp. SLV-7 A. aceti
KCTC 12290T A. pomorum
KCTC 22319T Initial pH (%) 0.97 1.03 1.02 Initial pH 3.69 3.70 3.72 Fermentation peak
Acetic Acid Concentration (%) 3.39 2.87 3.29 Lowest pH 3.20 3.21 3.27 Acetic acid yield (%) 31.00 24.00 29.60

Example  4: Acetobacter SLV Production of rice wine vinegar using -7 strain

Acetobacter (0.5%) and alcohol (6%) were injected into the reactor and incubated in YPGDE medium containing 1% of acetic acid for 48 hours. sp . SLV-7 strain was inoculated 10%. The reactor temperature was maintained at 30 ° C and acetic acid fermentation was carried out at 250 rpm for 10 days. A. using aceti strain KCTC 12290T were compared to produce a vinegar in the same conditions.

As a result, A. aceti 12290T strain is 3% acidity content, Acetobacter sp. The SLV-7 strain was found to have an excellent total acid content of 5.4%.

Example  5: Acetobacter SLV Preparation of persimmon vinegar using -7 strain

After washing the commercially available senses, the water was removed and left for about 10 days. Then, the faeces and seeds were removed and the alcohol fermentation was performed at 30 ° C for 10 days. After alcohol fermentation, the cells were cultured in YPGDE medium containing 1% of acetic acid for 48 hours. Acetobacter sp. SLV-7 culture broth was inoculated to conduct acetic acid fermentation. A. using aceti strain KCTC 12290T were compared to produce a vinegar in the same conditions.

As a result, A. aceti 12290T strain acidity content of 2%, Acetobacter sp. The SLV-7 strain was found to be excellent in total acid content of 3.8%.

The present invention has been described with reference to the preferred embodiments. It will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention as defined by the appended claims. Therefore, the disclosed embodiments should be considered in an illustrative rather than a restrictive sense. The scope of the present invention is defined by the appended claims rather than by the foregoing description, and all differences within the scope of equivalents thereof should be construed as being included in the present invention.

Agricultural Biotechnology Research Institute KACC92002P 20141110

<110> CHUNG ANG University industry Academic Cooperation Foundation <120> Acetobacter sp. SLV-7 as a novel strain with high acetic acid          producing ability and use thereof <130> PN160068 <160> 4 <170> KoPatentin 3.0 <210> 1 <211> 15 <212> DNA <213> Artificial Sequence <220> <223> synthesized nucleotide <400> 1 gtggtggtgg tggtg 15 <210> 2 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> synthesized nucleotide <400> 2 agagtttgat cmtggctcag 20 <210> 3 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> synthesized nucleotide <400> 3 tacggytacc ttgttacgac tt 22 <210> 4 <211> 1476 <212> RNA <213> Acetobacter sp. <400> 4 agagtttgat cctggctcag agcgaacgct ggcggcatgc ttaacacatg caagtcgcac 60 gaaggtttcg gccttagtgg cggacgggtg agtaacgcgt aggtatctat ccatgggtgg 120 gggataacac tgggaaactg gtgctaatac cgcatgacac ctgagggtca aaggcgtaag 180 tcgcctgtgg aggagcctgc gtttgattag ctagttggtg gggtaaaggc ctaccaaggc 240 gatgatcaat agctggtttg agaggatgat cagccacact gggactgaga cacggcccag 300 actcctacgg gaggcagcag tggggaatat tggacaatgg gggcaaccct gatccagcaa 360 tgccgcgtgt gtgaagaagg tcttcggatt gtaaagcact ttcgacgggg acgatgatga 420 cggtacccgt agaagaagcc ccggctaact tcgtgccagc agccgcggta atacgaaggg 480 ggctagcgtt gctcggaatg actgggcgta aagggcgtgt aggcggtttg tacagtcaga 540 tgtgaaatcc ccgggcttaa cctgggagct gcatttgata cgtgcagact agagtgtgag 600 agagggttgt ggaattccca gtgtagaggt gaaattcgta gatattggga agaacaccgg 660 tggcgaaggc ggcaacctgg ctcattactg acgctgaggc gcgaaagcgt ggggagcaaa 720 caggattaga taccctggta gtccacgctg taaacgatgt gtgctagatg ttgggtgact 780 tagtcattca gtgtcgcagt taacgcgtta agcacaccgc ctggggagta cggccgcaag 840 gttgaaactc aaaggaattg acgggggccc gcacaagcgg tggagcatgt ggtttaattc 900 gaagcaacgc gcagaacctt accagggctt gaatgtagag gctgcaagca gagatgtttg 960 tttcccgcaa gggacctcta acacaggtgc tgcatggctg tcgtcagctc gtgtcgtgag 1020 atgttgggtt aagtcccgca acgagcgcaa cccctatctt tagttgccat caggttgggc 1080 tgggcactct agagagactg ccggtgacaa gccggaggaa ggtggggatg acgtcaagtc 1140 ctcatggccc ttatgtcctg ggctacacac gtgctacaat ggcggtgaca gtgggaagct 1200 aggtggtgac accatgctga tctctaaaag ccgtctcagt tcggattgca ctctgcaact 1260 cgagtgcatg aaggtggaat cgctagtaat cgcggatcag catgccgcgg tgaatacgtt 1320 cccgggcctt gtacacaccg cccgtcacac catgggagtt ggtttgacct taagccggtg 1380 agcgaaccgc aaggacgcag ccgaccacgg tcgggtcagc gactggggtg aagtcgtaac 1440 aaggtagccg taggggaacc tgcggctgga tcacct 1476

Claims (10)

Acetobacter sp. Strain SLV-7 (Accession No. KACC 92002P), which has ethanol resistance, ability to produce acetic acid, and which can grow at 20 to 37 占 폚.
delete The strain according to claim 1, wherein the strain grows at an ethanol concentration of 0 to 8%.
The strain according to claim 1, wherein the strain grows at an acetic acid concentration of 0 to 4.5%.
A composition for fermentation with acetic acid comprising at least one selected from the group consisting of the strain of the genus Escherichia coli SLV-7 (Accession No. KACC 92002P), the culture of the strain, the concentrate of the strain or the culture and the dried product thereof.
(a) culturing the strain of the genus acetobacterium SLV-7 of claim 1 (accession number KACC 92002P) in a medium containing acetic acid to produce a herbaceous plant;
(b) fermenting the prepared herbaceous plant by inoculating the medium containing rice wine; And
(c) filtering the resultant of step (b).
delete delete [7] The method of claim 6, wherein the fermenting step is performed at 20 to 37 [deg.] C.
9. A makgeolli vinegar produced by the method of any one of claims 6 to 9.

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