KR101589153B1 - Novel Gluconacetobacter saccharivorans sp. CV1 having Alcohol-Resistance and the Producing Method of a Brewing Vinegar using the Same - Google Patents

Abstract

The present invention relates to a gluconacetobacter ( Gluconacetobacter) saccharivorans) using CV1 (KACC 17057) strain, and it relates to a process for preparing a brewed vinegar for producing a brewed vinegar.

Description

[0001] The present invention relates to a glucone acetobacterium Saccharovorans strain CV1 having alcohol resistance and a method for producing a fermented vinegar using the same. CV1 having Alcohol-Resistance and the Producing Method of a Brewing Vinegar using the Same}

The present invention relates to a glucone acetobacterium saccharolvance CV1 strain having alcohol resistance and a method for producing a fermented vinegar using the same.

Vinegar, which is a fermented food that has been in use for a long time, has been widely used not only as a seasoning in cooking but also as a food preservative and medicine. Vinegar is produced by fermenting microorganisms with saccharides or starch-rich cereals, fruits, and liquors. The fermented fermented vinegar has the aroma of the raw materials and the aroma produced during the fermentation process. In addition to acetic acid, volatile and nonvolatile Various organic acids, sugars, amino acids, esters and the like.

Generally, the vinegar is prepared by converting sugar into alcohol using yeast, converting alcohol into acetic acid by using acetic acid bacteria (Representative of Acetobacter) For example, Korean Patent Publication No. 2000-0058004 discloses a novel acetobacterium strain and a method for producing vinegar using the strain.

However, the strain of the genus Acetobacter used in the conventional invention has a limitation in the fermentation of acetic acid at a high concentration, and thus it is necessary to develop a new strain of acetic acid bacteria capable of fermenting acetic acid at a high concentration.

KR 10-2000-0058004 A

SUMMARY OF THE INVENTION The present invention has been made to solve the above-mentioned problems occurring in the prior art,

It is an object of the present invention to provide a novel strain having excellent ability to produce acetic acid from a high alcohol concentration and a method for producing the vinegar using the same.

According to an aspect of the present invention,

Gluconacetobacter saccharivorans CV1 (KACC 17057) strain having an alcohol resistance is provided.

The present invention also provides a process for producing a fermented vinegar, characterized in that the strain is cultivated in a medium and brewing vinegar is obtained from the obtained culture medium.

The present invention also provides a brewed vinegar produced using the strain.

According to the method for producing a brewed vinegar according to the present invention,

It is possible to grow all the sections from 15 ° C to 37 ° C irrespective of the culture temperature, and it is possible to grow various alcohols (5 -10%). As a result, it is possible to produce 9.3% of high acidity brewing vinegar from 10% alcohol.

1 is a photograph of a strain of the present invention photographed by a phase contrast microscope.
2 is a graph showing acetic acid production ability according to the alcohol concentration of the strain of the present invention.
3 is a graph showing acetic acid production ability according to the temperature of the strain of the present invention.
4 is a graph showing acetic acid production ability according to the pH value of the strain of the present invention.
5 is a view showing the nucleotide sequence of the strain of the present invention.
Fig. 6 is a diagram showing the system aquisition site of the strain of the present invention based on the 16S rDNA gene base sequence.

Hereinafter, the present invention will be described in detail.

The present invention relates to a gluconacetobacter ( Gluconacetobacter) saccharivorans) CV1 (a method for producing vinegar, characterized in that KACC 17057) strain, and use them to obtain a brewed vinegar from the culture broth in the culture medium, thus obtained.

The microorganism used in the present invention is Gluconacetobacter Saccharivorans strain (hereinafter referred to as CV1 strain), and the CV1 strain was identified by the inventors of the present invention from the vinegar obtained from a vinegar farmhouse in Jindo, Jindo, Jeollanam-do and was deposited with KACC of Rural Development Administration (KACC 17057 ). In the present invention, the deposited strain may be used.

The present invention also provides a method for producing a brewed vinegar, which comprises culturing the strain of Gluconacetobacter saccharivorans in a medium and obtaining a brewed vinegar from the obtained culture medium.

The method of producing vinegar vinegar of the present invention utilizes acetic acid fermentation, and aerobically oxidizes alcohol by the action of acetic acid bacteria using oxygen in air to produce acetic acid through acetaldehyde. That is, the acetic acid bacterium oxidizes alcohol to produce acetic acid. Alcohol is aerobically converted to acetaldehyde by an alcohol hydrogenation enzyme, and then by an aldehyde dehydrogenase, acetic acid, that is, vinegar.

In the method for producing a fermented vinegar according to the present invention, the CV1 strain is used as the acetic acid bacteria, and the CV1 strain is preferably inoculated in an amount of (5 to 10%) by weight per 100 parts by weight of the fermented alcohol. If the amount of the CV1 strain added is less than 5% by weight, fermentation is not sufficiently achieved. If the amount of the CV1 strain is more than 15% by weight, the effect of addition is not increased and economical efficiency is low.

In the method for producing a fermented vinegar of the present invention, the fermentation is preferably carried out at a temperature of 15 to 37 캜, preferably 20 to 30 캜, for 10 to 20 days. If the incubation temperature is lower than 15 ° C or higher than 37 ° C, the propagation ability of the acetic acid bacteria sharply decreases so that the temperature is preferably within the above range.

In the method for producing a vinegar of the present invention, the culturing is performed by fermenting for 10 to 20 days, wherein the pH value of the culture medium is preferably 3 or more. If the pH value is less than 3, there is a problem that the breeding power of the strain is rapidly weakened.

The method for producing a fermented vinegar of the present invention can culture a strain in a medium having an alcohol concentration of 10%. In the conventional method of producing vinegar, it was difficult to grow acetic acid bacteria in an alcohol concentration of 6% or more. However, since the CV1 strain used in the method for producing vinegar vinegar of the present invention is a strain resistant to alcohol, acetic acid can be produced even in the presence of a high concentration of alcohol.

In the production method of the present invention, yeast extract, glucose, calcium carbonate (CaCO ₃ ), agar, and ethanol are used as a plate medium for isolating acetic acid-producing bacteria, May be carried out in a liquid medium containing yeast extract, glucose, glycerin, magnesium sulfate (MgSO ₄ .7H ₂ O), ethanol and acetic acid.

In addition, in the production method of the present invention, the strain CV1 is selected from the group consisting of sucrose, xylose, lactose, inositol, mannose, mannitol, Rhamnose Sorbitol, Galactose, Arabinose, Fructose, Amygdalin, Maltose, Lactate, Glycerol, Gluconate, , Melibiose can be used as the carbon source of the AE liquid medium.

The brewing vinegar of the present invention can be produced by the above-described method for producing brewing vinegar, and the brewing vinegar can be recovered from the culture broth cultured by the above-mentioned production method using a conventional recovery method.

Hereinafter, the present invention will be described in more detail with reference to examples. However, the embodiments of the present invention described below are illustrative only and the scope of the present invention is not limited to these embodiments. The scope of the present invention is indicated in the claims, and moreover, includes all changes within the meaning and range of equivalency of the claims.

Example

Experimental conditions

The vinegar used in this experiment was collected from a farmhouse in Jindo, Jeonnam Province and used as an analytical sample at 4 ℃.

Yeast extract 0.5%, glucose 3.0%, CaCO ₃ 1.0%, agar 2.0%, ethanol 5.0%) was used as a plate medium for the isolation of acetic acid - producing bacteria. glucose 0.5%, glycerin 1.0%, MgSO ₄ · 7H ₂ O 0.02%, ethanol 5.0%, acetic acid 1.0%) were used. AE medium (yeast extract 0.2%, peptone 0.3%, glucose 0.5%, ethanol 3%, acetic acid 3%) was used to confirm the availability of the isolates.

Example  1. Isolation and Identification of Strain

To isolate the CV1 strain of the present invention, 100 μl of the collected vinegar was plated on a solid medium and cultured at 30 ° C for 5 days. The clear zone in which CaCO ₃ was dissolved was firstly screened, and a single acetic acid bacterium colony was subcultured and purified.

The mycological identification of the strain CV1 isolated from the above was performed by examining the morphological characteristics and biochemical characteristics (API 20NE Kit) of the acetic acid bacteria.

2.1. Morphological characteristics of the strain

To observe the morphology of strain CV1, the cells were incubated in solid medium for 48 hours. The cells were streaked out with 60, 70, 80, 90, 95 and 100% ethanol and observed with a phase contrast microscope (× 1,000).

The morphological characteristics of strain CV1 isolated according to the present invention were analyzed with A. pasteurianus and G. hansenii , which are known strains, and the results are shown in Table 1, indicating that they have gram negative, there was. In particular, as a result of a microscopic examination (× 1,000) using a phase contrast microscope, the separated strain CV1 (cultured at 30 ° C. for 2 days in a GYG agar medium) was a rod long rod having an elongated outer shape as shown in FIG. 1, And it was confirmed to be acetic acid bacteria which can be used for the production of traditional brewing vinegar.

Characteristic KCTC 12289
A. pasteurianus KCCM 40230
G. hansenii Isolated strain
CV1 Gram staining - - - Cell shape Rod Long rod Long rod Cell size (μm) 0.3-0.4 x 0.7-0.8 0.6-0.7 x 1.4-1.8 0.2-0.3 × 4-6 Motility - - - Colony characteristics
(Solid medium) - - - Shape Entire, circular convex to flat Entire, circular convex to flat Entire, circular convex to flat Color Pale white Pale white Pale white Surface Smooth to rough Smooth to rough Smooth to rough Transparence Opacity Opacity Opacity

2.2. Biochemical characteristics of strains

In order to investigate the biochemical properties of the isolates, experiments were carried out using API 20NE Kit. For identification using API kit (BioMerieux, France), 100 μl of the cultured isolate was inoculated into the API kit, and reacted at 30 ° C for 24 hours, followed by biochemical test and assimilation test. Biochemical tests were carried out by suspending the test bacteria in physiological saline, inoculating them into a cupule containing a dried substrate, culturing, adding coloring reagents, and determining the metabolism by color change. The assimilation test can be carried out by inoculating the test microorganisms suspended in the minimal medium into a cupule of a kit containing carbohydrates as the only carbon source, and then analyzing the turbidity of the test bacteria for the availability of these sugars. Cellulose production was incubated at 30 ℃ in AE liquid culture medium to observe whether or not these cells were formed. Based on the results of these morphological and biochemical characteristics, isolated acetic acid bacteria were identified according to the criteria of Bergey 's Manual of Systematic Bacteriology.

As shown in Table 2 below, the CV1 strain isolated from the biochemical test results was similar to the genus of Acetobacter than the genus of Gluconacetobacter, and the assimilation test results showed the same results as those of the acetic acid bacteria .

Characterization G. hansenii A. pasteurianus CV1 Catalase + + + Nitrate reduction - - - Indole production - + + Glucose fermentaion - - - Arginine dihydrolase - - - Urease - - - Escullin hydrolysis + + - Gelatin hydrolysis - - - β-glucosidase - - - Assimilation D-glucose - - - D-arabihose - - - D-mannose - - - D-mannitol - - - N-acetyl-glucosamine - - - D-maltose - - - Potassium gluconate - - - Capric acid - - - Adipic acid - - - Malic acid - - - Trisokium citrate - - - Phenylacetic acid - - -

2.3. Analysis of phylogenetic trees from isolated acetic acid bacteria

The sequence of 16S rDNA was analyzed to make phylogenetic tree of isolated acetic acid bacteria. Genomic DNA was extracted from the recovered cells using choromosomal DNA (gDNA prep kit) in ASW-YP liquid medium for 2 days. The 16S rRNA fragment was amplified by PCR using universal primers 27F (5'-AGAGTTTGATCCTGGCTCAG-3 ') and 1492R (5'-GGTTACCTTGTTACGACTT-3') as templates. The amplified PCR product was purified by electrophoresis and purified with a PCR product purification kit (Nucleogene, Korea), and sequenced by cycle sequencing using ABI BigDye terminator cycle sequencing ready reaction kit V3.1 (ABI) Lasergene 7).

The homology of the determined nucleotide sequences was determined by the BLAST search provided by the National Center for Biotechnology Information (NCBI; www.ncib.nlm.nih/gov/), and the similarity with the standard strains (similarity) Investigation and securing of 16S rRNA gene sequence of related taxa were compared with Genebank rebosomal DNA sequence. The homology between the nucleotide sequences was examined by multiple sequence alignment between the nucleotide sequences obtained using the Clustal W program. The phylogenetic tree of the isolates was constructed using the neighbor-joining method using the MEGA 4 program and the Kimura-nei model. 1,000 bootstrap resamplings were performed to check the stability of the system.

To elucidate the phylogenetic tree of isolated CV1 acetic acid bacteria, phylogenetic tree for each acetic acid bacterium was prepared by using neighbor-joining method using Mega 4 program and Kimura-nei model to determine the genotype and species name Respectively. The stability of the phylogenetic tree was examined by 1,000 bootstrap resampling. As a result, the nucleotide sequence of 16S rDNA of CV1 acetic acid was composed of 1,355 bp, which is shown in FIG. As shown in FIG. 6, the strain of 1,355 bp was highly homologous with the strain of the genus Gluconacetobacter, and more than 99% of the nucleotide sequence of Gluconacetobacter saccharivorans LMG 1582 And showed the highest homology. Therefore, the CV1 of the present invention is a Gluconacetobacter Saccharivorans CV1.

Example  2. High organic acid Generation  comparison analysis

Acetobacteria were isolated and plated on a solid medium (yeast extract 0.5%, glucose 3.0%, CaCO3 1.0%, ethanol 5.0%, agar 2.0%) with platinum at 1 spot and cultured at 30 ° C for 5 days. The size of the formed clear zone was measured and shown. After that, the ethanol content of the liquid medium was adjusted to 5, 6, 7, 8, 9, and 10% concentration, and cultured for 20 days to confirm the alcohol resistance and high organic acid production ability.

In order to investigate the growth characteristics of the isolated acetic acid bacteria, cultivation was carried out for 14 days at a culture temperature of 15 ° C, 20 ° C, 25 ° C, 30 ° C, 35 ° C and 37 ° C, And their growth state was measured by absorbance at 660 nm. The results are shown in FIG.

In addition, the pH of the culture medium was changed to 2.0, 2.4, 2.7, 3.0 and 3.4 in the liquid culture medium and cultured for 14 days to confirm the ability of acetic acid to be produced according to each pH value. The results are shown in Fig.

Acid bacteria producing organic acids at the highest 10% alcohol concentration were found to be 9.3% of total acids by CV1, and it was possible to grow in all the ranges from 15 ℃ to 37 ℃ regardless of the culture temperature. When the pH value was 3.0 to 3.4, the growth was vigorous. On the other hand, when the pH value was 2.0, no growth was observed.

Example  3. Isolation of isolate Carbon source Usability  Confirm

Separated acetic acid bacteria ( Gluconacetobacter saccharivorans CV1), various carbon sources such as lactose, fructose, maltose, mannitol, sucrose, sorbitol, glycerol and lactate were added to the AE liquid medium supplemented with 2% ethanol and acetic acid, As shown in Table 3 below, it was found that sucrose, mannose, fructose, glycerol, and mannitol showed excellent growth as a result of investigating whether the isolated strains utilize various carbon sources while culturing at 30 ° C for 14 days.

Carbon sources
AE (2a / 2e) broth
- Glucose replaced by CV1 Carbon sources
AE (2a / 2e) broth
- Glucose replaced by CV1 Sucrose +++ Xylose ++ Lactose ++ Inositol ++ Mannose +++ Mannitol +++ Rhamnose ++ Sorbitol ++ Galactose ++ Arabinose + Fructose +++ Amygdalin ++ Maltose ++ Lactate + Glycerol +++ Gluconate + Melibiose ++

Agricultural Biotechnology Research Institute KACC17057 20121029

Claims (7)

A composition for fermentation with acetic acid capable of fermenting acetic acid at an alcohol concentration of 10% or more and a temperature of 15 to 37 占 폚, comprising Gluconacetobacter saccharivorans CV1 (KACC 17057) having alcohol resistance. Which comprises fermenting an alcoholic fermentation product having an alcohol concentration of not less than 10% with Gluconacetobacter saccharivorans strain CV1 (KACC 17057) at 15 to 37 占 폚. delete delete The method of claim 2,
Wherein the alcohol fermented product has a pH value of 3 or more and 3.4 or less. The method of claim 2,
The strain may be selected from the group consisting of Sucrose, Xylose, Lactose, Inositol, Mannose, Mannitol, Rhamnose, Sorbitol, Galactose, Selected from the group consisting of arabinose, fructose, amygdalin, maltose, lactate, glycerol, gluconate and melibiose. Wherein the carbon source is used as the carbon source. A brewed vinegar prepared using the composition of claim 1.

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