KR20180122783A - Lactobacillus brevis having improved ability for reducting off-flavor of vinegar and productivity of anthocyanin, and method for manufacturing black rice vinegar using the same - Google Patents

Abstract

In the present invention, a novel Lactobacillus brevis strain having excellent ability for reducing vinegar flavor and anthocyanin-producing ability and a method for producing black rice vinegar using the same are disclosed. In one aspect, the Lactobacillus brevis strain of the present invention has a high esterase activity to alleviate pungent vinegar flavor naturally produced during alcohol and acetic acid fermentation, and has excellent anthocyanin and polyphenol producing ability, thereby manufacturing vinegar rich in nutrients.

Description

[0001] The present invention relates to a lactobacillus brevis strain having an improved anthocyanin-producing ability and an improved anthocyanin-producing ability, and a method for producing a black rice vinegar using the same. BACKGROUND ART }

The present invention discloses a novel Lactobacillus brevis strain having excellent vinegar-pickling activity and anthocyanin-producing ability, and a method for producing a black rice vinegar using the same.

Vinegar and sake are fermentation broths obtained by fermentation of ethanol and sugar with alcohol and acetic acid, and are representative fermented foods having a unique direction and sour taste produced during the fermentation process. In particular, it contains acetic acid as a main component and a small amount of volatile or nonvolatile organic acids, saccharides, amino acids and esters. Recently, the veterinary effects of various vinegar have been scientifically clarified, and consumption has gradually increased and become high-grade, and it has been attracting attention not only as a variety of functional materials such as vinegar drinks but also as a health food in simple seasoning applications. From this point of view, the production of vinegar made from fruits and cereals is attracting attention.

However, the main problem in the fermentation process of vinegar is that the irritating odor is an obstacle to the popularity of vinegar with excellent functionality. This irritating odor is attributed not only to the smell of acetic acid, which is the main component of vinegar, but also to ester compounds, including aldehydes and ethyl acetoates, which occur during the alcohol and acetic acid fermentation process.

Ethyl acetate is produced naturally by esterification by fermentation microorganisms during the fermentation process of acetic acid and alcohol. Various methods for removing such vinegar have been attempted. JP 7075543 discloses a method in which vinegar is evaporated at a temperature of 40 to 70 ° C and 60 to 160 mmHg, and JP 3123480 discloses a method of adsorbing vinegar fermented with cereals, fruits and / or sugars to a nonionic porous synthetic resin , The use of high pressure steam (JP 60137281), dehydrating sheet comprising semi (Japanese, 06-133756, 06-339365) and addition of masking agent (2000-312574, 2002-125673, 2009-065842) -Permeable external membranes (US 4765997) have been patented, but the reduction effect of vinegar is not sufficient. (JP 11065775) by the addition of yeast extract containing lactic acid and a method using an aldehyde dehydrogenase of yeast cell wall (Takakashi et al., Agric. Biol. Chem., 44, In addition, the removal method of vinegar using microorganisms has been rarely attempted.

N. Takahashi et al. Enzymic Improvement of Food Flavor. V. Oxidation of Aldehydes in Soybean Extracts in an NAD + -Regenerating System Made up of Aldehyde Dehydrogenase and Diaphorase, Agric. Biol. Chem., 44, 1669-1675. 1980

In one aspect, an object of the present invention is to provide a novel strain excellent in estrase activity.

In one aspect, an object of the present invention is to provide a strain which is improved in odor and capable of producing vinegar having high anthocyanin and total polyphenol contents.

The present invention relates to a novel Lactobacillus brevis nov. Strain which produces a highly active esterase. The present invention has been accomplished on the basis of this invention by producing AGRO-04 by fermenting AGRO-04 in parallel with fermentation to reduce vinegar odor and maintaining the balance of flavor and taste by fermentation and stabilizing atociane from black rice.

In one aspect, the present invention provides a Lactobacillus brevia strain wherein the esterase activity of the strain is 2.0 units / ml or more when cultured in an MRS broth medium for 48 hours at a temperature of 35 ° C.

In one aspect, the present invention relates to a lactobacillus Brevibus strain, a culture thereof, a lysate thereof, or a mixture thereof, wherein the lactase activity of the strain is 2.0units / ml or more when cultured in an MRS broth medium at a temperature of 35 ° C for 48 hours. Or an extract thereof.

In one aspect, the present invention relates to a method for producing vinegar, which comprises culturing a strain of Lactobacillus brevis, wherein the esterase activity of the strain is 2.0 units / ml or more when cultured in an MRS broth medium for 48 hours .

In one aspect, the Lactobacillus brevis strain of the present invention has a high estrase activity and can improve the vinegar odor and has an excellent anthocyanin-producing ability. Therefore, the vinegar having improved odor and nutrient-rich vinegar Can be manufactured.

Brief Description of the Drawings Fig. 1 is a schematic diagram of a novel Lactobacillus brevis nov. The result of electrophoresis of AGRO-04 DNA.
FIG. 2 is a graph showing the activity of Lactobacillus brevis nov. AGRO-04 (denoted as L6) and the genomic DNA of the acetic acid bacteria (expressed as A4 and A5) used in vinegar production.
FIG. 3 is a photograph of Lactobacillus brevis nov. The result of culturing AGRO-04 on phenol red olive oil agar medium shows the olive oil decomposition activity of the strain.
Fig. 4 shows the results of confirming the presence or absence of the esterase enzyme of each strain.
FIG. 5 shows the results of confirming the total acetic acid concentration according to the fermentation time.

Hereinafter, the present invention will be described in detail.

In one aspect, the present invention is a Lactobacillus brevis strain having an esterase activity of 2.0 units / ml or more when cultured in an MRS broth medium at a temperature of 35 ° C. for 48 hours.

In the same aspect, the strain is selected from the group consisting of Lactobacillus brevis nov. AGRO-04 strain. The novel Lactobacillus brevis nov. Producing the highly active estrase, a novel strain of the present invention. AGRO-04 was isolated and identified by the present inventors.

In this aspect, the strain may comprise the sequence of SEQ ID NO: 1.

Further, in one aspect, the present invention is a composition comprising the strain, the culture thereof, a lysate thereof, or an extract thereof.

The composition may be a composition for producing vinegar. In addition, in one aspect, the vinegar may include cereal fermented vinegar.

The cereal may include rice, wheat, barley, corn, oats and the like, and in one embodiment, the cereal may be black rice.

Further, in one aspect, the present invention is a method for producing vinegar, comprising culturing a strain.

Hereinafter, the present invention will be described in detail with reference to Examples and Experimental Examples. The examples and experimental examples are for the purpose of illustrating the present invention and are not intended to limit the present invention.

<Example: Preparation of black rice vinegar by parallel fermentation of lactic acid bacteria>

1. Black rice pretreatment process

Purified water having a concentration of 100 ppm of 5% lactic acid in black rice was added at a volume of about 3 to 10 times as much as the weight of black rice. After 24 hours of immersion, germinated brown rice was added at a concentration of about 100 to 120 mesh Size. When pulverized into a powder form without increasing the amount, the liquefaction and saccharification processes are more easily carried out by the growth and the enzyme.

2. Liquefaction and saccharification processes and alcohol fermentation

Purified water was added to the pulverized black rice at a volume of about 6 times as much as the weight of black rice, and the resulting mixture was stirred at 100 ° C. for 2 hours to grow SACCHAROMYCES CEREVISIA nov. AGRO-02 was added to 5 parts by weight (log 9 cfu / g) and 100 parts by weight of glucoamylase (Saczyme®, Gusmer Enterprises, Inc.) in an amount of 0.4-0.5% (w / v) Alcohol fermentation process was carried out simultaneously. The optimum amount of enzyme used was 20 ~ 30 Brix of soluble solids content.

The alcohol fermentation broth was prepared at 25 ~ 35 ℃ for 95 ~ 120 hours to obtain an alcohol fermentation broth having a sugar content of 11 ~ 12brix and an alcohol content of 17% (v / v). Diatomaceous earth was added to the alcoholic fermentation broth by 1 weight% based on the weight of the alcohol fermentation broth, followed by compression with a filter and filtration to remove impurities and debris. At this time, the yeast was inactivated by heating at 60 DEG C for 30 minutes, and the sugar content and alcohol content of the alcohol fermentation broth were maintained due to the inactivation of the yeast.

3. Acetic acid fermentation process of lactic acid bacteria in parallel

In the above alcohol fermentation broth, gluconacetobacterisaccharoborance nov. AGRO-03 (GLUCONACETOBACTR SACCHARIVORANS) was inoculated in an amount of 5 parts by weight based on 100 parts by weight of the alcohol fermentation broth at a concentration of log 9 cfu / g to conduct acetic acid fermentation. At this time, Lactobacillus brevis nov. AGRO-04 was inoculated with the same amount of acetic acid bacteria. This was performed in parallel to minimize the complexity of the fermentation step. The mixture was subjected to acetic acid fermentation at 25 to 30 ° C for about 5 days to prepare an acetic acid fermentation broth having an acidity of 12 to 17% and a sugar content of 11 to 13 Brix. The acidity was measured by neutralizing 0.1N NaOH with phenolphthalein solution as an indicator and converting it into acetic acid. The acetic acid fermentation broth was filtered at high temperature (85 ° C, 30 seconds) and diatomaceous earth to produce transparent and clear germinated brown rice vinegar, and the vinegar reduction effect was measured using this.

<Experimental Example>

Experimental Example 1. Lactobacillus brevis nov. Characteristics of AGRO-04

1. DNA sequence analysis

(1) DNA extraction

One colony cultured on an agar plate was inoculated into 100 μl digestion buffer (10 mM Tris-HCl, 50 mM NaCl, 10 mM EDTA and 1% SDS) and 0.5 μl proteinase K (20 mg / ml) using a sterilized platinum loop And the mixture was placed in a 1.5 ml centrifuge tube and allowed to stand at 55 ° C for 2 hours. After completion of the reaction, 200 μl of DNA binding solution (6 M GuSCN, 10 mM Tris-HCl pH 6.4, 20 mM EDTA pH 8.0, 1% Triton X-100) was added, mixed well and placed in a 96-well glass fiber filter plate (H25, Millipore, USA) was placed in vacuum (Millipore, USA) and vacuum (-25 inches, mmHg) was applied for 1 minute. The filter was washed twice with 90% ethanol and vacuumed (-25 inches, mmHg) for about 2 minutes until the filter was completely dry. After 1 minute, the cells were immersed in a 96-well V-bottom plate (Corning, USA) for 1 minute under vacuum (-25 inches, mmHg ) Was added to elute the DNA and stored at 0 ° C until use in the next experiment. DNA was quantitated at 260 and 280 nm using a Beckman DU 650 Spectrophotometer (Beckman, USA) and DNA purity was determined at a ratio of 260/280 nm. Electrophoresis was performed on 0.7% agarose gel The state of the DNA was confirmed with the naked eye, and electrophoresis photographs are shown in Fig. It can be seen from FIG. 1 that the DNA was extracted.

(2) Polymerase chain reaction (PCR) and DNA cloning

PCR of 16s ribosomal RNA gene Using 10 ng of extracted DNA as a template DNA, 1 unit of hot-start Taq polymerase (GenetBio, Korea), 4 ㎕ 10X PCR buffer, 2.5 mM MgCl2, 200 uM dNTP, Forward and reverse primers were mixed and sterilized distilled water was added to make a total of 40 μl PCR reaction solution.

PCR was performed by using GeneAmp PCR System 9700 Thermal Cycler (Applied Biosystems, USA) for denaturation at 96 ° C for 10 min, denaturation at 94 ° C for 30 sec, annealing at 55 ° C for 30 sec, and extension at 72 ° C for 1 min and 30 sec 30 cycles were performed and final extension was performed at 72 ° C for 30 minutes. After the PCR reaction was completed, the amplified product was stored at -20 ° C until use. The primers used in the PCR are shown in Table 1 below.

The primer sequence (5'-3 ') SEQ ID NO: Forward: AGAGTTTGATCMTGGCTCAG 2 Reverse: AAGGAGGTGATCCANCCRCA 3

After the PCR reaction, the polymerase chain reaction products were electrophoresed in 1% agarose gel (EtOAc) containing EtBr, and the PCR amplification was confirmed by a gel-bed system (Vilber Lourmat, France) The results are shown in Fig. It can be confirmed that PCR amplification is performed in FIG.

The cloning of 16s ribosomal DNA from amplified samples was cloned according to Enzynomics' manual using Enzynomics TOP cloner PCR cloning kit. Plasmid-transformed plasmids were extracted using AccuPrep Plasmid DNA Extraction Kit from BIONEER.

(3) DNA sequencing

After cycling PCR, 1 μl of 125 mM EDTA solution (pH 8.0) was added to the test tube containing the reactant, and 80 μl of 100% ethanol at -20 ° C was added thereto. The mixture was mixed up and down and stored at -20 ° C for 5 minutes. Then, centrifugation was performed at 14,000 × g for 10 minutes to remove ethanol, followed by drying at room temperature for 20 minutes. After drying, 15 μl of Hi-Di formamide (Applied Biosystems, USA) was added, and the mixture was treated at 95 ° C for 3 minutes, and then immediately quenched on ice to prepare a sample for sequencing. The prepared sample was sequenced using an Applied Biosystems 3130xl Genetic Analyzer (Applied Biosystems, USA) and confirmed to have the nucleotide sequence shown in SEQ ID NO: 1.

DNA sequencing was performed by DNA assemble and alignment using DNASIS® Max 3.0 (MiraiBio, USA) and genbank blast searching was performed to compare the DNA sequences of the microorganisms already reported in the gene bank. Maisse Serabijia nov. AGRO-02 was 99% similar to the standard strains Saccharomyces cerevisiae strain ySR127, Saccharomyces cerevisiae strain NCIM3186, Saccharomyces cerevisiae S288c RDN37-1, Saccharomyces cerevisiae YJM996, Saccharomyces cerevisiae YJM987 and Saccharomyces cerevisiae YJM984, and no perfectly matched microorganism was found.

Glucone Acetobacter Ska Riborance nov. AGRO-03 is a standard strain, Gluconacetobacter sp. SC-01, Gluconacetobacter saccharivorans strain AP3, Komagataeibacter saccharivorans strain LMG 1582, and Gluconacetobacter saccharivorans strain AP3 were 99% similar to each other and no perfectly matched microorganisms were found.

Experimental Example 2. Lactobacillus brevis nov. Estera measurement of AGRO-04

Lactobacillus brevis nov. Culture of AGRO-04

Lactobacillus brevis nov. AGRO-04 was cultured in MRS broth at 35 DEG C for 48 hours. (de Man, Rogosa & Sharpe, 1960).

Lactobacillus brevis nov. Preparation of AGRO-04 extract.

The cultured Lactobacillus brevis nov. AGRO-04 was centrifuged and the cells were collected, washed three times with 0.1 M phosphate buffer (pH 8.0), and suspended in 10 ml of physiological saline. Ballotini beads were placed and sonicated for 15-30 min using Soniprobe type I 130 A (Dawe Instruments Ltd.). The crushed cells were removed by centrifugation and allowed to stand at 20 ° C for 12 hours for clarification.

Lactobacillus brevis nov. Measurement of esterases in AGRO-04 extract

The esterase assay was performed using electrophoresis and spectrophotometer. The degree of degradation was measured at 405 nm using p-nitrophenylbutyrate (pNPB) as a substrate.

As a result, the esterase activity of the strain of the present invention is as shown in Table 2 below.

 lactic acid bacteria strains activity (units / ml) Lactobacillus casei 0.67 Lactobacillus plantarum 1.25 Lactobacillus brevis 1.78 Lactobacillus brevis nov.AGRO-04 2.16

Experimental Example 3. Effect of Vinegar Reduction by Sensory Test

Odor, Sweet taste, Sour taste, and Overall scores of the students who had knowledge about the vinegar of the parallel fermentation without parallel fermentation of lactic acid bacteria were evaluated by preference scale method (1 = very disliked, 5 = Normal, 9 = very good, Table 3).

Fermentation with lactic acid bacteria Black rice  Sensory evaluation of vinegar Treat. Sensory characteristics (0-9) Color acceptability Odor acceptability Sweet taste acceptability Sour taste acceptability Overall acceptability S1 ^‡ 5.33 ± 0.23 ^† 5.07 ± 0.22 6.67 + 0.13 5.92 + 0.18 5.67 ± 0.23 ^† S2 6.50 ± 0.17 5.60 ± 0.21 6.83 ± 0.16 4.17 ± 0.27 6.20 ± 0.17 S3 6.92 + 0.16 5.95 + 0.19 7.00 + - 0.17 4.42 + 0.16 7.17 ± 0.25 S4 6.00 0.20 5.90 + - 0.23 7.13 ± 0.14 4.92 ± 0.27 6.55 ± 0.27 S5 5.43 + 0.14 5.68 ± 0.28 7.35 ± 0.17 4.07 ± 0.17  7.45 0.26 ^† Each value represents the mean ± SE (n = 3)
^† Mean separation within each columns by Duncan's multiple range test at 5% level.
^{‡ S1: non-Lactic acid bacteria} S2: Lactobacillus casei, S3: Lactobacillus plantarum, S4: Lactobacillus brevis S5: Lactobacillus brevis nov. AGRO-04

Experimental Example 4. Measurement of anthocyanin and polyphenol contents

Lactobacillus Brevis nov. 5 ml of FROIN-Ciocalteau reagent was added to 5 ml of 10-fold dilution of AGRO-04 fermented black rice vinegar and 3 ml of 10% Na ₂ CO ₃ solution (w / v) And then colorimetrically determined at 700 nm. The total polyphenol content was determined from a calibration curve prepared using tannic acid as a reference material. The content of anthocyanin was measured by Lactobacillus brevis nov. 10 ml of black vinegar fermented in parallel with AGRO-04 strain was mixed with 40 ml of 0.1% HCl-80% MeOH solution (v / v) and extracted with shaking for 24 hours. The extracted pigment was centrifuged at 3,000 × g for 10 minutes, and the supernatant was quantified by colorimetry at 528 nm in 100 ml of 0.1% HCl-80% MeOH solution (v / v). Anthocyanin content was measured from a calibration curve prepared using cyanidin-3-glucoside as a reference material (Table 4).

Extraction processes Total polyphenol (/ / ml) Anthocyanin (/ / l) L. Casei 17.84 ± 2.02 † 508.44 + 32.26 L. plantarum 24.52 + - 3.01 1231.25 + - 44.32 L. brevis 36.10 ± 2.00 1527.69 + - 40.83 L. brevis nov.AGRP-04  40.15 ± 2.27 1676.23 + - 14.92

<110> RYU, II Hwan          JEON, Byung Hoon <120> LACTOBACILLUS BREVIS HAVING IMPROVED ABILITY FOR REDUCTING          OFF-FLAVOR OF VINEGAR AND PRODUCTIVITY OF ANTHOCYANIN, AND METHOD          FOR MANUFACTURING BLACK RICE VINEGAR USING THE SAME <130> PF170126 <160> 1 <170> KoPatentin 3.0 <210> 1 <211> 1449 <212> DNA <213> Lactobacillus brevis <400> 1 cctaatacat gcaagtcgaa cgagcttccg ttgaatgacg tgcttgcact gatttcaaca 60 atgaagcgag tggcgaactg gtgagtaaca cgtgggaaat ctgcccagaa gcaggggata 120 acacttggaa acaggtgcta ataccgtata acaacaaaat ccgcatggat tttgtttgaa 180 aggtggcttc ggctatcact tctggatgat cccgcggcgt attagttagt tggtgaggta 240 aaggcccacc aagacgatga tacgtagccg acctgagagg gtaatcggcc acattgggac 300 tgagacacgg cccaaactcc tacgggaggc agcagtaggg aatcttccac aatggacgaa 360 agtctgatgg agcaatgccg cgtgagtgaa gaagggtttc ggctcgtaaa actctgttgt 420 taaagaagaa cacctttgag agtaactgtt caagggttga cggtatttaa ccagaaagcc 480 acggctaact acgtgccagc agccgcggta atacgtaggt ggcaagcgtt gtccggattt 540 attgggcgta aagcgagcgc aggcggtttt ttaagtctga tgtgaaagcc ttcggcttaa 600 ccggagaagt gcatcggaaa ctgggagact tgagtgcaga agaggacagt ggaactccat 660 gtgtagcggt ggaatgcgta gatatatgga agaacaccag tggcgaaggc ggctgtctag 720 tctgtaactg acgctgaggc tcgaaagcat gggtagcgaa caggattaga taccctggta 780 gtccatgccg taaacgatga gtgctaagtg ttggagggtt tccgcccttc agtgctgcag 840 ctaacgcatt aagcactccg cctggggagt acgaccgcaa ggttgaaact caaaggaatt 900 gcgggggcc cgcacaagcg gtggagcatg tggtttaatt cgaagctacg cgaagaacct 960 taccaggtct tgacatcttc tgccaatctt agagataaga cgttcccttc ggggacagaa 1020 tgacaggtgg tgcatggttg tcgtcagctc gtgtcgtgag atgttgggtt aagtcccgca 1080 acgagcgcaa cccttattat cagttgccag cattcagttg ggcactctgg tgagactgcc 1140 ggtgacaaac cggaggaagg tggggatgac gtcaaatcat catgcccctt atgacctggg 1200 ctacacacgt gctacaatgg acggtacaac gagtcgcgaa gtcgtgaggc taagctaatc 1260 tcttaaagcc gttctcagtt cggattgtag gctgcaactc gcctacatga agttggaatc 1320 gctagtaatc gcggatcagc atgccgcggt gaatacgttc ccgggccttg tacacaccgc 1380 ccgtcacacc atgagagttt gtaacaccca aagccggtga gataaccttc gggagtcagc 1440 cgtctaagg 1449

Claims (8)

Lactobacillus brevis strain wherein the esterase activity of the strain is 2.0 units / ml or more when cultured in an MRS broth medium at a temperature of 35 캜 for 48 hours. The method according to claim 1,
The strain may be selected from the group consisting of Lactobacillus brevis nov. Lactobacillus brevis strain, AGRO-04 strain. The method according to claim 1,
Wherein the strain comprises the sequence of SEQ ID NO: 1. A composition comprising the strain of any one of claims 1 to 3, a culture thereof, a lysate thereof, or an extract thereof. 5. The method of claim 4,
Wherein the composition is for making vinegar. 6. The method of claim 5,
Wherein the vinegar comprises cereal fermented vinegar. The method according to claim 6,
Wherein the cereal comprises black rice. A method for producing vinegar, comprising culturing the strain of any one of claims 1 to 3.

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