KR20170078265A - LACTOBACILLUS BREVIS nov. AGRO-04 AS A NOVEL STRAIN AND USE THEREOF - Google Patents
LACTOBACILLUS BREVIS nov. AGRO-04 AS A NOVEL STRAIN AND USE THEREOF Download PDFInfo
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Abstract
The present invention relates to a novel strain Lactobacillus brevis nov. AGRO-04 and its use. More specifically, the present invention relates to a novel strain Lactobacillus brevis nov (Lactobacillus brevis) which is excellent in estrase producing ability and capable of reducing irritating vinegar stalks and strengthening anthocyanin components in black rice. . AGRO-04 and its use. The novel strain of the present invention, Lactobacillus brevis nov. AGRO-04 has excellent esterase-producing ability and thus remarkably reduces irritant vinegar stalks and raises anthocyanin content in vinegar, thus making it possible to produce vinegar having excellent sensory characteristics and health promoting effect.
Description
The present invention relates to a novel strain Lactobacillus brevis nov. AGRO-04 and its use. More specifically, the present invention relates to a novel strain Lactobacillus brevis nov (Lactobacillus brevis) which is excellent in estrase producing ability and capable of reducing irritating vinegar stalks and strengthening anthocyanin components in black rice. . AGRO-04 and its use.
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 obstructing 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 alcohol and acetic acid fermentation.
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 3123480 Method of adsorbing vinegar fermented with cereals, fruits and / or sugars to nonionic porous synthetic resin, adding specific raw materials or moromi (Japan, 06-133756, 06-339365) and adding masking agent (2000- The application of high pressure steam (JP 60137281) and removal of the dehydrating sheet by semi-permeable external membranes (US 4765997) have been patented, but the reduction effect of vinegar is insufficient not. (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.
DISCLOSURE OF THE INVENTION The present inventor has found a novel lactic acid bacterium having an esterase activity in order to solve the above problems of the prior art and completed the present invention.
SUMMARY OF THE INVENTION Accordingly, an object of the present invention is to provide Lactobacillus brevis nov. AGRO-04.
Another object of the present invention is to provide a method for producing Lactobacillus brevis nov. AGRO-04 as an active ingredient.
Another object of the present invention is to provide a method for producing Lactobacillus brevis nov. AGRO-04 is used to provide vinegar with reduced vinegar odor.
A final object of the present invention is to provide a fermented alcohol fermentation product comprising lactobacillus and Lactobacillus brevis nov. AGRO-04, which comprises the step of fermenting the vinegar with reduced vinegar content.
In order to achieve the object of the present invention, the present invention provides Lactobacillus brevis nov. AGRO-04 is provided.
In order to achieve the above-mentioned other objects, the present invention provides a method for producing Lactobacillus brevis nov. AGRO-04 as an active ingredient.
In order to achieve the above-mentioned further object, the present invention relates to a process for producing lactic acid bacteria and Lactobacillus brevis nov. AGRO-04 provides fermented vinegar.
In order to achieve the above-mentioned object, the present invention relates to a fermentation product obtained by fermenting an alcohol fermentation product with lactic acid bacteria and Lactobacillus brevis nov. AGRO-04, which comprises the step of fermenting the vinegar with reduced vinegar.
The novel strain of the present invention, Lactobacillus brevis nov. AGRO-04 has excellent esterase-producing ability and thus remarkably reduces irritant vinegar stalks and raises anthocyanin content in vinegar, thus making it possible to produce vinegar having excellent sensory characteristics and health promoting effect.
Brief Description of the Drawings Figure 1 is a schematic representation of a novel Lactobacillus brevis nov. It is the result of electrophoresis of DNA extracted from AGRO-04.
Figure 2 shows the results of a novel Lactobacillus brevis nov. Strain producing PCR-amplified highly active esterase. The result of electrophoresis of AGRO-04 DNA.
Fig. 3 shows the results of determination of the estrase production ability of various lactic acid bacteria by electrophoresis.
Hereinafter, the present invention will be described in detail.
The present invention relates to Lactobacillus brevis nov., Which has excellent esterase producing ability. AGRO-04 is provided.
The present invention relates to Lactobacillus brevis nov. AGRO-04 as an active ingredient.
The present invention relates to a process for producing lactic acid bacteria and Lactobacillus brevis nov. AGRO-04 provides fermented vinegar.
The present invention relates to a process for the fermentation of alcoholic fermentation products with lactic acid bacteria and Lactobacillus brevis nov. AGRO-04, which comprises fermenting the vinegar with reduced vinegar odor.
The strain is selected from the group consisting of Lactobacillus brevis nov. AGRO-04 It is possible to cultivate under general culture condition, and further, it is excellent in the ability to produce esterase.
Hereinafter, the present invention will be described in more detail with reference to Examples. It is to be understood that the scope of the present invention is not limited by these examples in accordance with the gist of the present invention, and it is to be understood by those skilled in the art that the present invention is not limited thereto It will be obvious.
<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 and dipped for 24 hours. Thereafter, germinated brown rice was grown at a size of about 100 to 120 mesh . 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 then the mixture was stirred at 100 ° C for 2 hours to grow Saccharomyces cerevisiae nov. (9 log cfu / g) and 0.4-0.5% (w / v) of glucoamylase (Saczyme ㄾ, Gusmer Enterprises, Inc.) relative to 100 parts by weight of the black rice added with AGRO- The process and the alcohol fermentation process were carried out simultaneously. The amount of the glucoamylase enzyme used was selected so that the soluble solids content was 20 to 30 Brix.
Alcohol fermentation was carried out at 25 ~ 35 ℃ for 95 ~ 120 hours to obtain an alcohol fermentation broth having a sugar content of 11-12 brix 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
To the alcoholic fermentation broth, Gluconacetobacter saccharivorans nov. AGRO-03 was inoculated at a concentration of 9 log cfu / g in an amount of 5 parts by weight based on 100 parts by weight of the alcoholic fermentation broth, thereby conducting acetic acid fermentation. At this time, Lactobacillus brevis nov. AGRO-04 was inoculated at the same dose as the above-mentioned 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 clear and clear germinated brown rice vinegar.
<Experimental Example>
Experimental Example 1: Lactobacillus brevis (
Lactobacillus brevis
) nov. Characterization 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 300 μL was transferred to HTS, Millipore, USA, and vacuum (-25 inches. MmHg) was applied for 1 minute in clean air (Millipore, USA). 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 template DNA, 1 unit of hot-start Taq polymerase (GenetBio, Korea), 4 ㎕ 10X PCR buffer, 2.5 mM MgCl 2 , 200 uM dNTP and 5 pM Of forward and reverse primers were mixed and sterilized distilled water was added to make a total of 40 μl of PCR reaction solution.
PCR was performed by denaturing at 96 ° C for 10 minutes, denaturing at 94 ° C for 30 seconds, annealing at 55 ° C for 30 seconds, and extension at 72 ° C for 1 minute and 30 seconds using GeneAmp PCR System 9700 Thermal Cycler (Applied Biosystems, USA) 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.
After the PCR reaction, PCR products were amplified by gel electrophoresis in a 1% agarose gel containing EtBr (SeaKem® LE agarose, USA), and then PCR amplification was carried out using a Gelek 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. After centrifugation at 14,000 ㅧ g for 10 minutes, the ethanol was removed and dried 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 samples were sequenced using an Applied Biosystems 3130xl Genetic Analyzer (Applied Biosystems, USA).
DNA sequence alignment 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. No matching microorganisms were found.
Experimental Example 2. Lactobacillus brevis nov. Estera measurement of AGRO-04
Various lactic acid bacteria were cultured in MRS broth (de Man, Rogosa & Sharpe, 1960) at 30 ° C for 18 hours. Then, the lactic acid bacteria were collected by centrifugation, washed three times with cold physiological saline or 0.1M phosphate buffer (pH 0.8), and suspended in saline. Ballotini beads were added to the suspension of the lactic acid bacteria, and the lactic acid bacteria were physically decomposed for 15 to 30 minutes. Ultrasonic pulverization was performed using Soniprobe type I 130A (Dawe Instruments Co., Ltd.) Were purified by centrifugation and stored at -20 ° C. Bovine plasma albumin was used as a reference material and it was found by biuret micro-method (Itzhaki & Gill, 1964 ) that the extract contained 10 to 20 mg of protein per ml.
The extract was subjected to electrophoresis analysis (Lund, 1965) on a polyacrylamide gel. The extract was filtered with 3MM Whatman filter paper, and loaded onto a polyacrylamide gel. The results are shown in FIG. The extracted esterase was quantitatively analyzed using Bradford Protein Assay (He, 2011), and the results are shown in Table 2 below.
It can be seen from Table 1 that the ability of Lactobacillus brevis nov. AGRO-04 to produce estrase is remarkably high.
Experimental Example 3. Determination of Vinegar Reducing Effect 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 = Usually, 9 = very good). The evaluation results are shown in Table 3 below.
† 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.
In Table 4, Lactobacillus brevis nov. The antioxidant contents of polyphenols and anthocyanins in black rice vinegar fermented with AGRO-04 were much higher than those of black rice vinegar.
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| KR20220101230A (en) * | 2021-01-11 | 2022-07-19 | 전북대학교산학협력단 | Kombucha vinegar containing Rubus coreanus and manufacturing method thereof |
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| KR20220101230A (en) * | 2021-01-11 | 2022-07-19 | 전북대학교산학협력단 | Kombucha vinegar containing Rubus coreanus and manufacturing method thereof |
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