KR20220089354A - Novel kimchi lactic acid bacteria lactiplantibacillus plantarum afy-10 and alcoholic liquors comprising the same - Google Patents

Abstract

Disclosed herein is a novel kimchi lactic acid bacterium Lactiplantibacillus plantarum AFY-10. In addition, the present application discloses a method for producing beer using the Lacti plantibacillus plantarum AFY-10. The method comprises the steps of saccharifying the pulverized malt to prepare wort; After cooling the wort, the step of souring by inoculating the Lacti plantibacillus plantarum AFY-10 strain having an accession number KACC81141BP; After souring, adding hops and boiling; And after cooling the wort after boiling, inoculating yeast to alcohol fermentation.

Description

A novel kimchi lactic acid bacterium Lacti plant bacillus plantarum AFY-10 and alcoholic beverages containing the same

Disclosed herein is a novel kimchi lactic acid bacterium, Lacti plant bacillus plantarum AFY-10, and alcoholic beverages containing the same.

The type of beer varies depending on the fermentation method, such as whether it is a top fermentation method or a bottom fermentation method. In general, when producing ale or lager beer, lactic acid and other yeasts or microorganisms are thoroughly excluded, whereas when making sour beer, it is aged in oak barrels that help them reproduce, so it has a strong sour taste. will have

Lactic acid bacteria have been used to control the microbial community growing on the malt surface when acidifying wort for brewing sour beer or preparing acid malt. Along with yeast and lactobacilli, lactic acid bacteria have been used to make traditional sour beers such as Berliner Weisse, Belgian Gueuze and Lambic in Germany. The traditional sour beer manufacturing method is brewed in an open environment, and since wild-derived yeast and lactic acid bacteria or acetic acid bacteria are involved at the same time, the fermentation process is complicated, takes a long time, and it is difficult to manufacture beer of uniform quality. have. Recently, as an alternative method to overcome the shortcomings of these traditional methods, studies have been reported on using a lactic acid starter to make the quality uniform, and at which stage of the fermentation process to inoculate the lactic acid bacteria.

The lactic acid bacteria used in sour beer are usually Lactobacillus , Lactococcus , Leuconostoc , and Pediococcus genera. Sour beer is prepared by inoculating lactic acid bacteria before and after the boiling process of boiling wort with hops. Acidifying the wort after boiling has the advantage of releasing desirable volatile aroma components through lactic acid fermentation, while reducing or eliminating the isomerization of DMS (dimethyl sulfide). have it In addition, since lactic acid bacteria may cause spoilage of non-sour beer, it is preferred to sour before the boiling process to reduce cross-contamination when the temperature during the brewing process drops.

Recently, as the craft beer industry is increasing in Korea, various craft beers are being sold, and sour beer products using lactic acid bacteria are being launched. However, all microorganisms used to manufacture them depend on imports, and there are no domestic studies.

KR 10-2019-0000970 A

In one aspect, the present disclosure aims to provide a novel kimchi lactic acid bacterium Lactiplantibacillus plantarum AFY-10 strain.

In another aspect, the present disclosure aims to provide a liquor composition comprising the novel strain.

In another aspect, an object of the present disclosure is to provide a method for preparing alcoholic beverages using the novel strain.

In one aspect, the present disclosure provides a Lactiplantibacillus plantarum AFY-10 strain having an accession number of KACC81141BP.

In an exemplary embodiment, the strain may be a kimchi-derived strain.

In an exemplary embodiment, the strain may be for alcohol production.

In an exemplary embodiment, the alcoholic beverage may be beer.

In an exemplary embodiment, the beer may be sour beer.

In an exemplary embodiment, the strain may have an acid-producing ability in which the pH value of the medium is 3.41 or less.

In an exemplary embodiment, the strain may not produce extracellular polysaccharide (Exopolysaccharide, EPS) and diacetyl.

In an exemplary embodiment, the strain may be sensitive to hops.

In an exemplary embodiment, the strain may have β-glucosidase activity.

In another aspect, the present disclosure provides a lactic acid bacteria composition for souring for the production of alcoholic beverages comprising the strain.

In another aspect, the present disclosure relates to the strain; Or it provides a liquor composition comprising a lactic acid bacteria composition for souring for the manufacture of liquor containing the strain.

In an exemplary embodiment, the alcoholic beverage may be sour beer.

In an exemplary embodiment, the alcoholic beverage composition may have one or more of the following values:

a) a pH of 3.2 or less;

b) a diacetyl concentration of 0.6 mg/L or less;

c) a free amino nitrogen (FAN) concentration of 80 mg/L or less; and

d) a maltotriose concentration of at least 1.5 g/L.

In another aspect, the present disclosure provides a method of making a liquor composition comprising preparing the liquor composition.

In an exemplary embodiment, the manufacturing method comprises the steps of saccharifying the pulverized malt to prepare wort; After cooling the wort, the deposit number is KACC81141BP Lactiplantibacillus plantarum ( Lactiplantibacillus plantarum ) Souring (souring) by inoculating the strain AFY-10; After souring, adding hops and boiling; And after cooling the boiling wort, it may be one comprising the step of inoculating yeast to alcohol fermentation.

In an exemplary embodiment, the souring may be carried out at 35 to 45 °C.

In an exemplary embodiment, the souring may be performed for 16 to 48 hours.

In one aspect, the technology disclosed in the present disclosure is effective in providing a novel kimchi lactic acid bacterium Lactiplantibacillus plantarum ( Lactiplantibacillus plantarum ) AFY-10 strain.

In another aspect, the technology disclosed in the present disclosure is effective in providing a liquor composition comprising the novel strain.

In another aspect, the technology disclosed in the present disclosure has the effect of providing a method for preparing alcoholic beverages using the novel strain.

1 shows the 16S rRNA nucleotide sequence (SEQ ID NO: 1) of Lactiplantibacillus plantarum AFY-10.
Figure 2 shows the phylogenetic tree of three strains used for the production of sour beer based on the 16S rRNA gene nucleotide sequence.
Figure 3 shows a sour beer manufacturing process diagram according to an embodiment.
Figure 4 shows the change in the number of lactic acid bacteria, pH, lactic acid, acetic acid concentration according to souring fermentation time.
5 is a heat map showing the analysis results of fragrance components after souring and alcohol fermentation by HS-SPME-GC-TOF/MS.
6 shows the results of PLS-DA analysis using multivariate statistical analysis.

Hereinafter, the present disclosure will be described in detail.

In one aspect, the present disclosure provides a Lactiplantibacillus plantarum AFY-10 strain having an accession number of KACC81141BP.

In an exemplary embodiment, the strain may have a 16S rRNA base sequence of SEQ ID NO: 1.

In an exemplary embodiment, the strain may be a kimchi-derived strain.

In an exemplary embodiment, the strain may be for alcohol production.

In an exemplary embodiment, the alcoholic beverage may be beer.

In an exemplary embodiment, the beer may be sour beer. Sour beer is characterized in that it has a sour taste due to lactic acid, a major metabolite produced by lactic acid bacteria, and, for example, may have a pH of about 3.9 or less and a lactic acid concentration of about 3 to 6 g/L.

In the production of sour beer, the souring process is usually performed within 48 hours, so the acid-forming ability is one of the most important characteristics of lactic acid bacteria for sour beer production. The lactic acid bacteria according to the present disclosure have excellent acid-producing ability, and in an exemplary embodiment, the strain may have an acid-producing ability in which the pH value of the medium is 3.41 or less or 3.40 or less.

In an exemplary embodiment, the strain may not produce extracellular polysaccharide (Exopolysaccharide, EPS) and diacetyl. EPS is a viscous substance such as oil that degrades beer quality, and diacetyl causes the beer to become acidic, cloudy, and artificially buttery. Therefore, it is possible to further improve beer quality by using lactic acid bacteria that do not produce EPS and diacetyl in the production of sour beer.

In an exemplary embodiment, the strain may be sensitive to hops. Hop sensitivity as used herein means reduced growth, but not failure to grow, in the presence of hops. In general, the isoα-acids component of hops, which gives beer a bitter taste, is present in beer at about 17 to 55 ppm, and this component has an antibacterial effect on most bacteria, but some hop-resistant microorganisms have hop resistance. It is known to cause beer spoilage by having 8 to 20 times higher hop resistance than microorganisms without it. Therefore, hop-sensitive microorganisms rather than hop-tolerant microorganisms are more suitable for sour beer production.

In an exemplary embodiment, the strain has the β-glucosidase activity to further improve the aroma properties of sour beer. Specifically, ethyl decanoate (Ethyl decanoate), ethyl 9-decanoate (ethyl 9-decanoate), phenethyl acetate (phenetyl acetate), ethyl laurate (ethyl laurate) , to produce phenethyl alcohol, octanoic acid, ethyl palmitate, ethyl 9-hexadecenoate and n-decanoic acid In particular, phenethyl alcohol, which has a sweet and floral scent, had the highest VIP value of 5.07.

The Lactiplantibacillus plantarum AFY-10 strain disclosed herein has a high acid production rate, thereby rapidly reducing pH and providing low pH, high foam stability, low diacetyl content, as well as β-glucosidase activity It has the effect of expressing a large amount of fragrance components providing excellent sensory properties. The Lacti plantibacillus plantarum AFY-10 strain is suitable for the production of sour beer, and provides an effect of improving the problem of difficulty in producing beer of uniform quality during the production of conventional sour beer.

In an exemplary embodiment, when preparing sour beer with the Lacti plant bacillus plantarum AFY-10 strain, the pH of the wort is adjusted to 4.5, and then the Lacti plantibacillus plantarum AFY-10 strain is 1 x 10 ⁷ CFU When the wort is inoculated to a /ml concentration and the souring process is performed at 40 °C for 40 hours, it has the effect of reaching pH 3.5 within 16 hours.

In an exemplary embodiment, when preparing sour beer with the Lactiplantibacillus plantarum AFY-10 strain, after adjusting the pH of the wort to 4.5, the strain is inoculated into the wort to a concentration of 1 x 10 ⁷ CFU/ml. And when the souring process is performed at 40 ° C. for 40 hours, there is an effect of showing 4.0 g/L or more of lactic acid and 0.1 g/L or more of acetic acid.

In another aspect, the present disclosure provides a lactic acid bacteria composition for souring for the production of alcoholic beverages comprising the strain.

In another aspect, the present disclosure relates to the strain; Or it provides a liquor composition comprising a lactic acid bacteria composition for souring for the manufacture of liquor containing the strain.

In an exemplary embodiment, the alcoholic beverage composition may have one or more of the following values:

a) a pH of 3.2 or less, 3.16 to 3.18, or 3.17;

b) a diacetyl concentration of 0.6 mg/L or less, 0.55 mg/L or less, or 0.49 to 0.51 mg/L;

c) a free amino nitrogen (FAN) concentration of 80 mg/L or less, 73 to 77 mg/L, 74 to 76 mg/L, or 75 to 76 mg/L; and

d) a maltotriose concentration of at least 1.5 g/L, 1.6-2.1 g/L, 1.7-2.0 g/L, 1.8-2.0 g/L, or 1.9-2.0 g/L.

The FAN concentration affects the bitter taste, turbidity, foam stability, etc. of beer, thereby reducing beer quality and stability. When using a strain according to the present disclosure, the FAN concentration can be lowered to improve beer quality and stability

In another aspect, the present disclosure provides a method of making a liquor composition comprising preparing the liquor composition.

In an exemplary embodiment, the manufacturing method comprises the steps of saccharifying the pulverized malt to prepare wort; After cooling the wort, the deposit number is KACC81141BP Lactiplantibacillus plantarum ( Lactiplantibacillus plantarum ) Souring (souring) by inoculating the strain AFY-10; After souring, adding hops and boiling; And after cooling the boiling wort, it may be one comprising the step of inoculating yeast to alcohol fermentation.

In another aspect, the present disclosure provides a method for producing beer, comprising preparing beer using a Lactiplantibacillus plantarum AFY-10 strain having an accession number of KACC81141BP.

In an exemplary embodiment, the manufacturing method comprises the steps of saccharifying the pulverized malt to prepare wort; After cooling the wort, the deposit number is KACC81141BP Lactiplantibacillus plantarum ( Lactiplantibacillus plantarum ) Souring (souring) by inoculating the strain AFY-10; After souring, adding hops and boiling; And after cooling the boiling wort, it may be one comprising the step of inoculating yeast to alcohol fermentation.

In an exemplary embodiment, the Lacti plantibacillus plantarum AFY-10 strain may be inoculated after adjusting the pH of the wort to 4.5 or less.

In an exemplary embodiment, the souring may be carried out at 35 to 45 °C.

In an exemplary embodiment, the souring may be performed for 16 to 48 hours.

In an exemplary embodiment, the boiling may be carried out at 100 °C.

In an exemplary embodiment, the alcoholic fermentation may be carried out at 18 to 22 °C.

In an exemplary embodiment, the alcoholic fermentation may be carried out for 1 to 2 weeks.

In an exemplary embodiment, the manufacturing method may further include aging at 4° C. for 1 to 2 weeks after alcoholic fermentation.

Hereinafter, the present disclosure will be described in more detail through examples. These examples are only for illustrating the present disclosure, and it will be apparent to those of ordinary skill in the art that the scope of the present disclosure is not to be construed as being limited by these examples.

Example 1. Lactobacillus isolation and identification

Four types of traditional fermented kimchi were collected from a farm in Gangwon-do, spread on BCP (Bromocresol Purple, MRS agar (BD Difco)), and cultured at 37°C for 3 days under anaerobic conditions using Gaspak (BD). After visually comparing the cultured single colonies, morphological characteristics such as color and size were compared and selected, and kimchi lactic acid bacteria were obtained by pure separation using a streaking method under the same medium and conditions.

For molecular phylogenetic analysis of the isolated microorganism, genomic DNA was extracted using AccuPrep ^® genomic DNA extraction kit (Bioneer, Korea), and 785F (SEQ ID NO: 2, 5'-GGATTAGATACCCTGGTA-3') and 907R were requested by Macrogen. (SEQ ID NO: 3, 5'-CCGTCAATTCMTTTGAGTTT-3') After amplifying DNA by polymerase chain reaction (PCR) using a primer, the 16S rRNA gene was used with a DNA analyzer (3730xl DNA analyzer, Thermo Fisher Scientific). The nucleotide sequence was analyzed. The nucleotide sequence analysis results were compared with the GeneBank database of the Basic Local Alignment Search Tool (BLAST) provided by the National Center for Biotechnology Institute (NCBI) and homology using EzCloud (https://eztaxon-e.ezbiocloud.net). was later identified. The analyzed 16S rRNA gene nucleotide sequence was edited using SeqMan software (DNAStar) and then inputted into NCBI's Nucleotide BLAST to collect nucleotide sequence information with a relatively close genetic distance. The nucleotide sequences collected through the NCBI database were aligned using the BioEdit program, rearranged using MEGA 10.1, and a phylogenetic tree was drawn using the neighbor-joining method.

Example 2. Lactobacillus activity evaluation

Acid production, extracellular polysaccharide production, diacetyl production, hop resistance, and β-glucosidase activity of the lactic acid bacteria isolated and identified in Example 1 were evaluated for the search for sour beer lactic acid bacteria. All screening experiments were performed in 96-well plates, and cultured under anaerobic conditions using Gaspak (BD).

Acid production was evaluated using the principle that 0.01% of bromocresol green (Sigma Aldrich) was added to the MRS medium, and the pH of the medium changed from green to yellow as the pH of the medium decreased. For the evaluation of exopolysaccharide production, lactic acid bacteria were inoculated into DME Agar (8.5°P dry malt extract, 1.5% agar) with 2% sucrose and cultured at 37 °C for 48 hours to produce slime around the colonies. This produced strain was determined as an EPS production-positive strain. Diacetyl production was evaluated in Formulated fruit simulation medium (FSM) broth (1% glucose, 1% fructose, 0.5% sorbitol, 0.2% malic acid, 0.02% MgSO ₄ 7H ₂ O, 0.005% MnSO ₄ H ₂ O, 0.1 % K ₂ HPO ₄ , 0.01% EDTA, 0.2% ammonium citrate, 1% vegetable peptone, 0.1% Tween 80, pH 6.1) Inoculate 20 μl of lactic acid bacteria into 200 μl of medium and incubate at 30 ℃ for 48 hours, then fresh 96 - Transfer 100 μl to a well plate, aliquot 50 μL of 4% α-naphthol solution (w/v), suspend it, add 30% Potassium hydroxide, and react at 30° C. for 30 minutes to turn pink. It was judged to be production positive. Inoculate lactic acid bacteria on DME agar containing 30% iso-Hop (BarthHaas) so that the concentration of iso α-acid becomes 1, 5, 10, and 20 IBU (International Bitterness Unit, iso α-acid ppm) for hop resistance evaluation. and incubated at 37 °C for 48 hours. For evaluation of β-Glucosidase activity, 0.5% esculin and 1.0% ferric ammonium citrate were added to DME agar by applying the esculin agar method, and then inoculated with lactic acid bacteria and cultured at 37 °C for 48 hours. Strain producing a black ring (black zone) was determined as a positive strain.

No. strain name Species acid production EPS production Diacetyl production Hop
tolerance β-glucosidase activity One L193 Lactiplantibacillus pentosus 3.47 - - +++++/+ - 2 L196 Latilactobacillus curvatus 3.48 +++ - +++++/- - 3 L198 Lactiplantibacillus pentosus 3.45 - - +++++/- - 4 L208 Lactiplantibacillus pentosus 3.39 +++++ - +++++/- - 5 L209 Latilactobacillus curvatus 3.45 - - +++++/- - 6 L210 Lactiplantibacillus pentosus 3.50 - - +++++/- - 7 L213 Lactiplantibacillus pentosus 3.42 - - +++++/+ + 8 L217 Latilactobacillus curvatus 3.46 - - +++++/+ - 9 L221 Lactiplantibacillus plantarum 3.49 - - +++++/+ + 10 L222 Lactiplantibacillus pentosus 3.45 - - +++++/+ + 11 L229 Lactiplantibacillus pentosus 3.45 - - +++++/+ - 12 L237 Lactiplantibacillus plantarum 3.46 - - +++++/+ ++ 13 L240 Limosilactobacillus fermentum 3.42 - - +++++/+ ++ 14 L241 Lactiplantibacillus plantarum 3.43 - - +++++/+ - 15 L257 Lactiplantibacillus plantarum 3.46 - - +++++/+ - 16 L258 Lactiplantibacillus pentosus 3.47 - - +++++/+ - 17 L261 Lactiplantibacillus plantarum 3.45 - - +++++/+ + 18 AFY-10 Lactiplantibacillus plantarum 3.40 - - +++++/+ ++

1) Acid production: pH value

2) EPS production: as the production of slime substances, no result (-), 1 mm (+), 2 mm (++), 3 mm (+++), 4 mm (++++), 5 mm (+ ++++)

3) Diacetyl production : Reaction color, no result (-), slightly light pink (+), light pink (++), pale pink (+++), pink (+++++), dark pink (+ ++++)

4) Hop tolerance: colony size (5 IBU/10 IBU), no result (-), 1 mm (+), 2 mm (++), 3 mm (+++), 4 mm (++++) ), 5 mm (++++++)

5) β-glucosidase activity: black ring size, no result (-), 1 mm (+), 2 mm (++), 3 mm (+++), 4 mm (++++), 5 mm (++++++)

Acid-producing ability is one of the most important characteristics of lactic acid bacteria for sour beer, since souring is usually completed within 48 hours when souring. -10 strain and showed high acid-producing ability.

In addition, as a result of confirming the production of EPS and diacetyl to confirm whether the isolated lactic acid bacteria act as a beer spoilage microorganism, only the L196 and L208 strains produced EPS, and it was found that diacetyl was not produced in all strains. It has been reported that some lactobacilli, known as beer spoilage microorganisms, produce diacetyl, which acidifies, turbids, and has an artificial buttery flavor in beer, and reduces beer quality by producing EPS, an oily mucilage substance.

In addition, as a result of hop resistance evaluation, all 18 strains had excellent growth at a hop concentration of 5 IBU, but the growth of all strains at a double concentration of 10 IBU was remarkably reduced to 1/5 level. It is not that the isolated lactic acid bacteria cannot grow at all in an environment with hops, but it is considered that they have hop-sensitive, not hop-resistant, hop-sensitive since growth decreases when IBU is increased. Hop-sensitive strains are preferred over hop-resistant strains because the lactic acid bacteria used in sour beer should not act as a biological hazard for beer production. All 18 strains showed no hop resistance, suggesting no risk of beer spoilage.

On the other hand, β-glucosidase is an enzyme that breaks down glycosides and converts them to non-glycosides. In wine production, lactic acid bacteria with β-glucosidase activity are known to have the ability to convert unflavored glycosidic aroma precursors to fragrant non-glycosides. . In the production of sour beer, β-glucosidase activity of lactic acid bacteria was judged to have an effect on the generation of aroma components, so the β-glucosidase activity was evaluated. As a result, L213, L221, L222, L237, L240, L261 and AFY-10 lactic acid bacteria were found to have β-glucosidase activity.

Finally, L213, L240, and AFY-10 strains have high acid-producing ability, do not produce EPS and diacetyl, and exhibit sensitivity to hops and β-glucosidase activity. It was selected as a candidate strain for beer.

Example 3. sour beer making

Put the pulverized malt Pilsen Malt 2-Row (Briess malt & Ingredients co. USA) into a saccharification tank, pour distilled water slowly, and react at 65 ℃ for 45 minutes, 72 ℃ for 15 minutes, and 78 ℃ for 2 minutes to saccharify so that the specific gravity becomes 1.040. did After sterilizing the wort at 100° C. for 5 minutes, it was cooled to 40° C. using a cooler, and the pH of the wort was adjusted to 4.5 by adding 88% lactic acid (Sigma Aldrich). Kimchi lactic acid bacteria (L213, L240, AFY-10) and commercial lactic acid bacteria Lactiplantibacillus pre-cultured at 40 ℃ for 24 hours in DME broth (8.5°P dry malt extract) plantarum (Wildbrew Sour Pitch, Lallemnad) was inoculated into the wort so as to have a concentration of 1 x 10 ⁷ CFU/ml, respectively. The souring process was performed at 40 °C for 40 hours. After souring, hop pellets (Citra hop, 13.1% α-acids) were added at a concentration of 0.4 g/L and boiled at 100° C. for 60 minutes, and then the wort was cooled to less than 25° C. using a cooler. For alcoholic fermentation, commercially available yeast Saccharomyces cerevisiae US-05 (Fermentis) was inoculated at a concentration of 1 x 10 ⁷ CFU/ml and fermented at 20 ° C. for 14 days to prepare sour beer.

Example 4. sour Beer characterization

The properties of the sour beer prepared in Example 3 were evaluated and compared as follows.

The number of lactic acid bacteria was counted by inoculating 1 ml of a sample diluted with 0.85% NaCl solution on a lactic acid bacteria count plate (3M) and culturing for 2 days at 37°C using Gaspak (BD).

The pH was measured using a pH meter (Mettler Toledo). Plato was measured using a Plato measuring machine (Atago). For the alcohol content (ABV) and specific gravity, the sample stock solution was filtered with diatomaceous earth, 0.5 g of diatomaceous earth was added per 50 ml of the sample, and then filtered using filter paper (5A, Adventec), followed by an alcohol meter (ALEX 500, Anton Paar) ) was used for measurement.

The degree of fermentation was calculated by measuring the initial gravity (OG) and final gravity (FG) of the beer using an alcohol meter (ALEX 500, Anton Paar) and substituting it in the formula below.

Fermentation degree (%) = (OG-FG)/(OG-1) x 100

Diacetyl concentration was measured by a colorimetric method using a spectrophotometer (EPOCH2, BioTek) at a wavelength of 420 nm. After acidification by adding acetic acid to 10 ml of 0.175 M iso-niazide solution, 5 ml of 2.9 × 10 ^-4 M diacetyl solution and 1 ml of 0.15 M ZR(IV) salt were added. After adjusting the pH to 1.7 by mixing 3 M HCl and 4 M NaOH, the prepared solution was transferred to a 50 ml diametrical volumetric flask and diluted with purified water. After mixing with 25 ml of sample and 70 ml of saturated NaCl solution, it was distilled until 20-30 ml of isoniazide solution was collected. This value was compared with the calibration curve to determine the diacetyl content.

For foam stability, 50 ml of the sample stock solution was poured into a 100 ml burette, and after 30 seconds, the lower layer other than the foam was removed by opening the cock, then closing the cock again and allowing the foam to break for 230 seconds. At this time, the amount of broken bubbles (b) and the amount of remaining bubbles (c) were measured and calculated. The formula for foam stability is as follows.

Foam stability = 230/{2.303log[(b+c)/c]}

The FAN concentration was measured by the manual method using the Free Amino Nitrogen Assay Kit (Bio Assay Systems) after diluting the sample 50-fold with sterile distilled water. After adding 150 μl of a mixture of 150 μl of Reagent A and 5 μl of Reagent B to 5 μl of the sample diluted 50-fold, the mixture was reacted at 100° C. for 10 minutes. After cooling to room temperature, 100 μl of each was dispensed into a 96-well plate by centrifugation within 1 minute, and absorbance was measured with a spectrophotometer (EPOCH2, BioTek) at a wavelength of 575 nm. As a standard solution, glycine (Sigma Aldrich) was used.

The organic acid was used after filtering the sample stock solution with a 0.45 μm filter. HPLC (1260 infinity II, Agilent) was used as an analytical instrument, Kinetex C18 (4.6 X 250 mm, 5 μm, Phenomenex) was used for the column, and UHPLC C18 (AJ0-8768, Phenomenex) was used for the guard column. Chromatography was performed at a flow rate of 0.7 ml/min using a column temperature of 25° C., a sample injection amount of 10 μl, and a mobile phase of 25 mM potassium phosphate, and absorbance was measured at a wavelength of 210 nm with a UV detector.

As a result of souring before the boiling process of adding hops using lactic acid bacteria selected as candidate strains for sour beer, the change in the number of lactic acid bacteria, pH, lactic acid, and acetic acid concentration according to the souring fermentation time is shown in FIG.

Looking at the number of lactic acid bacteria, all four strains increased the number of lactic acid bacteria as the fermentation time increased. At the time of souring, L213 strain had the highest number of lactic acid bacteria at 1 x 10 ⁹ CFU/ml, AFY-10 strain 7 x 10 ⁸ CFU/ml, L240 strain 5 x 10 ⁸ CFU/ml, commercially available lactic acid bacteria Sour Pitch was found to be 2 x 10 ⁸ CFU/ml. AFY-10 strain was confirmed to grow more than three times higher despite being the same species as Sour Pitch used as a commercial strain.

Sour beer has a sour taste due to lactic acid, a major metabolite produced by lactic acid bacteria, and is known to have a pH of 3.3-3.9 and a lactic acid concentration of about 3-6 g/L. After all souring, the pH was all lower than 3.3. The fastest pH drop was the AFY-10 strain, which reached pH 3.5 within 16 hours. Sour Pitch took 4 hours more to reach pH 3.5 compared to AFY-10 strain at pH 3.5 in 20 hours of souring. The L213 and L240 strains had a pH of 3.37 and 3.33, respectively, after 40 hours, and the L240 strain dropped the pH at the lowest rate.

As a result of checking lactic acid and acetic acid according to fermentation time, the lactic acid of sour pitch and L240 strains were 2.24 g/L and 2.15 g/L, respectively, at 16 hours. The L213 strain and the L240 strain were similar to pH and also had a slower rate of lactic acid production compared to the AFY-10 and Sour Pitch strains. At 40 hours after souring, lactic acid above 3.0 g/L was produced, and AFY-10 (4.43) > sour pitch (3.65) > L213 (3.44) > L240 (3.17). Acetic acid was lower than 0.3 g/L in all four strains, and the 40-hour measurement result was confirmed in the order of L213 (0.27) > AFY-10 (0.17) > L240 (0.13) > Sour Pitch (0.05). AFY-10 strain was the same species as Sour Pitch, but the concentration of acetic acid was more than three times higher.

Analysis Sour Pitch L213 L240 AFY-10 ABV (%) 4.24±0.01 ^c 4.11±0.01 ^b 4.10±0.00 ^b 3.86±0.01 ^a pH 3.27±0.01 ^c 3.27±0.00 ^c 3.24±0.02 ^b 3.17±0.00 ^a Attenuation (%) 80.73±0.20 ^b 78.36±0.10 ^a 82.54±0.22 ^c 79.89±1.96 ^a Foam stability 22.30±6.78 ^b 30.01±5.40 ^b 12.62±0.91 ^a 48.58±1.24 ^c Diacetyl (mg/L) 0.63±0.00 ^b 0.72±0.01 ^d 0.65±0.01 ^c 0.50±0.00 ^a

The alcohol content of the commercial strain Sour Pitch was the highest at 4.24%, the sour beer prepared with L213 and L240 had no significant difference at 4.11% and 4.10%, respectively, and the alcohol content of the sour beer prepared with AFY-10 was 3.86. % was the lowest. Although the Sour Pitch and AFY-10 strains were the same strain, the alcohol content of sour beer prepared with the AFY-10 strain was lower. It is judged that the difference in lactic acid concentration after souring of AFY-10 and Sour Pitch two strains affects ethanol production in yeast, resulting in a difference in alcohol content. Although the alcohol content of the sour beer prepared with the AFY-10 strain was rather low, the alcohol content of the sour beer is generally 3 to 5%, so it was determined to be suitable for the production of sour beer.

The pH of sour beer prepared with AFY-10 was significantly lowest at 3.17.

The degree of fermentation (attenuation) is an indicator of how much sugar was consumed in the wort, and the sour beer prepared with the L240 strain had the highest significantly with 82.54%, and the sour beer prepared with the L213 strain and AFY-10 was 78.4, respectively. %, as low as 79.9%. The lactic acid and acetic acid results of the wort measured immediately after souring showed that the L213 strain produced high acetic acid and the AFY-10 strain produced high lactic acid when compared with other strains. It is judged that the fermentation step affected the yeast fermentation.

Foam safety is one of the important indicators of beer quality visually judged by consumers. AFY-10 (48.58) > L213 (30.01) > Sour Pitch (22.30) > L240 (12.62) was found in the order, AFY-10 Sour beer made with the strain had the significantly highest foam stability, and sour beer made with strain L240 had the lowest.

The concentration of diacetyl, known as an off-flavor in beer with a flavor similar to butter or butterscotch, is L213 (0.72) > L240 (0.65) > Sour Pitch (0.63) > AFY-10 (0.50) in the order of AFY-10 strain was found to be the lowest.

As described above, compared to commercially available lactic acid bacteria, the AFY-10 strain has a high acid production rate, low pH, high foam safety, and low diacetyl content, confirming that it is very suitable for sour beer production. Accordingly, the AFY-10 strain was named Lacti plantibacillus plantarum AFY-10 and deposited with the deposit number KACC 81141BP at the National Academy of Agricultural Sciences as of October 20, 2020. The 16S rRNA nucleotide sequence of Lactiplantibacillus plantarum AFY-10 of SEQ ID NO: 1 is shown in FIG. 1 .

Condition FAN (mg/L) Sour Pitch L213 L240 AFY-10 wort 172.43±0.78 ^d souring 283.16±0.52 ^f 273.67±0.52 ^f 204.07±0.90 ^e 294.24±1.48 ^f alcohol fermentation 109.15±2.22 ^c 83.84±0.39 ^b 49.04±0.20 ^a 75.93±1.22 ^ab

Free amino nitrogen (FAN) is reduced during protein and peptide metabolism in yeast, and as the concentration of FAN in the final beer increases, it affects bitter taste, turbidity, foam stability, etc., thereby reducing the safety of beer quality. . The FAN concentration was measured in the souring and alcoholic fermentation steps, respectively, and is shown in Table 3. The FAN of the wort was measured to be 172.43 mg/L, and after souring, the concentration increased in the order of AFY-10 (294.24) > Sour Pitch (283.16) > L213 (273.67) > L240 (204.07). The concentration of FAN detected in sour beer after alcoholic fermentation by yeast was different for each strain.

Condition strain Free sugar (g/L) Glucose Fructose Maltose Maltotriose souring Sour Pitch 5.57±0.02 ^d 5.55±0.01 ^d 50.12±0.05 ^d 10.93±0.03 ^e L213 4.46±0.02 ^a 5.27±0.02 ^c 51.61±0.53 ^e 11.12±0.31 ^e L240 4.75±0.00 ^b 5.14±0.03 ^a 43.46±0.38 ^c 8.51±0.23 ^d AFY-10 5.39±0.02 ^c 5.21±0.01 ^b 41.09±1.12 ^b 11.14±0.11 ^e alcohol fermentation Sour Pitch N.D. N.D. 0.27±0.00 ^a 0.49±0.01 ^a L213 N.D. N.D. 0.20±0.00 ^a 1.78±0.01 ^c L240 N.D. N.D. 0.26±0.00 ^a 1.42±0.02 ^b AFY-10 N.D. N.D. 0.25±0.00 ^a 1.97±0.01 ^c

In addition, the free sugars immediately after souring and alcoholic fermentation of sour beer with different types of lactic acid bacteria were measured and shown in Table 4. As a result of comparing the free sugars for 4 types of glucose, fructose, maltose, and maltotriose, it was confirmed that the concentrations were significantly different for each strain. In particular, the L240 strain showed the significantly lowest levels of fructose, maltose, and maltotriose remaining in the wort, so the fermentation degree of yeast was expected to be the highest in the alcoholic fermentation stage. Sour Pitch and AFY-10 strains showed significant differences in the concentrations of glucose, fructose, and maltose despite the same species, and only maltotriose had no significant difference. After alcoholic fermentation, all four strains consumed glucose and fructose, and the remaining maltose concentration was 0.20-0.27 g/L, and there was no significant difference. Maltotriose concentration was found to be different for each strain. L213 and AFY-10 strains had more than 1.5 g/L, and sour pitch showed the lowest content at 0.49 g/L. It is judged that this affects the alcoholic fermentation of yeast after souring depending on the type of lactic acid bacteria.

On the other hand, a comparison of the carbon source availability of AFY-10 strain and commercially available sour pitch lactic acid bacteria is shown in Table 5. Despite the same species, the two strains differed in their carbon source availability of D-Sorbitol, Methyl-αD-Mannopyranoside, and D-Melibiose.

No. test division AFY-10 Sour Pitch One GLY Glycerol - - 2 ERY Erythritol - - 3 DARA D-Arabinose - - 4 LARA L-Arabinose + + 5 RIB D-Ribose + + 6 DXYL D-Xylose - - 7 LXYL L-Xylose - - 8 ADO D-Adonitol - - 9 MDX Methyl-βD-Xylopyranoside - - 10 GAL D-Galactose + + 11 GLU D-Glucose + + 12 FRU D-Fructose + + 13 MNE D-Mannose + + 14 SBE L-Sorbose - - 15 RHA L-Rhamnose - - 16 DUL Dulcitol - - 17 INO Inositol - - 18 MAN D-Mannitol + + 19 SOR D-Sorbitol - + 20 MDM Methyl-αD-Mannopyranoside + - 21 MDG Methyl-αD-Glucopyranoside - - 22 NAG N-Acetylglucosamine + + 23 AMY Amygdalin + + 24 ARB Arbutin + + 25 ESC Esculin + + ferric citrate + + 26 SAL Salicin + + 27 CEL D-Cellobiose + + 28 MAL D-Maltose + + 29 LAC D-Lactose (bovine origin) + + 30 MEL D-Melibiose + - 31 SAC D-Saccharose (Sucrose) + + 32 TRE D-Trehalose + + 33 INU Inulin - - 34 MLZ D-Melezitose + + 35 RAF D-Raffinose - - 36 AMD Amidon (Starch) - - 37 GLYG Glycogen - - 38 XLT Xylitol - - 39 GEN Gentiobiose + + 40 TUR D-Turanose + + 41 LYX D-Lyxose - - 42 tags D-Tagatose - - 43 DFUC D-Fucose - - 44 LFUC L-Fucose - - 45 DARL D-Arabitol - - 46 LARL L-Arabitol - - 47 GNT Potassium Gluconate - - 48 2KG Potassium 2-Ketogluconate - - 49 5KG Potassium 5-Ketogluconate - -

Example 5. Analysis of fragrance components

The volatile aroma component of the sour beer prepared in Example 3 was analyzed by the SPME (solid phase microextraction) method. After filtering the sample solution through a 0.45 μm filter, take 5 ml of the sample in a 20 ml vial, add 1 g of Sodium Chloride (Sigma aldrich), and adsorb to 65 μm PDMS/DVB fiber (Sigma aldrich) at 60°C at 500 rpm for 30 minutes. After GC-TOF/MS (7890A, Agilent Technologies), it was analyzed. The column used ZB-WAX (30 m x 0.32 mm x 0.25 μm, Phenomenex) to adsorb the sample at 250 °C in the inlet for 5 minutes, and the moving gas was analyzed using He gas at a flow rate of 1.13 ml/min. The oven temperature was raised to 240 °C at a rate of 8 °C/min and maintained for 3 minutes, and the transmission line temperature was set at 250 °C. TOF/MS conditions were set at 230 °C, 15 spectra/s, and 35-501 m/z. The identified substances were identified using the National Institute of Standards and Technology (NIST) library.

For multivariate statistical analysis, PCA (Principal Component Analysis) and PLS-DA (Partial Least Squares-Discriminant Analysis) were performed using SIMCA software (SIMCA 14.0, Umetics). Significance variables were extracted by obtaining VIP (Variable Importance in Projection) through PLS-DA analysis. For more accurate results, a standard deviation of less than 1.0 was selected.

Looking at the multivariate statistical analysis results shown in FIG. 6 , the principal component 1 (PC 1) was 66.8% and the principal component 2 (PC 2) was 11.4%, explaining 78.2% of the total variation. The major component 1 of PLS-DA expressed in the positive direction of the x-axis was sour beer after souring, and there was no significant difference by strain, whereas the aroma component of sour beer after alcoholic fermentation expressed in the negative direction of the x-axis was the strain. It was confirmed that the star difference was relatively larger than before souring. In particular, the three strains of Kimchi Lactobacillus L213, L240, AFY-10 were expressed in the positive y-axis direction of the main component 2, and Sour Pitch, a commercially available lactic acid bacterium, was expressed in the negative y-axis direction. It was confirmed that the aroma components of sour beer were clearly distinguished. As described above, it was confirmed that a large amount of volatile fragrance components were generated in the alcoholic fermentation step after souring, and it was confirmed that the kimchi lactic acid bacteria according to the present disclosure and the commercially available sour pitch strain for sour beer were clearly distinguished.

statistical analysis

Each experimental result was expressed as the mean and standard deviation of the results of three repeated experiments, and the significance was verified at the p<0.05 level through Duncan's multiple range test with IBM SPSS statistics software (SPSS 12.0, IBM). For correlation analysis, only variables following a normal distribution were selected and expressed as Pearson's correlation coefficient.

Above, specific parts of the present disclosure have been described in detail, for those of ordinary skill in the art, these specific techniques are only preferred embodiments, and it is clear that the scope of the present disclosure is not limited thereby. something to do. Accordingly, it is intended that the substantial scope of the present disclosure be defined by the appended claims and their equivalents.

National Academy of Agricultural Sciences KACC81141BP 20201020

<110> KANGWON PROVINCE <120> NOVEL KIMCHI LACTIC ACID BACTERIA LACTIPLANTIBACILLUS PLANTARUM <130> 20P557/IND <160> 3 <170> KoPatentIn 3.0 <210> 1 <211> 1530 <212> RNA <213> Unknown <220> <223> Lactiplantibacillus plantarum AFY-10 <400> 1 cgtcagacga acgctggcgg cgtgcctaat acatgcaagt cgaacgaact ctggtattga 60 ttggtgcttg catcatgatt tacatttgag tgagtggcga actggtgagt aacacgtggg 120 aaacctgccc agaagcgggg gataacacct ggaaacagat gctaataccg cataacaact 180 tggaccgcat ggtccgagct tgaaagatgg cttcggctat cacttttgga tggtcccgcg 240 gcgtattagc tagatggtgg ggtaacggct caccatggca atgatacgta gccgacctga 300 gagggtaatc ggccacattg ggactgagac acggcccaaa ctcctacggg aggcagcagt 360 agggaatctt ccacaatgga cgaaagtctg atggagcaac gccgcgtgag tgaagaaggg 420 tttcggctcg taaaactctg ttgttaaaga agaacatatc tgagagtaac tgttcaggta 480 ttgacggtat ttaaccagaa agccacggct aactacgtgc cagcagccgc ggtaatacgt 540 aggtggcaag cgttgtccgg atttattggg cgtaaagcga gcgcaggcgg ttttttaagt 600 ctgatgtgaa agccttcggc tcaaccgaag aagtgcatcg gaaactggga aacttgagtg 660 cagaagagga cagtggaact ccatgtgtag cggtgaaatg cgtagatata tggaagaaca 720 ccagtggcga aggcggctgt ctggtctgta actgacgctg aggctcgaaa gtatgggtag 780 caaacaggat tagataccct ggtagtccat accgtaaacg atgaatgcta agtgttggag 840 ggtttccgcc cttcagtgct gcagctaacg cattaagcat tccgcctggg gagtacggcc 900 gcaaggctga aactcaaagg aattgacggg ggcccgcaca agcggtggag catgtggttt 960 aattcgaagc tacgcgaaga accttaccag gtcttgacat actatgcaaa tctaagagat 1020 tagacgttcc cttcggggac atggatacag gtggtgcatg gttgtcgtca gctcgtgtcg 1080 tgagatgttg ggttaagtcc cgcaacgagc gcaaccctta ttatcagttg ccagcattaa 1140 gttgggcact ctggtgagac tgccggtgac aaaccggagg aaggtgggga tgacgtcaaa 1200 tcatcatgcc ccttatgacc tgggctacac acgtgctaca atggatggta caacgagttg 1260 cgaactcgcg agagtaagct aatctcttaa agccattctc agttcggatt gtaggctgca 1320 actcgcctac atgaagtcgg aatcgctagt aatcgcggat cagcatgccg cggtgaatac 1380 gttcccgggc cttgtacaca ccgcccgtca caccatgaga gtttgtaaca cccaaagtcg 1440 gtggggtaac cttttaggaa ccagccgcct aaggtgggac agatgattag ggtgaagtct 1500 aaaaggggaa accaaaaatg cctccacatg 1530 <210> 2 <211> 18 <212> RNA <213> Artificial Sequence <220> <223> 785F primer <400> 2 ggattagata ccctggta 18 <210> 3 <211> 20 <212> RNA <213> Artificial Sequence <220> <223> 907R primer <400> 3 ccgtcaattc mtttgagttt 20

Claims (17)

Accession number KACC81141BP Lacti plantibacillus plantarum ( Lactiplantibacillus plantarum ) AFY-10 strain.
The method of claim 1,
The strain is a strain derived from kimchi.
The method of claim 1,
The strain is for alcohol production, the strain.
4. The method of claim 3,
The alcoholic beverage is beer, the strain.
5. The method of claim 4,
The beer is a sour beer, strain.
The method of claim 1,
The strain is that the pH value of the medium has an acid-producing ability showing 3.41 or less, the strain.
The method of claim 1,
The strain does not produce extracellular polysaccharide (Exopolysaccharide, EPS) and diacetyl,
The method of claim 1,
The strain is susceptible to hops (hop).
The method of claim 1,
The strain will have β-glucosidase activity, the strain.
10. The lactic acid bacteria composition for souring for the production of alcoholic beverages comprising the strain of any one of claims 1 to 9.
The strain of any one of claims 1 to 9; or
A liquor composition comprising a lactic acid bacteria composition for souring for the production of liquor containing the strain.
12. The method of claim 11,
The alcoholic beverage is sour beer, the alcoholic beverage composition.
12. The method of claim 11,
wherein the alcoholic beverage composition has at least one of the following values:
a) a pH of 3.2 or less;
b) a diacetyl concentration of 0.6 mg/L or less;
c) a free amino nitrogen (FAN) concentration of 80 mg/L or less; and
d) a maltotriose concentration of at least 1.5 g/L.
A method of making a liquor composition comprising preparing the liquor composition of claim 11 .
15. The method of claim 14, wherein the manufacturing method,
saccharifying the pulverized malt to prepare wort;
After cooling the wort, the deposit number is KACC81141BP Lactiplantibacillus plantarum ( Lactiplantibacillus plantarum ) Souring (souring) by inoculating the strain AFY-10;
After souring, adding hops and boiling; and
A method for producing an alcoholic beverage composition comprising the step of cooling the wort after boiling is completed, and then inoculating yeast to perform alcoholic fermentation.
16. The method of claim 15,
The souring is carried out at 35 to 45 ℃, the method for producing a liquor composition.
16. The method of claim 15,
The souring will be carried out for 16 to 48 hours, a method for producing a liquor composition.

Images