KR102059624B1 - Probiotic lactic acid bacteria with outstanding immune-modulatory activity, intestinal attachment ability, and baking quality - Google Patents

Abstract

The present invention relates to probiotic lactic acid bacteria, and more specifically, to probiotic lactic acid bacteria with outstanding immunomodulatory activity, intestinal attachment, and backing quality. The probiotic lactic acid bacteria of the present invention has especially outstanding immunomodulatory activity and intestinal attachment. When the probiotic lactic acid bacteria of the present invention is kneaded and fermented with baking yeast, bread having improved bread properties and flavor, extended expiration date, and immune activity based on dead cells can be manufactured.

Description

Probiotic lactic acid bacteria with outstanding immune-modulatory activity, intestinal attachment ability, and baking quality}

The present invention relates to probiotic lactic acid bacteria, and more particularly, to probiotic lactic acid bacteria excellent in immunomodulatory activity, intestinal adhesion and baking aptitude.

Lactobacillus is a mycelia commonly found in fermented foods, it is contained in a lot of fermented foods such as yogurt, kimchi, natto, also known as lactic acid bacteria. Representative lactic acid bacteria include Lb. casei , Lb.acidophilus , Lb. bulgaricus , Bifidobacterium longgum , Bifidobacte Leeum B. bifidum.

One of the main health functions of probiotics lactic acid bacteria is immunomodulatory function and activation of immune system and short-chain fatty acids such as acetic acid, propionic acid and butylic acid produced by lactic acid bacteria in the intestine The mechanism by which dendritic cell stimulation of intestinal epithelial tissue is induced by polysaccharide, a cell wall component of, has been known. In particular, the immunomodulatory function by cell wall polysaccharides is known to appear at similar levels in dead cells as well as live cells, and immunoregulatory function by ingesting dead cells is considered as a potentially important health maintenance means. Based on the antimicrobial activity of Lactobacillus microorganisms, it is used as a raw material for diarrhea treatment. In Japan, microbial products have been developed for immune-related effects such as pollen allergy. For example, the "FK-23" product released in Japan is composed of microorganisms heat-treated with lactic acid bacteria, and hepatitis C treatment as an immune-activating function, and the microorganisms of the "acidophilus L-92" strain also provide immune regulation Was developed.

Immune responses are largely divided into innate immunity and adaptive immunity. Innate immunity is a process in which macrophages and dendritic cells are involved in non-specific recognition and removal of antigens, whereas adaptive immune cells, represented by T- and B-cells, specifically react with antigens. do. Antigen-presenting cells include B-cells, macrophages, and dendritic cells, which capture and degrade antigens to present antigenic peptides on the cell surface through major histocompatibility complex (MHC). It delivers structural information to T-cell receptors. After capturing the antigen, the activated antigen presenting cells induce the expression of molecules such as CD80, CD86, and CD40, which in turn act on the activation of T-cells and the activity of antigen presenting cells.

For example, through CD40 / CD40 binding between B-cells and activated helper T-cells, B-cells produce antibody IgM, which can be altered by IgA, IgG, or IgE. Activated helper T-cells differentiate into Th1 and Th2 cells depending on the cytokines present. When macrophages and dendritic cells recognize microorganisms and secrete the cytokines IL-12, IL-6, and TNF-α, activated adjuvant T-cells differentiate into Th1 cells and the secretion ratio of IFN-γ: IL-4 Increases. Conversely, without microbial stimulation, cytokines induce activated adjuvant T-cells to differentiate into Th2 cells, and the secretion rate of IFN-γ: IL-4 decreases, resulting in B cells producing IgE. . This differentiation pathway to Th2 cells is known to proceed basically in the absence of IL-12 stimulation signals.

IgE antibodies are a necessary defense against external organism infection, but excessive secretion causes allergic symptoms such as atopy, rhinitis and sputum by mast cells secreting inflammatory mediators from the mucosal layer. According to the well-known hygiene theory, without proper microbial stimulation, the innate immune cells that produce IL-12 are reduced and Th2 cells are excessively activated, which promotes IgE production and increases allergic symptoms. Is observed. As a result, since Th1 and Th2 cells antagonize each other, for the treatment of allergy caused by an excessive immune response of Th2 cells, a treatment for inducing an immune response by Th1 cells has been developed.

Meanwhile, the sourdough starter, which is a fermented bread material, is filled with live yeast and bacteria, and a natural fermentation method using the starter is used as a baking method. When baking by natural fermentation method, various microorganisms are mixed, so rich flavor can be expected, but there may be a problem due to contamination of other microorganisms, and if the concentration of applied microorganism is low, fermentation time should be extended. Difficult to produce Currently, the method of manufacturing fermented bread commonly applied is a method of artificially adding yeast (yeast) at the time of kneading and using it as a starter. Small bakeries prefer a one-stage sourdough process, which is a 15 to 24 hour fermentation process, and large bakeries prefer a two-stage sourdough process. However, in general, fermented bread using yeast alone has a low shelf life and poor fragrance.

This sourdough fermentation technology, also known as acid kneading fermentation technology, is generally manufactured by mixing flour, water, and rye flour. Yeast and lactic acid bacteria produce alcohol and lactic acid, respectively, to flavor bread and improve palatability. Lactic acid bacteria related to sourdough fermentation are known as Streptococcus genus, Pediococcus genus, Lactobacillus genus, Leuconostoc genus and Weissella genus. On the other hand, traditional sourdough bread (depending on the natural fermentation method) has a problem that the various kinds of bacteria derived from the working environment is contaminated, resulting in poor flavor or rancid odor. In addition, there is a difficulty in reproducing an equal quality product due to space and time difference. Therefore, it is necessary to add lactic acid bacteria starter (starter) secured functionality and safety to the dough in order to avoid the opportunity of contamination of natural fermented bread and to ensure the equivalent quality. Some domestic bakery companies are using imported starters for this purpose, but strong acidity is provided so that it is not suitable for the baking preference of domestic consumers, or strain characteristics are easily changed during subculture. Therefore, it is necessary to develop a starter that is suitable for Korean taste and can be used stably at a baking site. In addition, lactic acid bacteria showing immunomodulatory activity in the intestine after ingestion into dead cells can be used to provide both baking aptitude and health functionality.

Korean Patent Registration No. 10-1551836 (2015.09.03) describes a new native natural lactic acid bacteria for baking isolated from Korean traditional yeast. Korean Patent Registration No. 10-1551837 (2015.09.03) describes a new native natural lactic acid bacteria for baking isolated from Korean traditional yeast.

The present invention is to discover and provide probiotic lactic acid bacteria excellent in immunomodulatory activity, intestinal adhesion and baking aptitude.

The present invention provides Lactobacillus plantarum SPC-SNU 72-2 (KCTC13315BP).

On the other hand, the present invention provides a dough for baking, characterized in that the Lactobacillus plantarum ( Lactobacillus plantarum ) SPC-SNU 72-2 (KCTC13315BP) is added to the flour and then fermented.

Meanwhile, the present invention provides Lactobacillus plantarum SPC-SNU 72-2 (KCTC13315BP) to flour, followed by fermentation and baking.

The probiotic lactic acid bacteria of the present invention are particularly excellent in immunomodulatory activity and intestinal adhesion. When the probiotic lactobacillus of the present invention and the dough fermentation with baker's yeast, improve the physical properties, flavor, shelf life of the bread, it can be produced bread having immunity to the bacterium.

1 is a result of performing the Lactobacillus plantarum specific PCR of the strains isolated from kimchi.
Figure 2 shows the results of confirming that the present invention Lactobacillus plantarum ( Lactobacillus plantarum ) SPC-SNU 72-2 and biogenic amine production ability for two strains.
Figure 3 is the result of measuring the hemolysis of the Lactobacillus plantarum SPC-SNU 72-2 and the test strain ( Lb. reuteri KCCM40717) of the present invention.
Figure 4 is a result of comparing the acid resistance according to the pH of the present invention Lactobacillus plantarum SPC-SNU 72-2.
5 is a result of comparing the bile resistance of lactic acid bacteria according to the bile concentration of the present invention Lactobacillus plantarum SPC-SNU 72-2.
6 is a result of evaluating intestinal adhesion capacity using Caco-2 cells, which are colonic epithelial cells.
Figure 7 is a result showing the culture characteristics of the present invention Lactobacillus plantarum SPC-SNU 72-2 in maltose medium.
8 is a result confirming the maltose capacity of the present invention Lactobacillus plantarum SPC-SNU 72-2.
9 is a bread to which the Lactobacillus plantarum KACC11451 is applied as a test strain, a bread to which Lallemand L73 is applied as a commercial strain of Lactobacillus plantarum, and a bread to which the present invention Lactobacillus plantarum SPC-SNU 72-2 is applied. to be.
10 is a result of confirming the gas generating power of the present species to which the present invention Lactobacillus plantarum SPC-SNU 72-2, commercial strain Lallemand L73, lactobacillus plantarum KACC11451 used as the test strain.
11 is a result of measuring the aging rate of the bread to which the present invention Lactobacillus plantarum SPC-SNU 72-2 is applied.
12 is a result of comparing the quantitative value of the volatile aroma components (alcohol, aldehyde, ketone, acid, etc.) detected per 1g of bread (sample) to which the present invention Lactobacillus plantarum SPC-SNU 72-2 is applied.

The present invention provides Lactobacillus plantarum SPC-SNU 72-2 (KCTC13315BP). The Lactobacillus plantarum ( Lactobacillus plantarum ) SPC-SNU 72-2 (KCTC13315BP) is a lactic acid bacterium isolated from kimchi, and is particularly excellent in intestinal adhesion, and in addition, it has excellent immunomodulatory activity. In addition, baking aptitude was also excellent when applied to baking.

On the other hand, the present invention provides a dough for baking, characterized in that the Lactobacillus plantarum ( Lactobacillus plantarum ) SPC-SNU 72-2 (KCTC13315BP) is added to the flour and then fermented. Flour dough containing Lactobacillus plantarum SPC-SNU 72-2 (KCTC13315BP), which is a new lactic acid bacterium isolated from kimchi, is superior to the control group and is suitable for baking.

On the other hand, the present invention provides Lactobacillus plantarum ( Lactobacillus plantarum ) SPC-SNU 72-2 (KCTC13315BP) is added to the flour and then fermented and baked to provide a bread, characterized in that the baking, Baking means baking. The bread prepared as described above has a softer texture, a lower hardness, and a slower aging rate than the bread manufactured with commercial yeast, and thus has excellent shelf life. Moreover, the flavor is good by releasing the fragrance component excellent in preference.

Hereinafter, the content of the present invention will be described in more detail through the following examples or experimental examples. However, the scope of the present invention is not limited only to the following Examples and Experimental Examples, but includes modifications of equivalent technical ideas.

< Example  One: The present invention Probiotic  Isolation and Identification of Lactic Acid Bacteria>

(One) Lactic acid bacteria isolated

10 g of kimchi and 90 ml of 0.85% NaCl were placed in a filter bag and homogenized for 3 minutes using a stomacher. Dilution was carried out in steps of 0.85% NaCl to the appropriate concentration, followed by plating on MRS (de Man Rogosa and Sharpe, Difco) solid medium containing 0.01% cycloheximide and BPB (Bromophenol blue). It was. In order to selectively select strains using the characteristic of colony color change by the acid produced by lactic acid bacteria, bromophenol-blue was added to the existing MRS medium, and cycloheximide was used to suppress fungi. Added. Culture conditions were anaerobic culture for 48 hours at 30 ℃, the white colony with a dark blue circle was considered to be Lactobacillus plantarum candidate strain and purely isolated culture.

(2) Lactobacillus Planta Room  Sympathy

Primers specific for Lactobacillus plantarum can be found in Dr. Naoki Kato, 2000, Rapid identification of 11 human intestinal Lactobacillus species by multiplex PCR assays using group- and species-specific primers derived from the 16S 23S rRNA intergenic spacer region. LplanF (5'-ATTCATAGTCTAGTTGGAGGT-3 ') and LplanR (5'- CCTGAACTGAGAGAATTATTGA-3') designed with reference to its flanking 23S rRNA) were used and the amplification size was 248 bp, pre-dine at 95 ° C for 3 minutes. 30 cycles of pre-denaturation, 30 seconds at 95 ° C., 30 seconds at 60 ° C., and 30 seconds at 72 ° C., followed by 30 cycles, and PCR was performed at 72 ° C. for 5 minutes for final elongation. After the electrophoresis, the PCR result was confirmed (FIG. 1). Figure 1 shows the results of performing the Lactobacillus plantarum specific PCR of the strains isolated from kimchi. As a result, bands of 248 bp were formed in 36 strains, and 16S rRNA sequencing was identified as Lactobacillus plantarum . The isolated strain was named SPC-SNU 72-2, and was deposited with the Korea Research Institute of Bioscience and Biotechnology and received the accession number 'KCTC13315BP' on August 24, 2017.

(3) Lactobacillus Plantarum  Biochemical properties

The sugar use characteristics of the isolated strain (SPC-SNU 72-2), the strain (KACC11451), and the commercial strain (Lallemand L73) were analyzed using API50CH and API 50CHL kit (Biomerieux). Shown in As a result, the isolated strain (SPC-SNU 72-2) is a new strain showing difference in L-arabinose and D-turanose metabolism when compared to the public strain and the commercial strain. It turned out.

Analysis of Sugar Usage Characteristics of Lactobacillus Planta Room SPC-SNU 72-2 SPC- SNU  72-2 Disclosure strain  KACC11451 Commercial strain
Lallemand  L73 control - - - Glycerol - - - Erythriol - - - D-arabinose - - - L-arabinose - + + D-ribose + + + D-xylose - - - L-xylose - - - D-adonitol - - - Methyl-βD-xylopyranoside - - - D-galactose + + + D-glucose + + + D-fructose + + + D-mannose + + + L-sorbose - - - L-rhamnose - - - Dulcitol - - - Inositol - - - D-mannitol + + + D-sorbitol + + + Methyl-α-D-mannopyranoside + + + Methyl-α-D-gucopyranoside - - - N-acetylglucosamine + + + Amygdalin + + + Arbutin + + + Esulin + + + Salicin + + + D-celiobiose + + + D-maltose + + + D-lactose + + + D-melibiose + + + D-saccharose + + + D-trehalose + + + Inulin - - - D-melezitose + + + D-raffinose + + + Amidon - - - Glycogen - - - Xylitol - - - Gentiobiose + + + D-turanose - + + D-lyxose - - - D-tagatose - - - D-fucose - - - L-fucose - - - D-arabitol - - - L-arabitol - - - Potassium gluconate + + + Potassium 2-ketogluconate - - - Potassium 5-ketogluconate - - -

< Experimental Example  One: The present invention Probiotic  Human Intestinal Safety Analysis of Lactic Acid Bacteria>

(One) Biogenic amine Generating ability  Confirm

Biogenic amines are produced by microorganisms in fermented foods and induce food poisoning.To verify their safety, Multiplex PCR for colony direct detection of Gram-positive histamine- and tycamine-producing bacteria), HDC3 (5'-GATGGTATTGTTTCKTATGA-3 ') and HDC4 (5'-CAAACACCAGCATCTTC-3'), TD2 (5'-ACATAGTCAACCATRTTGAA-3 ') and TD5 (5'-CAAATGGAAGAAGAAGTAGG-3 '), And the amplification size is 1182bp, 436bp, respectively, pre-denaturation for 3 minutes at 95 ℃, 30 seconds at 95 ℃, 30 seconds at 52 ℃, 90 seconds at 72 ℃ 1 cycle, 30 cycles, multiplex PCR for 5 minutes at 72 ℃ for final elongation, electrophoresis was confirmed by the PCR results (Fig. 2). Figure 2 shows the results of confirming that the present invention Lactobacillus plantarum ( Lactobacillus plantarum ) SPC-SNU 72-2 and biogenic amine production ability for two strains. As a result, it was confirmed that the isolated Lactobacillus plantarum SPC-SNU 72-2 does not have a gene for generating two kinds (tyramine, histamine) belonging to the biogenic amine.

(2) Hemolysis

Lactic acid bacteria are generally known to be safe, but pathogenic lactic acid bacteria have been reported, so the safety verification of new lactic acid bacteria is required. To verify the safety, refer to the Korea Bio Venture Association's group standard (Method for measuring hemolysis of lactobacillus) and inoculate the Lactobacillus to be measured on TSA (Tryptic Soy Agar) or BHI (Brain Heart Infusion) medium containing 5% cotton blood , Hemolysis phenomenon appeared on the medium after incubation at 30 ℃ for 48 hours (Fig. 3). Figure 3 is the result of measuring the hemolysis of the Lactobacillus plantarum SPC-SNU 72-2 and the test strain ( Lb. reuteri KCCM40717) of the present invention.

As a result, hemolysis was not observed in both Lactobacillus plantarum SPC-SNU 72-2 and the strain ( Lb. reuteri KCCM40717), and there was no change in the color of the medium.

< Experimental Example  2: The present invention Probiotic  Human Intestinal Stability Analysis of Lactic Acid Bacteria>

(One) Acid resistance · Bile resistance  evaluation

end) Buffer preparation for gastric and bile reproduction

In view of the fact that a large number of lactic acid bacteria are killed by gastric acid and bile acids before the lactic acid bacteria enter the intestine, the intestinal viability of the lactic acid bacteria was examined prior to the functional studies. To adjust the intestinal pH, first, PBS buffer (based on 1 L, Sodium chloride 8g, Potassium chloride 0.2g, Sodium phosphate 1.44g, Potassium phosphate 0.24g, pH7.4) was prepared and autoclave, and then HCL was added. Gastric acid (pH 2.0 ~ 3.0) environment was made, bile salt (bile salt) was added to create a bile acid (0.3-0.4%) environment was used in the following experiment.

I) Acid resistance · Bile resistance  Measure

Prior to this experiment, the strains were activated twice and placed in 1 ml in a 1.5 ml tube and centrifuged at 12,000 rpm and 4 ° C. for 5 minutes. The supernatant was removed to recover the cells, and then washed twice with 0.85% NaCl to remove the remaining medium. PBS buffer solutions at pH 2.0 and 3.0 and 0.3% and 0.4% bile salt solutions were resuspended in the same amount as the initial culture (1 ml), respectively. Considering that food is introduced and passing through the stomach is 2 to 4 hours, the reaction time was set to 0, 90, and 180 minutes, and sampled by 100 μl for each hour. The sampling solution (100 μl) was used as a sample after the pH was restored to 900 μl of buffer. Diluted with 0.85% NaCl step by step, spread 20µl each in quartered MRS medium, and after 24 hours, the cell count (cell counting) to obtain the Log CFU / ㎖ value.

The Lactobacillus plantarum SPC-SNU 72-2 ( Lb. plantarum SPC-SNU 72-2) was reacted with pH 2 and 3 buffer solutions for 0, 90 and 180 minutes, respectively. Bacterial counts by reaction time showed a significant decrease. On the other hand, at pH 3.0, there was no significant change in the number of bacteria, and the viability was over 90%. Based on these results, Lactobacillus plantarum SPC-SNU 72-2 was used as a control for LGG ( Lb. rhamnosus GG) showed a higher survival rate than the strain, it was confirmed that it has resistance even in the environment of strong acids (Fig. 4). Figure 4 is a result of comparing the acid resistance according to the pH of the present invention Lactobacillus plantarum SPC-SNU 72-2.

Meanwhile, Lactobacillus plantarum SPC-SNU 72-2 was reacted with 0.3% and 0.4% bile salts for 0, 90 and 180 minutes, respectively, and the number of viable cells was measured. The bacterial counts decreased, but then remained unchanged. According to these results, Lactobacillus plantarum SPC-SNU 72-2 showed a higher survival rate than the LGG strain used as a control it was confirmed that the resistance in the environment of bile (Fig. 5). 5 is a result of comparing the bile resistance of lactic acid bacteria according to the bile concentration of the present invention Lactobacillus plantarum SPC-SNU 72-2.

(2) Intestinal fixability

 Polystylene membrane is a model widely used in small intestine absorption studies because it automatically differentiates when cells are cultured on the membrane and becomes morphologically and biochemically similar to human small intestinal epithelial cells. Intestinal adhesion of Lactobacillus plantarum SPC-SNU 72-2 was measured using one cell line, Caco-2 cells (FIG. 6). 6 is a result of evaluating intestinal adhesion capacity using Caco-2 cells, which are colonic epithelial cells.

As a result of evaluating the fixation ability of the intestinal Lactobacillus plantarum SPC-SNU 72-2 strain using Caco-2 cells, which are colonic epithelial cells, it was observed that about 800 cells were attached per 100 cells of Caco-2. As a result, Lactobacillus plantarum SPC-SNU 72-2 strain showed higher adhesion in colon epithelial cells than LGG ( Lb. rhamnosus GG) used as a control, it was confirmed that the intestinal fixation ability is very excellent.

< Experimental Example  3: Health Functional Analysis>

(One) Immune system

end) Macrophage Isolation and Culture

Balb / c 7-week-old mouse intraperitoneally injected with 2 ml of 3.5% thioglycollate media (BD, Sparks, MD, USA) 4 days after the cervical distal bone, 1% FBS (fetal bovine serum) : Hyclone, Utah, USA), 8 ml of DMEM (Hyclone) containing 1% penicillin-streptomycin (p / s) was injected to collect macrophages. After centrifugation at 1000 rpm for 10 minutes, macrophages were suspended in a medium containing 10% FBS, 1% p / s of DMEM, and the number of cells was measured using a Countess IIFL Automated Cell Counter (Life Technologies). Macrophages required for the experiment was incubated overnight in an incubator at 37 ℃, 5% CO ₂ conditions. Macrophages that did not adhere to the bottom were removed and each cell was treated for 24 hours by concentration, and then the supernatant was recovered.

I) Lactic Acid Bacteria Cultivation

Lactobacillus was inoculated in MRS broth medium and shaken for 24 hours under the optimum temperature of 30 ℃ or 37 ℃ conditions. The supernatant was removed by centrifugation and washed only 2-3 times with 0.85% NaCl to collect only pellets. This was quenched by heat treatment at 100 ° C. for 5 minutes using a water bath and then lyophilized. The lyophilisate produced was diluted by experimental concentration and used as a sample.

All) Cytokine measurement

Cytokine content was measured using an enzyme-linked immunosorbent assay (ELISA) method. TNF-α, IL-6 Duoset (R & D Systems, USA) and 1L-12 OptEIA set (BD, USA) were used and ELISA was performed according to the manufacturer's protocol.

① TNF -α, IL-6 Duoset  (R & D Systems, USA)

Each capture antibody was diluted in PBS and dispensed by 0.1 ml in 96-well immunoplate, and then reacted overnight at room temperature. After washing each well three times with a washing buffer (0.5% Tween / PBS), 0.3 ml of blocking buffer (1% BSA in PBS) was added to each well and reacted for 1 hour. Thereafter, each well was washed three times with a wash buffer, and the standard and the sample were diluted and dispensed 0.1 ml each, followed by reaction for 2 hours. Thereafter, each well was washed with a washing buffer, followed by diluting a secondary antibody conjugated with biotin in a blocking buffer, dispensing 0.1 ml of each well, and reacting at room temperature for 2 hours. Thereafter, the wells were washed three times with a washing buffer, and then diluted 0.1 ml of the Streptavidin-HRP solution in the blocking buffer, followed by reaction at room temperature for 20 minutes. Thereafter, each well was washed with a washing buffer, and then treated with 0.1 ml of TMB (BD pharmingen) solution per well, followed by reaction at room temperature for 30 minutes, and 2N H ₂ SO ₄ solution was added to each well by 0.05 ml. Completed. Absorbance was measured at 450 nm using a 96 well microplate reader.

② IL-12 OptEIA  set ( BD , USA)

Each capture antibody was diluted in PBS and dispensed by 0.1 ml in 96-well immunoplate, and then reacted overnight in refrigeration. After washing each well three times with washing buffer (0.5% Tween / PBS), 0.2 ml of blocking buffer (10% FBS, Welgene, Korea) was added to each well and reacted for 1 hour. Each well was washed three times with a wash buffer, and the standard and samples were diluted and dispensed by 0.1 ml, followed by reaction for 2 hours. After washing each well with a washing buffer, diluting the biotin-bound secondary antibody and Streptavidin-HRP solution in the blocking buffer and dispensing 0.1 ml each, and reacting for 1 hour at room temperature. Each well was treated with 0.1 ml / well TMB (BD pharmingen) solution washed with washing buffer, and then reacted at room temperature for 30 minutes, and 2N H ₂ SO ₄ solution was added to each well by 0.05 ml to complete the reaction. Absorbance was measured at 450 nm using a 96 well microplate reader.

As a result of confirming that the sample stimulates the pathogen-associated molecular pattern (PAMP) of macrophages, which are a kind of immune cells, to induce the secretion of immune-related cytokines, TNF-α, Lactobacillus plantarum SPC-SNU 72-2 strain, The secretion of IL-6 tended to increase with the concentration of bacteria, but unlike other cytokines, IL-12 was induced when 4-20 µg / ml bacteria were treated than the highest concentration of 100 µg / ml. The ability tended to be higher. As a result, three cytokine (TNF-α, IL-6, IL-12) inducing ability measurement in vitro conditions, Lactobacillus plantarum SPC-SNU 72-2 was higher than the control LGG The cytokine inducing ability has been confirmed, and is expected to produce immunomodulatory activity. This is shown in Tables 2 to 4 below.

TNF -α (concentration, pg / ml) Cell concentration
( μg / ml) 4 20 100 L. rhamnosus GG 0.00 ± 0.00 807.33 ± 856.71 6330.67 ± 1641.65 L. plantarum  SPC-SNU 72-2 12570.00 ± 4077.91 16620.00 ± 3276.98 16999.17 ± 2017.51

IL-6 (concentration, pg / ml) Cell concentration
( μg / ml) 4 20 100 L. rhamnosus GG 30.83 ± 51.26 13.33 ± 23.09 485.83 ± 104.83 L. plantarum  SPC-SNU 72-2 1243.61 ± 997.65 2140.83 ± 1665.15 2724.17 ± 1831.23

IL-12 (concentration, pg / ml) Cell concentration
( μg / ml) 4 20 100 L. rhamnosus GG 17.00 ± 29.44 25.44 ± 44.07 109.44 ± 62.21 L. plantarum  SPC-SNU 72-2 2721.33 ± 2272.79 2820.5 ± 2158.66 638 ± 576.04

< Experimental Example  4: Bakery Aptitude Evaluation>

(One) Lactobacillus Plantarum  maltose Utilization  Confirm

end) Growth curve

To make a 2% maltose-containing MRS medium (broth medium) having the composition shown in Table 5, and then inoculated by 10% (w / v) 72-2 isolated strain, test strain (KACC11451), commercial strain (Lallemand L73), respectively After incubation for 10 hours, the absorbance was measured at 600 nm to generate a growth curve (FIG. 7). Figure 7 is a result showing the culture characteristics of the present invention Lactobacillus plantarum SPC-SNU 72-2 in maltose medium. As a result, it was confirmed that the 72-2 isolate strain was faster in the maltose medium than the test strain and the commercial strain.

MRS broth medium containing 2% maltose Furtherance content Proteos peptone 10.0 g Beef extract 10.0 g Yeast extract 5.0 g maltose 20.0 g Sodium acetate 5.0 g Dipotassium Phosphate 2.0 g Ammonium citrate 2.0 g Polysorbate 80 1.0 g Magnesium sulfate 0.1g Manganese Sulfate 0.05g Distilled water 1L

I) Maltose Analysis

Inoculate 72% isolated strain, strain (KACC11451) and commercial strain (Lallemand L73) in maltose-containing MRS medium at 10% each and incubated for 10 hours. Used for analysis. Sugar analysis was performed using HPLC (SHISEIDO NANOSPACE S1-2). As HPLC conditions, the mobile phase flowed 80% ACN at a flow rate of 1 ml / min, and a sugar analysis column heated at 35 ° C. (Waters carbohydrate High performance 4 μm) and RI detector were used (FIG. 8). 8 is a result confirming the maltose capacity of the present invention Lactobacillus plantarum SPC-SNU 72-2. As a result of the experiment, 72-2 isolates were found to have better maltose capacity than test strains and commercial strains.

(2) Lactobacillus Planta Room (Lactobacillus plantarum Baking application of

In this experiment, Lactobacillus plantarum, test strain (KACC11451), and commercial strain (Lallemand L73) isolated from kimchi were applied to baking, and their characteristics were examined.

end) Preparation and Analysis of Lactic Acid Bacteria Fermented Dough

Lactic acid bacteria 100g, lactic acid bacteria number 2x10 ¹⁰ cfu / g, heated cooling water 100g mixed and fermented in a fermenter at 30 ℃ to prepare a lactic acid bacteria fermented dough, when the pH of the dough reaches pH 4.0 ± 0.2, after cooling the lactic acid bacteria The pH, TTA and bacterial count of the fermented dough were measured. On the other hand, the lactic acid bacteria used in this experiment was incubated in MRS medium for 30 ℃, 22 ± 2 hours, centrifuged and recovered the cells, the cells were washed with physiological saline several times, the first inoculation amount was 1 × 1g per dough Inoculated to be 10 ⁸ .

The experimental results are shown in Table 6 below. As a result, it took 4 hours to reach pH 4.0 ± 0.2 for both the strains and the pastes applied with the strains.

When the pH of the dough reaches pH 4.0 ± 0.2, measure the pH, TTA and number of bacteria of lactic acid bacteria fermented dough after cooling pH TTA ( 15 g  Standard, ml) Bacteria  ( cfu / g) Organic acid pH 6.6 pH 8.5 SPC-SNU 72-2
( Lb. plantarum KCTC13315BP)
Apply dough 3.87 8.60 11.01 1.67 x ^{10 9} Lactic acid 0.32%
Acetic acid 0.041% Commercial strain (Lb. plantarum Lallemand l73)
Apply dough 3.88 8.75 11.14 1.38 x ^{10 9} Lactic acid 0.35%
Acetic acid 0.051% Test strain (Lb. plantarum KACC 11451)
Apply dough 3.84 9.35 11.70 1.54 x ^{10 9} Lactic acid 0.76%
Acetic acid 0.076%

I) Bread manufacturing

Bread manufacturing method was carried out by the sponge (Sponge) method.

① Compounding process (medium seed) and primary fermentation

The ingredients as shown in Table 7 were added to a mixer (product name: SK101S MIXER: Japan), kneaded in two stages for two minutes and three stages for one minute, and mixed so that the final temperature of the dough was 25 ° C. In the fermentor, 27 ℃ primary fermentation for 4 hours.

Control (bread with commercial yeast only) Lb. plantarum  SPC-SNU 72-2
( KCTC13314B )
Applied bread Commercial strain
Lb. plantarum   Lallemand  L73
Applied bread Disclosure strain
Lb. plantarum
( KACC  11451)
Applied bread Zhongjong flour 70 70 70 70 Sac. cerevisiae
SPC 70-1 2 2 2 2 rating 42 42 42 42 Main species flour 30 20 20 20 Refined salt 2 2 2 2 White sugar 4 4 4 4 Skim milk powder 4 4 4 4 maintain 12 12 12 12 rating 23 13 13 13 Lactobacillus
Fermented species - 20 20 20

② Compounding process ( Main species ) And secondary fermentation

The first fermented dough and flour, white sugar, refined salt, whole milk powder, yeast, purified water with the ingredients as shown in Table 7 and kneaded in the first stage for 1 minute, the fermented product prepared in the middle seed process The mixture was added in 2 stages and mixed for 3 minutes and 3 stages for 2 minutes. Next, butter was added and kneaded in two stages for three minutes and three stages for three minutes, so that the final temperature of the dough composition was 27 ° C. After molding, the pan was panned and then subjected to secondary fermentation for 100 minutes at 38 ° C. and a relative humidity of 85%.

③ Firing and Cooling Process

The dough after the second fermentation process was baked for 37 minutes in a tech oven of 210 ° C. and 250 ° C. of a lower fire, and then taken out of a breadboard and cooled to an internal temperature of 32 ° C. at room temperature (FIG. 9). 9 is a bread to which the Lactobacillus plantarum KACC11451 is applied as a test strain, a bread to which Lallemand L73 is applied as a commercial strain of Lactobacillus plantarum, and a bread to which the present invention Lactobacillus plantarum SPC-SNU 72-2 is applied. to be.

(3) Sensory evaluation and measurement of bread properties

end) Measurement of the physical properties of bread

We tried to measure the physical properties (pH, total titratable acidity, chromaticity) of control (bread with commercial yeast only), bread with separate strain, bread with commercial strain (Lallemand L73), and bread with public strain (Lb. plantarum KACC11451). . The pH was measured by homogenizing 100 ml of distilled water and 15 g of a sample in a 250 ml beaker, followed by homogenization. Total titrable acid (TTA) was defined as the amount of NaOH solution consumed (ml) until titrated with 0.1 N NaOH solution to pH 6.6 and pH 8.5. Color measurement was measured by using a colorimeter (CR-400, KONIKA MINOLTA Co., Ltd.) of the bread cut to a thickness of about 20 mm, the results are shown in Table 8 below. As a result, the physical properties of the bread of the control and the addition of lactic acid bacteria were confirmed to be generally similar.

Measurement of the physical properties of bread Item Applied Product Sensory Evaluation and Product Characteristics Control Lb.plantarum
SPC-SNU 72-2
Applied bread Commercial strain Lb.plantarum Lallemand L73
Applied bread Disclosure strain
Lb.plantarum (KACC11451)
Applied bread Cost 4.88 4.85 4.85 4.87 Texture 7.4 7.8 7.8 7.7 *zest 7.3 7.6 7.6 7.6 pH 5.53 5.41 5.35 5.29 TTA (6.6 / 8.5) 2.27 / 4.87 3.25 / 5.11 3.29 / 5.18 3.25 / 5.27 Moisture content (%) 41.65% 41.51% 41.49% 41.54% Organic acid Lactic acid 0.015%
Acetic acid 0.037% Lactic acid 0.150%
Acetic acid 0.040% Lactic acid 0.152%
Acetic acid 0.043% Lactic acid 0.154%
Acetic acid 0.040% L 84.11 84.54 85.01 84.91 Hunter lab
color values a -2.16 -2.35 -2.11 -2.19 b 17.72 17.22 17.32 17.51

* 1: Very bad, 9: Very good

I) Gas of dough Generating force  Confirm

This study was designed to compare the gas generating powers of the control group (primary strains with commercial yeast), the strains with commercial strain (Lallemand L73), the strains with strain (KACC11451), and the strains with separate strain. Gas generation force measurement was measured 25g of the dough composition of the mixing process (main species) and measured for 10 hours at 30 ℃ temperature conditions through a gas generator (Fermometer) (Fig. 10). 10 is a result of confirming the gas generating force of the present species to which the present invention Lactobacillus plantarum SPC-SNU 72-2, commercial strain Lallemand L73, the lactobacillus plantarum KACC11451 used as the test strain. As a result, the gas generating powers of the control and the addition of lactic acid bacteria were generally similar, but the initial gas generating power of the lactic acid bacteria added group was found to be rather high.

All) Determination of Aging in Bread

In order to measure the aging rate according to the time change of the test and control of the bread prepared as described above, the hardness of the sample cut to about 20mm thickness after cooling the bread for 3 hours at room temperature (Texture analyser, Stable Micro Systems), and the values for hardness are shown in Table 9 below (FIG. 11). 11 is a result of measuring the aging rate of the bread to which the present invention Lactobacillus plantarum SPC-SNU 72-2 is applied. As a result of the hardness (Hardness) comparison according to the product storage, when the lactic acid bacteria were used, the overall hardness was low and the softness was excellent, and the overall aging rate was slower than the control.

Hardness measurement of bread Sample Hardness (N) 19 hours 63 hours 87 hours Control 1.652 2.615 2.680 Lb. plantarum 72-2 Fermented Seeds 1.495 2.515 2.544 Lb. plantarum Fermented Lallemand L73 1.532 2.509 2.530 Lb. plantarum (KACC 11451)
Fermented seed application 1.516 2.557 2.583

la) Lactobacillus  Application dough fragrance ingredient analysis

In the manufacture of bakery using lactic acid bacteria fermented dough compared to the control, in order to compare the expression of flavor components, it was analyzed using GC / MS system. Analysis of 1g of samples was performed, and GC / MS analysis conditions were as shown in Table 10 below. After GC / MS analysis, the overall quantitative values for alcohols, aldehydes, ketones, and acids were compared (FIG. 12), and in Table 11, for the 19 representative fragrance components, The relative proportions of each component are expressed as percentages. 12 is a result of comparing the quantitative value of the volatile aroma components (alcohol, aldehyde, ketone, acid, etc.) detected per 1g of bread (sample) to which the present invention Lactobacillus plantarum SPC-SNU 72-2 is applied.

As a result, the content of aroma component was higher in the experimental group compared to Lallemand L73, and the isolates were higher in alcohol and ketones . In addition, Lallemand L73 had a high content of hexanal, which causes greening, and the content of aromatic flower flavored 1-heptanol was high in 72-2 isolates.

GC / MS Analysis Conditions Analysis system Operating conditions GC / MS analysis GC Model name: Agilent 7890A
Inlet temperature: 230 ℃
Column: DB-WAX (60 m X 250 um X 0.25 uM)
Carrier gas: helium
Flow rate: 1 ml / min
Oven temperature program: from 40 ℃ (5 min) → 8 ℃ / min → 230 ℃ (10min)
MS detector: Agilent 5975C MSD (EI mode) SPME analysis Fiber: DVB / Carboxen / PSME (Supelco Co.)
Sample equilibration time
incubation temp. 85 ℃
-incubation time 30 min

SPC-SNU 72-2 KACC11451 Lallemand  L73 % SD % SD % SD alcohols Ethyl alcohol 0.30 0.02 0.21 0.04 0.28 0.02 One- Pentanol 3.20 0.09 3.39 0.08 3.59 0.14 3-Methyl-1- butanol 0.76 0.18 0.78 0.20 1.01 0.16 One- Hexanol 49.29 1.31 43.91 3.05 30.87 4.43 One- Heptanol 2.56 0.18 1.64 0.60 1.57 0.19 2- Ethylhexanol 2.17 0.19 2.42 0.15 2.49 0.45 One- Octanol 1.83 0.02 1.21 0.40 1.35 0.17 One- Nonanol 1.65 0.03 1.29 0.46 2.42 0.38 aldehydes Hexanal 13.03 1.87 20.72 0.22 27.44 4.75 (E) -2- Heptenal 4.88 0.16 4.08 0.63 5.31 0.55 2- Octenal 4.79 0.41 6.73 0.45 5.35 0.01 Nonenal 0.63 0.00 1.06 0.09 0.82 0.48 Benzaldehyde 0.48 0.03 0.50 0.13 0.75 0.08 ketones 2- Heptanone 0.64 0.46 0.43 0.12 1.58 0.29 Acetoin 0.73 0.29 0.50 0.09 1.19 0.09 2- Octanone 1.38 0.09 0.41 0.14 1.03 0.17 Phenyl methyl ketone 1.43 0.09 0.81 1.00 1.50 0.03 acids Hexanoic  acid 8.24 0.34 8.36 0.46 9.91 1.40 etc 2- Pentylfuran 2.04 0.03 1.55 0.35 1.54 0.25 sum 100 - 100 - 100 -

* Relative proportions of each component were shown as percentages for 19 representative fragrance components [Alcohol (8), Aldehyde (5), Ketone (4), Acid (1), and others (1).

E) sensory evaluation

Sensory evaluation of control (bread with commercial yeast only), bread with separate strain, bread with commercial strain (Lallemand L73), and bread with test strain ( Lb. plantarum KACC11451) showed that the experimental group with lactic acid bacteria was softer than the control. Excellent evaluation in terms of texture and flavor. The comparative evaluation in the experimental groups containing lactic acid bacteria did not show a big difference, but in the overall evaluation, the bread applied with the isolate strain SPC-SNU 72-2 received the highest rating.

Depositary: Korea Research Institute of Bioscience and Biotechnology

Accession number: KCTC13315BP

Trust Date: 2017824

Claims (3)

Lactobacillus plantarum SPC-SNU 72-2 (KCTC13315BP) exhibiting acid resistance even at pH 3.0.
Claim 1 Lactobacillus plantarum Lactobacillus plantarum SPC-SNU 72-2 (KCTC13315BP) The dough for baking, characterized in that prepared by adding to the flour and then fermented.
Claim 1 Lactobacillus plantarum ( Lactobacillus plantarum ) SPC-SNU 72-2 (KCTC13315BP) is added to flour, fermented, and then bread.

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