KR101769445B1 - New Lactobacillus plantarum isolated from tea leaf - Google Patents

Abstract

The present invention relates to a novel Lactobacillus < RTI ID = 0.0 > plantarum ) APsulloc 331269 (Accession No .: KCCM11182P). The present invention also relates to the use of Lactobacillus plantarum ) APsulloc 331269 strain or a culture thereof.

Description

New Lactobacillus plantarum isolated from tea leaf < RTI ID = 0.0 >

The present invention relates to novel Lactobacillus plantarum isolated from tea leaves.

The tea used for drinking is deactivated and dehydrated by oxidizing enzymes present in camellia sinensis buds or leaves of Camellia sinensis . Such tea contains vitamins, caffeine, tannins, flavonoids and essential oils, and is widely used in a variety of fields, such as food.

The present invention is to provide a novel Lactobacillus plantarum strain. And to provide a composition comprising the novel Lactobacillus plantarum strain or a culture thereof.

One aspect of the present invention relates to a method of treating Lactobacillus < RTI ID = 0.0 > plantarum ) APsulloc 331269.

Another aspect of the present invention provides a method of treating Lactobacillus < RTI ID = 0.0 > plantarum ) APsulloc 331269 strain or a culture thereof.

The novel Lactobacillus plantarum ( Lactobacillus < RTI ID = 0.0 > plantarum APsulloc 331269 is excellent in acid resistance, so it can survive in the stomach when consumed as food, and the intestinal reach rate may be higher. Lactobacillus plantarum APsulloc 331269 is excellent in intestinal solubility because of its excellent bile acidity. In addition, Lactobacillus plantarum APsulloc 331269 has excellent antimicrobial activity and is thus effective in inhibiting harmful bacteria. Lactobacillus plantarum APsulloc 331269 has a lower proportion of D-lactic acid among the produced lactic acid than existing Lactobacillus plantarum, so that adults or infants who are vulnerable to lactic acid can also freely take it. Therefore, the novel Lactobacillus plantarum APsulloc 331269 according to the present invention can be widely used in various fields exemplified in the food field.

Lactic acid bacteria refers to bacteria that decompose saccharides such as glucose to produce lactic acid, and are also referred to as lactic acid bacteria. Lactic acid produced by lactic acid fermentation of lactic acid bacteria can inhibit the growth of pathogens and harmful bacteria, and such properties are used in the manufacture of foods such as dairy products, kimchi, and brewed foods. Lactic acid bacteria are also important bacteria used as enteric agents because they live in the intestines of mammals and prevent abnormal fermentation by germs.

Lactobacillus plantarum is a lactic acid bacterium belonging to the genus Lactobacillus, and it is known that kimchi is fermented mainly and grows when it becomes sour. The optical isomers of lactic acid to be produced are D type and L type. Lactobacillus plantarum can be widely used in various foods that require fermentation, so it would be useful if Lactobacillus plantarum, which has excellent acid resistance, bile acid and antibacterial properties, is developed.

Hereinafter, the present invention will be described in detail.

One aspect of the present invention relates to a method of treating Lactobacillus < RTI ID = 0.0 > plantarum ) APsulloc 331269 (Accession No .: KCCM11182P).

The APsulloc 331269 was grown on a Camellia sinensis ) leaves, belonging to Lactobacillus plantarum . Specifically, APsulloc 331269 includes the steps of ripening tea leaves with a salt of 5 to 15% by weight based on the weight of the tea leaves; The marinated tea leaves are mixed with a sugar solution, for example, 0.1% to 3% of fructo-oligosaccharides, and cultured at 25 to 35 ° C for 1 to 5 days; And a step of culturing for 1 to 5 days under anaerobic conditions at 25 to 35 캜 by taking a culture medium having a pH of less than 5.

In accordance with one aspect of the present invention, Lactobacillus < RTI ID = 0.0 > plantarum ) APsulloc 331269 has excellent acid resistance. When a lactic acid bacterium such as Lactobacillus plantarum is taken as a probiotic agent, it is preferable that the intestinal permeability is high in order to exhibit the effect specific to the lactic acid bacteria. In order for the intestinal reach rate to be high, the survival rate in the stomach should be high due to the acid secretion first. The above fasting pH is about 1.2 ~ 2.0, but it is known that when the food is consumed, the pH is about 2 ~ 3. Lactobacillus plantarum APSulloc 331269 according to the present invention has excellent acid resistance at pH 2.5 to 4.0, specifically pH 2.5 to 3.5, more specifically pH 2.5 to 3.0. On the other hand, when food is consumed, the average uptake time of food is known to be about 1 to 3 hours. Lactobacillus plantarum APSulloc 331269 according to the present invention has excellent acid resistance performance at pH 2.5 to 4.0, specifically pH 2.5 to 3.5, more specifically pH 2.5 to 3.0, for 0.5 to 5 hours, specifically 1 to 4 hours I have. Therefore, Lactobacillus plantarum APsulloc 331269 according to the present invention can survive at the lower pH during the stomach period, so that the intestinal reaching rate of the food is high.

In accordance with one aspect of the present invention, Lactobacillus < RTI ID = 0.0 > plantarum ) APsulloc 331269 has excellent bile acid abilities. The food passed through the stomach is transferred to the small intestine, where bile acid is secreted to help digest food. Staphylococcus aureus strains resistant to bile acids are known to have good colonization potential. Lactobacillus plantarum APsulloc 331269 is excellent in intestinal solubility because of its excellent bile acidity.

In accordance with one aspect of the present invention, Lactobacillus < RTI ID = 0.0 > plantarum ) APsulloc 331269 has the ability to produce lactic acid. Generally lactic acid produced by lactic acid bacteria has L-type and D-type. Among these, D-lactic acid is slower in metabolism than L-lactic acid, so that the concentration of D-lactic acid in the blood can cause lactic acid toxicity. Therefore, adults who are susceptible to lactic acid, or infants and young children, preferably do not take lactic acid bacteria that produce a large amount of D-lactic acid.

According to another aspect of the present invention, Lactobacillus plantarum APsulloc 331269 is capable of producing lactic acid having 75% or less, specifically 70% or less of the D-form of the produced lactic acid.

According to another aspect of the present invention, Lactobacillus plantarum APsulloc 331269 is capable of producing lactic acid at 17.5 g / L or less, specifically 17.0 g / L or less.

Thus, Lactobacillus plantarum AP1231269 according to the present invention produces lactic acid having a lower proportion of D-form than the conventional Lactobacillus plantarum, so that adults and infants who are vulnerable to lactic acid can freely take it.

One aspect of the present invention relates to a method of treating Lactobacillus < RTI ID = 0.0 > plantarum ) APsulloc 331269. Another aspect of the present invention provides a composition comprising Lactobacillus plantarum APsulloc 331269 strain, an extract thereof, or a culture thereof.

One aspect of the present invention provides a food composition comprising a strain of Lactobacillus plantarum APsulloc 331269, an extract thereof or a culture thereof.

The food composition may be a health food composition, and may be a fermented food composition requiring fermentation, such as tea, dairy, kimchi, and brewed foods.

The formulation of the food composition is not particularly limited, but may be formulated into tablets, pills, soft and hard capsules, granules, drinks, caramels, diet bars, tea bags and the like. The food composition of each formulation can be blended with the ingredients commonly used in the field in addition to the active ingredient without difficulty by those skilled in the art depending on the purpose of formulation or use, and synergistic effect can be obtained when the composition is applied simultaneously with other ingredients.

Determination of the dosage of the active ingredient is within the level of ordinary skill in the art, and its daily dose is, for example, 10 ⁵ to 10 ¹³ CFU / day, more specifically 10 ⁶ to 10 ¹⁰ CFU / day for Lactobacillus plantarum But may vary depending on various factors such as the age, health condition, complications, etc. of the subject to be administered.

An aspect of the present invention provides a cosmetic composition comprising a strain of Lactobacillus plantarum APsulloc 331269, an extract thereof, or a culture thereof. The cosmetic composition may be provided in any formulation suitable for topical application. For example, it may be provided as a solution, an emulsion obtained by dispersing an oil phase in an aqueous phase, an emulsion obtained by dispersing an oil phase in water, a suspension, a solid, a gel, a powder, a paste, a foam or an aerosol composition. Compositions of such formulations may be prepared according to conventional methods in the art.

The cosmetic composition may contain, in addition to the above-mentioned substances, other ingredients which can give rise to a synergistic effect on the main effect, so long as they do not impair the main effect. The cosmetic composition according to the present invention may comprise a material selected from the group consisting of vitamins, high molecular peptides, high molecular weight polysaccharides and sphingolipids. The cosmetic composition according to the present invention may also contain a moisturizing agent, an emollient agent, a surfactant, an ultraviolet absorber, an antiseptic, a bactericide, an antioxidant, a pH adjuster, an organic and inorganic pigment, a fragrance, . The blending amount of the above components can be easily selected by those skilled in the art within a range not to impair the objects and effects of the present invention. The blending amount thereof can be 0.01 to 5% by weight, specifically 0.01 to 3% by weight based on the total weight of the composition have.

One aspect of the present invention provides a pharmaceutical composition comprising a strain of Lactobacillus plantarum APsulloc 331269, an extract thereof or a culture thereof. The pharmaceutical composition may be used for the prevention or treatment of intestinal diseases such as irritable bowel syndrome, constipation, diarrhea and the like.

The pharmaceutical composition according to one aspect of the present invention may be administered orally, parenterally, rectally, topically, transdermally, intravenously, intramuscularly, intraperitoneally, subcutaneously, and the like. Formulations for oral administration may be in the form of tablets, pills, soft and hard capsules, granules, powders, granules, solutions, emulsions or pellets, It is not. Formulations for parenteral administration may be, but are not limited to, solutions, suspensions, emulsions, gels, injections, drops, suppositories, patches or spray formers. The formulations may be readily prepared according to conventional methods in the art and may be prepared by conventional means including, but not limited to, surfactants, excipients, wetting agents, emulsifying accelerators, suspending agents, salts or buffers for controlling osmotic pressure, colorants, spices, stabilizers, preservatives, Other commonly used adjuvants may be used.

The effective ingredients of the pharmaceutical composition according to one aspect of the present invention will vary depending on the age, sex, weight, pathological condition and severity of the subject to be administered, route of administration, or judgment of the prescriber. Determination of the amount of application based on these factors is within the level of ordinary skill in the art and its daily dose is, for example, from 0.1 mg / kg / day to 5000 mg / kg / day, more specifically from 50 mg / kg / day to 500 mg / / Day, but is not limited thereto.

The Lactobacillus < RTI ID = 0.0 > plantarum ) APsulloc 331269 was deposited with the Korean Culture Center of Microorganisms on March 28, 2011 as microorganism deposit number KCCM11182P.

Name of depository: Korea Microorganism Conservation Center (Domestic)

Accession number: KCCM11182P

Checked on: 20110328

Hereinafter, the present invention will be described in further detail with reference to the following Examples and Experimental Examples. Lactobacillus plantarum ) APsulloc 331269 will be described in detail. However, the following examples and experimental examples are provided for illustrative purposes only in order to facilitate understanding of the present invention, and the scope and scope of the present invention are not limited thereto.

[Example 1] Preparation of Lactobacillus plantarum) separation of APsulloc 331269

200g of tea leaves are washed twice with primary distilled water to remove foreign matter. Shake off the water of the washed tea leaves and mix with the salt equivalent to 8% of the weight of the tea leaves and leave at room temperature for 3 hours. Mixed tea leaves in 1000 mL of 1% fructose oligosaccharide solution and incubate in a 32 ° C incubator for 3 days. Three days confirmed that the pH of the culture solution drop to less than 5 and less than pH 5 after taking them deep nose galactosidase is cultured in MRS agar Bashile ^® (Difco Lactobacilli MRS Agar ^®) medium. At this time, the culture is carried out in a chamber of anaerobic condition at 32 ° C for 2 days, and then a colony showing white colonies is taken.

In the same manner as above, Lactobacillus plantarum ) APsulloc 331269 was isolated from tea leaves.

[Example 2] Lactobacillus < RTI ID = 0.0 > plantarum ) Identification of APsulloc 331269

(1) Culture of the strain

APsulloc 331269 isolated in Example 1 is streaked onto MRS agar plates and incubated at 37 ° C for 2 days. A single colony obtained is inoculated into 10 mL of MRS broth and incubated overnight at 37 ° C to prepare APsulloc 331269 culture.

(2) Analysis of the fermentation pattern of APsulloc 331269

The culture of the APsulloc 331269 strain prepared as described in (1) above was inoculated 0.5% in 10 mL of MRS broth and incubated overnight at 37 ° C. The culture was centrifuged at 8,000 rpm for 5 minutes, and the supernatant was removed to collect only the cells, followed by the addition of 2 mL of 0.85% saline buffer. Followed by an API 50CHL kit (Biomerieux) according to the manufacturer's protocol. The details are as follows.

First, a suspension of the strain was added to 5 ml of the API suspension medium to measure the amount of suspension required to have a turbidity of about 2 McFarland Standard 2 (Biomerieux). Two times the amount of the measured suspension was added to 10 mL of API 50CHL medium, and the mixture was shaken. The mixture was placed in a cupule containing different substrates, and the mineral oil was dropped one by one. The mixture was incubated at 37 ° C for 2 days, and the sugar fermentation pattern was analyzed.

Lactobacillus ( Lactobacillus) The results of the identification of APsulloc 331269 using the result of sugar fermentation of APsulloc 331269 and the results of the comparison with the standard strain of KCTC3108 ( plantarum ) (KCTC3108) are shown in the following table.

temperament KCTC3108 APsulloc 331269 temperament KCTC3108 APsulloc 331269 24h 48h 24h 48h 24h 48h 24h 48h Control group - - - - Essecurin + + + + Glycerol - - - - Salincin + + + + Erythritol - - - - Cellobiose ? + + + D-arabinose - - - - Maltose + + + + L-arabinose + + - - Lactose + + + + Ribos + + + + Melibiose + + + + D-xylose - - + + D-Sakaros (Sucross) + + + + L-xylose - - - - Trehalose + + + + Adonitol - - - - Inulin - - - - beta -methyl-D-xylose - - - - Melechitos + + + + Galactose + + + + Raffinos - - + + Glucose + + + + Amidon (starch) - - - - Fructose + + + + Glycogen - - - - Mannos + + + + Xylitol - - - - Sorbos - - - - Gentiobios - - + + Rams North - - - - D-Turanos + + + + Dallitol - - - - D-Rick sauce - - - - Inositol - - - - D-tagatose - - - - Mannitol + + + + D-fucose - - - - Sorbitol + + + + L-fucose - - - - alpha -methyl-D-mannose ? + ? + D-arabitol ? - - - alpha -methyl-D-glucoside - - - - L-arabitol - - - - N-acetyl-glucosamine + + + + Gluconic acid ? + ? + Amigalline + + + + 2-ketogluconate - - - - Arbutin + + + + 5-ketogluconate - - - -

+: Substrate decomposition, -: Not decomposing the substrate,?: Not determinable

Strain Dependent name % index T index KCTC Lactobacillus Florata Room 99.9 0.8 3108 Lactobacillus pentosus 0.1 0.29 APsulloc 331269 Lactobacillus Florata Room 99.4 0.79 Lactobacillus pentosus 0.5 0.51

As can be seen from the above, APsulloc 331269 inhibits Lactobacillus < RTI ID = 0.0 > plantarum) agreement on (% index) appeared in more than 99% can be seen that belongs to the Lactobacillus Planta Room (Lactobacillus plantarum). In addition, APsulloc 331269 can be confirmed to be a strain different from the standard strains (KCTC3108), L-arabinose and raffinose using different properties.

(3) Analysis of enzyme activity pattern of APsulloc 331269

The culture of the APsulloc 331269 strain prepared as described in (1) above was inoculated 0.5% in 10 mL of MRS broth and incubated overnight at 37 ° C. The culture was centrifuged at 8,000 rpm for 5 minutes, and the supernatant was removed to collect only the cells, followed by the addition of 2 mL of 0.85% saline buffer. And then proceeded according to the manufacturer's protocol using the API ZYM kit (Biomerieux). The details are as follows.

First, a suspension of the strain was added to 5 ml of the API suspension medium to measure the amount of suspension required to have a turbidity of about 2 McFarland Standard 2 (Biomerieux). Two times the amount of the measured suspension was added to 10 mL of API 50CHL medium, and the mixture was shaken. 65 상기 of the mixture was added to each cupule and incubated at 37 캜 for 4 hours. One drop of ZYM A reagent and ZYM B reagent was dropped to each of the spools. After 5 minutes, the scale was scored from 0 to 5 according to the intensity of the color, and the result was judged to be positive if it was 3 or more.

Lactobacillus ( Lactobacillus) plantarum strain (KCTC3108) as a standard strain, the results of the enzyme activity pattern of APsulloc 331269 are as follows.

enzyme KCTC3108 APsulloc 331269 Score result Score result Control group 0 - 0 - Alkaline phosphatase 0 - One - Estradiol One - One - Estera lase lipase One - One - Lipase 0 - One - Leucine arylamidase 5 + 4 + Valine arylamidase 4 + 3 + Cristin arylamidase One - 2 - Trypsin 0 - One - α-chymotrypsin 0 - One - Sanfors patija One - 3 + Naphthol-AS-BI-phosphohydrolase One - 3 + ? -galactosidase One - 3 + beta -galactosidase 5 + 5 + β-glucuronidase One - 2 - ? -glucosidase 3 + 5 + N-acetyl -? - glucosaminidase 0 - 4 + ? -mannosidase 0 - One - alpha-fucosidase 0 - 0 -

As can be seen from the above, APsulloc 331269 is a standard strain (KCTC3108) and an acid phosphatase, Naphthol-AS-BI-phosphohydrolase,? -Galactoside There is a difference in the enzyme activity intensity of α-galactosidase and N-acetyl-β-glucosaminidase. Therefore, APsulloc 331269 is different from the standard strain.

In addition, APsulloc 331269 was negative for β-glucuronidase activity, which is known to be one of the typical carcinogenic enzymes that can induce cancer by acting on carcinogenic precursors in the intestines and transforming them into carcinogens . Therefore, APsulloc 331269 can be used for a food composition.

(4) Antimicrobial resistance evaluation of APsulloc 331269

20 mL of sterilized MRS agar was added to a petri dish (100 mm in diameter), and the culture medium was prepared by cooling in a clean bench. The APsulloc 331269 culture prepared in (1) was inoculated 0.5% in 10 mL of MRS broth and cultured at 37 DEG C for 6 hours. And diluted so that the absorbance was about 0.08 to 0.13 at 625 nm. The diluted solution was thoroughly soaked in a sterilized cotton swab and then evenly spread on the MRS agar plate previously prepared. Antibiotic susceptibility evaluation disks were dropped onto the plate at appropriate intervals. After culturing at 37 ° C for 24 hours, the diameter of the clear zone was measured. Antibiotic susceptibility evaluation The disk concentration and evaluation criteria and the evaluation results are as follows.

Antibiotic Zone diameter interpretation Ingredients density Resistant Yes (R) Medium (I) Sensitivity (S) Ampicillin 10 g 13 14-16 ≥ 17 Ceftazidim 30 ㎍ ≤ 14 15-17 ≥18 Chloramphenicol 30 ㎍ ≤12 13-17 ≥18 Siprofloxacin 5 g ≤15 16-20 ≥ 21 Clindamycin 2 g ≤ 14 15-20 ≥ 21 Erythromycin 15ug 13 14-22 ≥23 Gentamicin 120 g ≤6 7-9 ≥10 Imipenem 10 g 13 14-15 ≥ 16 Streptomycin 10 g ≤11 12-14 ≥15 Neomycin 30 ㎍ ≤12 13-16 ≥ 17 Nitrofurantoin 300 g ≤ 14 15-16 ≥ 17 penicillin 10U ≤ 14 - ≥15 Polymyxin B 300U ≤8 9-11 ≥12 Tetracycline 30 ㎍ ≤ 14 15-18 ≥ 19 Trimetoprim 5 g ≤10 11-15 ≥ 16 Vancomycin 30 ㎍ ≤ 14 15-16 ≥ 17

Lactobacillus ( Lactobacillus) The results of the antimicrobial resistance test of APsulloc 331269, which was compared with the strain (KCTC3108) as a standard strain, are as follows.

Antibiotic KCTC3108 APsulloc 331269 Zone (mm) result Zone (mm) result Ampicillin 29 S 21 S Ceftazidim 23 S 10 I Chloramphenicol 24 S 22 S Siprofloxacin - R - R Clindamycin 12 R 9 S Erythromycin 26 S 28 S Gentamicin 20 S 18 S Imipenem 39 S 39 S Streptomycin - R - R Neomycin 11 R 9 R Nitrofurantoin - R 27 S penicillin 24 S 12 R Polymyxin B - R - R Tetracycline 17 I 17 I Trimetoprim - R - R Vancomycin - R - R

R: resistant, I: intermediate, S: susceptible.

As can be seen above, APsulloc 331269 showed differences in antibiotic resistance patterns of the standard strain (KCTC3108) and ceftazidime, clindamycin, nitrofurantoin and peniciilin. Therefore, APsulloc 331269 is different from the standard strain.

APsulloc 331269 belongs to Lactobacillus plantarum and is a new strain different from Lactobacillus plantarum standard strain (KCTC3108) in the comprehensive analysis of sugar fermentation pattern, enzyme activity pattern and antibiotic resistance pattern.

[Experimental Example 1] Evaluation of acid resistance

(1) Evaluation of acidic performance by pH

10 mL of MRS broth was inoculated with 0.5% APsulloc 331269 culture and incubated overnight at 37 ° C. After adjusting the pH to 2.0, 2.5, 3.0, and 3.5 with HCl, 5 ml of the sterilized MRS broth was inoculated with 50 상기 of the culture, followed by incubation at 37 캜 for 1 hour. After 1 hour, the cells were diluted with a peptone saline buffer and the number of bacteria per mL was measured. The viability of the culture was determined by counting the number of cultures and then multiplying by 0.01 to control (control) and 100% of control. Lactobacillus ( Lactobacillus) plantarum strain (KCTC3108) as a standard strain.

pH KCTC3108 APsulloc 331269 cfu / ml Survival rate (%) cfu / ml Survival rate (%) Control group 2.9x10 ⁷ 100 5.0 x 10 ⁷ 100 2.0 3.0x10 ¹ 0 3.6 x 10 ² 0 2.5 2.5x10 ⁷ 87.4 5.6 x 10 ⁷ 130.7 3.0 3.2 x 10 ⁷ 110.3 6.5 x 10 ⁷ 112.7 3.5 2.8 x 10 ⁷ 97.7 6.2 x 10 ⁷ 124.0

As can be seen from the above, APsulloc 331269 showed a higher survival rate than the standard strain at pH 2.5 to 3.5. That is, APsulloc 331269 has excellent acid resistance. Considering that the pH at the time of ingestion of food is 2.0 to 3.0, it can be seen that when the APsulloc 331269 is included in the food composition, the above survival rate will be high.

(2) Evaluation of acid-resistant performance over time

10 mL of MRS broth was inoculated with 0.5% APsulloc 331269 culture and incubated overnight at 37 ° C. 5 ml of the sterilized MRS broth was inoculated with 50 μl of a culture solution adjusted to pH 2.5 with HCl, and then cultured at 37 ° C for 3 hours. After 1 hour and 3 hours, the cells were diluted with a peptone saline buffer and the number of bacteria per mL was measured. The viable cell count was calculated by multiplying 0.01 by the number of bacteria in the culture medium. The control group was used as control, and the viability of the control group was calculated as 100%. Lactobacillus ( Lactobacillus) plantarum strain (KCTC3108) as a standard strain.

time KCTC3108 APsulloc 331269 (city) cfu / ml Survival rate (%) cfu / ml Survival rate (%) 0 3.3 x 10 ⁷ 100 7.1 x 10 ⁷ 100 One 2.9x10 ⁷ 86.9 7.6 x 10 ⁷ 107.1 3 2.2 x 10 ⁷ 67.3 5.6 x 10 ⁷ 78.8

As can be seen from the above, it can be confirmed that APsulloc 331269 survives more than 70% even after 1 hour and 3 hours at pH 2.5. That is, APsulloc 331269 has excellent acid resistance for a long time. Considering that the average uptake time during food ingestion is 1 to 3 hours, it can be seen that when APsulloc 331269 is included in the food composition, the survival rate will be high even during the stay.

Therefore, APsulloc 331269 can survive above the lower pH during the uptake period, so it can survive above the food intake, and thus the intestinal reach rate is high.

[Experimental Example 2] Evaluation of bile acid content in bile

10 mL of MRS broth was inoculated with 0.5% APsulloc 331269 culture and incubated overnight at 37 ° C. 0.3% and 0.5% ox gall were added to the MRS agar plates, respectively. As a control (control), MRS agar without bovine bile was used. The above culture broth was diluted and stained on MRS agar medium and then cultured at 37 캜 for 2 days. After the colonies were counted, the survival rate (%) of APsulloc 331269 was calculated using the number of control cells as 100%. Lactobacillus ( Lactobacillus plantarum strain (KCTC3108) as a standard strain.

Ox Bile Concentration (%) KCTC3108 APsulloc 331269 cfu / ml Survival rate (%) cfu / ml Survival rate (%) 0 3.3 x ^{10 9} 100 3.3 x ^{10 9} 100 0.3 3.2x10 ⁹ 97 3.2x10 ⁹ 98.1 0.5 3.0x10 ⁹ 91.9 3.0x10 ⁹ 95.3

As can be seen from the above, APsulloc 331269 showed an excellent bile acid resistance with a survival rate of 95% or more at 0.3% and 0.5% of oxal bile concentration, which is superior to the standard strain. It is known that the strain having excellent bile acid resistance is also excellent in intestinal fixation ability, so that APsulloc 331269 has excellent intestinal fixation ability and intestinal reachability.

[Experimental Example 3] Evaluation of lactic acid production ability

10 mL of MRS broth was inoculated with 0.5% APsulloc 331269 culture and incubated overnight at 37 ° C. The culture was centrifuged at 8000 rpm for 15 minutes to recover only the supernatant. The recovered supernatant was treated at 80 ° C for 15 minutes to stop the enzyme reaction. The supernatant, which was heat-treated with distilled water, was diluted 100-fold. Lactic acid / L-lactic acid UV method kit (R-biopharm) according to the manufacturer's protocol. The details are as follows.

1 mL of kit solution 1 (glycylglycine buffer / L-glutamate), 0.2 mL of solution 2 (NAD solution) and 0.02 mL of GPT suspension solution 3 were added to the cuvette in this order. 0.1 mL of the supernatant prepared above was added to the cuvette. In the control group, 1 mL of the third distilled water was added, and 0.9 mL of the third distilled water was added to the sample. After 5 minutes, the absorbance (A1) was measured at 340 nm. After adding 0.02 mL of D-LDH solution 4, the mixture was incubated for 30 minutes and absorbance (A2) was measured at 340 nm. 0.02 mL of L-LDH solution 5 was added thereto, followed by thorough mixing. After reacting for 30 minutes, absorbance (A3) was measured at 340 nm. The concentrations of D-lactic acid and L-lactic acid in the samples were calculated according to the calculation method. Lactobacillus ( Lactobacillus) plantarum strain (KCTC3108) as a standard strain.

Lactic acid
KCTC3108 APsulloc 331269 ratio(%) Concentration (g / L) ratio(%) D type 72 11.7 69 L type 28 5.2 31 Sum 100 16.9 100

As can be seen from the above, APsulloc 331269 produced both D-lactic acid and L-lactic acid, and the proportion of D-lactic acid in the produced lactic acid was small. Therefore, foods containing them can be freely consumed by adults and infants who are vulnerable to lactic acid.

[Experimental Example 4] Evaluation of antibacterial activity

10 mL of MRS broth was inoculated with 0.5% APsulloc 331269 culture and incubated overnight at 37 ° C. The culture was centrifuged at 8000 rpm for 5 minutes, and only the supernatant was recovered. 10 mL of the supernatant was prepared by sterilizing with a 0.22 L syringe filter.

Meanwhile, Salmonella typhimurium (Salmonella typhimurium and Staphylococcus aureus were each plated on a Tryptic soy agar and incubated at 37 ° C overnight. BHI broth colonies were inoculated and cultured overnight.

BHI agar was sterilized, and 20 mL of each was added to petri dish to prepare a cold culture medium. The cultures of Salmonella typhimurium and Staphylococcus aureus were diluted 50 times with sterile physiological saline and then evenly spread on the prepared medium with a sterilized cotton swab. The sterilized paper disk was raised and 100 占 퐇 of the APsulloc 331269 culture supernatant prepared above was dropped on the paper disk. Allowed to stand at room temperature for about 3 hours to be absorbed, and then cultured at 37 DEG C for 24 hours. The size of the clear zone was measured and the Lactobacillus ( Lactobacillus) plantarum strain (KCTC3108) as a standard strain.

Test strain Diameter of clear zone (mm) KCTC no. Strain KCTC3108 APsulloc 331269 2514 Salmonella typhimurium 8.8 9.3 1621 Stapiro Cocus Aleus 10 10.8

As can be seen from the above, APsulloc 331269 has superior antimicrobial effect against Salmonella typhimurium and Staphylococcus aureus than standard strains.

Korea Microorganism Conservation Center (Domestic) KCCM11182P 20110328

Claims (9)

Wherein the total amount of D-lactic acid and L-type lactic acid to be produced is 17 g / L or less and the ratio of the D-type lactic acid concentration in the total of the D-type lactic acid and L-type lactic acid concentration is 70% , Lactobacillus plantarum APsulloc 331269 (Accession No .: KCCM11182P).
The method according to claim 1,
Lactobacillus planta Room APsulloc 331269 is a tea plant ( Camellia Sinensis ) leaves. Lactobacillus plantarum APsulloc.
delete The method according to claim 1,
Lactobacillus plantarum APsulloc 331269 is characterized in that it has acid resistance performance at pH 2.5 to pH 4.0 for 0.5 to 5 hours.
The method according to claim 1,
Lactobacillus plantarum APsulloc 331269 has Lactobacillus plantarum APsulloc 331269, which has a bile acid capacity in the range of 0.3 to 0.5 wt.% Of bile in 2 days.
delete delete A fermented food composition comprising Lactobacillus plantarum APsulloc 331269 strain or a culture thereof according to any one of claims 1, 2, 4 and 5.
delete