KR100771209B1 - Antioxidant activity Lactobacillus plantarum ?? - Google Patents

Abstract

The present invention relates to Lactobacillus spissis NC (Gene Bank Accession No. KCTC 10869 BP) excellent in antioxidant activity, and more particularly to a novel lactic acid bacterium excellent in acid resistance and antioxidant activity. Lactobacillus spis NC according to the present invention is excellent in physiological function has an effect that can be widely used in cosmetics, food, medicine, household goods.

Antioxidant, Acid Resistant, Lactobacillus spis NC, Accession No. KCTC 10869 BP

Description

Antioxidant activity Lactobacillus plantarum NC

1 is a graph showing the evaluation results of the electron donating ability of lactic acid bacteria including Lactobacillus spissis NC according to the present invention.

Figure 2 is a graph showing the iron reduction ability evaluation results of lactic acid bacteria including Lactobacillus spissis NC according to the present invention.

The present invention relates to Lactobacillus spissis NC (Gene Bank Accession No. KCTC 10869 BP) excellent in antioxidant activity, and more particularly to a novel lactic acid bacterium excellent in acid resistance and antioxidant activity. Lactobacillus spis NC according to the present invention is excellent in physiological function can be widely used in cosmetics, food, medicine, household goods.

In general, lactic acid bacteria are widely present in nature and are microorganisms that produce lactic acid by using carbohydrates such as lactose, and for a long time, lactic acid bacteria have been directly or indirectly used in food.

Although Pasteur first isolated lactic acid bacteria in 1858, it was the first attempt to isolate bacillus from Bulgarian milk and name it Bulgarian bacillus , because it was the concept of a bad microorganism that caused wine to become a disease rather than a probiotics. would. The actions of lactic acid bacteria include normalizing the intestinal flora, reducing lactose intolerance, lowering blood cholesterol levels, anticancer effects, strengthening the immune system, and improving the nutritional value of food.

Lactic acid bacteria using foods including kimchi improves the shelf life of the food by the metabolite lactic acid, and has the advantage of improving the flavor and texture of the food. Lactic acid bacteria ingested through fermented kimchi enters the intestinal tract and enters the intestinal epithelial cells, thereby inhibiting and antagonizing pathogenic microorganisms, enhancing immune activity, reducing cancer incidence, and reducing carcinogenic enzymes. It helps a lot. Therefore, lactic acid bacteria are used in dairy products, meat products, and vegetables.

The present inventors have succeeded in separating strains having excellent physiological efficacy and high antioxidant activity as compared to conventional kimchi lactic acid bacteria while studying kimchi lactic acid bacteria.

An object of the present invention is to provide a strain having superior physiological efficacy and high antioxidant activity compared to conventional kimchi lactic acid bacteria.

The lactic acid bacteria according to the present invention were collected from kimchi and serially diluted with physiological saline, and then smeared in a solid medium containing bromocresol purple and incubated anaerobicly for 24 hours at 37 ° C. to obtain yellow colonies. . Lactobacillus spysis NC was found to have excellent acid resistance and very high antibacterial activity.

The characteristics of the Lactobacillus spysis NC strain according to the present invention are as follows and the sugar fermentation characteristics are shown in Table 1.

1. Morphology of bacteria: rod type

2. Colony color: milky white

3. Optimum temperature for growth: 37 ℃

4. Mobility: None

5. Gram Dyeing: Positive

6. Catalase: negative

7. Oxygen Effects: Organized Anaerobic

Table 1 Sugar Fermentability Test

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Glycerol +

Erythritol-

D-Arabinose-

L-Arabinose-

Ribose +

D-Xylose-

L-xylose-

Adonitol-

b-methyl-xyloside-

Galactose +

D-glucose +

D-fructose +

D-manose +

L-sorbose-

Rhamnose-

Dulcitol-

Inositol-

Mannitol +

Sorbitol +

a-Methyl-D-mannoside-

a-Methyl-D-glucoside-

N-Acethyl glucosamine +

Amygdaline +

Arbutine +

Esculine +

Salicine +

Cellobiose +

Maltose +

Lactose +

Melibiose +

Saccharose +

Trehalose +

Inuline-

Melezitose +

D-Raffinose +

Amidon-

Glycogene-

Xylitol-

b-Gentibiose +

D-Turanose +

D-Lyxose-

D-Tagatose +

D-fucose-

L-Fucose-

D-Arabitol-

L-Arabitol-

Gluconate +

2-ceto-gluconate-

5-ceto-gluconate-

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Based on the above results, the selected lactic acid bacteria Lactobacillus spicy NC was identified as Lactobacillus plantarum .

(1) acid resistance evaluation

After inoculating Lactobacillus spis NC of the present invention into 10 ml MS liquid medium and incubating at 37 ° C. for 18 hours, a certain amount of the culture solution was inoculated again in a pH 2.0 buffer solution and incubated for 60 minutes. A certain amount of the culture solution was taken and diluted in a constant ratio in the dilution buffer solution. After inoculating a predetermined amount of the above diluted solution in MLS agar medium, the number of colonies grown by incubating for 48 hours at 37 ℃ was counted. The survival rate was over 50%.

(2) antioxidant activity evaluation

The electron donating ability (DPPH) or iron reduction ability (FRAP) assay, which is widely used for the evaluation of antioxidant activity, is known as a simple and accurate method for measuring antioxidant capacity in fruits or vegetables or extracts thereof (Takao T , Kitatani F, Watanabe N, Yagi A. Sakata KA. 1994.Simple screening method for antioxidants and isolation of several antioxidants produced by marine bacteria from fish and shellfish.Biosci.Biotech.Biochem. 58: 1780-1783; Benzie IFF, Strain JJ. 1996. The ferric reducing ability of plasma (FRAP) as measurement of "antioxidant power": The Frap assay.Anal. Biochem. 239: 70-76)).

As a result of evaluating electron donating ability of lactic acid bacteria isolated from Kimchi, NC strain showed significantly better electron donating ability than other lactic acid bacteria. NC, L-55 and L-67 showed relatively high antioxidant capacities, while 1213 and LB-2 showed relatively low antioxidant capacities. No significant difference was found in the measurement of electron donating ability using DPPH. However, FRAP assay showed differences in antioxidant capacity of GP1B and L-65.

The above results indicate that Lactobacillus spis NC is a new lactic acid bacterium with acid resistance, electron donating ability, and iron reducing ability.

Hereinafter, the present invention will be described in detail with reference to the following Examples.

Example 1 (Isolation of Acid Resistant Strains)

Samples were collected from various sources, such as kimchi, fermented milk products, newborn feces, and adult feces, and then mixed well by putting 1 g in 10 ml of pH 3.0 buffer solution and calibrating pH to 2.3 with HCl stock solution. After standing at 37 ° C. for 2 hours, a sample was taken, diluted with a dilution buffer solution, and plated on an MS agar plate medium prepared by adjusting the pH to 5.4, and incubated at 37 ° C. for 48 hours. After colonization, the colonies were identified as gram-positive, catalase-negative, rod-shaped rods that did not form spores, and were separated into Lactobacillus having acid resistance. The isolated Lactobacillus was named Lactobacillus spis NC through identification of buggy's manual and carbohydrate decomposition experiment.

Example 2 (acid resistance evaluation)

After inoculating a certain amount of Lactobacillus spis NC of the present invention into 10ml MS liquid medium and incubating at 37 ° C. overnight, a predetermined amount of the culture solution was initially inoculated at a pH of 3.5, 3.0 and 2.5 buffer solution of about 10 ⁶ cfu / ml, respectively. Inoculated at concentration. After standing at 37 ° C. for 1 hour, 1 ml was taken and diluted in each diluted saline solution. A certain amount of dilution was smeared on MS agar plate medium and the number of colonies shown by incubating for 48 hours at 37 ° C. was counted and compared. The results are shown in Table 2 below.

TABLE 2 Acid resistance evaluation test of Lactobacillus spis NC

pH Viable cell count (cfu / ml) Early after pH treatment 3.5 3.0 2.5 1.21 x 10 ⁶ 5.3 x 10 ⁶ 4.4 x 10 ⁶ 3.6 x 10 ⁶ 2.1 x 10 ⁶ 2.8 x 10 ⁶

Example  3 (electronic Donation ability  evaluation)

Electron donating ability (EDA) on DPPH was measured by DPPH (2, 2-diphenyl-1-picrylhydrazyl) free radical scavenging method with some modifications to Takao et al. (1). An appropriate amount of ethanol (3-4 mL) was added to 0.8 mL of the prepared DPPH solution (0.04 mM), followed by vigorous shaking for 10 seconds to adjust the amount of ethanol having an absorbance value of 0.95 to 0.99. After adjusting the appropriate amount of ethanol, 0.8 mL of DPPH solution and 0.2 mL of sample solution were mixed for 10 minutes at room temperature and the absorbance was measured at 525 nm. Control experiments were carried out in the same manner, taking 0.2 mL of ethanol instead of the sample. EDA was calculated by the following equation 1 to obtain the absorbance of the sample added furniture and the non-added furniture.

[Equation 1]

% Electron donating ability = {1-(absorbance of sample / absorbance of control)} × 100

The electron donating ability evaluation results of lactic acid bacteria including Lactobacillus spysis NC according to the present invention are shown in FIG. 1.

Example 4 (Iron Reduction Capability Evaluation)

The FRAP assay (ferric reducing ability of plasma) of Benzie and strain (2) was used to determine the total antioxidant potential of the sample. FRAP reagent was mixed with 25 mL of acetate buffer, 2.5 mL of TPTZ solution, 2.5 mL of FeCl ₃ · 6H ₂ O solution (10: 1: 1), and 3 mL of fresh FRAP reagent solution was added to the test tube. Maintained. 100 μl of sample and 300 μl of H ₂ O were added and left for 4 minutes. The amount of blue Fe (II) -tripyridyltriazin compound produced by reducing iron was measured at 593 nm, and FeSO ₄ · 7H ₂ O (0.1-1 mM / L) was used to prepare a standard curve and calculate the concentration of Fe (II) -tripyridyltriazin.

FRAP value was calculated as the absorbance of 4 minutes minus the absorbance of the control.

The iron reduction ability evaluation results of lactic acid bacteria including Lactobacillus spesis NC according to the present invention is shown in FIG.

As described above, according to the Lactobacillus spis NC according to the present invention, it is excellent in acid resistance and antioxidant activity and excellent in physiological function and can be widely used in cosmetics, food, medicine, household goods, and the like.

Claims (2)

Isolated from acidic samples containing at least one of kimchi, fermented dairy products, neonatal feces, and adult feces, identified as stick-shaped bacilli, which are acid and antioxidant resistant and do not form gram-positive, catalase-negative, spore-free forms. Lactobacillus plantarum NC (Accession No .: KCTC 10869 BP). A method for producing a cosmetic having acid resistance and antioxidant activity using Lactobacillus plantarum NC (KCTC 10869 BP) according to claim 1.

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