KR101485182B1 - Novel Lactobacillus plantarum from kimchi with inhibiting activities on pathogenic microorganism and use thereof - Google Patents

Abstract

The present invention relates to a lactic acid bacterium which inhibits the growth of pathogenic bacteria and its use, and more particularly to a lactobacillus plantarum DSR 330 which inhibits the growth of pathogenic bacteria and is excellent in acid resistance, salt tolerance, . In particular, the Lactobacillus plantarum DSR 330 or the culture thereof according to the present invention can be usefully used for a dressing, a probiotic agent, a feed, a food additive, an antibacterial agent, a pharmaceutical composition and a fermented food.

Lactobacillus plantarium, acid resistance, salt tolerance, anti-juice, antibacterial

Description

Novel Lactobacillus plantarum isolated from kimchi which suppresses the growth of pathogenic bacteria and its use (Novel Lactobacillus plantarum from kimchi with inhibitory activities on pathogenic microorganism and use thereof)

The present invention relates to a lactic acid bacterium which inhibits the growth of pathogenic bacteria and its use, and more particularly to a lactobacillus plantarum DSR 330 which inhibits the growth of pathogenic bacteria and is excellent in acid resistance, salt tolerance, .

Lactic acid bacteria, which produce lactic acid which is beneficial to humans without producing harmful substances such as indole, sucraldehyde, phenol, amine and ammonia, are present in the most abundant density in the intestines of animals. Especially, (Barnes, FM et al . , Amer . J. Clin . Nutr . , 33, 2426-2433, 1980; Salanitro, JP et al . , Appl . Environ . Microbiol . , 33, 79-84, 1977 ).

Examples of the lactic acid bacteria include Carnobacterium, Enterococcus, Lactobacillus, Lactococcus, Leuconostoc, Pediococcus, Lactobacillus, Streptococcus, Tetragenococcus, Vagococcus, Weissella and Bifidobacterium strains, etc. (Stiles, ME et al . , Int. J Food Microbiol. , 36, 1-29, 1997). In particular, the strain of the genus Lactobacillus is excellent in safety and is used in various studies. It normally produces a uniform microflora in the intestines of an animal including humans, and enhances immunity, neutralization and synthesis of harmful toxins, increase digestion and absorption of nutrients, It is well known that it represents activity, and it is widely administered to animals as well as humans.

On the other hand, pathogenic bacteria include Salmonella spp., Staphylococcus aureus, enterotoxigenic E. coli, B. cereus, S. cerevisiae, Yersinia enterocolitica and Listeria monocytogenes. Especially, Staphylococcus aureus is widely known as a bacterium which causes not only food poisoning but also purulent diseases such as skin pox, otitis and cystitis.

To date, synthetic or natural antibiotics have been used to prevent or treat infections caused by these pathogenic bacteria. However, the use of synthetic antibiotics is strictly regulated because of its resistance and residuals, and it also destroys intestinal beneficial microflora, thus losing the preventive effect against pathogenic bacteria infection by normal microflora. Although alkaloids, flavonoids, phytoalexins, antimicrobial peptides, organic acids and fatty acids are known as natural antibiotics, most of them are not commercialized due to problems of color, stability, narrow antibacterial spectrum and shape In fact.

Due to these problems, lactic acid bacteria can be widely used as a natural antimicrobial agent against the pathogenic bacteria, but in order to exhibit a substantial antimicrobial effect, the following characteristics are required. Specifically, the following characteristics are required to be tolerated: various substances secreted from the intestinal digestive organs It should have excellent acid-tolerance and biliary properties, and should have excellent ability to survive in the intestines. Superior ability to inhibit pathogenic bacteria by low pH, nutrient consumption, reduction of potential difference, production of hydrogen peroxide and production of antimicrobial substances (eg bacteriocin). In particular, antimicrobial agents using lactic acid bacteria inhibit the growth of pathogenic bacteria, reduce the amount of antibiotics that cause various problems, and are safe for the human body, so they can be usefully used in the food manufacturing process.

Therefore, there is a desperate need to develop a novel natural antibacterial agent, lactic acid bacteria, which has antimicrobial effect against the pathogenic bacteria and excellent in acid resistance, salt tolerance, biliary properties and antibiotic resistance.

On the other hand, kimchi is a traditional fermented food in Korea and is a representative source of lactic acid bacteria contributing to the intestinal health of Koreans. Recently, it has been known that kimchi has various useful functions such as anti-mutation and anticancer effects due to the complex function of kimchi materials such as Chinese cabbage and radish and various lactic acid bacteria participating in kimchi fermentation. The export volume is steadily increasing. Microorganisms involved in the fermentation for flavoring the kimchi include L. plantarum, L. brevis, Lactococcus, Leuconostoc, Streptococcus (Lactobacillus, Lactobacillus, Streptococcus, and Pediococcus. By fermentation stage, the type and composition ratio of microorganisms forming the entire microflora are changed.

In recent years, studies have been made to control the taste and fermentation of kimchi by adding lactic acid bacteria derived from Kimchi and adding it as a starter in the manufacture of kimchi. In Korean Patent No. 1989-4894, sodium hypochlorite A method for producing a kimchi having an inhibitory effect against rancidity and an improved preservability by adding a strain of Lactobacillus stearothermophilus and a strain of Lactobacillus stearothermophilus was disclosed in Korean Patent No. 0181009, Which is not only good in taste but also inhibits the growth of the strain of the genus Lactobacillus to delay rancidity. However, since the lactic acid resistance of the above-mentioned prior art rucono-Stokes senteloid is weak, the fermentation of the kimchi gradually progresses and the number of the strains is decreased, so that there is a problem that the fresh taste of the kimchi can not be maintained continuously. Therefore, there is an urgent need to develop a new lactic acid bacteria starter which can improve the quality of kimchi and add functionality for health.

Accordingly, the present inventors have searched for new lactic acid bacteria that can inhibit the growth of pathogenic bacteria from kimchi, which is a representative fermented food of Korean, and have been studying new bacteria that inhibit pathogenic bacteria and have excellent resistance to acidic, salt tolerance, biliary and antibiotic resistance The present invention has been completed by isolating and identifying Kimchi lactic acid bacteria.

Accordingly, an object of the present invention is to provide Lactobacillus plantarum DSR 330 which inhibits the growth of pathogenic bacteria and is excellent in acid resistance, salt tolerance, bile resistance and antibiotic resistance, and uses thereof.

In order to achieve the above object, the present invention provides a method for inhibiting the growth of pathogenic bacteria and having excellent resistance to antibiotics, acid resistance at pH 2.5 to 5, salt tolerance at 2 to 15% of NaCl and 0.3 to 3% of oxgall Lactobacillus plantarum having biliary properties is provided.

In order to accomplish still another object of the present invention, the present invention provides a composition for rectal preparation, a prodrug, a feed, an antibiotic, a food additive and a pharmaceutical composition containing the lactobacillus plantarum or a culture thereof.

According to another aspect of the present invention, there is provided a fermented food comprising the lactobacillus plantarium or a culture thereof.

The present invention also provides a method for producing a fermented food using the lactobacillus plantarum or a culture thereof as a starter.

Hereinafter, the present invention will be described in detail.

The Lactobacillus plantarum of the present invention is characterized by inhibiting the growth of pathogenic bacteria and having acid resistance, salt resistance, bile resistance and antibiotic resistance. In particular, the Lactobacillus plantarum DSR 330 of the present invention, isolated from kimchi, can be isolated from pathogenic bacteria such as Staphylococcus aureus, E. coli, B. cereus, Salmonella typhimurium At least one microorganism selected from the group consisting of Salmonella typimurium, Salmonella choleraesuis, Yersinia enterocolitica and Listeria monocytogenes, It is characterized by acid resistance at a pH of 2.5 to 5, salt resistance at 2 to 15% of NaCl and biliary properties of 0.3 to 3% of oxgall (see Examples 1-2 to 1-7).

The Lactobacillus plantarum of the present invention survived more than 90% at a pH of 2.5 to exhibit a strong acid tolerance, survive more than 97% of NaCl at 15%, exhibit a strong salt tolerance and survival rate of more than 95% at 3% of oxgall, It has my biliary properties.

Since the lactobacillus plantarum of the present invention is excellent in acid resistance, salt tolerance and bile resistance, when the above-mentioned strain is orally ingested, the acid environment in which the pH is lowered to 3 or less and the digestive enzymes secreted from the stomach and small intestine And cholanic acid may increase the probability of reaching the growth with excellent growth.

Since the lactobacillus plantarium of the present invention is excellent in antibiotic resistance, it can grow in the presence of an antibiotic and can perform beneficial functions in vivo (see Examples 1-6).

The Lactobacillus plantarum of the present invention also showed excellent antibacterial activity. In particular, the lactic acid bacteria of the present invention are useful as pathogenic bacteria such as Staphylococcus aureus, E. coli, B. cereus, Salmonella typimurium, Salmonella cholera, choleraesuis, Yersinia enterocolitica, and Listeria monocytogenes. (See Examples 1-7).

Lactobacillus plantarum strains which have the characteristics of suppressing the growth of pathogenic bacteria and excellent in acid resistance, salt tolerance, biliary properties and antibiotic resistance are provided for the first time in the present invention.

Therefore, the lactic acid bacterium of the present invention can be used for the health promotion of humans and animals, that is, for the application of formulations, probiotics, antibacterials, feeds, foods or pharmaceutical compositions. The composition may contain a crushed cell wall fraction of Lactobacillus plentarium of the present invention, a live cell, a dead cell, a dried bacterium or a culture, as an active ingredient, and may further include an excipient or a carrier. The culture includes a culture medium itself cultured in a liquid medium, a filtrate (centrifuged supernatant) obtained by removing the strain by filtration or centrifugation of the culture medium, and the like. The content of lactobacillus plantarum in the composition may vary depending on the use of the composition and the formulation.

The pharmaceutical composition refers to a composition capable of treating or preventing an infection of a pathogenic bacterium, wherein the pathogenic bacterium is selected from the group consisting of Staphylococcus aureus, E. coli, B. cereus, Refers to at least one bacterium selected from the group consisting of Salmonella typimurium, Salmonella choleraesuis, Yersinia enterocolitica and Listeria monocytogenes, Including but not limited to all foodborne illnesses, skin pneumonia, otitis media, cystitis, and the like, including all diseases caused by bacterial infections.

The formulations, probiotics, antibacterial or pharmaceutical compositions according to the present invention can be prepared and administered in a variety of formulations and methods. For example, Lactobacillus plantarum or culture thereof may be mixed with carriers and flavoring agents commonly used in the pharmaceutical field to form tablets, troche, capsules, elixir, May be formulated and administered in the form of a syrup, powder, suspension or granule, and the like. Examples of the carrier include binders, lubricants, disintegrants, excipients, solubilizers, dispersants, stabilizers, suspending agents and the like. The administration method may be oral, parenteral or application, but is preferably orally administered. The dose may be appropriately selected depending on the degree of absorption, inactivation rate and excretion rate of the active ingredient in the body, age, sex, condition, etc. of the subject.

In addition, the composition for feed according to the present invention can be produced in the form of a fermented feed, a compounded feed, a pellet form, a silage or the like. The fermented feed can be prepared by fermenting an organic material by adding lactobacillus plentarium and various microbial bacteria or enzymes of the present invention. The fermented feed can be prepared by mixing various kinds of general feeds and lactobacillus plantarum of the present invention . The pellet-shaped feed can be prepared by formulating the fermented feed or the compound feed into a pelletizer, and the silage can be produced by fermenting the selected feed with the lactobacillus plantarum according to the present invention.

The lactobacillus plantarum or the culture thereof according to the present invention can be used as a food additive for foods such as kimchi, beverages, baby foods, and dairy products. In addition, the lactobacillus plantarum of the present invention can be used as a starter for the production of fermented foods. The fermented food includes cheese, kimchi, fermented reproductive products and the like. The fermented food using the lactobacillus plantarium of the present invention can be produced according to a conventional method known in the art. For example, Lactobacillus plantarum according to the present invention or a mixed lactic acid bacterium containing two or three kinds of lactam bacteria according to the present invention is processed into cereal powder such as brown rice and yulmu and fermented at an appropriate temperature, and various agricultural products such as white rice, glutinous rice, Fermented reproductive products can be produced by appropriately blending them so as to have superior nutritional balance and palatability. In particular, the lactobacillus plantarum according to the present invention can be used for producing kimchi. Preferably, the kimchi can be prepared by adding a common kimchi seasoning such as red pepper powder, garlic, ginger, green onion, mungbean, sugar, etc. to the cabbage pickled with salt and then adding a culture of Lactobacillus plentarium of the present invention. The culture of the lactobacillus platelium of the present invention is preferably added in an amount of 0.1 to 5 wt%, and most preferably 1 wt%, based on the total weight of the kimchi.

The Lactobacillus plantarum according to the present invention can be cultured in a large amount by a conventional method for culturing Lactobacillus sp. Microorganisms. As the culture medium, a medium composed of carbon source, nitrogen source, vitamins and minerals can be used. For example, MRS (Man-Rogosa-Sharp) medium or cabbage juice medium can be used. The Chinese cabbage juice medium can be used by crushing, juicing and sterilization of the pickled Chinese cabbage. The microorganism can be cultured under usual culture conditions of Lactobacillus microorganism. For example, the microorganism can be cultured at 25 ° C to 37 ° C for about 18 hours to 48 hours, more preferably at 37 ° C for about 20 hours. The culture medium in the culture broth can be removed and centrifugation or filtration can be carried out to recover only the concentrated cells, and these steps can be carried out as required by those skilled in the art. The concentrated cells may be frozen or lyophilized according to a conventional method so as not to lose their activity.

As described above, the novel lactic acid bacteria Lactobacillus plantarum DSR 330 of the present invention has an activity of inhibiting the growth of pathogenic bacteria, and is characterized by excellent acid resistance, salt resistance, bile resistance and antibiotic resistance. Therefore, the lactic acid bacterium of the present invention can be variously used for the preparation of pharmaceutical preparations, probiotics, antibacterials, feeds, food additives, pharmaceutical compositions and fermented foods. In particular, when Lactobacillus plantarum DSR 330 according to the present invention is used as a starter for fermentation of kimchi, a good kimchi taste can be maintained for a long time and functionality can be added to the kimchi. In addition, And is effective for the treatment and prevention of the disease.

Hereinafter, the present invention will be described in detail by way of examples.

However, the following examples are illustrative of the present invention, and the present invention is not limited to the following examples.

≪ Example 1 >

Isolation of Lactobacillus plantarum strain

<1-1> Isolation of lactic acid bacteria from kimchi

20 kinds of kimchi prepared by a conventional kimchi preparation method were selected and the kimchi was used as a lactic acid bacteria-isolated kimchi sample. The kimchi samples were diluted 10 times with 0.85% saline, and 10 ml of MRS agar medium (MRS agar, Difco .; 10 g of bovine peptone, 10 g of beef extract, 5 g of yeast extract, 20 g of glucose, 801 g of citric acid, 2 g of citric acid, 2 g of dibasic potassium phosphate, 5 g of sodium acetate, 0.1 g of manganese sulfate, 0.05 g of magnesium sulfate and 15 g of agar / 1 l of distilled water). Plates were then incubated for 2 days in a 25 &lt; 0 &gt; C incubator. Each of the resulting colonies was inoculated into MRS agar plates and cultured at 25 DEG C for 2 days to isolate a total of 200 colonies of lactic acid bacteria.

<1-2> Pathogenic bacteria  Isolation of lactic acid bacteria inhibiting growth

Among the 200 lactic acid bacteria isolated in Example <1-1>, Staphylococcus aureus KCCM 12214, Escherichia coli KCCM 11234 (hereinafter referred to as "KCCM") were selected as a pathogenic bacterium in order to select lactic acid bacteria inhibiting the growth of pathogenic bacteria E. coli KCCM 11234), Bacillus cereus KCCM 11774 and Salmonella typimurium KCCM 40253 were mixed in a nutrient medium (5.0 g of bupropion peptone, 1.0 g of beef extract, 2.0 g of yeast extract 2.0 g, 5.0 g of sodium chloride, 15 g of agar / 1 L of distilled water) and cultured at 37 DEG C for 24 hours. The cultured cells were scraped from the medium, washed twice with phosphate buffered saline (PBS, pH 7.4), suspended in 20% glycerol and stored at -20 ° C. Were inoculated on MRS liquid medium, cultured at 37 ° C for 24 hours, centrifuged, suspended in 10% skim milk, and stored at -20 ° C.

Thereafter, 1% of pathogenic bacteria was inoculated on a nutrient agar and dried for 20 minutes. Then, disc papers were placed on the nutrient agar, and then 50 μl of each of the 200 lactic acid culture supernatants was added dropwise and cultured at 37 ° C for 24 hours The best isolate among the lactic acid bacteria forming the inhibitory ring was firstly selected. Then, the first selected strains were selected again in the same manner as described above to finally select the DSR 330 which suppresses the growth of pathogenic bacteria to the best (see FIG. 2).

<1-3> Acid resistance test

DSR 330 lactic acid bacteria which suppress the growth of pathogenic bacteria isolated in the above Example <1-2> are best treated with hydrochloric acid at pH 2.5, 3.0, 3.5, 4.0, 4.5 and 5, and MRS broth (MRS broth, Difco .; 10 g of bactopeptone, 10 g of beef extract, 5 g of yeast extract, 20 g of glucose, 801 g of tween, 2 g of citric acid, 2 g of dibasic potassium phosphate, 5 g of sodium acetate, 0.1 g of manganese sulfate, g / distilled water 1 liter). Then, the viable cells were counted while incubating at 37 DEG C for 2 hours.

As a result, the selected strains were active at pH 2.5 to 5, particularly 90% or more at pH 2.5, and showed a survival rate of about 95% or more at pH 3, showing strong acid resistance (see FIG. 3).

<1-4> Salt resistance test

The DSR 330 lactic acid bacteria isolated in Example <1-2> were inoculated in 10 ml of MRS broth adjusted to 2, 5, 8, 10 and 15% with sodium chloride (NaCl), respectively. Then, the growth of the strain was observed at the salt concentration while culturing at 37 캜 for 2 hours. As a result, the selected strains were active at 2-15% of sodium chloride, and showed a survival rate of 97% or more even at 15% of sodium chloride (see FIG. 4).

<1-5> Testing my bubbles

The DSR 330 lactic acid bacteria isolated in Example <1-2> were inoculated in 10 ml of MRS broth adjusted to 0.3, 0.5, 1 and 3% by oxgall, respectively. Then, the growth of the strain at the artificial bile concentration was observed while culturing at 37 캜 for 2 hours. As a result, the selected strains were active at 0.3 to 3.0% of oxgall, and showed a survival rate of 95% or more even at 3% of oxgall (see FIG. 5).

<1-6> Antibiotic resistance test

The DSR 330 lactic acid bacteria isolated in Example <1-2> were inoculated on a MRS solid medium, and a filter (diameter 6 mm) containing antibiotics was placed on the plate and cultured for 24 hours to measure the size of the inhibitory ring formed by antibiotics. The smaller the inhibitory ring size, the greater the resistance to antibiotics. Table 1 below shows the degree of antibiotic resistance to DSR 330.

The antibiotic resistance measurement results of the lactic acid bacteria according to the present invention Antibiotic Lactobacillus plantarum DSR 330 (mm) Tetracycline (30 占 퐇) 22 Erythromycin (15 μl) 11 Ampicillin (10 μl) 26 Penicillin (10 IU / IE / UI) 24 Separocin (30 μl) 21

As shown in Table 1, the DSR 330 strain of the present invention is excellent in antibiotic resistance, and its activity is retained even when it is used in a formulating, probiotic, feed, food additive, antibacterial, pharmaceutical composition and fermented food together with an antibiotic It can be seen that

<1-7> Antimicrobial activity test

The DSR 330 lactic acid bacteria isolated in Example <1-2> were subcultured to MRS broth for antibacterial activity test, and Staphylococcus aureus KCCM 12214, a pathogenic bacterium, was added to a nutrient agar. , E. coli KCCM 11234, B. cereus KCCM 11774 and Salmonella typimurium KCCM 40253, Salmonella choleraesuis KCCM 40763, Bacillus cereus KCCM 11204, 1% of Yersinia enterocolitica KCCM 41657, Staplyococcus aureus KCCM 11335 and Listeria monocytogenes KCCM4307 were inoculated and dried for 20 minutes, And 50 μl of the culture supernatant of the above-mentioned DSR 330 lactic acid bacteria was added dropwise and cultured at 37 ° C. for 24 hours to measure the size of the inhibitory circle Groups are shown in Table 2.

The test results of the antibacterial activity of the lactic acid bacteria according to the present invention Pathogenic bacteria Lactobacillus plantarum DSR 330 (mm) Salmonella cholera suis 15 Bacillus cereus KCCM 11774 20 Jessinia enterrocolitica 30 Staphylococcus aureus KCCM 12214 13 Listeria monocytogenes 24 Staphylococcus Eureus KCCM 11335 11 Escherichia coli 11 Salmonella typhimurium 12.5 Bacillus cereus KCCM11204 16

As shown in Table 2 above, the DSR 330 strain of the present invention is excellent in the antibacterial activity against the pathogenic bacteria, and particularly for pathogenic bacteria such as Y. niea enterocolitica, Listeria monocytogenes and Bacillus cereus KCCM 11774, Has an excellent antibacterial activity exceeding 20 mm, and thus it can be used effectively as an antimicrobial agent against the pathogenic bacteria.

<1-8> Identification of selected lactic acid bacteria

a. Classification of Burgess analysis according to bacteriology manual

After isolating the lactic acid bacteria selected in Example <1-2> into single colonies, morphological and biochemical characteristics were examined according to Bergy's manual of systematic bacteriology and Gram staining was performed. As a result, it was confirmed that the isolated strain was a gram-positive strain and had a bacterium form (see Table 3 and FIG. 6).

Morphological and biochemical characteristics of lactic acid bacteria according to the present invention characteristic DSR 330 Gram + Colony form Rod Colony color Yellow Motility - Catalase (Catalase) - Fermentation form Homo Spore - Growth at 15 ℃ + Growth in 4% NaCl +

b. Analysis using API system

Thereafter, the strain was identified as an API system (La Balme-les-Grottes, France). First, the sterilized platinum microbial colonies were each taken and suspended in 2 ml of sterile distilled water. Again, the bacterial solution was suspended in 5 ml of sterilized distilled water to a concentration of MccFaland Standard Solution No. 2 supplied by API 50CH kit (BioMerieux, France). The supernatant was added to the liquid medium of the API 50CH kit and homogenized, and 200 μl of each of the 50 tubes of the API 50CH kit was inoculated. The tube was overlaid with mineral oil and then cultured at 30 DEG C for 48 hours. The culture solution was analyzed by an API system to identify 49 types of carbohydrate fermentation patterns, and the results were entered into an ATB identification computer system.

As a result, the DSR 330 lactic acid bacteria selected in Example <1-2> were found to be strains belonging to the genus Lactobacillus having the carbohydrate fermentation pattern shown in Table 4 below.

The analysis of carbohydrate fermentation pattern of lactic acid bacteria according to the present invention Control group - D-Manno + Salincin + Gentio Vioz + Glycerol - L-Solbos - Cellobiz + D-Turanz _ Erythritol - Rannoz + Malts + D-rings - D-arabinose - Dissytol - Laktos + D-Tagatoz - L-arabinose + Inositol - Melibiose + D-fucose - Riboze + Mannitol + Saka Rose + L-fucose - D-xylose - Sorbitol + Trehalose + D-arabitol - L-xylose - D-mannoside - Inulin + L-arabitol - Adonitol - D-glucoside - Melrietto + Gluconate + Xyloside - Glucosamine + D-Rapinoz + 2-Setto-gluconate - Galactos + Amigalline + Amidon - 5-Seto-gluconate - D-glucose + Arbutin + Glycogen - D-Prostose + Esculin + Xylitol +

c. 16S rDNA sequencing

The nucleotide sequences of the 16S rDNAs of the selected lactic acid bacteria were analyzed according to a conventional method known in the art. As a result, the 16S rDNA nucleotide sequence (SEQ ID NO: 1) of the DSR 330 Lactobacillus was 99% identical to the 16S rDNA nucleotide sequence of Lactobacillus plantarum (see FIG. 1).

 Therefore, the present inventors named DSR 330 Lactobacillus as "Lactobacillus plantarium DSR 330 " and deposited it on August 10, 2007 in the Korean Culture Center of Microorganisms (Accession No .: KFCC- 11393P).

&Lt; Example 2 >

Production of Kimchi Using Lactic Acid Bacteria of the Present Invention

The cabbage was salted with salt, so that the cabbage had a salinity of 2.3%. (2.5 wt.%), Garlic (2 wt.%), Ginger (0.5 wt.%), Green onion (2 wt.%), Rape seeds (9 wt.%) And sugar Kimchi seasoning was mixed. Lactobacillus plantarum DSR 330 of the present invention was then added as a starter to 1 wt% (about 1 x 10 ⁵ strain) of the total weight of the kimchi. At this time, starter lactic acid bacteria were not added to the negative control group. Then, the prepared kimchi was aged at 10 DEG C for 6 days.

<Experimental Example 1>

The acidity of kimchi and the number of lactic acid bacteria were measured

<1-1> Measurement of acidity

The acidity of each kimchi prepared in Example <2> was measured. 100 g of each kimchi which had been aged at 10 캜 for 10 days was crushed and filtered with gauze to prepare a kimchi solution. Then, the acidity of each kimchi was measured in order to compare the aged state of kimchi. The acidity was measured by NaCl titration method. After the kimchi juice was prepared from each kimchi, the pH of 5 ㎖ of Kimchi juice was measured. Thereafter, 0.1 N NaOH was titrated to determine the amount of 0.1 N NaOH added at the time when the pH became 8.2, and the acidity (%) was calculated by the following equation (1).

Acidity (%) = {0.00908 × 1N NaOH factor ^* × 1N NaOH addition amount} / sample weight

^* 1N NaOH factor = 1

The acidity measurement results of the kimchi prepared using the lactic acid bacteria of the present invention Lactic acid bacteria added as a starter Acidity (%) Lactobacillus platarium DSR 330 0.60 No starter added (negative control) 0.72

As a result, as shown in Table 5, the kimchi prepared using Lactobacillus plantarum DSR 330 strain had a relatively lower acidity than the kimchi prepared without starter, and the lactic acid bacteria showed an acidity in the latter half of the fermentation And it was found that the quality of Kimchi could be improved by gradually increasing the maturity.

<1-2> Measurement of the number of lactic acid bacteria in Kimchi

The number of lactic acid bacteria in each kimchi prepared in Example <2> was measured. Each kimchi which had been aged at 10 ° C for 6 days was diluted 10 times with 0.85% saline, and 10 g of MRS agar medium (MRS agar, Difco .; buprope peptone, 10 g of beef extract, 5 g of yeast extract, g, tween 80 1 g, citric acid 2 g, potassium diphosphate 2 g, sodium acetate 5 g, manganese sulfate 0.1 g, magnesium sulfate 0.05 g, agar 15 g / distilled water 1 ℓ) And then smoothed with a glass plate. Each of the agar plates in which the kimchi diluted solution had been smoothed was cultured in a constant temperature incubator at 27 ° C for 12 days, and the number of colonies formed was counted as each bacterial count. The results are shown in Table 6.

The result of measuring the number of lactic acid bacteria in kimchi prepared using the lactic acid bacteria of the present invention Lactic acid bacteria added as a starter (log cfu / ml) Total lactic acid bacteria number Lactobacillus platarium DSR 330 9.35 No starter added (negative control) 8.71

As a result, as shown in Table 6, it was confirmed that the total number of lactic acid bacteria was significantly increased in the kimchi prepared by adding the lactic acid bacteria of the present invention to the kimchi prepared without the starter.

<Experimental Example 2>

Sensory evaluation of prepared kimchi

 The sensory evaluation of each of the kimchi prepared in Example <2> was performed. After 10 days of fermentation at 10 ℃ for 6 days, 10 kimchi samples were taken at 10 ℃ for 2 days. The results were analyzed by t-test. The results are shown in Table 7 below.

The sensory evaluation results of the kimchi prepared using the lactic acid bacteria of the present invention Lactic acid bacteria added as a starter A comprehensive taste already Exterior Sour taste Texture Lactobacillus platarium DSR 330 3.9 1.0 3.5 3.7 3.8 No starter added (negative control) 3.1 1.0 3.1 3.2 3.4

As a result, as shown in Table 7, it was evaluated that the kimchi prepared by adding the lactic acid bacteria of the present invention had better overall sensory characteristics than the kimchi prepared without starter.

&Lt; Example 3 >

Production of Dongchimi using the Lactic Acid Bacteria of the Present Invention

Radish and Chinese cabbage were pickled with salt so that the cabbage had a salinity of 4.0%. The pickled Chinese cabbage (6%) and radish (38%) were mixed with Dongchimi sauce composed of garlic (3 wt%), ginger (0.2 wt%) and glucose (5 wt%). Lactobacillus plantarum DSR 330 of the present invention was added as a starter to 1 wt% (about 1 x 10 ⁵ strain) of the total weight of the dongchimi, and fermented at 25 ° C for 5 days to prepare a dongchimi. At this time, the case where the starter lactic acid bacteria were not added was used as a negative control.

<Experimental Example 3>

Determination of acidity and lactic acid bacteria count of dongchimi prepared

<3-1> Measurement of acidity

The dongchimi soup prepared in Example <3> was filtered with gauze to prepare a Dongchimi liquid. The pH of 5 ml of Dongchimi liquid was measured and the acidity was calculated in the same manner as in Experimental Example <1-1> Are shown in Table 8 below.

The acidity measurement results of Dongchimi prepared using the lactic acid bacteria of the present invention Lactic acid bacteria added as a starter Acidity (%) Lactobacillus platarium DSR 330 0.73 No starter added (negative control) 0.40

As a result, as shown in Table 8, the acidity of the dongchimi prepared using the lactic acid bacteria of the present invention was faster than that of the dongchimi prepared without using the starter. Therefore, it was confirmed that the lactic acid bacteria of the present invention rapidly induce the fermentation of Dongchimi, indicating a proper acidity.

<3-2> Measurement of the number of lactic acid bacteria in Dongchimi

The dongchimi prepared in Example 3 was diluted 10-fold with 0.85% saline, and the total number of lactic acid bacteria was measured in the same manner as in Experimental Example <1-2>. The results are shown in Table 9.

As a result of measuring the number of lactic acid bacteria in Dongchimi prepared using the lactic acid bacteria of the present invention Lactic acid bacteria added as a starter (log cfu / ml) Total lactic acid bacteria number Lactobacillus platarium DSR 330 9.21 No starter added (negative control) 7.69

As a result, as shown in Table 9, it was confirmed that the total number of lactic acid bacteria was increased in the dongchimi prepared by adding the lactic acid bacteria of the present invention to the dongchimi prepared without the starter.

<3-3> Measurement of antibacterial activity of dongchimi prepared

 Salmonella choleraesuis KCCM40763, Yersinia enterocolitica KCCM 41657, Listeria monocytogenes KCCM4307, Staphylococcus aureus KCCM 12214, E. coli, and the like are examples of pathogenic bacteria. E. coli KCCM 11234), Salmonella typimurium KCCM 40253 and B. cereus KCCM 11774 were mixed in a nutrient medium (5.0 g of bupropion peptone, 1.0 g of beef extract, 2.0 g of yeast extract, 5.0 g of sodium chloride, Agar 15 g / distilled water 1 L), cultured at 37 ° C. for 24 hours, inoculated on a nutrient agar and dried for 20 minutes, and then a disk paper was placed on the nutrient agar. In the same manner as in Example 3 50 占 퐇 of the prepared Dongchimi solution was added dropwise and incubated at 37 占 폚 for 24 hours to measure inhibition rings. The results are shown in Table 10

As a result of the antibacterial activity test of Dongchimi according to the present invention Pathogenic bacteria Dongchimi prepared using Lactobacillus plantarum DSR 330 (mm) Salmonella cholera suis 16 Bacillus cereus KCCM 11774 23 Jessinia enterrocolitica 28 Staphylococcus aureus KCCM 12214 15 Listeria monocytogenes 26 Staphylococcus Eureus KCCM 11335 13 Escherichia coli 15 Salmonella typhimurium 14 Bacillus cereus KCCM11204 20

As shown in Table 10, the dongchimi prepared by using the DSR 330 strain of the present invention is excellent in antibacterial activity against the pathogenic bacteria. In particular, it has excellent antimicrobial activity against the pathogenic bacterium, and in particular, is resistant to E. coli TCC, Listeria monocytogenes, Bacillus cereus KCCM 11774 and Bacillus cereus For the pathogenic bacteria such as KCCM11204, the size of the inhibitory ring exceeded 20 mm, indicating that it has excellent antibacterial activity.

&Lt; Preparation Example 1 &

Production of a feed composition using the lactic acid bacterium of the present invention

The Lactobacillus plantarum DSR 330 lactic acid bacterium of the present invention was inoculated on a medium containing 2.0% glucose, 1.0% peptone, 1.0% yeast extract, 0.2% ferric phosphate, 0.05% magnesium sulfate and 0.05% cysteine, Were cultivated aerobically. The culture medium of each strain and the excipient degreasing solution were simply mixed at a weight ratio of 1: 1, followed by drying at 40 ° C or lower, and the dried mixture was pulverized to prepare a feed composition. At this time, the prepared feed composition contained the lactic acid bacteria of the present invention at a concentration of 1 × 10 ⁹ cfu / g or more.

&Lt; Preparation Example 2 &

Production of a Probiotic Compound Using the Lactic Acid Bacteria of the Present Invention

The Lactobacillus plantarum DSR 330 lactic acid bacterium of the present invention was inoculated on a medium containing 2.0% glucose, 1.0% peptone, 1.0% yeast extract, 0.2% ferric phosphate, 0.05% magnesium sulfate and 0.05% cysteine, Were cultivated aerobically. The culture broth of each of the above strains was inoculated at 10% by weight in a solid culture medium in which a defatted steel, soybean meal, corn and molasses were mixed at a weight ratio of 3: 1: 1: 1, Lt; / RTI &gt; The fermentation broth composition was prepared by anaerobically fermenting the fermentation broth with stirring at 40 ° C for 3 days. The broth composition prepared herein contained 1 × 10 ⁹ cfu / g or more of the lactic acid bacteria of the present invention.

&Lt; Preparation Example 3 &

Production of a food additive composition using the lactic acid bacterium of the present invention

The Lactobacillus plantarum DSR 330 lactic acid bacteria of the present invention was inoculated into a medium containing 2.0% glucose, 1.0% peptone, 1.0% yeast extract, 0.2% phosphoric acid, 0.05% magnesium sulfate, 0.05% cysteine, And cultured for 2 days aerobically. 60% by weight of the culture solution was added to a mixture of milk powder or skim milk and starch or lactose at a weight ratio of 1: 1, and the mixture was rapidly frozen at -70 캜 or lower. Followed by freeze-drying to prepare a composition for food addition. At this time, the temperature of the hot plate for increasing the efficiency of moisture evaporation during lyophilization was set to 30 ° C, and the prepared food additive composition contained 1 × 10 ⁹ cfu / g or more of the lactic acid bacteria of the present invention.

< Manufacturing example  4>

Production of a pharmaceutical composition using the lactic acid bacterium of the present invention

Each of the bacterial composition powders obtained in Preparation Examples 1, 2 and 3 was molded into tablets, pills and capsules according to a conventional method to prepare a pharmaceutical composition. The composition prepared herein had a composition of 1 × 10 ⁹ cfu / g or more.

1 is a dendrogram comparing the 16S rDNA base sequence of the lactic acid bacterium of the present invention with the base sequence on the gene bank.

FIG. 2 is a graph comparing the antibacterial activity against pathogenic bacteria of lactic acid bacteria of the present invention and other lactic acid bacteria isolated from kimchi.

3 is a graph showing the acid resistance of the Lactobacillus plantarum DSR 330 lactic acid bacteria of the present invention.

4 is a graph showing the salt tolerance of Lactobacillus plantarum DSR 330 (Lactobacillus plantarum DSR 330) of the present invention.

FIG. 5 is a graph showing the biliary properties in the Lactobacillus plantarum DSR 330 of the present invention. FIG.

FIG. 6 is a photograph of the Lactobacillus plantarum DSR 330 (Lactobacillus plantarum DSR 330) of the present invention observed under a microscope.

<110> DAESANG FNF CORPORATION <120> Novel Lactobacillus plantarum from kimchi with inhibiting          activities on pathogenic microorganism and use thereof <130> NP07-0116 <160> 1 <170> Kopatentin 1.71 <210> 1 <211> 858 <212> DNA <213> Lactobacillus plantarum <400> 1 gggggatcgg gtctatatga agtcgacgaa ctctggtatt gattggtgct tgctcatgat 60 ttacatttga gtgagtggcg aactggtgag taacacgtgg gaaacctgcc cagaagcggg 120 ggataacacc tggaaacaga tgctaatacc gcataacaac ttggaccgca tggtccgagc 180 ttgaaagatg gcttcggcta tcacttttga atggtcccgc ggcgtattag ctagatggtg 240 gggtaacggc tcaccatggc aatgatacgt agccgacctg agagggtaat cggccacatt 300 gggactgaga cacggcccaa actcctacgg gaggcagcag tagggaatct tccacaatgg 360 acgaaagtct gatggagcaa cgccgcgtga gtgaagaagg gtttcggctc gtaaaactct 420 gttgttaaag aagaacatat ctgagagtaa ctgttcaggt attgacggta tttaaccaga 480 aagccacggc taactacgtg ccagcagccg cggtaatacg taggtggcaa gcgttgtccg 540 gatttattgg gcgtaaagcg agcgcaggcg gttttttaag tctgatgtga aagccttcgg 600 ctcaaccgaa gaagtgcatc ggaaactggg aaacttgagt gcagaagaag acagtggaac 660 tccatgtgta gcggtgaaat gcgtagatat atggaagaac accagtgncg aaagcggctg 720 tctggtctgt aactgacgct gaagctcgna agtatgggta gccaacaaga ttagataccc 780 ctggtagtcc ataccgtaaa cgatgaatgc taagtgttgg gaaggtttcc gcccttcagt 840 gctgccacta aagccata 858

Claims (10)

Lactobacillus plantarum DSR330 which inhibits the growth of pathogenic bacteria and has acid resistance at pH 2.5 to 5, salt tolerance at 2 to 15% of NaCl, biliary and antibiotic resistance at 1 to 3% of Oxgall (KFCC11393P) Wherein said pathogenic bacterium is selected from the group consisting of B. cereus, Salmonella choleraesuis , Yersinia enterocolitica , and Listeria monocytogenes . Lactobacillus plantarum DSR330 (KFCC11393P). delete A dressing composition comprising the Lactobacillus plantarum DSR330 (KFCC11393P) of claim 1 or a culture thereof. 11. A probiotic composition containing the Lactobacillus plantarum DSR330 (KFCC11393P) of claim 1 or a culture thereof. A feed composition comprising the Lactobacillus plantarum DSR330 (KFCC11393P) of claim 1 or a culture thereof. A composition for food addition containing the Lactobacillus plantarum DSR330 (KFCC11393P) of claim 1 or a culture thereof. B. cereus, Salmonella choleraesuis , Yersinia enterocolitica containing the Lactobacillus plantarum DSR330 (KFCC11393P) or a culture thereof of claim 1, And Listeria monocytogenes . 2. The antimicrobial composition according to claim 1, wherein the antimicrobial agent is selected from the group consisting of Listeria monocytogenes and Listeria monocytogenes . B. cereus, Salmonella choleraesuis , Yersinia enterocolitica containing the Lactobacillus plantarum DSR330 (KFCC11393P) or a culture thereof of claim 1, And Listeria monocytogenes . &Lt; RTI ID = 0.0 &gt; A &lt; / RTI &gt; pharmaceutical composition for the treatment or prophylaxis of an infectious disease of one or more pathogenic bacteria selected from the group consisting of Listeria monocytogenes . A fermented food containing the Lactobacillus plantarum DSR330 (KFCC11393P) of claim 1 or a culture thereof. A method for producing a fermented food product using the Lactobacillus plantarum DSR330 (KFCC11393P) of claim 1 or a culture thereof as a starter.

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