KR20080110397A - Novel lactic acid bacteria having acid resistance, bile-salt resistance and anti-bacterial effect and composition containing the same - Google Patents

Abstract

A novel Lactobacillus plantarum CLP0707, a fermented product using the lactobacillus, and a method for culturing the Lactobacillus plantarum CLP0707 are provided to improve acid resistance, bile acid resistance and the resistance against digestive enzyme and to obtain antibacterial effect. A Lactobacillus plantarum CLP0707 (deposition number : KCCM10868P) shows an excellent antibacterial effect and good acid resistance and bile acid resistance. A fermented product containing the Lactobacillus plantarum CLP0707 or its culture. The Lactobacillus plantarum CLP0707 is cultured by culturing the Lactobacillus plantarum CLP0707 in a culture medium comprising a carbon source, a nitrogen source, vitamin and mineral at 15-45 deg.C for 10-72 hours.

Description

Novel lactic acid bacteria having acid resistance, bile-salt resistance and anti-bacterial effect and composition containing the same

1 is a result of measuring the antimicrobial activity of Escherichia coli of Lactobacillus plantarum CLP0707 according to the present invention.

Figure 2 is the result of measuring the antimicrobial activity of Staphylococcus aureus of Lactobacillus plantarum CLP0707 according to the present invention.

Figure 3 is the result of measuring the antimicrobial activity against Listeria bacteria of Lactobacillus plantarum CLP0707 according to the present invention.

Figure 4 is a result of measuring the antimicrobial activity of Salmonella against Lactobacillus plantarum CLP0707 according to the present invention.

The present invention relates to a novel lactic acid bacteria having excellent acid resistance and bile acid resistance and having an antimicrobial effect, and more particularly, the present invention has a high intestinal viability and excellent antimicrobial effect due to its resistance to acid resistance, bile acid resistance and digestive enzymes. It relates to a composition comprising a Lactobacillus plantarum CLP0707, a novel microorganism capable of inhibiting the growth of harmful microorganisms and the lactic acid bacteria or lactic acid bacteria culture.

Probiotics is derived from the Greek word for for life. Probiotics were defined in 1965 as "microbial-releasing substances that stimulate the growth of other microorganisms," and in 1974 they were defined as "organisms or substances that have a beneficial effect on host animals." Later, in 1989, Fuller introduced the concept of living microorganisms as a redefinition of probiotics: "probiotics that benefit the host by improving the intestinal flora". In such probiotics, aerobic bacteria, anaerobic bacteria, lactic acid bacteria, and yeasts are used, but mainly lactic acid bacteria are used as the main strains.

Lactobacillus is widely present in nature and uses carbohydrates anaerobicly to produce lactic acid. The natural environment in which lactobacillus is found is diverse, not only in the gut of humans or animals, but also in various vegetables and fruits, and plays an important role in the fermentation process in fermented foods such as kimchi, yogurt in Korea, and sauerkraut in Germany. In charge of.

These lactic acid bacteria enters the intestinal epithelial cells and enters the intestinal epithelial cells to prevent intestinal settlement of harmful microorganisms and secrete antimicrobial substances, thereby inhibiting the growth of harmful microorganisms and improving diarrhea and constipation, as well as enhancing immune activity and anticancer activity. To lower serum cholesterol. In general, these lactic acid bacteria are known to be added directly or indirectly to food to improve the shelf life of food by their metabolites lactic acid and improve the flavor and texture of food. The major lactic acid bacteria are reported to be Lactobacillus acidophilus , Lactobacillus casei , Lactobacillus fermentum , Lactobacillus plantarum , etc. Currently, it is widely used as a spawn and probiotic of fermented dairy products all over the world.

Recently, as the functionalities of plant-derived lactic acid bacteria isolated from fermented foods such as vegetables and fruits, or fruit and vegetable such as kimchi, are emerging as a new functional material. These plant-derived lactic acid bacteria include Lactobacillus plantarum , Lactobacillus paracasei , Lactobacillus brevis , etc. among the genus Leukonostock, Pediococcus and Lactobacillus. do. Plant-derived lactic acid bacteria have strong viability even in harsh environments such as low pH and high salt concentrations. Also, plant-derived lactic acid bacteria have less nutritional requirements than animal-derived lactic acid bacteria and are easy to cultivate. Also, new products can be developed in the fermented beverage market, which is one of the fermented milk products. Do.

In order for these lactic acid bacteria to act as probiotics, they must have the following characteristics.

First, they must survive the digestive system,

Second, it should have a beneficial effect on the host,

Third, it can proliferate or settle in the intestines,

Fourth, the food must be satisfied that does not damage the quality of the food.

In order for the lactic acid bacteria to be effective as probiotics as described above, it is important to have acid resistance and bile acid resistance. Ingested lactic acid bacteria pass through gastric juice and bile acids to reach the intestine. In general, the bacteria are killed or deactivated in this process, so the lactic acid bacteria administered are resistant to these acids and digestive enzymes in order to function in the intestines. Must have Therefore, only lactic acid bacteria having excellent acid resistance and bile resistance can be used to substantially perform useful functions for the human body.

Korean Patent Publication No. 2006-80130 discloses a product using antifungal active Lactobacillus plantarum AF1 isolated from kimchi and a culture solution of the above bacteria, but has excellent acid resistance, bile resistance, and resistance to digestive enzymes. There is no disclosure of strains having an antimicrobial effect.

The present inventors have newly isolated Lactobacillus plantarum lactic acid bacteria having excellent acid resistance and bile resistance and antibacterial activity. The Lactobacillus plantarum CLP0707 strain newly isolated and identified by the present inventors has superior acid resistance and bile resistance compared to other known strains, and has resistance to digestive enzymes such as pepsin and pancreatin. The survival rate in the body is expected to be high. In addition, this strain can be expected to produce antimicrobial substances against Escherichia coli and staphylococci, thereby inhibiting harmful microorganisms, normalizing the intestinal flora, and intestinal action.

An object of the present invention is to provide a novel lactic acid bacteria having excellent acid resistance and bile acid resistance, resistance to digestive enzymes, and antibacterial effect.

Another object of the present invention to provide a fermentation product comprising the lactic acid bacteria or its culture.

Another object of the present invention is to provide a composition for formal use, probiotic, feed additives, antibacterial or food additives comprising the lactic acid bacteria or cultures thereof.

Another object of the present invention is to provide a method for culturing the lactic acid bacteria.

In order to achieve the object of the present invention, the present invention provides a novel lactic acid bacteria Lactobacillus plantarum CLP0707 (Accession No .: KCCM10868P), excellent in acid resistance and bile acid resistance.

The present invention also provides an antimicrobial, formal, probiotic or feed additive composition and fermentation product comprising the culture of lactic acid bacteria or lactic acid bacteria.

The present inventors newly isolated and identified lactic acid bacteria having excellent anti-acid and bile acid resistance and antibacterial activity among the lactic acid bacteria naturally present from fruits. The lactic acid bacteria newly isolated and identified by the present inventors are excellent in acid resistance and bile acid resistance, and exhibit antimicrobial activity against harmful bacteria, and thus can be expected to inhibit growth and formal effects of harmful microorganisms.

Hereinafter, the novel lactic acid bacteria of the present invention and a composition comprising the same will be described in more detail.

Lactobacillus plantarum CLP0707 (Accession No .: KCCM10868P), a novel lactic acid bacterium, useful for use in compositions such as probiotics, antimicrobials, feeds, etc., having excellent acid resistance and bile acid resistance and showing antibacterial activity. to provide.

Lactic acid bacteria according to the present invention were isolated and identified by the following method. The present inventors isolated only the lactic acid bacteria from the fermentation broth obtained by fermenting the fruit to pH 3.0 using a lactic acid bacterium selection medium, and isolates strains excellent in acid resistance and bile acid resistance.

Finally, the selected lactic acid bacteria were subjected to molecular classification analysis based on the 16S rDNA sequence. As a result, the strain isolated in the present invention was identified as Lactobacillus plantarum. The present inventors named the Lactobacillus plantarum CLP0707 strain isolated in the present invention and the Korea Microorganism Conservation Center located in Yurim Building, 361-221, Hongje 1-dong, Seodaemun-gu, Seoul, Korea, April 30, 2007 under the Treaty of Budapest. (KCCM) was deposited with accession number KCCM10868P.

Lactobacillus CLP0707 isolated and identified for the first time in the present invention has the following characteristics.

First, the lactic acid bacteria according to the present invention show excellent acid resistance and bile acid resistance, and have high utility as a probiotic.

Secondly, the lactic acid bacteria according to the present invention show excellent resistance to digestive enzymes such as pepsin and pancreatin, so that the intestinal viability is excellent.

Third, the lactic acid bacteria and its culture medium according to the present invention has the effect of inhibiting the growth of harmful microorganisms. Harmful microorganisms that can be inhibited by the novel lactic acid bacteria growth according to the present invention include E. coli , Staphylococcus aureus , etc., specifically Salmonella gallinarum ( Salmonella gallinarum ), Salmonella enteritidis ( Salmonella enteritidis , Salmonella typhimurium , Staphylococcus aureus , enterotoxigenic E. coli , Listeria monocytogenes ( Listeria) monocytogenes ), Campylobacter jejuni ), Clostridium perfringens , and combinations thereof, but is not limited thereto.

The lactic acid bacteria CLP0707 according to the present invention can be cultured in large quantities by a conventional lactic acid culture method. As the culture medium, a medium consisting of a carbon source, a nitrogen source, vitamins and minerals can be used. For example, a Man-Rogosa-Sharp (MRS) liquid medium and a liquid medium to which milk is added can be used. The carbon source may be one selected from the group consisting of starch, glucose, sucrose, galactose, fructose and mixtures thereof, and preferably glucose. The nitrogen source may be selected from the group consisting of ammonium sulfate, ammonium nitrate, sodium nitrate, glutamic acid, casamino acid, yeast extract, peptone, tryptone, soybean meal, and mixtures thereof. Minerals include sodium chloride, dipotassium phosphate, magnesium sulfate And mixtures thereof.

Each of the carbon source, nitrogen source and mineral in the microbial culture medium may use 10 to 100 g, 5 to 40 g and 0.5 to 4 g per liter.

Vitamins added to the conventional culture medium may be selected from the group consisting of vitamin A, vitamin B, vitamin C, vitamin D, vitamin E and mixtures thereof. The vitamin may be added to a conventional culture medium at the same time as the above-mentioned medium, such as carbon sources, nitrogen sources, minerals, or the like, to the medium prepared by sterilization.

The culture can be carried out under conventional lactic acid culture conditions, for example, can be incubated for about 10-72 hours at about 15-45 ℃. Centrifugation or filtration may be performed to remove the culture medium in the culture and recover only the concentrated cells, and this step may be performed according to the needs of those skilled in the art. The concentrated cells can be preserved so as not to lose their activity by freezing or lyophilizing according to a conventional method.

In addition, the microorganism according to the present invention may be improved or improved to have equivalent activity or better activity by conventional physicochemical mutation methods and the like known in the art.

On the other hand, CLP0707 of the present invention is excellent in acid resistance and bile acid resistance, and has antibacterial activity against harmful bacteria can be provided in the form of spawn, fermentation composition, probiotic, antimicrobial composition and the like of fermented food.

In the above, 'formal use' refers to a use for treating and ameliorating various symptoms caused by abnormal fermentation of intestinal bacterial flora of humans or animals. The symptoms include, but are not limited to, infectious diarrhea caused by harmful microorganisms, gastroenteritis, inflammatory bowel disease, neuro enteritis syndrome, small intestine microbial hypergrowth and intestinal diarrhea.

The 'probiotic' is a microorganism that has the effect of preventing or treating a specific pathological condition as a living organism, that is, a microorganism that can survive in the gastrointestinal tract when ingested by humans or animals. In general, probiotics have the effect of treating and ameliorating all symptoms caused by abnormal fermentation of the intestinal flora, and when administered to humans and animals, they proliferate and settle in the gut wall of the intestine to prevent harmful microorganisms from settling. It produces and lowers the intestinal pH to inhibit the growth of harmful microorganisms.

As used herein, 'antibacterial' refers to an activity that inhibits or inhibits the growth of harmful microorganisms. Preferably, the harmful microorganism may be a microorganism present in the digestive tract in vivo and causing various diseases related to the digestive tract. The microorganisms include Salmonella gallinarum , Salmonella enteritidis , Salmonella typhimurium , Staphylococcus aureus , Enterotoxigenic E. coli , Listeria monocyto. Listeria monocytogenes ), Campylobacter jejuni , Clostridium perfringens , and combinations thereof, but is not limited thereto.

The above-mentioned compositions may contain the crushed cell wall fraction of lactic acid bacteria, live bacteria, dead bacteria, dry bacteria or cultures thereof according to the present invention as an active ingredient and further include a suitable excipient or carrier. The culture is culture medium itself in which the lactic acid bacteria according to the present invention were cultured in a suitable liquid medium, filtrate (filtered or centrifuged supernatant) from which the strain was removed by filtration or centrifugation, the culture solution was sonicated or the culture solution Cell lysates obtained by treating lysozyme.

In addition, the composition may be prepared and administered in various formulations and methods according to methods known in the art. For example, the lactic acid bacterium of the present invention or its culture may be mixed with a carrier commonly used in the pharmaceutical field, such as tablets, troches, capsules, elixirs, syrups. It may be prepared and administered in the form of powder, suspension or granule. Such carriers include, but are not limited to, binders, suspending agents, disintegrants, excipients, solubilizers, dispersants, stabilizers, suspending agents, pigments and flavorings. In addition, the dosage may be appropriately selected depending on the degree of absorption, inactivation rate, excretion rate, age, sex, condition and severity of disease of the active ingredient in the body. In the case of a probiotic composition, the number of viable cells in the composition may be administered to be 1 × ^{10 5} to 1 × ^{10 11} cells / g, preferably 1 × 10 ⁷ cells / g or more.

In addition, the Lactobacillus plantarum CLP0707 or a culture thereof of the present invention may be provided in the form of a food composition or a food additive composition. The food composition of the present invention may be prepared in the form of powder, granules, tablets, capsules or beverages by mixing the lactic acid bacteria or cultures thereof with a suitable carrier, excipient or auxiliary ingredient. Furthermore, the lactic acid bacteria of the present invention can be used as a seed for the preparation of fermented products. The fermentation products include fruit and vegetable fermented beverages, fermented meat products such as ham and sausage, fermented raw food products, fermented milk products such as cheese, buttermilk, yogurt, whey, and kimchi. The fermented product may be prepared according to methods known in the art using the lactic acid bacteria of the present invention. The production of the fermented product is not limited to the following examples, but, for example, in the case of the fruit vegetable fermented beverage, the fruit, such as apples, pears, tangerines, and the like, is inoculated with lactic acid bacteria of the present invention after mixing with sugar water such as purified water and sugar. To be prepared through fermentation at a temperature of 20 ~ 37 ℃. In addition, in the case of fermented reproductive products can be prepared by mixing the lactic acid bacteria powder of the present invention to cereal powder, fruit vegetable powder, mushroom powder such as brown rice and yulmu. In addition, after mixing the lactic acid bacteria of the present invention to the grain powder and fermentation at a suitable temperature, it can be prepared by properly mixing the fruit and mushroom powder in consideration of nutritional balance and palatability.

In addition, Lactobacillus plantarum CLP0707 or a culture thereof according to the present invention may be provided in the form of a feed composition or a feed additive composition. The feed composition can be used as a substitute for the existing antibiotics to inhibit the growth of harmful intestinal bacteria and maintain the intestinal flora in a stable condition to improve the health of the livestock, improve the weight gain and meat quality of the livestock and increase the milk yield and immunity . Feed composition of the present invention is not limited to this, but can be prepared in the form of fermented feed, compound feed, pellet form and silage (silage) and the like. The fermented feed may be prepared by fermenting an organic material by adding the lactic acid bacteria and various microorganisms or enzymes of the present invention, the blended feed may be prepared by mixing various kinds of lactic acid bacteria of the general feed and the present invention. In addition, the feed in the form of pellets can be prepared by applying heat and pressure to the fermented feed or blended feed in a pellet machine, silage can be prepared by fermenting the green feed with the lactic acid bacteria of the present invention.

In addition, the compositions according to the present invention are suitable for ingestion by animals, including humans, together with the lactic acid bacteria of the present invention, and other types of known activities having the activity of inhibiting the growth of harmful microorganisms and improving the balance of the intestinal flora. It may further comprise a microorganism. Examples of such microorganisms include, but are not limited to, Bifidobacterium longum , Bifidobacterium breve , Bifidobacterium bifidum , Lactobacillus ashdophilus ( Lactobacillus acidophilus , Lactobacillus casei , Lactobacillus reuteri , Lactococcus lactis and Saccharomyces cerevisease ), and the like.

Hereinafter, the specific method of the present invention will be described in detail with reference to Examples, but the scope of the present invention is not limited only to these Examples.

Example  1: Isolation and Identification of Lactic Acid Bacteria

In order to separate fruit-derived lactic acid bacteria having excellent acid resistance, first, fruits, such as apples, pears, bananas, and tangerines, were washed and pulverized, and then fermented to pH 3.0 at 30 ° C. by adding a predetermined weight of purified water and sugar. The fermentation broth was plated on BCP (Brom Cresol Purple) flat medium (Difco, USA) to separate colonies forming yellow colonies. The isolated colonies were selected strains excellent acid resistance through the evaluation of acid resistance, it was confirmed that the selected lactic acid bacteria Lactobacillus plantarum through the following example and named CLP0707.

<1-1> Morphological Analysis and Biochemical Properties

Selected lactic acid bacteria of the present invention were cultured in MRS plate medium (Difco, USA) and the morphology of the colonies was observed. Colony forms of the selected lactic acid bacteria are shown in Table 1, and biochemical characteristics are shown in Table 2 below.

Morphology of Colonies in MRS Flat Medium shape circle size 1.5-2mm color cream color Opacity opacity bump Convex surface lubricity

Biochemical Features of CLP0707 Gram dyeing positivity Aerobic growth positivity Anaerobic growth positivity Catalase voice Spore formation voice motility voice

<1-2> 16S rDNA analysis

16S rDNA sequencing was performed to identify the selected lactic acid bacteria and the results are shown in SEQ ID NO: 1. Since the lactic acid bacteria CLP0707 of the present invention showed 99% sequence homology with 16S rDNA of Lactobacillus plantarum , it was confirmed that the strain CLP0707 isolated from the present invention was Lactobacillus plantarum . Therefore, the inventors of the present invention deposited the strain isolated in the present invention with the accession number KCCM10868P to the Korea Microorganism Conservation Center, which is an international depository organization under the Budapest Treaty on April 30, 2007.

<1-3> Sugar Fermentation Characteristics of Selected Lactic Acid Bacteria

Sugar fermentation characteristics were investigated according to the experimental method of the supplier using API 50 CHL kit (Bio Merioux). The sugar fermentation characteristics of the lactic acid bacteria CLP0707 are as shown in Table 3. As a result of investigation of sugar fermentation properties, CLP0707 was identified as Lactobacillus plantarum, which could confirm the results of 16S rRNA sequencing.

Sugar Fermentation Characteristics of Lactic Acid Bacteria Party reaction Party reaction Glycerol - Salinity + Erythritol - Cellobios + D-Arabinose - maltose + L-Arabinose + Lactose + Ribose + Melibiose + D-Xylose - Sucrose + L-Xylose - Trehalose + Adonitol - Inulin - Methyl-xyloxide - Melezitos + Galactose + D-Raffinose + D-glucose + Starch - D-fructose + Glycogen - D-Mannose + Xylitol - L-sorbos - Genthiobios + Rhamnos - D-turanoose + Dulcitol - D-Lithus - Inositol - D-tagatose - Mannitol + D-fucose - Sorbitol + L-fucose - Methyl-D-mannoside + D-Arabitol - Methyl-D-glucoside - L-Arabitol - N-acetyl glucosamine + Gluconate + Amigdalin + 2 Keto-Gluconate - Arbutin + 5 Keto-Gluconate - Esculin + +: Fermented,-: not fermented

Example 2 :

<2-1> Comparison of Acid Resistance of Lactobacillus Plantarum CLP0707 and Other Strains According to the Present Invention

In order to compare acid resistance between CLP0707 and other lactic acid strains, four kinds of lactic acid strains, which are widely used in fermentation of dairy products, were selected and standard strains were distributed at the Korea Institute of Bioscience and Biotechnology. CLP0707 and pre-strained strains were incubated at 37 ° C. for 16 hours after inoculation in sterile MRS liquid medium (Difco, USA). Then, 1% of the lactic acid bacteria culture medium was inoculated in MRS liquid medium adjusted to pH 3.0 with HCl, and incubated at 37 ° C for 1 hour. Each sample immediately after lactic acid inoculation and after 1 hour incubation was collected, diluted in sterile saline solution, plated in MRS plate medium, incubated at 37 ° C. for 72 hours, and the number of colonies on the plate medium was counted to determine the number of viable cells. The survival rate of the lactic acid bacteria was expressed as a percentage by comparing the number of viable cells immediately after the inoculation to 100% and the number of viable cells immediately after the inoculation and after 1 hour incubation (Table 4).

Survival at pH 3.0 Name Survival rate (%) CLP0707 ( Lactobacillus plantarum ) 100.0 ATCC15707 ( Bifidobacterium longum ) 0.0 KCTC3635 ( Lactobacillus delbrueckii subsp.Bulgararicus ) 49.7 KCTC3109 ( Lactobacillus casei ) 80.6 KCTC3164 ( Lactobacillus acidophilus ) 96.8

As a result, the lactic acid bacteria CLP0707 selected in the present invention was not affected at all at pH 3.0 was very excellent acid resistance, even when compared with other known lactic acid species was able to confirm higher acid resistance.

<2-2> According to the invention Lactobacillus Plantarum CLP0707 Pepsin enzyme resistance and acid resistance

In order to investigate the pepsin enzyme resistance and acid resistance of CLP0707, experiments were conducted by the following method. CLP0707 was incubated at 37 ° C. for 16 hours after inoculation in sterile MRS liquid medium. Then, after sterilization by adjusting to pH 3.0 and pH 2.0 with HCl, 1% of the lactic acid bacteria culture was inoculated in MRS liquid medium in which pepsin was added at a concentration of 1%, and incubated at 37 ° C for 1 hour. Each sample immediately after lactic acid inoculation and 1 hour incubation was collected, diluted in sterile saline solution, plated in MRS plate medium, incubated at 37 ° C. for 72 hours, and the number of colonies on the plate medium was counted to determine the number of bacteria. The survival rate of the lactic acid bacteria was expressed as a percentage by comparing the number of bacteria immediately after the inoculation to 100% and the number of bacteria immediately after the inoculation and after 1 hour incubation (Table 5).

Pepsin Enzyme and Acid Resistance Evaluation of CLP0707 Condition Survival rate (%) pH 3.0, without pepsinase 100.0 pH 3.0, 1% pepsinase 100.0 pH 2.0, pepsinase 1% 97.0

As a result, the lactic acid bacteria CLP0707 selected in the present invention was not affected by the survival rate even in the medium to which the pepsin enzyme was added, and the survival rate was 97% even at pH 2.0, and it was confirmed that the acid resistance and the resistance to the pepsin enzyme were very excellent. The characteristics of the CLP0707 shows a high survival rate even at low pH due to gastric secretion of digestive enzymes and gastric acid secretion can be expected to reach a high intestinal at the time of ingestion.

Example  3: according to the invention Lactobacillus Plantarum CLP0707 of Bile Acid Resistance  Research

The bile acid resistance of Lactobacillus plantarum CLP0707 and known lactic acid bacteria according to the present invention was examined by the following method. That is, the strain and known lactic acid bacteria according to the present invention were inoculated in sterile MRS liquid medium, incubated at 37 ° C. for 16 hours, and then diluted to smear plate with MRS plate medium containing 0.3% oxgall, respectively. Incubated at 72 ° C. for 72 hours. At this time, each strain was plated on MRS plate medium containing no bile acid as a control and incubated in the same manner. The survival rate of lactic acid bacteria was expressed as a percentage by comparing the number of colonies of the control group with the number of colonies of the lactic acid bacteria as 100% (Table 6).

Survival in Oxgal 0.3% Added Medium Name Survival rate (%) CLP0707 ( Lactobacillus plantarum ) 91.4 ATCC15707 ( Bifidobacterium longum ) 54.7 KCTC3635 ( Lactobacillus delbrueckii subsp.Bulgararicus ) 0.0 KCTC3109 ( Lactobacillus casei ) 84.2 KCTC3164 ( Lactobacillus acidophilus ) 1.6

As a result, Lactobacillus plantarum CLP0707 according to the present invention showed very good bile acid resistance compared to known strains.

Example  4: according to the invention Lactobacillus Plantarum CLP0707 Investigation of Pancreatin Enzyme Resistance in Rats

Lactobacillus plantarum CLP0707 according to the present invention was examined for resistance to pancreatin, a pancreatic enzyme present in the small intestine. To this end, the strain according to the present invention was inoculated in sterile MRS liquid medium and incubated at 37 ° C. for 24 hours. The culture was inoculated in MRS liquid medium containing 0.1% pancreatin and then incubated at 37 ° C. for 1 hour. At this time, MRS liquid medium containing no pancreatin was used as a control. After completion of the culture, the survival rate of the lactic acid bacteria according to the present invention was calculated. Survival rate measures viable count after incubation is complete Compared with the number of viable cells immediately after the inoculation, the percentage was expressed, and the survival rate of the control group was 100%.

Resistance to Pancreatic Enzymes of Lactobacillus Plantarum CLP0707 According to the Present Invention Concentration of Pancreatin (%) Survival rate (%) 0.0 100.0 0.1 100.0

As a result, the Lactobacillus plantarum CLP0707 according to the present invention did not reduce the survival rate at all even in the medium containing the pancreatic enzyme showed excellent resistance.

To summarize the results so far, it can be confirmed that the CLP0707 of the present invention not only has excellent acid resistance and bile acid resistance, but also has strong resistance to digestive enzymes such as pepsin and pancreatin. This feature of CLP0707 is thought to increase the intestinal reach rate when ingested, so that it can function as a probiotic.

Example  5: according to the invention Lactobacillus Plantarum CLP0707 Investigation of antimicrobial activity

Probiotics lactic acid bacteria are known to produce antibacterial substances such as bacteriocin depending on the strain, as well as inhibit the growth of other microorganisms by lowering the surrounding pH through the production of lactic acid. Therefore, the antibacterial activity of the lactic acid bacteria Lactobacillus plantarum CLP0707 of the present invention was investigated.

The indicators used in the experiment were E. coli , Staphylococcus aureus , Salmonella typhimurium , and Listeria monocytogenes , all of which were distributed at the Korea Research Institute of Bioscience and Biotechnology. .

The experiment consisted of the following procedure. CLP0707 was inoculated in sterile MRS liquid medium and incubated at 37 ° C. for 16 hours. MRS plate medium was prepared, 5 μl of CLP0707 culture was added dropwise to the center of plate medium, and then cultured at 37 ° C. for 24 hours. The indicators were inoculated in Tryptic soy liquid medium (Difco, USA), incubated for 16 hours at 37 ° C, and then sterilized and cooled to 45-50 ° C in 1% of Tryptic soy agar medium (0.75%, Difco, USA). Inoculation. Agar medium inoculated with the indicator bacteria was inoculated and cultured on an MRS plate medium in which CLP0707 was inoculated in advance and cultured. Indicated.

Size of low ring produced by CLP0707 Indicator Diameter of low ring (mm) KCTC2441 ( Escherichia coli ) 25 KCTC1621 ( Staphylococcus aureus ) 25 KCTC3710 ( Listeria monocytogenes ) 27 KCTC2514 ( Salmonella typhimurium ) 20

As a result, it was confirmed that CLP0707 inhibits the growth of harmful microorganisms. The following experiment was conducted to confirm that the antibacterial activity of CLP0707 is not caused by lactic acid but by the production of antimicrobial substances.

CLP0707 was inoculated in sterile MRS liquid medium and incubated at 37 ° C. for 16 hours, and the culture solution was centrifuged at 8000 rpm for 5 minutes to remove the cells. In order to exclude the antimicrobial activity due to the low pH of the culture medium was adjusted to pH 6.5, and then concentrated 10-fold using a centrifugal vacuum concentrator. The concentrated culture solution was filtered through a 0.22㎛ filter and used for antibacterial activity.

Indicated bacteria was used as the Escherichia coli (E.coli) and Staphylococcus aureus (Staphylococcus aureus). The indicator bacteria were inoculated in Tryptic soy liquid medium and incubated at 37 ° C. for 16 hours, and then inoculated in 1% of Tryptic soy agar medium sterilized and cooled to 45-50 ° C. Agar medium inoculated with the indicator bacteria was inoculated in a Petri dish by dispensing 15 ml of the agar medium, and then placed on a sterile paper disk, and 100 μl of the culture solution prepared thereon was incubated at 37 ° C. for 24 hours to observe the formation of low-ring rings (Table 9).

Size of low ring produced by CLP0707 Indicator Diameter of low ring (mm) Pre-Concentrate Concentrated culture KCTC2441 ( Escherichia coli ) 17 25 KCTC1621 ( Staphylococcus aureus ) 12 28

As a result, the concentrated culture medium was observed to produce a low-ring ring against Escherichia coli and Staphylococcus aureus, and the low-ring ring was also seen in the culture medium adjusted to 6.5 before concentration. Thus, it was found that CLP0707 produces other antimicrobial substances in addition to lactic acid.

The present invention provides a novel lactic acid bacteria having excellent acid resistance and bile acid resistance and having antibacterial activity, and a composition containing the lactic acid bacteria and / or lactic acid bacteria culture. Lactic acid bacteria, lactic acid bacteria culture or composition according to the present invention is excellent in acid resistance, bile acid resistance and digestive enzyme resistance, and has an antibacterial effect can be usefully used in food, medicine, cosmetics, feed and the like.

<110> CTCBIO INC. <120> Novel lactic acid bacteria having acid resistance, bile-salt          resistance and anti-bacterial effect and composition containing          the same <160> 1 <170> KopatentIn 1.71 <210> 1 <211> 1444 <212> DNA <213> Lactobacillus plantarum <220> <221> gene (222) (1) .. (1444) <223> CLP0707 16S rDNA sequence <400> 1 tacatgcaag tcgaacgaac tctggtattg attggtgctt gcatcatgat ttacatttga 60 gtgagtggcg aactggtgag taacacgtgg gaaacctgcc cagaagcggg ggataacacc 120 tggaaacaga tgctaatacc gcataacaac ttggaccgca tggtccgagc ttgaaagatg 180 gcttcggcta tcacttttgg atggtcccgc ggcgtattag ctagatggtg gggtaacggc 240 tcaccatggc aatgatacgt agccgacctg agagggtaat cggccacatt gggactgaga 300 cacggcccaa actcctacgg gaggcagcag tagggaatct tccacaatgg acgaaagtct 360 gatggagcaa cgccgcgtga gtgaagaagg gtttcggctc gtaaaactct gttgttaaag 420 aagaacatat ctgagagtaa ctgttcaggt attgacggta tttaaccaga aagccacggc 480 taactacgtg ccagcagccg cggtaatacg taggtggcaa gcgttgtccg gatttattgg 540 gcgtaaagcg agcgcaggcg gttttttaag tctgatgtga aagccttcgg ctcaaccgaa 600 gaagtgcatc ggaaactggg aaacttgagt gcagaagagg acagtggaac tccatgtgta 660 gcggtgaaat gcgtagatat atggaagaac accagtggcg aaggcggctg tctggtctgt 720 aactgacgct gaggctcgaa agtatgggta gcaaacagga ttagataccc tggtagtcca 780 taccgtaaac gatgaatgct aagtgttgga gggtttccgc ccttcagtgc tgcagctaac 840 gcattaagca ttccgcctgg ggagtacggc cgcaaggctg aaactcaaag gaattgacgg 900 gggcccgcac aagcggtgga gcatgtggtt taattcgaag ctacgcgaag aaccttacca 960 ggtcttgaca tactatgcaa atctaagaga ttagacgttc ccttcgggga catggataca 1020 ggtggtgcat ggttgtcgtc agctcgtgtc gtgagatgtt gggttaagtc ccgcaacgag 1080 cgcaaccctt attatcagtt gccagcatta agttgggcac tctggtgaga ctgccggtga 1140 caaaccggag gaaggtgggg atgacgtcaa atcatcatgc cccttatgac ctgggctaca 1200 cacgtgctac aatggatggt acaacgagtt gcgaactcgc gagagtaagc taatctctta 1260 aagccattct cagttcggat tgtaggctgc aactcgccta catgaagtcg gaatcgctag 1320 taatcgcgga tcagcatgcc gcggtgaata cgttcccggg ccttgtacac accgcccgtc 1380 acaccatgag agtttgtaac acccaaagtc ggtggggtaa ccttttagga accagccgcn 1440 taat 1444  

Claims (8)

Lactobacillus plantarum CLP0707 (Accession Number: KCCM10868P) which has excellent acid resistance and bile acid resistance and has an antibacterial effect. A fermentation product comprising the Lactobacillus plantarum CLP0707 of claim 1 or a culture thereof. Claim 1 Lactobacillus plantarum CLP0707 or a formal composition comprising the culture thereof. A probiotic composition comprising the Lactobacillus plantarum CLP0707 of claim 1 or a culture thereof. Claim 1 Lactobacillus plantarum CLP0707 or a feed composition comprising a culture thereof. The antimicrobial composition of claim 1 comprising Lactobacillus plantarum CLP0707 or a culture thereof. A food additive composition comprising the Lactobacillus plantarum CLP0707 of claim 1 or a culture thereof. The method of culturing Lactobacillus plantarum CLP0707 according to claim 1, wherein the Lactobacillus plantarum CLP0707 is incubated at 15-45 ° C. for 10-72 hours in a medium consisting of a carbon source, a nitrogen source, vitamins and minerals.

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