KR101488770B1

KR101488770B1 - Lactobacillus fermentum PL9036 isolated from healthy senior citizens in the Korean longevity villages

Info

Publication number: KR101488770B1
Application number: KR20130109916A
Authority: KR
Inventors: 이연희; 백경수; 신은주; 홍현진; 이학미; 이민영
Original assignee: (주) 피엘바이오
Priority date: 2013-09-12
Filing date: 2013-09-12
Publication date: 2015-02-04

Abstract

The present invention relates to Lactobacillus fermentum PL9036 and a use thereof. In particular, the Lactobacillus fermentum PL9036 and a culture medium thereof have no resistance transfer, have excellent attachment to enterocytes, acid resistance, and bile acid tolerance, and have effects of suppressing intestinal harmful pathogens, thereby enhancing immunity and anti-oxidation. Therefore, Lactobacillus fermentum PL9036 can be usefully used for probiotics for improving and promoting the intestinal health and defecation, or as a health food.

Description

Lactobacillus fermentum PL9036 {Lactobacillus fermentum PL9036 isolated from the longevity villages}

The present invention is a Lactobacillus momentum spread (Lactobacillus jangsuin developed from living in Korea longevity villages to help improve bowel activity and promoting fermentum PL9036 strains and probiotic use of the strains.

It is known that the relationship between food intake and health has been important since ancient times, and it has been found that the distribution of intestinal microorganisms varies depending on the food to be consumed. Intestinal microorganisms are closely related to human health and disease, and determine the immunity of intestines (Round JL, Mazmanian SK. 2009. The gut microbiota shapes intestinal immune responses during health and disease, Nature Reviews Immunology 9, 313-323) , And the results of a study in which obesity is determined (Turnbaugh PJ, et al., 2006. The obesity-associated gut microbiome with increased capacity for energy harvest. Nature 444, 1027-1031) Is closely related to the constitution of.

It is determined by the lactobacillus that comes into the body for the first few months after birth and the lactobacillus that comes in the body is not kept in the intestines. For example, Lactobacillus ( Lactobacillus , Lactobacillus , Lactobacillus, acidophilus is rarely found in Asians and, in contrast, Lactobacillus ( Lactobacillus) , commonly found in Asians, plantarum ) is not well known to Westerners. These lactic acid bacteria are found in Western dairy products and fermented foods of Asians, respectively.

Most of the lactic acid bacteria that have been commercialized and sold in Korea are lactic acid bacteria that have been developed separately from westerners or their foods, and lactic acid bacteria that are not found in the intestines of everyday Korean people. Therefore, we have developed a Korean lactic acid bacterium by isolating the lactic acid bacteria from adults who are living in a typical long belt of Korea and have healthy intestinal activity, and identify the various functions of the lactic acid bacteria as a probiotic, , Which is a problematic antibiotic resistant metastatic disease, has been developed and used as an effective probiotic agent.

Probiotics are defined as single or multiple strains of live microorganisms that have beneficial effects on the host by improving intestinal microbial properties when ingested in modest amounts. However, recently, Salminen et al. (Salminen et al., 1999. Probiotics: how should they be defined Trends in food Science & Technology, 10, 107-110) is defined as a living microbial formulation contained in foods and feedstuffs or food additives designed to promote the health of humans or animals.

Probiotic lactic acid bacteria are significantly Lactobacillus bacteria (Lactobacillus), Lactococcus (Lactococcus), Streptococcus (Streptococcus), Lou Kono Stock (Leuconostoc), Peddie Oh Caucus (Pediococcus), and separated by Bifidobacterium (Bifidobacterium), ministers and urinary It promotes human health by preventing the microorganism contamination of the reproductive system and maintaining the intestinal health, constipation relief, inhibition of the growth of harmful bacteria, anticancer, immunity enhancement, cholesterol reduction, production of conjugated linoleic acid (CLA) and inhibition of Helicobacter pylori It plays an important role.

Preferable conditions for the probiotics should be safety first, tolerance to acid and bile, adherence to the intestinal epithelium, inhibition against pathogenic bacteria, , Should have an immunostimulating effect, and antibiotic resistance should be resistant to antibiotic resistance due to inherent resistance only.

Bacteria have antibiotic resistance in two ways. The first is tolerance inherent to the patient without being metastasized by the inherent resistance. For example, lactic acid bacteria are resistant to vancomycin due to other chemical structures and thick cell walls, other Gram-positive cell walls, and this inherent resistance is not caused by metastasis, I never do that. The second is resistance to extrinsic resistance. Exogenous resistance refers to the tolerance that a resistant gene binds to a plasmid or a transposon and enters into other external bacterium. In this case, the resistance gene of this resistant bacterium easily transmits tolerance to other bacteria.

Previously, it was known that lactic acid bacteria resistant to strong antibiotics were good, and antibiotic-resistant lactic acid bacteria that survived by treating various antibiotics were considered to be good lactic acid bacteria. However, resistance genes of lactic acid bacteria resistant to various antibiotics are now transferred to intestinal bacteria and cause tolerance problems Has been reported (Mathur, S. and Singh, R., 2005. Antibiotic resistance in food lactic acid bacteria review, International Journal of Food Microbiology, 105, 281-295). Therefore, in the western part of the world, antibiotic-resistant Lactobacillus has not been commercialized for a long time. In addition, Korea has recently introduced a clause on antibiotic resistance transfer in the criteria of food raw materials of microorganisms in a food processing plant, and is used as a criterion for judging the raw materials of microorganisms.

On the other hand, the distribution of intestinal microorganisms in the residents of the rural health and longevity villages in Korea and the residents of the forties and over in the urban area in 2011 was analyzed by the Food and Drug Administration in 2011, and Lactobacillus , Lactococcus , Lactobacillus was 0.56%: 1.355% and Lactococcus was 0.02%: 0.1% compared to the intestinal bacteria. Residents of longevity villages were three to five times higher than urban residents However, the lactic acid bacteria that are free of antibiotic resistant transformation and have all the necessary properties as antibiotic resistance against acid and bile, adherence to intestinal epithelium, inhibitory activity against pathogenic bacteria, antioxidative activity and immunity enhancing effect are known none.

Therefore, the present inventors have made efforts to find a safe lactic acid bacterium free from the possibility of an antibiotic resistance transformation. As a result, Lactobacillus fermentum strain was isolated from a healthy adult living in a place known as a longevity village of Korea. The strain and its culture The present inventors have demonstrated that the present invention has excellent antioxidant, anticancer and antioxidant properties, and has excellent intestinal cell adhesion, acid resistance and biliary cholesterol, Lactobacillus fermentum ) PL9036 strain should be provided as a probiotic agent and all of the above conditions are satisfied satisfactorily. Therefore, it has been confirmed that Lactobacillus perfumont PL9036 or its culture can be used as a probiotic or health food for improving and promoting defecation activity Respectively.

The object of the present invention is to provide Lactobacillus < RTI ID = 0.0 > ( Lactobacillus < / RTI > fermentum PL9036 strain.

Still another object of the present invention is to provide a probiotic composition comprising Lactobacillus perfumont PL9036 strain or a culture thereof as an active ingredient, and a health food for improving and promoting defecation activity.

In order to achieve the above object,

The present invention relates to Lactobacillus < RTI ID = 0.0 > ( Lactobacillus < / RTI > fermentum strain PL9036.

The present invention also provides a biocidal composition comprising Lactobacillus perfumont PL9036 strain or a culture thereof as an active ingredient.

In addition, the present invention provides a health food for promoting bowel movement and intestinal health state containing Lactobacillus fermentum PL9036 strain or a culture thereof as an active ingredient.

Lactobacillus < / RTI > deposited with accession number KACC91850P of the present invention fermentum ) PL9036 strain or culture thereof has excellent adherence to cells, acid resistance and bile acid resistance, has no risk of antibiotic resistance transformation, inhibits harmful pathogenic bacteria in the intestine, has immunity enhancement and antioxidant effect, Or as a health food.

1 is a diagram showing a relationship between the flexible buffer Lactobacillus 16s rRNA base sequence of the momentum (Lactobacillus fermentum) PL9036 strain and Lactobacillus standard strain of Bacillus momentum spread (Lactobacillus fermentum strain CECT562).
Fig. 2 is a diagram showing the relationship of the 16S rRNA base sequence of the Lactobacillus fermentum PL9037 strain and the Lactobacillus fermentum standard strain CECT562.
Fig. 3 is a graph showing the results of a standard strain of Lactobacillus fermentum PL9038 and Lactobacillus fermentum 16S rRNA base sequence of CECT562.
Fig. 4 is a diagram showing the relationship of the 16S rRNA nucleotide sequence of the lactobacillus fermentum PL9039 strain and the standard strain CECT562 of Lactobacillus perfumant.
Fig. 5 is a graph showing the results of a standard strain of Lactobacillus fermentum PL9040 and Lactobacillus fermentum 16S rRNA base sequence of CECT562.
6 is a graph showing the inhibitory ring size inhibiting the growth of harmful bacteria in Lactobacillus perfumant PL9036 strain.
Fig. 7 is a diagram showing resistance of paraquat of Lactobacillus perfumant PL9036 strain. Fig.
FIG. 8 is a diagram showing resistance of Lactobacillus perfumant PL9036 strain to hydrogen peroxide. FIG.

Hereinafter, the present invention will be described in detail.

The Lactobacillus fermentum PL9036 strain has the 16S rRNA base sequence described in SEQ ID NO: 1.

In a specific example of the present invention, the present inventors isolated a strain from a fecal sample of a healthy 80-year-old or older healthy female living on a longevity belt and regularly conditioning it, and identified the 16S rRNA base sequence analysis as a standard of Lactobacillus fermentum The strain CECT562 ( Lactobacillus fermentum strain CECT562) and 99.45% homology. Gram staining confirmed that the strain had a typical Lactobacillus fermentum-like Gram-positive bacterium (see FIG. 1). The strain Lactobacillus perfumeum was named Lactobacillus perfumant PL9036 on Jul. 25, 2013 (Accession No. KACC91850P) at the National Institute of Agricultural Science and Technology.

Further, the present inventors identified additional strains from the fecal samples and identified four types of Lactobacillus fermentum strains showing 99% or more homology with the standard strain CECT562 of Lactobacillus perfumant through 16S rRNA sequencing analysis And named Lactobacillus fermentum PL9037, PL9038, PL9039 and PL9040, respectively (Figs. 2 to 5).

The Lactobacillus fermentum PL9037 strain has the 16S rRNA base sequence described in SEQ ID NO: 2, and the Lactobacillus fermentum PL9038 strain has the 16S rRNA nucleotide sequence shown in SEQ ID NO: 3, and the lactobacillus fermentum PL9039 strain 4 has the 16S rRNA base sequence shown in SEQ ID NO: 4, and the Lactobacillus perfumont PL9040 has the 16S rRNA base sequence shown in SEQ ID NO: 5.

The present invention also provides a probiotic composition comprising as an active ingredient a lactobacillus fermentum PL9036 strain deposited with Accession No. KACC91850P or a culture thereof.

The culture medium of the microorganism includes various antimicrobial organic acids and non-protein antimicrobial substances produced by the microorganisms.

The strain may be selected from the group consisting of gentamicin, kanamycin, streptomycin, neomycin, tetracycline, erythromycin, clindamycin, chloramphenicol, endogenous and exogenous resistance to antibiotics such as ampicillin, synercid, quinupristin and dalfopristin combination, linezolid, trimethoprim, ciprofloxacin and rifampicin, Potential), and for vancomycin that is not susceptible to metastatic transformation.

The strain has excellent intestinal cell adhesion, acid resistance and biliary cholesterol, and has antimicrobial activity, antioxidative activity, immunosuppressive activity and endotoxic shock inhibitory effects.

In a specific example of the present invention, the inventors of the present invention conducted experiments on Lactobacillus fermentum PL9036, Lactobacillus fermentum PL9037, PL9038, PL9039 and PL9040 strains according to the ISO guidelines to confirm antibiotic susceptibility of Lactobacillus perfumant strains Treatment of the antibiotics gentamycin, tetracycline, erythromycin, clindamycin, chloramphenicol, ampicillin, linaczide, linezolide and rifampicin and liquid dilution resulted in the Lactobacillus perfumont PL9036 strain being different from other Lactobacillus fermentum strains (See Table 1). There was no risk of antibiotic resistance due to sensitivity to all antibiotics.

Further, in order to confirm the inhibitory activity of the Lactobacillus perfumer strain against pathogens, the present inventors have found that Escherichia coli, which is a harmful bacterium, coli) O157: H7 ATCC43894, Salmonella typhimurium (Salmonella typhimurium) CCARM 8001, Salmonella entera itty display (Salmonella enteritidis) CCARM 8010, Enterococcus faecalis (Enterococcus faecalis) CCARM 0011, Staphylococcus aureus (Staphylococcus aureus ) CCARM 0045, Listeria monocytogenes ( Listeria monocytogenes CCARM 0019 and Lactobacillus fermentum strains were cultured to determine the presence or absence of inhibitory rings and to confirm that the Lactobacillus fermentum PL9036 strain and Lactobacillus fermentum PL9037, PL9038, PL9039 and PL9040 strains exhibited significant pathogenic inhibitory activity, In particular, Lactobacillus perfumant PL9036 strain kills all six kinds of harmful bacteria, thus confirming that it is excellent in inhibiting pathogens (see FIG. 6 and Table 2).

In order to confirm the safety of the lactobacillus fermentum strains, the present inventors have also found that, in order to confirm the safety of the lactobacillus fermentum strain, (Lactobacillus fermentum PL9036 strain), Lactobacillus fermentum PL9037, PL9038, PL9038, PL9038, PL9038, PL9038, PL9038, and PL9038 were tested for the production of harmful enzymes such as beta-glucuronidase and beta -glucosidase, And PL9040 strains, they did not show hemolysis, did not produce harmful substances and noxious enzymes, and showed negative stability in the gelatin liquefaction reaction, and thus showed the best stability (see Table 3).

In order to confirm the acid resistance and bile resistance of the lactobacillus fermentum strain, the present inventors measured the viable cell count after culture with artificial gastric juice and bile added to the strain. As a result, the Lactobacillus fermentum PL9036 strain was most likely to survive It was confirmed that it was excellent in acid resistance and brittle resistance (see Table 4).

Further, in order to confirm the ability of Lactobacillus perfumant PL9036 strain to adhere to intestinal cells, the number of adhered lactic acid bacteria was measured by treating the intestinal cell line with lactic acid bacteria and using Gram stain and serial dilution method. As a result, It was confirmed that Bacillus pertumum PL9036 strain had excellent adhesion ability.

In order to confirm the immunostimulating effect of the Lactobacillus perfumant PL9036 strain, the present inventors also examined the effect of Lipopolysaccharide (LPS) and Lactobacillus fermentum PL9988 on the macrophage cell line, and proinflammatory cytokine TNF- α, IL-6, and IL-1β were measured. As a result, when the lactobacillus fermentum PL9988 strain was treated, it was found that the immunoregulation effect was increased by increasing the concentration. In addition, TNF-α, IL-6, and IL-1β concentrations were increased when LPS alone was treated with LPS and Lactobacillus fermentum PL9036, -1β in the culture medium, the Lactobacillus perfumont strain PL9036 was found to have an endotoxic shock-minimizing effect.

In order to confirm the antioxidant effect of Lactobacillus perfumant PL9036 strain, the present inventors conducted an experiment to measure antioxidative functions. As a result, it was found that the Lactobacillus perfumant PL9036 strain contains 10 mM of paraquat, which produces a superoxide anion, When 100 mM was added, growth inhibition was not observed in all of the cells. In addition, it was confirmed that the Lactobacillus perfumant PL9036 strain had a survival rate of 57.8% with respect to hydrogen peroxide and showed resistance to hydrogen peroxide. Thus, it was confirmed that the Lactobacillus perfumant PL9036 strain had excellent antioxidant ability 7, Fig. 8 and Table 5).

Therefore, the Lactobacillus perfumant PL9036 strain or its culture of the present invention is excellent in adherent cell adhesion, acid resistance and biliary cholesterol, has no risk of antibiotic resistance transformation, inhibits harmful pathogenic bacteria in the intestines, Toxin shock suppression effect, and antioxidative effect, it can be usefully used as a biocide composition for improving bowel activity and intestinal health.

The composition of the present invention may be prepared according to a conventional method for producing a prod- uct composition, and may be generally in the form of a culture suspension or a dry powder. In addition, one or two or more pharmaceutically acceptable conventional carriers or one or two or more additives may be selected from the above-mentioned Lactobacillus perfumont PL9036 strain as a main component or an effective amount of the culture solution thereof to prepare a composition of a conventional formulation can do.

The carrier may be selected from one or more selected from among diluents, lubricants, binders, disintegrants, sweeteners, stabilizers and preservatives. Examples of the additives include one or more of flavorings, vitamins and antioxidants Can be used.

In the present invention, the carrier and additives may be any pharmaceutically acceptable ones. Specific examples of the diluent include lactose monohydrate, trehalose, cornstarch, soybean oil, Microcrystalline cellulose or mannitol may be preferably used and the lubricant may be magnesium stearate or talc. The binder may be polyvinylpyrrolidone (PVP) or hydroxypropyl It is preferable to select from hydroxypropylcellulose (HPC). The disintegrant is preferably selected from carboxymethylcellulose calcium (Ca-CMC), sodium starch glycollate, polacrylin potassium or cross-linked polyvinylpyrrolidone And the sweetening agent is selected from white sugar, fructose, sorbitol or aspartame. Examples of the stabilizer include sodium carboxymethylcellulose sodium (Na-CMC), beta-cyclodextrin (beta -cyclodextrin) white bee's wax or xanthan gum and the preservative is selected from the group consisting of methyl p-hydroxy benzoate, methlparaben, propyl p-hydroxybenzoate, propylparaben, or potassium sorbate potassium sorbate).

The present invention also provides a health food containing as an active ingredient a lactobacillus fermentum PL9036 strain deposited with Accession No. KACC91850P or a culture thereof.

In addition, the composition according to the present invention is suitable for ingesting together with the lactic acid bacteria of the present invention, further inhibiting the growth of harmful microorganisms upon ingestion, and further adding other known microorganisms having an activity of improving intestinal microflora .

The strain has excellent intestinal cell adhesion, acid resistance and biliary excretion, and enhances intestinal health activity through antibacterial ability, antioxidative effect, immunity enhancing activity, endotoxin shock inhibition and intestinal pathogenic microorganism growth inhibition.

The food may be selected from the group consisting of ice cream, milk, soy milk, yoghurt, dairy products including cheese, meat, sausage, bread, chocolate, candy, snacks, confectionery, pizza, ramen, other noodles, And an alcoholic beverage.

Therefore, the Lactobacillus perfumant PL9036 strain of the present invention is excellent in the adhesiveness to intestinal cells, acid resistance and biliary cholesterol, has no danger of antibiotic resistance transformation, inhibits harmful pathogenic bacteria in the intestines, has immunity enhancing effect, endotoxic shock inhibition And antioxidant effect, it can be useful as a health food for improving bowel activity and intestinal health.

Examples of the health food of the present invention include folk remedies for the purpose of improving the intestinal function of tea, jellies, juices, extracts and beverages containing the Lactobacillus sp. Strain or the culture thereof as an active ingredient. The health food for preventing diseases according to the present invention, which has been processed into various forms as described above, is easy to take without any adverse effects on the human body and can be stored for a long period of time.

When the strain of the genus Lactobacillus or a culture thereof of the present invention is used as a food additive, the strain or a culture thereof may be directly added, used together with other food or food ingredients, and suitably used according to a conventional method. The amount of the active ingredient to be mixed can be suitably determined according to the intended use (prevention, health or therapeutic treatment). Generally, in the production of food or beverage, it is added in an amount of 15 parts by weight or less, preferably 10 parts by weight or less, based on 100 parts by weight of the lactobacillus strain or the culture broth of the present invention. However, in the case of long-term consumption intended for health and hygiene purposes or health control purposes, the amount may be less than the above range, and there is no problem in terms of safety. Therefore, the active ingredient may be used in an amount of more than the above range.

There is no particular limitation on the kind of the food. Examples of the food to which the Lactobacillus sp. Strain or the culture thereof according to the present invention can be added include meat, sausage, bread, chocolate, candy, snacks, confectionery, pizza, ramie noodles, other noodles, ice confectionery, ice cream, milk , Dairy products including milk substitute, cream, butter, buttermilk, yogurt, yoghurt, cheese, various soups, drinks, tea, drinks, alcoholic beverages and vitamin complexes. do.

The health beverage composition of the present invention may contain various flavors or natural carbohydrates as an additional ingredient such as ordinary beverages. The above-mentioned natural carbohydrates are sugar saccharides such as monosaccharides such as glucose and fructose, disaccharides such as maltose and sucrose, polysaccharides such as dextrin and cyclodextrin, and sugar alcohols such as xylitol, sorbitol and erythritol. Examples of sweeteners include natural sweeteners such as tau Martin and stevia extract, synthetic sweeteners such as saccharin and aspartame, and the like. The ratio of the natural carbohydrate is generally 0.01 to 0.04 g, preferably about 0.02 to 0.03 g per 100 ml of the lactobacillus strain or culture thereof of the present invention.

The Lactobacillus sp. Strain or culture thereof according to the present invention may further contain various nutrients, vitamins, electrolytes, flavors, colorants, pectic acid and salts thereof, alginic acid and its salts, organic acids, protective colloid thickening agents, pH adjusting agents, , Preservatives, glycerin, alcohols, carbonating agents used in carbonated drinks, and the like. In addition, the probiotics of the present invention may contain flesh for the production of natural fruit juices, fruit juice drinks and vegetable drinks. These components may be used independently or in combination. Although the ratio of such additives is not critical, the strain of the present invention or its culture is generally selected in the range of 0.01 to 0.1 parts by weight per 100 parts by weight.

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

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

< Example 1> Isolation of strain

A sample of feces from healthy elderly people aged 80 years or older who regularly refresh themselves at eight villages in Jeollabuk-do, known as the longevity belt of Korea, was provided to arrive at the laboratory within 6 hours of refrigerated state. The feces samples were diluted with sterile physiological saline and then diluted in De Man Rogosa medium (MRS, Difco, Becton Dickinson, Sparks, MD, USA), plated and cultured in a 37 ° C incubator for 48 hours. In the case of colonies of the same type observed in the same person, only up to 2 colonies were isolated from the same colonies (Lee, HM and Lee Y, 2008, Letters in Applied Microbiology 46, 676-681) And the strain was isolated. The isolated colonies were screened for catalase reaction, and then stained with Gram stain to isolate Gram-positive and rod-shaped strains.

< Example 2> Identification of the strain

For the identification of the strains isolated in Example 1, 16S rRNA gene sequencing was performed.

Specifically, the nucleotide sequence EzTaxon-e server (. Http://eztaxon-e.ezbiocloud.net/; Kim et al, 2012) and the online BLAST algorithm at the National Center for Biotechnology Information Web server (http: // The strains were identified using the Nucleotide Sequence Listing (ncbi.nlm.nih.gov ), and the molecular phylogenetic analysis was performed based on the 16S rRNA sequence.

As a result, as shown in Figure 1, the Lactobacillus of the present invention spread momentum (Lactobacillus fermentum ) strain PL9036 has the 16S rRNA base sequence shown in SEQ ID NO: 1 and is a standard strain of Lactobacillus fermentum CECT562 ( Lactobacillus fermentum strain CECT562) and 99.45% of 16S rRNA homology (Fig. 1). In addition, the strain was treated with Lactobacillus < RTI ID = 0.0 > fermentum ) PL9036 and deposited on July 25, 2013 as the accession number KACC91850P to the National Institute of Agricultural Science and Technology, National Institute of Agricultural Science and Technology.

In addition, strains were further isolated and identified by the same method.

As a result, as shown in FIG. 2 to FIG. 5, the Lactobacillus perfume strain CECT562, which has a 16S rRNA base sequence which is different from the lactobacillus fermentum PL9036 strain of the present invention and is represented by SEQ ID NO: 2 to SEQ ID NO: 5, Four Lactobacillus fermentum strains showing 99% or more of 16S rRNA homology were isolated and identified (FIGS. 2 to 5), respectively, and designated Lactobacillus fermentum PL9037, PL9038, PL9039 or PL9040, respectively.

< Example 3> Lactobacillus fermentum PL9036 Staining of the strain

In order to confirm the shape of Lactobacillus fermentum PL9036 strain isolated from Example 1, Gram staining was performed.

Specifically, in order to analyze the morphology of the lactobacillus fermentum PL9036 strain isolated and identified in the above <Example 1> and <Example 2>, the strain was cultured in MRS agar plate medium at 30 ° C. for 24 hours, The bacteria were streaked onto a glass slide, and the morphology and colony morphology of the bacteria were observed through a microscope. Gram staining experiments were performed using a Gram stain kit (Sigma Diagnostics, kit HT 90-A) manufactured by Sigma.

As a result, it was confirmed that the shape of the lactobacillus fermentum PL9036 strain of the present invention is the appearance of a gram-positive bacterium which is a typical lactobacillus fermentum.

< Example 4> Lactobacillus fermentum PL9036 Identification of susceptibility of strains to antibiotics

Liquid dilution was performed to determine the antibiotic susceptibility of Lactobacillus fermentum PL9036 strain identified from <Example 2>. Although there is a suggestion of the international organization for standardization (ISO), the clinical and laboratory standards institute (CSLI), and the EUCAST (European committee on antimicrobial susceptibility testing) methods for lactic acid bacteria susceptibility testing, the tolerance standards for various lactic acid bacteria and antibiotics are almost established . Therefore, susceptibility tests of isolated Lactobacillus fermentum strains were performed at the same time to determine the relative resistance.

In particular, the test for antimicrobial susceptibility of lactic acid bacteria was conducted in accordance with the ISO guidelines (ISO10932: 2010 (E), Milk and milk products-Determination of the minimal inhibitory concentration (MIC) of antibiotics to bifidobacteria and non-enterococcal lactic acid bacteria (LAB) (PH 6.7) of LSM medium (IST broth (Iso-Sensitest, 90%; Oxoid) and MRS broth (10%)) according to the manufacturer's instructions. Antibiotics may be used in combination with other antimicrobial agents such as gentamicin, tetracycline, erythromycin, clindamycin, chloramphenicol, ampicillin, vancomycin, linezolid and rifampicin in a concentration range of 0.5 to 256 占 퐂 / ml. The antibiotics to be tested were prepared in 2 × and 50 μl / well of each microplate was dispensed into each well. Lactobacillus cultured overnight was inoculated into wells containing antibiotics, and the microplate inoculated with the strain was incubated at 37 ° C for 48 hours under anaerobic conditions and the results were observed.

As a result, as shown in Table 1, by confirming that the lactomycin fermentum strains of the present invention showed high resistance to vancomycin at a minimal inhibitory concentration (minimum inhibitory concentration = 128 ug / ml or more) Mentham strains were found to be caused by a thick inner wall, which is a common feature of lactic acid bacteria. In addition, the lactobacillus fermentum PL9036 of the present invention was confirmed to have sensitivity to all antibiotics unlike the other lactobacillus fermentum PL9037, PL9038, PL9039 and PL9040 strains having more than one resistance to gentamycin, erythromycin and clindamycin , And the lactobacillus fermentum PL9036 strain showed no risk of antibiotic resistance transformation (Table 1).

The results of the minimum inhibitory concentration (MIC) measurement of Lactobacillus frumumthum strains discrimination
number Minimum inhibitory concentration (MIC) GEN TET ERY CLI CHL AMP VAN SYN LIN RIF PL9036 8 8 2 0.25 4 0.5 > 128 0.5 4 ≤0.125 PL9037 16 8 8 4 4 0.5 > 128 0.5 4 0.25 PL9038 8 4 8 4 8 0.5 > 128 0.5 4 ≤0.125 PL9039 16 8 8 0.25 8 0.5 > 128 0.5 8 ≤0.125 PL9040 16 4 4 0.5 8 One > 128 0.5 4 ≤0.125

(GEN: gentamicin, TET: tetracycline, ERY: erthromycin, CLI: clindamycin, CHL: chloramphenicol, AMP: ampicillin, VAN: vancomycin, SYN: Linerzide, LIN:

< Example 5> Lactobacillus fermentum PL9036 Pathogen of the strain Inhibition Confirm

The following experiment was conducted to determine the inhibitory activity of the Lactobacillus perfumant PL9036 strain isolated from Example 1 above.

Specifically, the lactic acid bacteria were cultured in an MRS liquid medium for 25 hours and then centrifuged to prepare a supernatant. Escherichia coli) O157: H7 ATCC 43894, Salmonella typhimurium (Salmonella typhimurium) CCARM 8001, Salmonella entera ET display (Salmonella enteritidis) CCARM 8010, Enterococcus faecalis (Enterococcus faecalis) CCARM 0011, Staphylococcus aureus (Staphylococcus aureus) CCARM 0045, L. monocytogenes cytokines jeneseu (Listeria monocytogenes ) Six harmful bacteria including CCARM 0019 were inoculated into MH solid medium with a turbidity of McFaland standard 0.5, punched in a sterilized test tube, and 1 ml of the above lactic acid culture supernatant was mixed with 200 μl of 3% agar . Listeria monocytogenes was cultured at 30 ° C and the other harmful bacteria were cultured at 37 ° C to confirm the presence or absence of inhibitory rings.

As a result, as shown in Fig. 6 and Table 2, it was confirmed that the lactobacillus fermentum PL9036 strain showed inhibitory ability against all six kinds of harmful bacteria, and thus it was confirmed that the lactobacillus fermentum PL9036 strain had inhibitory effect against various pathogens (Fig. 6 and Table 2).

The inhibitory ring size of Lactobacillus fermentum strains discrimination
number Suppression of harmful bacteria EC ST SE EF SA LM PL9036 19 21.5 22 27 16.5 26 PL9037 18 20 22 21 - 29 PL9038 17.5 21.5 21.5 21 - 28 PL9039 16.5 21.5 20.5 20 19 27 PL9040 16.5 19.5 20 20 19.5 26

(EC: Escherichia coli O157: H7 ATCC 43894, ST: Salmonella typhimurium CCARM 8001, SE: Salmonella entereraitidis CCARM 8010, EF: Enterococcus faecalis CCARM 0011, SA: Staphylococcus aureus CCARM 0045, LM: Listeria monocytogenes CCARM 0019)

< Example 6> Lactobacillus fermentum PL9036 Confirmation of the safety of the strain

In order to measure the pathogen safety of Lactobacillus fermentum PL9036 strain isolated from Example 1, a hemolytic phenomenon and toxic substances and noxious enzyme production tests were carried out. In addition, the bacterial pathogenicity is often influenced by the cell invasion ability, and the gelatin liquefaction test was performed to confirm the protein degradation ability because the cell invasion requires the protein degrading ability.

Specifically, the test bacteria were inoculated into the blood agar medium and incubated at 37 ° C for 24 hours to confirm hemolysis. In addition, the test bacteria were inoculated in MRS gelatin medium containing 0.3 g of beef extract, 0.5 g of peptone, 12 g of gelatin and 100 ml of MRS medium, followed by culturing at 35 ° C for 6 weeks, After cooling for 4 hours at 4 ° C with the control group not inoculated, gelatin liquefaction was confirmed. When the medium was not solidified even after refrigerating, it was regarded as a positive reaction. In addition, it is also possible to use harmful substances such as ammonia (urease), indole and phenylpyruvic acid, and some intestines such as beta-glucuronidase and beta-glucosidase It was confirmed whether or not a harmful enzyme produced by the microorganism was produced.

As a result, as shown in Table 3, the lactobacillus fermentum PL9036 strain of the present invention all showed negative in the gelatin liquefaction reaction, and the harmful metabolites ammonia, indole and phenylpyruvic acid, the noxious enzyme beta-glucuronidase And did not produce beta-glucosidase.

On the other hand, unlike the Lactobacillus fermentum PL9036 strain, the Lactobacillus perfumant PL9039 strain causes α-type hemolysis, while the Lactobacillus perfumant PL9037, Lactobacillus perfumant PL9038, and Lactobacillus perfumant PL9040 strain cause harmful metabolites or harmful enzymes It was confirmed that the Lactobacillus fermentum PL9036 strain was the most safe (Table 3).

Safety test results of Lactobacillus fermentum strains discrimination
number Hemolysis
phenomenon Generate harmful substances Production of harmful enzymes ammonia Indole Phenylpyruvic acid β-glucuronidase ? -glucosidase PL9036 × × × × × × PL9037 × ○ × × × × PL9038 × × ○ × ○ × PL9039 ○ × × × × × PL9040 × × × ○ × ○

< Example 7> Lactobacillus fermentum PL9036 Strain Acid resistance And My bile Confirm

In order to measure the acid resistance and bile resistance of the lactobacillus fermentum PL9036 strain isolated from Example 1, acid resistance and bile resistance test were conducted.

Specifically, the acid resistance test was carried out in an artificial gastric juice supplemented with 1000 U / ㎖ of pepsin in pH 3.0 medium for measurement in an environment similar to the digestive tract condition. Separated lactic acid bacteria were cultured in liquid medium for 24 hours, respectively. Cells were recovered by centrifugation and washed three times with sterile physiological saline. The same amount as the supernatant was added to the artificial gastric juice, and the reaction was carried out for 90 minutes under the same condition as that of the lactic acid bacteria culture. The resultant was diluted compared to the control group and smoothed on an MRS plate to count the viable cell counts.

In addition, the bacteriostatic test was carried out for each lactic acid bacterium treated for 90 minutes under artificial gastric condition. To the bacteriostatic solution, 0.3% bile (pork bile extract, Sigma) and 1000 U / Respectively. After centrifuging the artificial gastric juice, the artificial bile juice was added in the same amount as the supernatant, and further reacted for 90 minutes. The resultant was further diluted compared to the control group, and plated on an MRS plate to count viable cells.

As a result, as shown in Table 4, Lactobacillus fermentum PL9036, PL9037, PL9038 and PL9039 strains had significant viability even after treatment with artificial gastric juice and artificial bile juice, , It was confirmed that the lactobacillus fermentum PL9036 strain was excellent in acid resistance and bile acid tolerance (Table 4).

Acid resistance and endosperm concentration of lactobacillus fermentum strain discrimination
number MRS Artificial gastric juice gall 0 minutes 90 minutes 180 minutes 90 minutes 90 minutes PL9036 3.50 x 10 ⁸ 1.82 × 10 ⁹ 2.12 x 10 ⁹ 7.80 x 10 ⁸ 4.00 x 10 ⁸ PL9037 3.50 x 10 ⁸ 4.10 × 10 ⁹ 4.40 × 10 ⁹ 7.20 x 10 ⁸ 3.10 x 10 ⁸ PL9038 3.60 x 10 ⁸ 4.20 × 10 ⁹ 4.70 × 10 ⁹ 6.80 x 10 ⁸ 3.70 x 10 ⁸ PL9039 3.70 x 10 ⁸ 4.30 × 10 ⁹ 4.60 × 10 ⁹ 8.40 x 10 ⁸ 3.60 x 10 ⁸ PL9040 3.60 x 10 ⁸ 3.90 × 10 ⁹ 4.30 × 10 ⁹ 6.50 x 10 ⁸ 3.80 × 10 ⁸

< Example 8> Lactobacillus Fermantum PL9036 For the intestinal cells of the strain Attachment Confirm

In order to measure the adherence of Lactobacillus perfumant PL9036 strain isolated from Example 1 to human intestinal cells, the following experiment was conducted.

Specifically, human intestinal cell line Caco-2 cells (Korean Cell Line Bank, Korea) were inoculated with 20% inactivated feral bovine serum (FBS, Gibco), 1% (v / v) antibiotic- antimycotics, 37 ℃ using MEM (Eagles, Gibco) medium containing Gibco), 7% CO ₂ Lt; / RTI > The lactic acid bacteria (1 x 10 ⁸ CFU / ml) cultured in the above Caco-2 cells and MRS broth of 70% or more were washed three times with 10 mM PBS (pH 7.0), suspended in 1 ml of MEM medium, I put it on the plate. 37 ° C, CO ₂ After incubation for 1 hour in the incubator, the plates were washed three times with 10 mM PBS to remove unattached lactic acid bacteria. 100 ml of 35% formaldehyde, 16 g Na ₂ HPO ₄ , and NaH ₂ PO ₄ .H ₂ O, and then stained with Gram stain as in Example 3, and then observed with a microscope. The reaction plate was washed three times with 10 mM PBS, and 1 ml of 0.1% Triton X-100 was added thereto. Cells and lactic acid bacteria suspensions were obtained using a scrapper, and the cells were washed with MRS broth plates And the number of lactic acid bacteria was counted to confirm the number of lactic acid bacteria attached to the human intestinal cells. The adhesion test was carried out three times and averaged.

As a result, it was confirmed that the lactobacillus fermentum PL9036 strain was adhered to the intestinal cells, so that it was confirmed that the lactobacillus fermentum PL9036 could grow in the intestinal tract and grow under intestinal conditions.

< Example 9> Lactobacillus Fermantum PL9036 Identification of the immune enhancement effect of the strain

When non-pathogenic bacteria (lactic acid bacteria) stimulate macrophage cells, TNF-α, a proinflammatory cytokine, is secreted, resulting in an increased level of cytokine mRNA and secretion of IL-10 . Therefore, in order to confirm the concentration of TNF-?, IL-6 and IL-1? Secreted from macrophages by the lactobacillus fermentum PL9036 strain isolated from Example 1, the following experiment was conducted.

Specifically, RPMI1640 (Roswell Park Memorial Institute-1640, Gibco BRL, Grand island, USA) supplemented with 10% fetal bovine serum (Gibco) and 1% antibiotic-antifungal agent (Gibco) ) And inoculated in a 96-well plate at 1.0 × 10 ⁵ / well, and cells were stimulated with 1 μg / ml of lipopolysaccaride (LPS). After washing three times with each one lactic acid bacteria subcultured in MRS medium with PBS and the final number of bacteria 1.0 × 10 was suspended in RPMI1640 so that the ⁸ CFU / well and dispensed to the plate 37 ℃ for 24 hours, CO ₂ Lt; / RTI > The culture supernatant obtained by reacting only the LPS alone, the culture supernatant obtained by reacting only the lactic acid bacteria, and the culture supernatant obtained by reacting LPS and lactic acid bacteria in the same manner was obtained. The immunopositive cytokines TNF-a, IL-6 and The concentration of IL-1β was analyzed by the manufacturer's procedure using a TNF-α cytokine kit (Biosource, CA, USA). All the experiments were conducted three times and averaged.

As a result, when the macrophage line was stimulated with LPS and treated with Lactobacillus perfumont PL9036, the secretion of TNF-α, IL-6 and IL-1β was significantly reduced as compared with the LPS alone treatment group, Lactobacillus fermentum PL9036 strain has an immunopotentiating effect and, in particular, reduces excessive LPS-induced TNF-α, IL-6 and IL-1β secretion, thereby minimizing LPS-induced endotoxic shock in bacterial infections .

< Example 10> Lactobacillus Fermantum PL9036 Identification of antioxidative effect of strain

<10-1> Lactobacillus Fermantum PL9036 Strain In the paraquat Confirm resistance to

In order to confirm the antioxidative effect of the lactobacillus fermentum PL9036 strain isolated from <Example 1>, resistance test against paraquat was carried out.

Specifically, a suspension was prepared so that the strain cultured overnight at 10 ⁷ CFU / ml in saline, 0.1 ml of the suspension was inoculated into a MRS solid medium, and a paper disk containing 10 μl of paraquat dissolved in physiological saline was placed After overnight incubation at 37 ° C, the growth inhibition rings were measured. 100 mM paraquat was added to the suspension, and the cells were cultured overnight. The bacterial growth was measured at an absorbance of 600 nm per hour to determine the ROS resistance ability Respectively. Lactobacillus fermentum PL9005 strain deposited with accession number KCCM-10250 was used as a control.

As a result, as shown in FIG. 7 and Table 5, in the case of Lactobacillus fermentum PL9005 deposited with Accession No. KCCM-10250, growth inhibitory rings were observed upon addition of 100 mM paraquat, but the lactobacillus fermentum PL9036 The strain was confirmed to be resistant to paraquat (Fig. 7 and Table 5) by confirming that the growth inhibitory ring was not observed at all when 10 mM and 100 mM of paraquat were added, so that the lactobacillus fermentum PL9036 strain was resistant to paraquat.

Growth inhibition by superoxide anion produced by papraquat Ring size Paraquat concentration
(mM) Lactobacillus fermentum PL9036 PL9005 0 0 0 10 0 0 100 0 16

(Unit (diameter): mm)

<10-2> Lactobacillus Fermantum PL9036 The hydrogen peroxide of the strain ( hydrogen Peroxide ) Resistance to

In order to confirm the antioxidant effect of the lactobacillus fermentum PL9036 strain isolated from the <Example 1>, a resistance test against hydrogen peroxide was carried out.

Specifically, the MRS medium was inoculated with 1% (v / v) Lactobacillus fermentum PL9036 strain cultured overnight, added with 0, 0.4, 0.7 and 1.0 mM hydrogen peroxide (30% After incubation for 8 hours, the hydrogen peroxide resistance ability was measured at an absorbance of 600 nm. Lactobacillus fermentum PL9005 strain deposited with accession number KCCM-10250 was used as a control.

As a result, as shown in Fig. 8 and Table 6, when 1.0 mM of hydrogen peroxide was treated and cultured for 8 hours, the survival rate of the lactobacillus fermentum PL9005 strain deposited with Accession No. KCCM-10250 was 0% The survival rate of the Lactobacillus perfume strain PL9036 of the present invention was confirmed to be 57.8%, confirming that the lactobacillus fermentum PL9036 strain was excellent in resistance to hydrogen peroxide (FIG. 8 and Table 6). Therefore, it was confirmed that the Lactobacillus fermentum PL9036 strain had excellent antioxidant ability through the results of Example 10 above.

Resistance to hydrogen peroxide in Lactobacillus fermentum strains discrimination
number
Hydrogen peroxide concentration (mM) 0 0.4 0.7 One 0 8 hours 0 8 hours 0 8 hours 0 8 hours PL9036 0.045 4.390 0.032 4.190 0.024 3.850 0.016 2.540 PL9005 0.031 0.627 0.023 0.332 0.019 0.089 0.011 0.000

Hereinafter, a preparation example for a food containing the strain of the present invention and a culture solution thereof as an active ingredient is exemplified.

< Formulation example 1> Manufacturing of food

Foods comprising the Lactobacillus perfumont PL9036 strain of the present invention and a culture thereof were prepared as follows.

<1-1> Preparation of cooking seasoning

Lactobacillus fermentum PL9036 strain of the present invention and 1 to 12 parts by weight of the culture medium of the present invention were mixed with 100 parts by weight of cooking sauce to prepare cooked sauce for improving the function of the bowel.

<1-2> soup And juicy ( gravies )

1 to 12 parts by weight of the lactobacillus fermentum PL9036 strain of the present invention and its culture were added to 100 parts by weight of soup and juice to prepare a meat processing product for improving intestinal function, a soup noodle and a juice.

<1-3> Dairy products ( dairy products )

Lactobacillus fermentum PL9036 strain of the present invention and 1 to 12 parts by weight of the culture thereof were added to 100 parts by weight of milk, and various dairy products such as butter and ice cream were prepared using the milk.

<1-4> Solar Produce

The lactobacillus fermentum PL9036 strain of the present invention and its culture solution were reduced in pressure in a vacuum concentrator, concentrated and dried, and dried with a hot-air drier. The resulting dried product was pulverized to a size of 60 mesh with a pulverizer to obtain a dry powder.

The cereals, seeds, and the lactobacillus fermentum PL9036 strain of the present invention and a culture thereof were compounded to prepare an aliquot.

< Formulation example 2> Manufacturing of beverages

<2-1> Production of carbonated beverage

5 to 10 parts by weight of sugar, 0.05 to 0.3 parts by weight of citric acid, 0.005 to 0.02 parts by weight of caramel, 0.1 to 1 part by weight of vitamin C, the lactobacillus fermentum PL9036 strain of the present invention and 10 parts by weight of the culture solution thereof were mixed, The syrup is sterilized at 95 to 98 ° C for 20 to 180 seconds and mixed with the cooling water at a ratio of 1: 4. Then, 0.5 to 0.82 parts by weight of carbon dioxide is injected into the syrup, .

<2-2> Production of functional beverage

Lactobacillus perfumont PL9036 strain of the present invention and 0.12 part by weight of citric acid were added to 0.1 part by weight of vitamin C, 5.8 parts by weight of fructose, 3.8 parts by weight of white sugar, 0.12 part by weight of citric acid, 0.03 part by weight of malic acid, 0.04 part by weight of sodium citrate, And 10 parts by weight of the culture solution were mixed and completely dissolved in the metered water. The dissolved beverage was adjusted to the purified water to be weighed so that the total amount was 100 parts by weight.

<2-3> Manufacture of health drinks

0.5 parts by weight of fructose, 2 parts by weight of oligosaccharide, 2 parts by weight of sugar, 0.5 part by weight of salt, and 75 parts by weight of water, 10 parts by weight of the lactobacillus fermentum PL9036 strain of the present invention and 10 parts by weight thereof, And then packaged in small containers such as glass bottles and plastic bottles to produce health drinks.

<2-4> Preparation of vegetable juice

A vegetable juice for health promotion was prepared by adding 10 g of the lactobacillus fermentum PL9036 strain of the present invention and the vacuum dried product of the culture solution to 1 L of tomato or carrot juice.

<2-5> Preparation of fruit juice

10 g of the lactobacillus fermentum PL9036 strain of the present invention and the vacuum dried product of the culture medium of the present invention were added to 1 liter of apple or grape juice to prepare fruit juice for health promotion.

< Formulation example 3> Preparation of fermented milk

Using the skim milk powder, the raw material oil having the non-skimmed milk solids content adjusted to 8 to 20 parts by weight was sterilized at 72 to 75 캜 for 15 seconds. After the sterilized raw material oil was cooled to a predetermined temperature, Lactobacillus perfumant PL9036 strain was inoculated at a concentration of 10 ⁶ CFU / ml until the pH reached 4 to 5. After completion of the culture, the culture was cooled. 0.1 to 20 parts by weight of dietary fiber, 0.5 to 30 parts by weight of glucose, 0.1 to 15 parts by weight of oligosaccharide, 0.001 to 10 parts by weight of calcium and 0.0001 to 5 parts by weight of vitamins were dissolved to prepare syrup . The syrup thus produced was sterilized, cooled, mixed with the culture solution at a predetermined ratio, and stirred to prepare a fermented milk by packing in a homogenized container.

< Formulation example 4> lactic acid bacteria Spindle Produce

Lactobacillus fermentum PL9036 strain was inoculated to MRS medium at a concentration of 10 ⁶ CFU / ml and subjected to pH-control fermentation at 37 ° C for 18 to 24 hours. After completion of the culture, the cells were recovered by centrifugation at 10,000 xg at 4 ° C. The recovered cells were mixed with the same amount of 5% skim milk, 2.5% whey and 5% sucrose, and then powdered through a freeze dryer. The dry powder of Lactobacillus fermentum PL9036 thus prepared was diluted with trehalose and prepared to have a viable cell count of 1 x 10 < ¹¹ > CFU / g or more.

< Formulation example 5> Preparation of lactic acid bacteria preparation

A lactic acid bacterial preparation such as a lactic acid bacterium food, a dressing agent and the like was prepared using the lactic acid bacterium produced in <Formulation Example 4>. 10 parts by weight of oligosaccharide, 20 parts by weight of anhydrous glucose, 5 parts by weight of crystalline fructose, 2 parts by weight of vitamin C, 2 parts by weight of fruit powder, 20 parts by weight of a dry powder of Lactobacillus fermentum PL9036 (viable cell count of 1 x 10 ¹⁰ CFU / 5 parts by weight of aloe, 15 parts by weight of dietary fiber, and 18 parts by weight of diaphoresis were mixed and sprayed on a stick or bottle by a predetermined amount. The lactic acid bacterial preparation thus prepared maintained a viable cell count of 5 × 10 ⁸ CFU / g or more.

National Institute of Agricultural Science KACC91850P 20130725

<110> plbio <120> Lactobacillus fermentum from PT9036 isolated from employed or used citizens in the Korean longevity villages <130> 13P-07-31 <160> 5 <170> Kopatentin 2.0 <210> 1 <211> 500 <212> DNA <213> Lactobacillus fermentum <400> 1 aattgattga tggtgcttgc acctgattga ttttggtcgc caacgagtgg cggacgggtg 60 agtaacacgt aggtaacctg cccagaagcg ggggacaaca tttggaaaca gatgctaata 120 ccgcataaca acgttgttcg catgaacaac gcttaaaaga tggcttctcg ctatcacttc 180 tggatggacc tgcggtgcat tagcttgttg gtggggtaac ggcctaccaa ggcgatgatg 240 catagccgag ttgagagact gatcggccac aatgggactg agacacggcc catactccta 300 cgggaggcag cagtagggaa tcttccacaa tgggcgcaag cctgatggag caacaccgcg 360 tgagtgaaga agggtttcgg ctcgtaaagc tctgttgtta aagaagaaca cgtatgagag 420 taactgttca tacgttgacg gtatttaacc agaaagtcac ggctaactac ctgccagcag 480 ccgcggtaat acgtaggtgg 500 <210> 2 <211> 900 <212> DNA <213> Lactobacillus fermentum <400> 2 tgcaagtcga acgcgttggc ccaattgatt gatggtgctt gcacctgatt gattttggtc 60 gccaacgagt ggcggacggg tgagtaacac gtaggtaacc tgcccagaag cgggggacaa 120 catttggaaa cagatgctaa taccgcataa cagcgttgtt cgcatgaaca acgcttaaaa 180 gatggcttct cgctatcact tctggatgga cctgcggtgc attagcttgt tggtggggta 240 acggcctacc aaggcgatga tgcatagccg agttgagaga ctgatcggcc acaatgggac 300 tgagacacgg cccatactcc tacgggaggc agcagtaggg aatcttccac aatgggcgca 360 agcctgatgg agcaacaccg cgtgagtgaa gaagggtttc ggctcgtaaa gctctgttgt 420 taaagaagaa cacgtatgag agtaactgtt catacgttga cggtatttaa ccagaaagtc 480 acggctaact acgtgccagc agccgcggta atacgtaggt ggcaagcgtt atccggattt 540 attgggcgta aagagagtgc aggcggtttt ctaagtctga tgtgaaagcc ttcggcttaa 600 ccggagaagt gcatcggaaa ctggataact tgagtgcaga agagggtagt ggaactccat 660 gtgtagcggt ggaatgcgta gatatatgga agaacaccag tggcgaaggc ggctacctgg 720 tctgcaactg acgctgagac tcgaaagcat gggtagcgaa caggattaga taccctggta 780 gtccatgccg taaacgatga gtgctaggtg ttggagggtt tccgcccttc agtgccggag 840 ctaacgcatt aagcactccg cctgggggag tacgaccgca aggttgaaac tcaaaggaat 900 900 <210> 3 <211> 1050 <212> DNA <213> Lactobacillus fermentum <400> 3 ggcccaattg attgatggtg cttgcacctg attgattttg gtcgccaacg agtggcggac 60 gggtgagtaa cacgtaggta acctgcccag aagcggggga caacatttgg aaacagatgc 120 taataccgca taacagcgtt gttcgcatga acaacgctta aaagatggct tctcgctatc 180 acttctggat ggacctgcgg tgcattagct tgttggtggg gtaacggcct accaaggcga 240 tgatgcatag ccgagttgag agactgatcg gccacaatgg gactgagaca cggcccatat 300 cctacgggag gcagcagtag ggaatcttcc acaatgggcg caagcctgat ggagcaacac 360 cgcgtgagtg aagaagggtt tcggctcgta aagctctgtt gttaaagaag aacacgtatg 420 agagtaactg ttcatacgtt gacggtattt aaccagaaag tcacggctaa ctacgtgcca 480 gcagccgcgg taatacgtag gtggcaagcg ttatccggat ttattgggcg taaagagagt 540 gcaggcggtt ttctaagtct gatgtgaaag ccttcggctt aaccggagaa gtgcatcgga 600 aactggataa cttgagtgca gaagagggta gtggaactcc atgtgtagcg gtggaatgcg 660 tagatatatg gaagaacacc agtggcgaag gcggctacct ggtctgcaac tgacgctgag 720 actcgaaagc atgggtagcg aacaggatta gataccctgg tagtccatgc cgtaaacgat 780 gagtgctagg tgttgggagg gtttccgccc ttcagtgccg gagctaacgc attaagcact 840 ccgcctgggg agtacgaccg caaggttgaa actcaaagga attgacgggg gcccgcacaa 900 gcggtggagc atgtggttta attcgaagct acgcgaagac cttaccaggt cttgacatct 960 gcgccaaccc tagagatagg gcgtttcctt cgggaacgca atgacaggtg gtgcatggtc 1020 gtcgtcagct cgtgtcgtga gaatgtgggt 1050 <210> 4 <211> 924 <212> DNA <213> Lactobacillus fermentum <400> 4 ggtgcttgca cctgattgat tttggtcgcc aacgagtggc ggacgggtga gtaacacgta 60 ggtaacctgc ccagaagcgg gggacaacat ttggaaacag atgctaatac cgcataacag 120 cgttgttcgc atgaacaacg cttaaaagat ggcttctcgc tatcacttct ggatggacct 180 gcggtgcatt agcttgttgg tggggtaacg gcctaccaag gcgatgatgc atagccgagt 240 tgagagactg atcggccaca atgggactga gacacggccc atactcctac gggaggcagc 300 agtagggaat cttccacaat gggcgcaagc ctgatggagc aacaccgcgt gagtgaagaa 360 gggtttcggc tcgtaaagct ctgttgttaa agaagaacac gtatgagagt aactgttcat 420 acgttgacgg tatttaacca gaaagtcacg gctaactacg tgccagcagc cgcggtaata 480 cgtaggtggc aagcgttatc cggatttatt gggcgtaaag agagtgcagg cggttttcta 540 agtctgatgt gaaagccttc ggcttaaccg gagaagtgca tcggaaactg gataacttga 600 gtgcagaaga gggtagtgga actccatgtg tagcggtgga atgcgtagat atatggaaga 660 acaccagtgg cgaaggcggc tacctggtct gcaactgacg ctgagactcg aaagcatggg 720 tagcgaacag gattagatac cctggtagtc catgccgtaa acgatgagtg ctaggtgttg 780 gagggtttcc gcccttcagt gccggagcta acgcattaag cactccgcct ggggagtacg 840 accgcaaggt tgaaactcaa aggaattgac gggggcccgc acaagcggtg gagcatgtgg 900 tttaattcga agctacgcga agaa 924 <210> 5 <211> 911 <212> DNA <213> Lactobacillus fermentum <400> 5 tgcaagtcga acgcgttggc ccaattgatt gatggtgctt gcacctgatt gattttggtc 60 gccaacgagt ggcggacggg tgagtaacac gtaggtaacc tgcccagaag cgggggacaa 120 catttggaaa cagatgctaa taccgcataa caacgttgtt cgcatgaaca acgcttaaaa 180 gatggcttct cgctatcact tctggatgga cctgcggtgc attagcttgt tggtggggta 240 atggcctacc aaggcgatga tgcatagccg agttgagaga ctgatcggcc acaatgggac 300 tgagacacgg cccatactcc tacgggaggc agcagtaggg aatcttccac aatgggcgca 360 agcctgatgg agcaacaccg cgtgagtgaa gaagggtttc ggctcgtaaa gctctgttgt 420 taaagaagaa cacgtatgag agtaactgtt catacgttga cggtatttaa ccagaaagtc 480 acggctaact acgtgccagc agccgcggta atacgtaggt ggcaagcgtt atccggattt 540 attgggcgta aagagagtgc aggcggtttt ctaagtctga tgtgaaagcc ttcggcttaa 600 ccggagaagt gcatcggaaa ctggataact tgagtgcaga agagggtagt ggaactccat 660 gtgtagcggt ggaatgcgta gatatatgga agaacaccag tggcgaaggc ggctacctgg 720 tctgcaactg acgctgagac tcgaaagcat gggtagcgaa caggattaga taccctggta 780 gtccatgccg taaacgatga gtgctaggtg ttggagggtt tccgcccttc agtgccggag 840 ctaacgcatt aagcactccg cctggggagt acgaccgcaa ggttgaaact caaaggaatt 900 gacgggggcc c 911

Claims

Antibiotics deposited with accession number KACC91850P Lactobacillus fermentum PL9036 strain with no resistance to foreign material and no risk of resistance metastasis.

2. The Lactobacillus perfumont PL9036 strain according to claim 1, wherein said strain has the 16S rRNA base sequence set forth in SEQ ID NO: 1.

Lactobacillus fermentum PL9036 strain deposited with accession number KACC91850P and a culture thereof as an active ingredient.

4. The antimicrobial prophylactic composition according to claim 3, wherein the composition has no antibiotic agent resistance and no risk of resistance metastasis.

Antibiotic deposited with accession number KACC91850P Lactobacillus fermentum PL9036 strain lacking the extrinsic resistance and no risk of resistance metastasis and a culture solution thereof as an active ingredient.

Antibiotics deposited with accession number KACC91850P Lactobacillus fermentum PL9036 strain lacking the extrinsic resistance and no risk of resistance metastasis and a culture solution thereof as an active ingredient.

The composition according to claim 6, wherein the composition inhibits the secretion of TNF-α, IL-6 or IL-1β and has an immunopotentiating activity.

Antibiotics deposited with accession number KACC91850P Antibacterial, antioxidant or immunosuppressive health food containing Lactobacillus perfumont PL9036 strain or culture thereof as an active ingredient, which has no resistance to extraneous substances and has no risk of resistance to metastasis.

9. The method of claim 8, wherein the food is selected from the group consisting of ice cream, milk, soy milk, yoghurt, milk products including cheese, and soy milk products, meat, sausage, bread, chocolate, candy, snacks, confectionery, pizza, , Various soups, beverages, tea, drinks, and alcoholic beverages. The antifungal, antioxidant or immunosuppressive health food according to claim 1,