Medium composition for selectively culturing lactobacillus plantarum and use thereof

Abstract

The present invention relates to a medium composition for selectively culturing Lactobacillus plantarum and a method for selectively isolating Lactobacillus plantarum using same. When using the medium composition for selectively culturing Lactobacillus plantarum and the method for selectively isolating Lactobacillus plantarum using same, it is possible to successfully isolate and quantify, from not only lactic acid bacteria belonging to the genus Streptococcus, the genus Lactococcus, and the genus Bifidobacterium, but also from other species of lactic acid bacteria belonging to the same genus Lactobacillus), only L. plantarum.

Description

Medium composition for selective cultivation of Lactobacillus plantarum and use thereof

[0001] This patent application claims priority to Republic of Korea Patent Application No. 10-2023-0134689, filed with the Korean Intellectual Property Office on October 10, 2023, the disclosure of which is incorporated herein by reference.

[0002] The present invention relates to a medium composition for selectively culturing Lactobacillus plantarum and a method for selectively isolating Lactobacillus plantarum using the same.

[0003]

[0004] Probiotics are beneficial microorganisms that are non-pathogenic to the human body and, when administered in appropriate amounts, provide health benefits. Currently, probiotics are widely used in various forms, including fermented foods, health supplements, pharmaceuticals, and cosmetics. Recently, the connection between probiotics and the human microbiome has become known, and the microbiome-related effects of probiotics, such as improving the intestinal environment, alleviating inflammation, and treating autoimmune diseases, are receiving renewed attention. Consequently, the probiotics product market is growing rapidly, with the domestic probiotics market expected to reach approximately KRW 900 billion in 2022.

[0005] Since probiotics are living microorganisms, quantifying the viable probiotic strains within a product is essential to ensuring its quality. Therefore, identifying and quantifying the viability of specific probiotic strains is considered a critical challenge in the field of probiotics technology.

[0006] Most probiotics known to date are lactic acid bacteria. Lactic acid bacteria are microorganisms that use sugar as an energy source to produce lactic acid. They are widely distributed throughout nature and have a close, direct and indirect impact on human life. Representative lactic acid bacteria used in probiotic products include Lactobacillus and Bifidobacterium. The isolation, analysis, and quantification of Lactobacillus and Bifidobacterium species are typically performed using the MRS (De Man, Rogosa, and Sharpe) medium. However, MRS medium can only measure the total number of lactic acid bacteria, and the nutritional characteristics of lactic acid bacteria within the same genus are closely related, making selective culture and isolation difficult. Accordingly, various studies have been attempted to control temperature conditions and atmospheric conditions (anaerobic or aerobic) during cultivation or to change the composition of the culture medium for selective cultivation and isolation of specific lactic acid bacteria species. However, most studies have focused only on methods to isolate specific species of lactic acid bacteria among 3 to 5 species of lactic acid bacteria (Food Sci Technol, 2019), and thus their utility is minimal.

[0007] Therefore, research is needed on methods to selectively isolate and quantify specific lactic acid bacteria among a larger number of lactic acid bacteria species.

[0008]

[0009] The present inventors have conducted extensive research efforts to develop a method for selectively cultivating and isolating Lactobacillus plantarum from a multitude of lactic acid bacteria species. As a result, the present invention was completed by establishing a medium composition and culture conditions that enable the selective cultivation, isolation, and quantification of only Lactobacillus plantarum from 15 other lactic acid bacteria species.

[0010] Accordingly, an object of the present invention is to provide a medium composition for selective cultivation of Lactobacillus plantarum containing D-arabitol as a carbon source.

[0011] Another object of the present invention is to provide a method for selectively isolating Lactobacillus plantarum, comprising a step of culturing a lactic acid bacteria sample in a medium composition for selectively culturing Lactobacillus plantarum.

[0012]

[0013] The present invention provides the inventions 1 to 33 below.

[0014] 1. A medium composition for selective cultivation of Lactobacillus plantarum containing D-arabitol as a carbon source.

[0015] 2. A medium composition for selective culture of Lactobacillus plantarum, wherein the composition does not contain glucose.

[0016] 3. A medium composition for selective culture of Lactobacillus plantarum, wherein the concentration of D-arabitol in the medium composition in 1 or 2 is 0.5 g / L to 50 g / L.

[0017] 4. A medium composition for selective culture of Lactobacillus plantarum, further comprising a glycopeptide antibiotic, an aminoglycoside antibiotic, or a combination thereof, in any one of 1 to 3.

[0018] 5. A medium composition for selective culture of Lactobacillus plantarum, wherein the glycopeptide antibiotic in 4 is vancomycin or teicoplanin.

[0019] 6. A medium composition for selective culture of Lactobacillus plantarum, wherein in 4 or 5, the aminoglycoside antibiotic is at least one antibiotic selected from the group consisting of streptomycin, gentamycin, tobramycin, and amikacin.

[0020] 7. A medium composition for selective culture of Lactobacillus plantarum, wherein the concentration of the glycopeptide antibiotic in the medium composition is 10 mg / L to 2000 mg / L in any one of 4 to 6.

[0021] 8. A medium composition for selective culture of Lactobacillus plantarum, wherein the concentration of the aminoglycoside antibiotic in the medium composition is 50 mg / L to 1200 mg / L in any one of 4 to 7.

[0022] 9. A medium composition for selective culture of Lactobacillus plantarum, further comprising soy peptone, yeast extract, ammonium citrate, dipotassium phosphate, and magnesium sulfate, in any one of 1 to 8.

[0023] 10. A medium composition for selective cultivation of Lactobacillus plantarum, wherein soy peptone is included in the medium composition at a concentration of 10 g / L to 30 g / L.

[0024] 11. A medium composition for selective cultivation of Lactobacillus plantarum, wherein in 9 or 10, yeast extract is included in the medium composition at a concentration of 10 g / L to 30 g / L.

[0025] 12. A medium composition for selective cultivation of Lactobacillus plantarum, wherein ammonium citrate is included in the medium composition at a concentration of 0.5 g / L to 5 g / L in any one of 9 to 11.

[0026] 13. A medium composition for selective cultivation of Lactobacillus plantarum, wherein in any one of 9 to 12, dipotassium phosphate is included in the medium composition at a concentration of 0.3 g / L to 3 g / L.

[0027] 14. A medium composition for selective cultivation of Lactobacillus plantarum, wherein in any one of 9 to 13, magnesium sulfate is included in the medium composition at a concentration of 0.3 g / L to 3 g / L.

[0028] 15. A medium composition for selective culture of Lactobacillus plantarum, further comprising manganese sulfate, Tween-80 (polysorbate 80), L-cysteine ​​HCl or a combination thereof, in any one of 9 to 14.

[0029] 16. A medium composition for selective cultivation of Lactobacillus plantarum, wherein manganese sulfate is included in the medium composition at a concentration of 0.01 g / L to 0.1 g / L.

[0030] 17. A medium composition for selective cultivation of Lactobacillus plantarum, wherein in 15 or 16, Tween-80 (polysorbate 80) is included in the medium composition at a concentration of 0.01 g / L to 0.1 g / L.

[0031] 18. A medium composition for selective cultivation of Lactobacillus plantarum, wherein in any one of 15 to 17, L-cysteine ​​hydrochloride (L-cysteine ​​HCl) is included in the medium composition at a concentration of 0.01 g / L to 0.1 g / L.

[0032] 19. A medium composition for selective culture of Lactobacillus plantarum, wherein the pH of the composition is 5.0 to 7.0 in any one of 1 to 18.

[0033] 20. A medium composition for selective culture of Lactobacillus plantarum, wherein the selective culture is performed under temperature conditions of 20°C to 45°C in any one of 1 to 19.

[0034] 21. A medium composition for selective culture of Lactobacillus plantarum, wherein the selective culture is performed at a temperature of 20°C to 45°C in any one of 1 to 20.

[0035] 22. A medium composition for selective culture of Lactobacillus plantarum, wherein the selective culture is performed under aerobic conditions in any one of 1 to 21.

[0036] 23. A method for producing a medium composition for selective cultivation of Lactobacillus plantarum, comprising a step of adding and mixing D-arabitol as a carbon source.

[0037] 24. A method for producing a medium composition for selective culture of Lactobacillus plantarum, comprising the step of adding and mixing soy peptone, yeast extract, ammonium citrate, dipotassium phosphate, and magnesium sulfate in 23.

[0038] 25. A method for producing a medium composition for selective culture of Lactobacillus plantarum, comprising the step of additionally adding and mixing a glycopeptide antibiotic, an aminoglycoside antibiotic, or a combination thereof in 23 or 24.

[0039] 26. A method for producing a medium composition for selective culture of Lactobacillus plantarum, comprising the step of adding and mixing manganese sulfate, Tween-80 (polysorbate 80), L-cysteine ​​HCl, or a combination thereof, in any one of 23 to 25.

[0040] 27. A method for selective isolation or cultivation of Lactobacillus plantarum, comprising the step of (a) culturing a lactic acid bacteria sample in any one of the medium compositions 1 to 22.

[0041] 28. A method for selective isolation or cultivation of Lactobacillus plantarum, comprising the step of (b) determining whether a colony is formed in the medium composition in 27.

[0042] 29. A method for selective isolation or cultivation of Lactobacillus plantarum, wherein the cultivation in step (a) is performed under aerobic conditions in 27 or 28.

[0043] 30. A method for selectively isolating or culturing Lactobacillus plantarum, wherein the aerobic conditions in 29 are conditions in which the concentration of oxygen is 8% to 25%.

[0044] 31. A method for selective isolation or cultivation of Lactobacillus plantarum, wherein the cultivation in step (a) is performed under conditions of pH 5.0 to pH 7.0 in any one of 27 to 30.

[0045] 32. A method for selective isolation or cultivation of Lactobacillus plantarum, wherein the cultivation of step (a) described above is performed at a temperature of 20°C to 45°C in any one of 27 to 31.

[0046] 33. A method for selective isolation or cultivation of Lactobacillus plantarum, wherein the cultivation of step (a) described above is performed for 4 to 60 hours in any one of 27 to 32.

[0047]

[0048] According to one aspect of the present invention, the present invention provides a medium composition for selective cultivation of Lactobacillus plantarum, which comprises D-arabitol as a carbon source.

[0049] The present inventors have conducted extensive research efforts to develop a method for selectively cultivating and isolating Lactobacillus plantarum from a number of other lactic acid bacteria species. As a result, they have established a medium composition and culture conditions that enable the selective cultivation, isolation, and quantification of only Lactobacillus plantarum from 15 other lactic acid bacteria species.

[0050] As used herein, "selective culture" refers to culturing a strain or microorganism under conditions that allow only that strain or microorganism to survive or grow, thereby allowing only the desired strain or microorganism to survive or grow. The term "selective culture" may be used with the same meaning as "selection culture."

[0051] Lactobacillus plantarum (L. plantarum) is a lactic acid bacterium of the genus Lactobacillus. It is mainly isolated from milk, cheese, butter, kefir, fermented sausages, fermented potatoes, grains, acidic bread dough, pickled vegetables such as pickles and kimchi, and tomatoes, and is one of the most widely distributed lactic acid bacteria in nature. In particular, Lactobacillus plantarum is known as a bacterium that acts in the later stages of kimchi fermentation.

[0052] In this specification, the term 'Lactobacillus plantarum (L. plantarum)' refers to all strains belonging to the species Lactobacillus plantarum (L. plantarum) of the genus Lactobacillus, and is not limited to referring to a specific strain.

[0053] In one embodiment of the present invention, the Lactobacillus plantarum described above may be at least one Lactobacillus plantarum selected from the group consisting of L. plantarumKCTC 3108, L. plantarumQ180, L. plantarumCKDB008, L. plantarumCKDB001, and L. plantarumCDKB002, but is not limited thereto.

[0054] The above-mentioned L. plantarum KCTC 3108 is a representative standard strain (type strain) of L. plantarum, and is a standard strain for scientific research and classification of L. plantarum. L. plantarum KCTC 3108 was isolated from kimchi and is widely used commercially.

[0055] In this specification, the term 'type strain' means a representative example of a specific microbial species or strain, generally a strain that serves as a standard for the species or strain, and a microorganism that represents the characteristics and properties of the species or strain.

[0056] As demonstrated in the examples described below, when using the composition and culture conditions of the medium of the present invention, only L. plantarum can be selectively isolated and quantified from lactic acid bacteria belonging to the genera Streptococcus, Lactococcus and Bifidobacterium, as well as from other species of lactic acid bacteria belonging to the same genus Lactobacillus.

[0057] As demonstrated in the examples described below, the inventors of the present invention have found that L. plantarum can utilize D-arabitol as a carbon source, and confirmed that when glucose is removed from the culture medium and D-arabitol is added, only L. plantarum can be selectively isolated.

[0058] In one embodiment of the present invention, the medium composition for selective cultivation of Lactobacillus plantarum of the present invention described above may not contain glucose. However, the present invention is not limited thereto, and the medium composition for selective cultivation of Lactobacillus plantarum of the present invention may contain only D-arabitol as the main carbon source.

[0059] In one embodiment of the present invention, the concentration of the above-described D-arabitol in the medium composition may be 0.5 g / L to 50 g / L. More specifically, the concentration of the above-described D-arabitol in the medium composition is 0.5 g / L to 50 g / L, 0.5 g / L to 40 g / L, 0.5 g / L to 30 g / L, 0.5 g / L to 25 g / L, 0.5 g / L to 20 g / L, 0.5 g / L to 10 g / L, 1 g / L to 50 g / L, 1 g / L to 40 g / L, 1 g / L to 30 g / L, 1 g / L to 25 g / L, 1 g / L to 20 g / L, 1 g / L to 10 g / L, 1.5 g / L to 50 g / L, 1.5 g / L to 40 g / L, 1.5 g / L to 30 g / L, 1.5 g / L to 25 g / L, 1.5 g / L to 20 g / L, 1.5 g / L to 10 g / L, 2 g / L to 50 g / L, 2 g / L to 40 g / L, 2 g / L to 30 g / L, 2 g / L to 25 g / L, 2 g / L to 20 g / L, or 2 g / L to 10 g / L, but is not limited thereto.

[0060] As confirmed in the examples described below, Lactobacillus plantarum uses D-arabitol as a carbon source, and the number of lactic acid bacteria of Lactobacillus plantarum increases in a concentration-dependent manner with D-arabitol.

[0061] In one embodiment of the present invention, the medium composition for selective culture of Lactobacillus plantarum of the present invention may additionally include a glycopeptide antibiotic, an aminoglycoside antibiotic, or a combination thereof, and more specifically, may additionally include a combination of the above-described glycopeptide antibiotic and aminoglycoside antibiotic, but is not limited thereto.

[0062] As demonstrated in the examples described below, the inventors of the present invention confirmed that when a combination treatment with a glycopeptide antibiotic and an aminoglycoside antibiotic is performed, Lactobacillus plantarum can be isolated from a greater number of lactic acid bacteria than when each antibiotic is treated alone, and confirmed that an unpredictable effect occurs when antibiotics of each series are treated in combination.

[0063]

[0064] In one embodiment of the present invention, the glycopeptide antibiotic described above may be vancomycin or teicoplanin, and more specifically, vancomycin, but is not limited thereto.

[0065] In one embodiment of the present invention, the aminoglycoside antibiotic described above may be at least one antibiotic selected from the group consisting of streptomycin, gentamycin, tobramycin, and amikacin, and more specifically, may be streptomycin, but is not limited thereto.

[0066] As demonstrated in the examples described below, the inventors of the present invention additionally applied antibiotics that inhibit other lactic acid bacteria without affecting L. plantarum to the medium composition in order to establish a medium composition for selective cultivation of Lactobacillus plantarum. Specifically, the inventors confirmed that L. plantarum has resistance to vancomycin, a glycopeptide antibiotic, and streptomycin, an aminoglycoside antibiotic, and treated the medium composition with vancomycin, streptomycin, or a combination thereof.

[0067] When lactic acid bacteria were cultured using a medium composition treated with vancomycin alone, Lactobacillus plantarum was isolated from other lactic acid bacteria species, but strains belonging to L. fermentum, L. rhamnosus, L. paracasei, and L. mesenteroides were also isolated. When streptomycin was treated alone, Lactobacillus plantarum was isolated from other lactic acid bacteria species, but strains belonging to L. fermentum, L. rhamnosus, and L. paracasei were also isolated. On the other hand, when vancomycin and streptomycin were treated in combination, it was confirmed that L. plantarum could be selected from the remaining lactic acid bacteria strains with a survival rate of less than approximately 0.01%, excluding strains belonging to L. fermentum. From this, it was found that the combined treatment with vancomycin and streptomycin produced an effect that could not be predicted from the case of treating each antibiotic alone.

[0068] In one embodiment of the present invention, the concentration of the glycopeptide antibiotic in the medium composition may be 10 mg / L to 2000 mg / L, and more specifically, 10 mg / L to 2000 mg / L, 10 mg / L to 1750 mg / L, 10 mg / L to 1600 mg / L, 10 mg / L to 1500 mg / L, 10 mg / L to 1200 mg / L, 10 mg / L to 750 mg / L, 10 mg / L to 100 mg / L, 100 mg / L to 2000 mg / L, 100 mg / L to 1750 mg / L, 100 mg / L to 1600 mg / L, 100 mg / L to 1500 mg / L, 100 mg / L to 1200 mg / L, 100 mg / L to 750 mg / L, 500 mg / L to 2000 mg / L, 500 mg / L to 1750 mg / L, 500 mg / L to 1600 mg / L, 500 mg / L to 1500 mg / L, 500 mg / L to 1200 mg / L, 500 mg / L to 750 mg / L, 750 mg / L to 2000 mg / L, 750 mg / L to 1750 mg / L, 750 mg / L to 1600 mg / L, 750 mg / L to 1750 mg / L, 750 mg / L to 1200 mg / L, 1200 mg / L to 2000 mg / L, 1200 mg / L to 1750 mg / L, 1200 mg / L It may be, but is not limited to, 1600 mg / L or 1200 mg / L to 1750 mg / L, most specifically 1600 mg / L.

[0069] In one embodiment of the present invention, the concentration of the aminoglycoside antibiotic in the medium composition may be 50 mg / L to 1200 mg / L, and more specifically, 50 mg / L to 1200 mg / L, 50 mg / L to 1000 mg / L, 50 mg / L to 700 mg / L, 50 mg / L to 500 mg / L, 50 mg / L to 450 mg / L, 50 mg / L to 400 mg / L, 50 mg / L to 350 mg / L, 50 mg / L to 300 mg / L, 50 mg / L to 250 mg / L, 50 mg / L to 200 mg / L, 250 mg / L to 1200 mg / L, 250 mg / L to 1000 mg / L, 250 mg / L to 700 mg / L, 250 mg / L to 500 mg / L, 250 mg / L to 450 mg / L, 250 mg / L to 400 mg / L, 250 mg / L to 350 mg / L, 250 mg / L to 300 mg / L, 300 mg / L to 1200 mg / L, 300 mg / L to 1000 mg / L, 300 mg / L to 700 mg / L, 300 mg / L to 500 mg / L, 300 mg / L to 450 mg / L, 300 mg / L to 400 mg / L, 300 mg / L to 350 mg / L, 350 mg / L to 1200 mg / L, 350 mg / L to 1000 mg / L, 350 mg / L to 700 mg / L, 350 mg / L to 500 mg / L, 350 mg / L to 450 mg / L, 350 mg / L to 400 mg / L, 400 mg / L to 1200 mg / L, 400 mg / L to 1000 mg / L, 400 mg / L to 700 mg / L, 400 mg / L to 500 mg / L or 400 mg / L to 450 mg / L, most specifically 400 mg / L, but is not limited thereto.

[0070] In one embodiment of the present invention, the medium composition for selective culture of Lactobacillus plantarum of the present invention described above may additionally include soy peptone, yeast extract, ammonium citrate, dipotassium phosphate, and magnesium sulfate.

[0071] In one embodiment of the present invention, the medium composition for selective culture of Lactobacillus plantarum of the present invention described above contains soy peptone in the medium composition at a concentration of 10 g / L to 30 g / L, more specifically, at a concentration of 10 g / L to 25 g / L, 10 g / L to 22.5 g / L, 10 g / L to 20 g / L, 15 g / L to 30 g / L, 15 g / L to 25 g / L, 15 g / L to 22.5 g / L, 15 g / L to 20 g / L, 17.5 g / L to 30 g / L, 17.5 g / L to 25 g / L, 17.5 g / L to 22.5 g / L or 17.5 g / L to 20 g / L, and more specifically, at a concentration of 20 It may be included in a concentration of g / L, but is not limited thereto.

[0072] In one embodiment of the present invention, the medium composition for selective culture of Lactobacillus plantarum of the present invention described above contains yeast extract in a concentration of 10 g / L to 30 g / L in the medium composition, more specifically, in a concentration of 10 g / L to 25 g / L, 10 g / L to 22.5 g / L, 10 g / L to 20 g / L, 15 g / L to 30 g / L, 15 g / L to 25 g / L, 15 g / L to 22.5 g / L, 15 g / L to 20 g / L, 17.5 g / L to 30 g / L, 17.5 g / L to 25 g / L, 17.5 g / L to 22.5 g / L or 17.5 g / L to 20 g / L, more specifically, in a concentration of 20 g / L. May include, but is not limited to:

[0073] In one embodiment of the present invention, the medium composition for selective culture of Lactobacillus plantarum of the present invention described above may contain ammonium citrate in the medium composition at a concentration of 0.5 g / L to 5 g / L, more specifically at a concentration of 0.5 g / L to 5 g / L, 0.5 g / L to 3 g / L, 0.5 g / L to 2 g / L, 1 g / L to 5 g / L, 1 g / L to 3 g / L, or 1 g / L to 2 g / L, and even more specifically at a concentration of 2 g / L, but is not limited thereto.

[0074] In one embodiment of the present invention, the medium composition for selective culture of Lactobacillus plantarum of the present invention described above may include dipotassium phosphate in the medium composition at a concentration of 0.3 g / L to 3 g / L, more specifically at a concentration of 0.3 g / L to 3 g / L, 0.3 g / L to 1.5 g / L, 0.3 g / L to 1 g / L, 0.75 g / L to 3 g / L, 0.75 g / L to 1.5 g / L, or 0.75 g / L to 1 g / L, and even more specifically at a concentration of 1 g / L, but is not limited thereto.

[0075] In one embodiment of the present invention, the medium composition for selective culture of Lactobacillus plantarum of the present invention described above may include magnesium sulfate in the medium composition at a concentration of 0.3 g / L to 3 g / L, more specifically at a concentration of 0.3 g / L to 3 g / L, 0.3 g / L to 1.5 g / L, 0.3 g / L to 1 g / L, 0.75 g / L to 3 g / L, 0.75 g / L to 1.5 g / L, or 0.75 g / L to 1 g / L, and even more specifically at a concentration of 1 g / L, but is not limited thereto.

[0076] In one embodiment of the present invention, the medium composition for selective culture of Lactobacillus plantarum of the present invention described above may additionally include manganese sulfate, Tween-80 (polysorbate 80), L-cysteine ​​hydrochloride (L-cysteine ​​HCl) or a combination thereof in addition to soy peptone, yeast extract, ammonium citrate, dibasic potassium phosphate and magnesium sulfate, but is not limited thereto.

[0077] In one embodiment of the present invention, the medium composition for selective culture of Lactobacillus plantarum of the present invention described above may include manganese sulfate in the medium composition at a concentration of 0.01 g / L to 0.1 g / L, more specifically at a concentration of 0.01 g / L to 0.1 g / L, or 0.01 g / L to 0.075 g / L, or 0.01 g / L to 0.05 g / L, and even more specifically at a concentration of 0.05 g / L, but is not limited thereto.

[0078] In one embodiment of the present invention, the medium composition for selective culture of Lactobacillus plantarum of the present invention described above may include Tween-80 (polysorbate 80) in the medium composition at a concentration of 0.01 g / L to 0.1 g / L, more specifically at a concentration of 0.01 g / L to 0.1 g / L, 0.01 g / L to 0.075 g / L, or 0.01 g / L to 0.05 g / L, and even more specifically at a concentration of 0.05 g / L, but is not limited thereto.

[0079] In one embodiment of the present invention, the medium composition for selective culture of Lactobacillus plantarum of the present invention described above may include L-cysteine ​​hydrochloride (L-cysteine ​​HCl) in the medium composition at a concentration of 0.01 g / L to 0.1 g / L, more specifically at a concentration of 0.01 g / L to 0.1 g / L, 0.01 g / L to 0.075 g / L, or 0.01 g / L to 0.05 g / L, and even more specifically at a concentration of 0.05 g / L, but is not limited thereto.

[0080] According to another aspect of the present invention, the present invention provides a method for producing a medium composition for selective culture of Lactobacillus plantarum, comprising the step of mixing soy peptone, yeast extract, glucose, ammonium citrate, dipotassium phosphate, and magnesium sulfate.

[0081] In one embodiment of the present invention, in the mixing step, in addition to soy peptone, yeast extract, ammonium citrate, dibasic potassium phosphate, and magnesium sulfate, manganese sulfate, Tween-80 (polysorbate 80), L-cysteine ​​HCl, or a combination thereof may be additionally added and mixed.

[0082] In one embodiment of the present invention, the method for producing a medium composition for selective culture of Lactobacillus plantarum of the present invention described above may additionally include a step of removing glucose from the mixture produced through the above-described steps.

[0083] In one embodiment of the present invention, the method for producing a medium composition for selectively culturing Lactobacillus plantarum of the present invention described above may additionally include a step of adding D-arabitol to the medium composition after the step of removing glucose described above.

[0084] In one embodiment of the present invention, the above-mentioned D-arabitol is present in the medium composition at a concentration of 0.5 g / L to 5 g / L, more specifically, 0.5 g / L to 50 g / L, 0.5 g / L to 40 g / L, 0.5 g / L to 30 g / L, 0.5 g / L to 25 g / L, 0.5 g / L to 20 g / L, 0.5 g / L to 10 g / L, 1 g / L to 50 g / L, 1 g / L to 40 g / L, 1 g / L to 30 g / L, 1 g / L to 25 g / L, 1 g / L to 20 g / L, 1 g / L to 10 g / L, 1.5 g / L to 50 g / L, 1.5 g / L to 40 g / L, 1.5 g / L to 30 g / L, 1.5 g / L to 25 g / L, 1.5 g / L to 20 g / L, 1.5 g / L to 10 g / L, 2 g / L to 50 g / L, 2 g / L to 40 g / L, 2 g / L to 30 g / L, 2 g / L to 25 g / L, 2 g / L to 20 g / L, or 2 g / L to 10 g / L, but is not limited thereto.

[0085]

[0086] In the case of the method for producing a medium composition for selective culture of Lactobacillus plantarum of the present invention, as a method for producing a medium composition for selective culture of Lactobacillus plantarum, which is an aspect of the present invention described above, overlapping contents between the two inventions are commonly applied, and their description is omitted to avoid excessive complexity of the present specification.

[0087]

[0088] According to another aspect of the present invention, the present invention provides a method for selectively isolating Lactobacillus plantarum or a method for selectively culturing Lactobacillus plantarum, comprising the step of (a) culturing a lactic acid bacteria sample in the above-described medium composition for selectively culturing Lactobacillus plantarum. In one embodiment of the present invention, the culturing in step (a) may be performed under aerobic conditions, but is not limited thereto.

[0089] In this specification, the term 'aerobic conditions' means that the growth of microorganisms occurs in an environment where oxygen is sufficiently supplied, and means conditions where the oxygen concentration is approximately 21%.

[0090] In one embodiment of the present invention, the above-described aerobic conditions may be conditions in which the concentration of oxygen is 8% to 25%, 8% to 23%, 8% to 22%, 8% to 21%, 12% to 25%, 12% to 23%, 12% to 22%, 12% to 21%, 17% to 25%, 17% to 23%, 17% to 22%, 17% to 21%, 20% to 25%, 20% to 23%, 20% to 22%, or 20% to 21%, but are not limited thereto.

[0091] As used herein, the term "anaerobic conditions" refers to microorganisms that grow in an environment where oxygen is absent or has a low concentration. Obligate anaerobes refer to bacteria that cannot grow in the presence of oxygen, while opportunistic anaerobes refer to microorganisms that can grow even in the presence of oxygen, unlike obligate anaerobes.

[0092] In one embodiment of the present invention, the anaerobic condition described above may be a condition in which the concentration of oxygen is 0% or more but less than 8%, 0% or more but less than 5%, 0% or more but less than 3%, 0% or more but less than 2%, 0% or more but less than 1%, 0% or more but less than 0.5%, 0.05% or more but less than 8%, 0.05% or more but less than 5%, 0.05% or more but less than 3%, 0.05% or more but less than 2%, 0.05% or more but less than 1%, 0.05% or more but less than 0.5%, 0.1% or more but less than 8%, 0.1% or more but less than 5%, 0.1% or more but less than 3%, 0.1% or more but less than 2%, 0.1% or more but less than 1%, or 0.1% or more but less than 0.5%, but is not limited thereto.

[0093] In one embodiment of the present invention, the culturing of the above-described step (a) can be performed under conditions of pH 5.0 to pH 7.0, and more specifically, pH 5.0 to pH 7.0, pH 5.0 to pH 6.5, pH 5.0 to pH 6.0, pH 5.0 to pH 5.8, pH 5.0 to pH 5.6, pH 5.0 to pH 5.4, pH 5.0 to pH 5.2, pH 5.2 to pH 7.0, pH 5.2 to pH 6.5, pH 5.2 to pH 6.0, pH 5.2 to pH 5.8, pH 5.2 to pH 5.6, pH 5.2 to pH 5.4, pH 5.4 to pH 7.0, pH 5.4 to pH 6.5, pH 5.4 to pH 6.0, pH 5.4 to pH 5.8, pH It can be performed under conditions of pH 5.4 to pH 5.6, pH 5.6 to pH 7.0, pH 5.6 to pH 6.5, pH 5.6 to pH 6.0, pH 5.6 to pH 5.8, and most specifically, it can be performed under conditions of pH 5.6, but is not limited thereto.

[0094] In one embodiment of the present invention, the culturing of the above-described step (a) can be performed under a temperature condition of 20°C to 45°C, and more specifically, 20°C to 45°C, 20°C to 40°C, 20°C to 35°C, 20°C to 32.5°C, 20°C to 30°C, 20°C to 27.5°C, 20°C to 25°C, 25°C to 45°C, 25°C to 40°C, 25°C to 35°C, 25°C to 32.5°C, 25°C to 30°C, 25°C to 27.5°C, 27.5°C to 45°C, 27.5°C to 40°C, 27.5°C to 35°C, 27.5°C to 32.5°C, 27.5°C to 30°C, 30°C to It can be performed under temperature conditions of 45°C, 30°C to 40°C, 30°C to 35°C, 30°C to 32.5°C, 32.5°C to 45°C, 32.5°C to 40°C, 32.5°C to 35°C, 35°C to 45°C or 35°C to 40°C, and most specifically, it can be performed under temperature conditions of 30°C, but is not limited thereto.

[0095] In one embodiment of the present invention, the culturing of the above-described step (a) may be performed for 4 hours to 60 hours, more specifically, for 4 hours to 60 hours, 4 hours to 55 hours, 4 hours to 50 hours, 4 hours to 48 hours, 16 hours to 60 hours, 16 hours to 55 hours, 16 hours to 50 hours, 16 hours to 48 hours, 24 hours to 60 hours, 24 hours to 55 hours, 24 hours to 50 hours, 24 hours to 48 hours, 30 hours to 60 hours, 30 hours to 55 hours, 30 hours to 50 hours, 30 hours to 48 hours, 45 hours to 60 hours, 45 hours to 55 hours, 45 hours to 50 hours or 45 hours to 48 hours, and most specifically, for 48 hours. It can be performed, but is not limited to:

[0096] The above lactic acid bacteria sample is a mixed sample of multiple lactic acid bacteria including Lactobacillus plantarum.

[0097] The above lactic acid bacteria sample may include one or more lactic acid bacteria selected from the group consisting of Lactobacillus, Streptococcus, Leuconostoc, and Bifidobacterium.

[0098] The above Lactobacillus strain may be one or more selected from the group consisting of Lactobacillus acidophilus, Lactobacillus rhamnosus, Lactobacillus casei, Lactobacillus plantarum, Lactobacillus reuteri, Lactobacillus fermentum, Lactobacillus brevis, Lactobacillus helveticus, Lactobacillus johnsonii, Lactobacillus salivarius, Lactobacillus paracasei, and Lactobacillus bulgaricus, but is not limited thereto.

[0099] The above Streptococcus strain may be one or more selected from the group consisting of Streptococcus pyogenes, Streptococcus pneumoniae, Streptococcus agalactiae, Streptococcus mutans, Streptococcus sanguinis, Streptococcus thermophilus, Streptococcus salivarius, Streptococcus bovis, Streptococcus oralis, and Streptococcus anginosus, but is not limited thereto.

[0100] The above bifidobacterium bacteria may be one or more selected from the group consisting of Bifidobacterium longum, Bifidobacterium bifidum, Bifidobacterium breve, Bifidobacterium infantis, Bifidobacterium adolescentis, Bifidobacterium animalis, Bifidobacterium lactis, Bifidobacterium pseudocatenulatum, Bifidobacterium dentium, and Bifidobacterium thermophilum, but is not limited thereto.

[0101] The above Leuconostoc fungus may be one or more selected from the group consisting of Leuconostoc mesenteroides, Leuconostoc lactis, Leuconostoc cremoris, Leuconostoc citreum, Leuconostoc gelidum, Leuconostoc fallax, Leuconostoc pseudomesenteroides, and Leuconostoc carnosum, but is not limited thereto.

[0102] In one embodiment of the present invention, the method for selectively isolating or culturing Lactobacillus plantarum comprises the step of (b) confirming whether a colony is formed in the medium composition.

[0103] In one embodiment of the present invention, the formation of colonies in the medium composition in the step (b) described above means the presence of Lactobacillus plantarum, indicating that Lactobacillus plantarum has been successfully selectively cultured.

[0104] In one embodiment of the present invention, in addition to confirming whether a colony is formed after culturing a lactic acid bacteria sample in the above-described medium composition, the presence of Lactobacillus plantarum or successful selective cultivation of Lactobacillus plantarum can be confirmed through methods such as biological tests using microbiological characteristics (e.g., analysis of expression of specific genes, enzyme activity, and physiological activity), counting of the number of lactic acid bacteria, and molecular biological analysis (e.g., detection of specific DNA sequences or RNA sequences).

[0105] In one embodiment of the present invention, the method for selectively isolating Lactobacillus plantarum additionally includes a step of (c) obtaining Lactobacillus plantarum.

[0106] Since the selective isolation method of Lactobacillus plantarum of the present invention is performed using the medium composition for selective culture of Lactobacillus plantarum, which is an embodiment of the present invention described above, the overlapping contents between the two inventions are commonly applied, and the description thereof is omitted to avoid excessive complexity of the present specification.

[0107]

[0108] The present invention provides a medium composition for selective cultivation of Lactobacillus plantarum containing D-arabitol as a carbon source.

[0109] In addition, the present invention provides a method for selectively isolating Lactobacillus plantarum, comprising the steps of culturing a lactic acid bacteria sample in a medium composition for selectively culturing Lactobacillus plantarum; and the step of confirming whether a colony is formed in the medium composition described above.

[0110] When using the medium composition for selective culture of Lactobacillus plantarum of the present invention and the selective isolation method of Lactobacillus plantarum using the same, only Lactobacillus plantarum can be successfully isolated and quantified from lactic acid bacteria belonging to the genus Streptococcus, Lactococcus, and Bifidobacterium as well as from other species of lactic acid bacteria belonging to the same genus Lactobacillus.

[0111]

[0112] Figures 1a and 1b show the results of confirming the sugar utilization of 20 types of lactic acid bacteria strains used in the experiment using the API 50 CH kit.

[0113]

[0114] Hereinafter, the present invention will be described in more detail through examples. These examples are intended solely to illustrate the present invention more specifically, and it will be apparent to those skilled in the art that the scope of the present invention is not limited by these examples, in accordance with the gist of the present invention.

[0115]

[0116] Example

[0117]

[0118] (Throughout this specification, "%" used to indicate the concentration of a particular substance is (weight / weight) % for solid / solid, (weight / volume) % for solid / liquid, and (volume / volume) % for liquid / liquid, unless otherwise stated.)

[0119]

[0120] Example 1: Lactic acid bacteria strains and strain culture medium composition

[0121] Example 1-1. Lactic acid bacteria strain

[0122] In this experiment, 20 strains of lactic acid bacteria isolated from various sources, including adult and infant feces and traditional fermented foods, were provided by CKDBiO (Korea) and the Korean Collection for Type Cultures (KCTC, Korea). The strains used are shown in Table 1. Except for Leuconostoc mesenteroides in Table 1, the remaining 15 strains belong to the genera Lactobacillus, Streptococcus, Lactococcus, and Bifidobacterium, and are notified strains listed in the Health Functional Food Raw Material Codex of the Ministry of Food and Drug Safety.

[0123] 연번균주명균주 약어KCTC 기탁번호 (기탁일)1Lactobacillus plantarumQ180LP1KACC91865BP (2013.10.18)2Lactobacillus plantarumCKDB008LP2KCTC13673BP (2018.10.23)3Lactobacillus plantarumCKDB001LP3-4Lactobacillus plantarumCKDB002LP4-5Lactobacillus plantarumKCTC 3108LP5-6Lactobacillus fermentumSRK414LFKCTC13686BP (2018.10.25)7Lactobacillus rhamnosusCKDB009LRKCTC13249BP (2017.04.12)8Lactobacillus paracaseiCKDB005LPC-9Lactobacillus caseiCKDB007LC-10Lactobacillus acidophilusCKDB007LAKCTC13117BP (2016.09.23)11Lactobacillus helveticusCKDB001LHKCTC13670BP (2018.10.23)12Lactobacillus reuteriCKDB019LRTKCTC13674BP (2018.10.23)13Lactobacillus bulgaricusCKDB001LBKCTC13669BP (2018.10.23)14Streptococcus thermophilusCKDB027STKCTC14471BP (2018.10.23)15Lactococcus lactisCKDB001LcL-16Leuconostoc mesenteroidesCKDB018LM-17Bifidobacterium lactisCDKB005BLKCTC13116BP (2016.09.23)18Bifidobacterium bifidumCKDB001BBKCTC13114BP (2016.09.23)19Bifidobacterium longumCKDB004BLGKCTC13671BP (2018.10.23)20Bifidobacterium breveCKDB002BRKCTC13893BP (2019.07.19).

[0124] Example 1-2. Composition of lactic acid bacteria strain culture medium

[0125] The medium composition used for strain culture was as follows: soy peptone 20 g / L, yeast extract 20 g / L, glucose 20 g / L, ammonium citrate 2 g / L, dipotassium phosphate 1 g / L, magnesium sulfate 1 g / L, manganese sulfate 0.05 g / L, Tween-80 (polysorbate 80) 0.05 g / L, L-cysteine ​​HCl 0.05 g / L. The medium with the above composition was named 'CKDB MRS medium'. The medium was sterilized at 121℃ for 20 minutes, and the lactic acid bacteria strain was prepared into powder through lyophilization and used in the experiment.

[0126]

[0127] Using the above strains and strain culture media, various condition studies were conducted as in the examples described below to establish optimal culture conditions for selectively isolating L. plantarumQ180, L. plantarumCDKB008, L. plantarumCDKB001, L. plantarumCDKB002, and L. plantarumKCTC 3108.

[0128]

[0129] Example 2: Evaluation of atmospheric, pH, and temperature conditions for culturing lactic acid bacteria strains

[0130] First, in order to determine the basic strain characteristics of L. plantarum, the pH conditions, temperature conditions, and atmospheric conditions (anaerobic or aerobic) for culture were confirmed as follows. The CKDB MRS medium was cultured for 48 hours each under a pH of 4.0 to 7.0, a culture temperature of 25℃ to 40℃, and aerobic / anaerobic conditions, and then the number of lactic acid bacteria was confirmed. The aerobic condition was tested by setting the oxygen concentration to approximately 21%, and the anaerobic condition was tested by setting the oxygen concentration to less than 0.5%. As a control group for the general culture conditions of lactic acid bacteria, the number of lactic acid bacteria was confirmed under the anaerobic culture conditions of 37℃ for 48 hours as notified in the Lactic Acid Bacteria Test Method in the Health Functional Food Code.

[0131] Each sample (1 g) was diluted in peptone buffer (Buffered Peptone Water; Kisan Bio, Korea) using the decimal dilution method, and then cultured on CKDB MRS agar using the pouring method according to the experimental conditions such as temperature and pH, and the number of colonies on the cultured plate was measured to express the viable cell count of lactic acid bacteria (lactic acid bacteria count) as CFU / g. Anaerobic conditions were created using an anaerobic pack (AnaeroPack, Mitsubishi, Japan) in a culture-only container.

[0132]

[0133] Example 2-1. Evaluation of atmospheric conditions for culturing lactic acid bacteria strains

[0134] Generally, lactic acid bacteria culture is performed under anaerobic conditions, but L-type lactic acid bacteria such as lactic acid bacteria of the genus Lactobacillus, lactic acid bacteria of the genus Streptococcus, and lactic acid bacteria of the genus Lactococcus are facultative anaerobes and can be cultured under aerobic conditions. Accordingly, atmospheric conditions that can select L. plantarumQ180, L. plantarumCDKB008, L. plantarumCDKB001, L. plantarumCDKB002, and L. plantarumKCTC 3108 using CKDB MRS medium were confirmed, and are shown in Table 2 below.

[0135] Confirmation of the number of lactic acid bacteria according to the culture atmosphere conditions (37℃, pH 6.5, 48 hours of culture, CFU / g) Classification Control group Experimental group Atmospheric conditions Anaerobic Aerobic LP15.0x10 11 5.2x10 11 LP25.5x10 11 5.4x10 11 LP33.5x10 11 3.4x10 11 LP44.2x10 11 4.2x10 11 LP53.8x10 11 3.8x10 11 LF3.2x10 11 3.2x10 11 LR3.2x10 11 3.1x10 11 LPC4.4x10 11 4.2x10 11 LC1.1x10 12 1.0x10 12 LA2.5x10 11 2.2x10 11 LH2.2x10 11 2.2x10 11 LRT3.3x10 11 3.0x10 11 LB2.3x10 11 2.3x10 11 ST5.8x10 11 5.9x10 11 LcL4.0x10 11 4.2x1011 LM2.1x10 11 2.0x10 11 BL1.2x10 12 4.0x10 10 BB3.2x10 11 1.2x10 10 BLG2.5x10 11 5.0x10 9 BR4.2x10 11 1.2x10 10

[0136] As shown in Table 2 above, there was no difference in the number of lactic acid bacteria in the L-type lactic acid bacteria between anaerobic and aerobic cultures. However, Bifidobacterium is an anaerobic bacterium, and the number of lactic acid bacteria in aerobic culture was less than about 5% of that in anaerobic culture, indicating that culture was inhibited under aerobic conditions. Therefore, the culture atmosphere conditions for selecting L. plantarum were set to aerobic conditions.

[0137]

[0138] Example 2-2. Evaluation of pH conditions for culturing lactic acid bacteria strains

[0139] Since pH is an important factor among the lactic acid bacteria culture factors, it was necessary to check the number of lactic acid bacteria according to the medium pH. Since the pH of the basic culture medium is approximately 6.5, the pH of CKDB MRS Agar medium was adjusted to 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, and 7.0 using HCl, and then cultured under anaerobic conditions at 37℃ for 48 hours. As a result of checking the medium, since the medium did not harden when the pH was 5.1 or lower, the medium pH conditions were readjusted to 5.2, 5.6, 6.0, 6.5, and 7.0, and the number of lactic acid bacteria was checked, which is shown in Table 3 below.

[0140] Confirmation of the number of lactic acid bacteria according to culture pH conditions (37℃, anaerobic, 48-hour culture, CFU / g) Classification Control group Experimental group pH 6.5 5.2 5.6 6.0 7.0 LP 15.0 x 10 11 5.0x10 11 5.1x10 11 4.9x1011 4.9x10 11 LP25.5x10 11 5.4x10 11 5.5x10 11 5.6x10 11 5.5x10 11 LP33.5x10 11 3.4x10 11 3.5x10 11 3.6x10 11 3.5x10 11 LP44.2x10 11 4.4x10 11 4.1x10 11 4.1x10 11 4.2x10 11 LP53.8x10 11 3.8x10 11 4.0x10 11 3.9x10 11 3.8x10 11 LF3.2x10 11 3.0x10 11 2.9x10 11 3.1x10 11 3.2x10 11 LR3.2x10 11 3.1x10 11 3.0x10 11 2.8x10 11 3.0x10 11 LPC4.4x10 11 4.5x10 11 4.5x10 11 4.4x10 11 4.3x10 11 LC1.1x10 12 1.0x10 12 9.8x10 11 1.1x10 12 1.0x10 12 LA2.5x10 11 1.2x10 11 1.3x10 11 2.2x10 11 2.3x10 11 LH2.2x10 11 1.0x10 11 1.1x10 11 2.2x10 11 2.2x1011 LRT3.3x10 11 2.0x10 11 2.3x10 11 2.9x10 11 3.2x10 11 LB2.3x10 11 1.0x10 11 1.9x10 11 2.2x10 11 2.3x10 11 ST5.8x10 11 4.5x10 11 4.3x10 11 5.3x10 11 5.4x10 11 LcL4.0x10 11 4.0x10 11 4.0x10 11 4.3x10 11 4.1x10 11 LM2.1x10 11 2.0x10 11 2.0x10 11 1.9x10 11 1.8x10 11 BL1.2x10 12 1.1x10 11 1.1x10 11 8.5x10 11 1.0x10 12 BB3.2x10 11 1.2x10 10 1.9x10 10 2.1x10 11 3.0x10 11 BLG2.5x10 11 9.2x10 9 1.0x10 10 1.2x10 11 2.6x10 11 BR4.2x10 11 2.0x10 10 5.0x10 10 1.3x10 11 4.4x10 11

[0141] As shown in Table 3 above, the number of lactic acid bacteria in each strain was confirmed, and there was no change in the number of lactic acid bacteria from pH 5.2 to pH 7.0 for L. plantarumQ180, L. plantarumCDKB008, L. plantarumCDKB001, L. plantarumCDKB002, and L. plantarumKCTC 3108. In other words, it was found that L. plantarumQ180, L. plantarumCDKB008, L. plantarumCDKB001, L. plantarumCDKB002, and L. plantarumKCTC 3108 were not significantly affected by pH when growing. On the other hand, among the L-series lactic acid bacteria, some lactic acid bacteria such as L. acidophilus, L. helveticus, L. reuteri, L. bulgaricus, and S. thermophilus, and four species of lactic acid bacteria in the genus Bifidobacteirum were affected and their growth decreased at pH 5.6 or lower. Therefore, the pH was set to 5.6 as a culture condition for selecting L. plantarum.

[0142]

[0143] Example 2-3. Evaluation of temperature conditions for culturing lactic acid bacteria strains

[0144] Since temperature conditions are also an important factor in lactic acid bacteria culture, it was necessary to confirm the number of lactic acid bacteria according to temperature conditions. Experiments were conducted at culture temperatures of 25°C, 30°C, 37°C, and 40°C, and the number of lactic acid bacteria under each condition is shown in Table 4 below.

[0145] Confirmation of the number of lactic acid bacteria according to culture temperature conditions (pH 6.5, anaerobic, 48-hour culture, CFU / g) Classification Control group Experimental group Culture temperature 37℃25℃30℃40℃LP15.0x10 11 4.1x10 11 5.2x10 11 5.0x10 11 LP25.5x10 11 4.4x10 11 5.5x10 11 5.6x1011 LP33.5x10 11 2.7x10 11 3.5x10 11 3.6x10 11 LP44.2x10 11 3.4x10 11 4.2x10 11 4.1x10 11 LP53.8x10 11 3.0x10 11 3.8x10 11 3.9x10 11 LF3.2x10 11 1.3x10 11 3.1x10 11 3.1x10 11 LR3.2x10 11 1.9x10 11 3.1x10 11 3.2x10 11 LPC4.4x10 11 1.0x10 11 3.2x10 11 4.2x10 11 LC1.1x10 12 9.9x10 11 7.0x10 11 1.0x10 12 LA2.5x10 11 9.3x10 10 2.1x10 11 2.3x10 11 LH2.2x10 11 1.0x10 11 1.1x10 11 2.2x10 11 LRT3.3x10 11 1.0x10 11 2.5x10 11 2.7x10 11 LB2.3x10 11 8.2x10 10 1.0x10 11 2.3x10 11 ST5.8x10 11 1.0x10 11 3.1x10 11 5.8x10 11 LcL4.0x10 11 3.0x10 11 3.1x1011 4.1x10 11 LM2.1x10 11 9.0x10 10 1.0x10 11 1.1x10 11 BL1.2x10 12 5.1x10 11 7.8x10 11 9.1x10 11 BB3.2x10 11 2.1x10 10 3.7x10 10 2.0x10 11 BLG2.5x10 11 1.0x10 10 2.9x10 10 2.1x10 11 BR4.2x10 11 2.1x10 10 7.9x10 10 3.0x10 11

[0146] As a result of confirming the number of lactic acid bacteria in Table 4, it was confirmed that the number of bacteria tended to decrease when cultured at 25℃ for all lactic acid bacteria. On the other hand, when cultured at 30℃ to 40℃, the strains L. plantarumQ180, L. plantarumCDKB008, L. plantarumCDKB001, L. plantarumCDKB002, L. plantarumKCTC3108, L. fermentumSRK414, and L. rhamnosusCKDB009 showed almost no change in the number of bacteria. Therefore, the culture temperature condition for selecting L. plantarum was set to 30℃.

[0147]

[0148] Example 2-4: Comprehensive evaluation of atmospheric, pH, and temperature conditions for culturing lactic acid bacteria strains

[0149] Through the above-described Examples 2-1 to 2-3, the conditions for selectively isolating L. plantarumQ180, L. plantarumCDKB008, L. plantarumCDKB001, L. plantarumCDKB002, and L. plantarumKCTC 3108 among lactic acid bacteria were established as aerobic conditions, pH 5.6, and temperature 30℃, respectively. An experiment was conducted to confirm whether L. plantarumQ180, L. plantarumCDKB008, L. plantarumCDKB001, L. plantarumCDKB002, and L. plantarumKCTC 3108 could be selectively isolated even when the above conditions were applied in combination. As a control group, CKDB MRS medium was used for culture under the basic conditions of anaerobic, pH 6.5, and 37℃. The number of lactic acid bacteria was confirmed and shown in Table 5 below.

[0150] Confirmation of lactic acid bacteria count according to the combined application of atmosphere, pH, and temperature (48-hour culture, CFU / g) Classification Control group Experimental group pH 6.5 5.6 Atmospheric conditions Anaerobic aerobic Culture temperature 37℃ 30℃ CFU / g, % lactic acid bacteria count lactic acid bacteria count survival rate LP 15.1x10 11 5.1x10 11 100LP25.4x10 11 5.4x10 11 100LP33.5x10 11 3.5x10 11 100LP44.2x10 11 4.1x10 11 98LP53.8x10 11 3.8x10 11 100LF3.4x10 11 3.3x10 11 97LR3.1x10 11 3.1x10 11 100LPC4.4x10 11 2.3x10 11 52LC1.1x10 12 3.0x10 11 27LA2.4x10 11 1.0x10 11 42LH2.1x1011 8.9x10 10 42LRT3.2x10 11 1.1x10 11 34LB2.1x10 11 8.0x10 10 38ST5.5x10 11 2.0x10 11 36LcL4.0x10 11 3.1x10 11 78LM2.1x10 11 2.0x10 10 10BL1.0x10 12 2.1x10 10 2BB3.1x10 11 1.0x10 10 3BLG2.3x10 11 9.0x10 9 4BR4.3x10 11 5.0x10 9 1

[0151] As shown in Table 5 above, the results of the lactic acid bacteria count confirmation showed that there was no change in the number of L. plantarumQ180, L. plantarumCDKB008, L. plantarumCDKB001, L. plantarumCDKB002, and L. plantarumKCTC 3108, so it was confirmed that the L. plantarum selective culture conditions were not abnormal.

[0152] In addition, most lactic acid bacteria showed a decrease in the number of lactic acid bacteria from about 50% to 1% under the above complex conditions, but L. fermentum SRK414 and L. rhamnosus CKDB009 showed no change in the number of bacteria, as did L. plantarum Q180, L. plantarum CDKB008, L. plantarum CDKB001, L. plantarum CDKB002, and L. plantarum KCTC 3108 (Table 5). Therefore, in order to efficiently select L. plantarum alone, we planned to conduct an experiment to additionally evaluate the number of lactic acid bacteria according to antibiotic treatment.

[0153]

[0154] Example 3: Evaluation of antibiotic treatment conditions for culturing lactic acid bacteria strains

[0155] Example 3-1. Antibiotic selection

[0156] To establish a selective medium for the efficient selection of L. plantarum, antibiotics were additionally applied under aerobic conditions, pH 5.6, and a temperature of 30°C. Antibiotics that had no effect on L. plantarum but inhibited other lactic acid bacteria were selected using the following method.

[0157] With reference to the 'Guidelines for Establishing Standards for Ensuring Quality of Lactic Acid Bacteria Products (Ministry of Food and Drug Safety, 2022)', the antibiotic resistance characteristics of lactic acid bacteria based on the European Food Safety Authority (EFSA) standards were confirmed and are shown in Table 6 below. The above antibiotic resistance characteristics of lactic acid bacteria are classified by species.

[0158]

[0159] EFSA's minimum inhibitory concentration (MIC) index (mg / L) Ampicillin Vancomycin Gentamicin Kanamycin Streptomycin Erythromycin Clindamycin Tetracycline Chloramphenicol Tylosin Lactobacillus Obligate heterofermentative 121616161144-Lactobacillus acidophilus group 121664161144-Lactobacillus obligate heterofermentative 2n.r1632641184-Lactobacillus reuteri 2n.r8646411164-Lactobacillus facultative heterofermentative4n.r1664641184-Lactobacillus plantarum / pentosus2n.r1664n.r12328-Lactobacillus rhamnosus4n.r1664321184-Lactobacillus casei / paracasei4n.r3264641144-Bifidobacterium2264n.r1281184-Pediococcus4n.r1664641184-Leuconostoc2n.r1616641184-Lactococcus lactis243264321148-Streptococcus thermophilus243264642244-Bacillus sppn.r44884488-Propionibacterium246464640.50.2522-Other Gram +1241680.50.2522-Enterococcus faecium24321024128444164

[0160] As shown in Table 6 above, L. plantarum was confirmed to be resistant to vancomycin and streptomycin. Therefore, to establish a screening method for L. plantarum, we conducted a study on the optimal treatment conditions for vancomycin and streptomycin antibiotics under existing culture conditions.

[0161]

[0162] Example 3-2. Evaluation of Vancomycin Treatment Conditions

[0163] Vancomycin is a glycopeptide antibiotic that acts exclusively on Gram-positive bacteria. All strains used in the experiment were Gram-positive. The resistance of each strain to vancomycin was confirmed using the following method.

[0164] Vancomycin concentrations of 20 mg / L, 200 mg / L, 1,000 mg / L, and 1,600 mg / L were applied to CKDB MRS medium, respectively, and cultured for 48 hours under aerobic conditions at pH 5.6 and 30°C, and the number of lactic acid bacteria was analyzed. As a control group, CKDB MRS medium was cultured for 48 hours under anaerobic conditions at pH 6.5 and 37°C. The number of lactic acid bacteria was confirmed by the same method as described in Example 2, and the results are shown in Table 7 below.

[0165] Lactic acid bacteria count according to vancomycin treatment dose (48-hour culture, CFU / g) Classification Control group Experimental group Culture conditions Anaerobic, pH 6.5, 37℃ Aerobic, pH 5.6, 30℃ Vancomycin (mg / L) -20 200 1,000 1,600 LP 15.1 x 10 11 5.4x10 11 5.0x10 11 4.9x10 11 5.3x10 11 LP25.5x10 11 5.5x10 11 5.5x10 11 5.3x10 11<h2 style=";text-align:left;direction:ltr">5.5x10<h2 style=";text-align:left;direction:ltr"> 11 <h2 style=";text-align:left;direction:ltr"> LP33.5x10<h2 style=";text-align:left;direction:ltr"> 11 <h2 style=";text-align:left;direction:ltr"> 3.5x10<h2 style=";text-align:left;direction:ltr"> 11 <h2 style=";text-align:left;direction:ltr"> 3.5x10<h2 style=";text-align:left;direction:ltr"> 11 <h2 style=";text-align:left;direction:ltr"> 3.6x10<h2 style=";text-align:left;direction:ltr"> 11 <h2 style=";text-align:left;direction:ltr"> 3.6x10<h2 style=";text-align:left;direction:ltr"> 11 <h2 style=";text-align:left;direction:ltr"> LP44.2x10<h2 style=";text-align:left;direction:ltr"> 11 <h2 style=";text-align:left;direction:ltr"> 4.3x10<h2 style=";text-align:left;direction:ltr"> 11 <h2 style=";text-align:left;direction:ltr"> 4.2x10<h2 style=";text-align:left;direction:ltr"> 11 <h2 style=";text-align:left;direction:ltr"> 4.1x10<h2 style=";text-align:left;direction:ltr"> 11 <h2 style=";text-align:left;direction:ltr"> 4.2x10<h2 style=";text-align:left;direction:ltr"> 11 <h2 style=";text-align:left;direction:ltr"> LP53.8x10<h2 style=";text-align:left;direction:ltr"> 11 <h2 style=";text-align:left;direction:ltr"> 3.6x10<h2 style=";text-align:left;direction:ltr"> 11 <h2 style=";text-align:left;direction:ltr"> 3.8x10<h2 style=";text-align:left;direction:ltr"> 11 <h2 style=";text-align:left;direction:ltr"> 3.8x10<h2 style=";text-align:left;direction:ltr"> 11 <h2 style=";text-align:left;direction:ltr"> 3.7x10<h2 style=";text-align:left;direction:ltr"> 11 <h2 style=";text-align:left;direction:ltr"> LF3.0x10<h2 style=";text-align:left;direction:ltr"> 11 <h2 style=";text-align:left;direction:ltr"> 3.1x10<h2 style=";text-align:left;direction:ltr"> 11 <h2 style=";text-align:left;direction:ltr"> 2.8x10<h2 style=";text-align:left;direction:ltr"> 11 <h2 style=";text-align:left;direction:ltr"> 3.0x10<h2 style=";text-align:left;direction:ltr"> 11 <h2 style=";text-align:left;direction:ltr"> 2.3x10<h2 style=";text-align:left;direction:ltr"> 11 <h2 style=";text-align:left;direction:ltr"> LR3.2x10<h2 style=";text-align:left;direction:ltr"> 11 <h2 style=";text-align:left;direction:ltr"> 3.4x10<h2 style=";text-align:left;direction:ltr"> 11 <h2 style=";text-align:left;direction:ltr"> 3.5x10<h2 style=";text-align:left;direction:ltr"> 11 <h2 style=";text-align:left;direction:ltr"> 3.3x10<h2 style=";text-align:left;direction:ltr"> 11 <h2 style=";text-align:left;direction:ltr"> 2.5x10<h2 style=";text-align:left;direction:ltr"> 11 <h2 style=";text-align:left;direction:ltr"> LPC4.7x10<h2 style=";text-align:left;direction:ltr"> 11 <h2 style=";text-align:left;direction:ltr"> 4.8x10<h2 style=";text-align:left;direction:ltr"> 11 <h2 style=";text-align:left;direction:ltr"> 4.7x10<h2 style=";text-align:left;direction:ltr"> 11 <h2 style=";text-align:left;direction:ltr"> 4.8x10<h2 style=";text-align:left;direction:ltr"> 11 <h2 style=";text-align:left;direction:ltr"> 1.9x10<h2 style=";text-align:left;direction:ltr"> 11 <h2 style=";text-align:left;direction:ltr"> LC1.0x10<h2 style=";text-align:left;direction:ltr"> 12 <h2 style=";text-align:left;direction:ltr"> 9.0x10<h2 style=";text-align:left;direction:ltr"> 11 <h2 style=";text-align:left;direction:ltr"> 7.3x10<h2 style=";text-align:left;direction:ltr"> 10 <h2 style=";text-align:left;direction:ltr"> < 10<h2 style=";text-align:left;direction:ltr"> 8 <h2 style=";text-align:left;direction:ltr"> < 10<h2 style=";text-align:left;direction:ltr"> 8 <h2 style=";text-align:left;direction:ltr"> LA3.0x10<h2 style=";text-align:left;direction:ltr"> 11 <h2 style=";text-align:left;direction:ltr"> < 10<h2 style=";text-align:left;direction:ltr"> 8 <h2 style=";text-align:left;direction:ltr"> < 10<h2 style=";text-align:left;direction:ltr"> 8 <h2 style=";text-align:left;direction:ltr"> < 10<h2 style=";text-align:left;direction:ltr"> 8 <h2 style=";text-align:left;direction:ltr"> < 10<h2 style=";text-align:left;direction:ltr"> 8 <h2 style=";text-align:left;direction:ltr"> LH2.1x10<h2 style=";text-align:left;direction:ltr"> 11 <h2 style=";text-align:left;direction:ltr"> < 10<h2 style=";text-align:left;direction:ltr"> 8 <h2 style=";text-align:left;direction:ltr"> < 10<h2 style=";text-align:left;direction:ltr"> 8 <h2 style=";text-align:left;direction:ltr"> < 10<h2 style=";text-align:left;direction:ltr"> 8 <h2 style=";text-align:left;direction:ltr"> < 10<h2 style=";text-align:left;direction:ltr"> 8 <h2 style=";text-align:left;direction:ltr"> LRT3.3x10<h2 style=";text-align:left;direction:ltr"> 11 <h2 style=";text-align:left;direction:ltr"> 3.3x10<h2 style=";text-align:left;direction:ltr"> 11 <h2 style=";text-align:left;direction:ltr"> 1.3x10<h2 style=";text-align:left;direction:ltr"> 10 <h2 style=";text-align:left;direction:ltr"> 1.0x10<h2 style=";text-align:left;direction:ltr"> 10 <h2 style=";text-align:left;direction:ltr"> < 10<h2 style=";text-align:left;direction:ltr"> 8 <h2 style=";text-align:left;direction:ltr"> LB2.1x10<h2 style=";text-align:left;direction:ltr"> 11 <h2 style=";text-align:left;direction:ltr"> < 108 < 10 8 < 10 8 < 10 8 ST5.5x10 11 < 10 8 < 10 8 < 10 8 < 10 8 LcL4.0x10 11 < 10 8 < 10 8 < 10 8 < 10 8 LM2.1x10 11 2.0x10 11 2.2x10 11 1.9x10 11 9.4x10 10 BL1.0x10 12 < 10 8 < 10 8 < 10 8 < 10 8 BB1.5x10 11 < 10 8 < 10 8 < 10 8 < 10 8 BLG2.1x10 11 < 10 8 < 10 8 < 10 8 < 10 8 BR3.9x10 11 < 10 8 < 10 8 < 10 8 < 10 8

[0166] As shown in Table 7 above, L. plantarumQ180, L. plantarumCDKB008, L. plantarumCDKB001, L. plantarumCDKB002, and L. plantarumKCTC 3108 are resistant to vancomycin, and even when treated with a high concentration of 1,600 mg / L of vancomycin, the number of bacteria was maintained, indicating that they were not affected by vancomycin.

[0167] In contrast, L. fermentum SRK414, L. rhamnosus CKDB009, L. paracasei CKDB005, and L. mesenteroides CKDB018 strains were partially inhibited only at a concentration of 1,600 mg / L vancomycin, and L. casei CKDB007 and L. reuteri CKDB019 strains were inhibited by less than 10% at a concentration of 1,000 mg / L or less of vancomycin.

[0168] The remaining 9 types of lactic acid bacteria were cultured inhibited by vancomycin, and the number of lactic acid bacteria was 10 8 CFU / g By confirming that it was not cultured above, it was determined that L. plantarum could be selected alone with a survival rate of less than about 0.1% for these 9 types of lactic acid bacteria.

[0169] In addition, from the above results, it was confirmed that the most effective concentration for L. plantarum selection media was 1,600 mg / L. However, since vancomycin could not be used to select only L. plantarum from all 20 lactic acid bacteria, it was confirmed that there are limitations to using vancomycin alone in L. plantarum selection media.

[0170]

[0171] Example 3-2. Evaluation of Streptomycin Treatment Conditions

[0172] Streptomycin is an antibiotic that acts on a wide range of microorganisms, including Gram-negative bacteria. The resistance of each strain to streptomycin was confirmed using the following method.

[0173] Streptomycin concentrations of 200 mg / L, 300 mg / L, 400 mg / L, 500 mg / L, and 1,000 mg / L were applied to CKDB MRS medium, respectively, and cultured under aerobic conditions at pH 5.6 and 30°C for 48 hours, and then the number of lactic acid bacteria was confirmed. As a control group, CKDB MRS medium was cultured under anaerobic conditions at pH 6.5 and 37°C for 48 hours. The number of lactic acid bacteria was confirmed in the same manner as described in Example 2, and the results are shown in Table 8 below.

[0174] Lactic acid bacteria count according to streptomycin treatment dose (48-hour culture, CFU / g) Classification Control group Experimental group Culture conditions Anaerobic, pH 6.5, 37℃ Aerobic, pH 5.6, 30℃ Streptomycin (mg / L) -200 300 400 500 1,000 LP 15.1 x 10 11 5.0x10 11 5.0x10 11 5.0x10 11 3.7x10 11 2.6x10 11 LP25.5x10 11 5.4x10 11 5.3x10 11 5.5x10 11 4.0x10 11 3.6x10 11 LP33.5x10 11 3.5x10 11 3.6x10 11 3.5x10 11 3.5x10 11 3.5x10 11 LP44.2x10 11 4.3x10 11 4.1x10 11 4.1x10 11 4.2x10 11 4.1x10 11 LP53.8x10 11 3.8x10 11 3.8x10 11 3.8x10 11 3.7x10 11 3.8x10 11 LF3.0x10<h2 style=";text-align:left;direction:ltr"> 11 <h2 style=";text-align:left;direction:ltr"> 3.3x10<h2 style=";text-align:left;direction:ltr"> 10 <h2 style=";text-align:left;direction:ltr"> 3.3x10<h2 style=";text-align:left;direction:ltr"> 10 <h2 style=";text-align:left;direction:ltr"> 3.1x10<h2 style=";text-align:left;direction:ltr"> 10 <h2 style=";text-align:left;direction:ltr"> < 10<h2 style=";text-align:left;direction:ltr"> 8 <h2 style=";text-align:left;direction:ltr"> < 10<h2 style=";text-align:left;direction:ltr"> 8 <h2 style=";text-align:left;direction:ltr"> LR3.2x10<h2 style=";text-align:left;direction:ltr"> 11 <h2 style=";text-align:left;direction:ltr"> 2.9x10<h2 style=";text-align:left;direction:ltr"> 11 <h2 style=";text-align:left;direction:ltr"> 2.9x10<h2 style=";text-align:left;direction:ltr"> 11 <h2 style=";text-align:left;direction:ltr"> 3.1x10<h2 style=";text-align:left;direction:ltr"> 11 <h2 style=";text-align:left;direction:ltr"> < 10<h2 style=";text-align:left;direction:ltr"> 8 <h2 style=";text-align:left;direction:ltr"> < 10<h2 style=";text-align:left;direction:ltr"> 8 <h2 style=";text-align:left;direction:ltr"> LPC4.7x10<h2 style=";text-align:left;direction:ltr"> 11 <h2 style=";text-align:left;direction:ltr"> 4.3x10<h2 style=";text-align:left;direction:ltr"> 11 <h2 style=";text-align:left;direction:ltr"> 4.3x10<h2 style=";text-align:left;direction:ltr"> 11 <h2 style=";text-align:left;direction:ltr"> 4.6x10<h2 style=";text-align:left;direction:ltr"> 11 <h2 style=";text-align:left;direction:ltr"> < 10<h2 style=";text-align:left;direction:ltr"> 8 <h2 style=";text-align:left;direction:ltr"> < 10<h2 style=";text-align:left;direction:ltr"> 8 <h2 style=";text-align:left;direction:ltr"> LC9.9x10<h2 style=";text-align:left;direction:ltr"> 11 <h2 style=";text-align:left;direction:ltr"> < 10<h2 style=";text-align:left;direction:ltr"> 8 <h2 style=";text-align:left;direction:ltr"> < 10<h2 style=";text-align:left;direction:ltr"> 8 <h2 style=";text-align:left;direction:ltr"> < 10<h2 style=";text-align:left;direction:ltr"> 8 <h2 style=";text-align:left;direction:ltr"> < 10<h2 style=";text-align:left;direction:ltr"> 8 <h2 style=";text-align:left;direction:ltr"> < 10<h2 style=";text-align:left;direction:ltr"> 8 <h2 style=";text-align:left;direction:ltr"> LA3.0x10<h2 style=";text-align:left;direction:ltr"> 11 <h2 style=";text-align:left;direction:ltr"> < 10<h2 style=";text-align:left;direction:ltr"> 8 <h2 style=";text-align:left;direction:ltr"> < 10<h2 style=";text-align:left;direction:ltr"> 8 <h2 style=";text-align:left;direction:ltr"> < 10<h2 style=";text-align:left;direction:ltr"> 8 <h2 style=";text-align:left;direction:ltr"> < 10<h2 style=";text-align:left;direction:ltr"> 8 <h2 style=";text-align:left;direction:ltr"> < 10<h2 style=";text-align:left;direction:ltr"> 8 <h2 style=";text-align:left;direction:ltr"> LH2.1x10<h2 style=";text-align:left;direction:ltr"> 11 <h2 style=";text-align:left;direction:ltr"> < 10<h2 style=";text-align:left;direction:ltr"> 8 <h2 style=";text-align:left;direction:ltr"> < 10<h2 style=";text-align:left;direction:ltr"> 8 <h2 style=";text-align:left;direction:ltr"> < 10<h2 style=";text-align:left;direction:ltr"> 8 <h2 style=";text-align:left;direction:ltr"> < 10<h2 style=";text-align:left;direction:ltr"> 8 <h2 style=";text-align:left;direction:ltr"> < 10<h2 style=";text-align:left;direction:ltr"> 8 <h2 style=";text-align:left;direction:ltr"> LRT3.3x10<h2 style=";text-align:left;direction:ltr"> 11 <h2 style=";text-align:left;direction:ltr"> < 10<h2 style=";text-align:left;direction:ltr"> 8 <h2 style=";text-align:left;direction:ltr"> < 10<h2 style=";text-align:left;direction:ltr"> 8 <h2 style=";text-align:left;direction:ltr"> < 10<h2 style=";text-align:left;direction:ltr"> 8 <h2 style=";text-align:left;direction:ltr"> < 10<h2 style=";text-align:left;direction:ltr"> 8 <h2 style=";text-align:left;direction:ltr"> < 10<h2 style=";text-align:left;direction:ltr"> 8 <h2 style=";text-align:left;direction:ltr"> LB2.1x10<h2 style=";text-align:left;direction:ltr"> 11 <h2 style=";text-align:left;direction:ltr"> < 10<h2 style=";text-align:left;direction:ltr"> 8 <h2 style=";text-align:left;direction:ltr"> < 10<h2 style=";text-align:left;direction:ltr"> 8 <h2 style=";text-align:left;direction:ltr"> < 10<h2 style=";text-align:left;direction:ltr"> 8 <h2 style=";text-align:left;direction:ltr"> < 10<h2 style=";text-align:left;direction:ltr"> 8 <h2 style=";text-align:left;direction:ltr"> < 10<h2 style=";text-align:left;direction:ltr"> 8 <h2 style=";text-align:left;direction:ltr"> ST5.5x10<h2 style=";text-align:left;direction:ltr"> 11 <h2 style=";text-align:left;direction:ltr"> < 10<h2 style=";text-align:left;direction:ltr"> 8 <h2 style=";text-align:left;direction:ltr"> < 10<h2 style=";text-align:left;direction:ltr"> 8 <h2 style=";text-align:left;direction:ltr"> < 10<h2 style=";text-align:left;direction:ltr"> 8 <h2 style=";text-align:left;direction:ltr"> < 10<h2 style=";text-align:left;direction:ltr"> 8 <h2 style=";text-align:left;direction:ltr"> < 10<h2 style=";text-align:left;direction:ltr"> 8 <h2 style=";text-align:left;direction:ltr"> LcL4.0x10<h2 style=";text-align:left;direction:ltr"> 11 <h2 style=";text-align:left;direction:ltr"> < 10<h2 style=";text-align:left;direction:ltr"> 8< 10 8 < 10 8 < 10 8 < 10 8 LM2.1x10 11 < 10 8 < 10 8 < 10 8 < 10 8 < 10 8 BL1.0x10 12 < 10 8 < 10 8 < 10 8 < 10 8 < 10 8 BB1.5x10 11 < 10 8 < 10 8 < 10 8 < 10 8 < 10 8 BLG2.1x10 11 < 10 8 < 10 8 < 10 8 < 10 8 < 10 8 BR3.9x10 11 < 10 8 < 10 8 < 10 8 < 10 8 < 10 8

[0175] As shown in Table 8 above, L. plantarumQ180, L. plantarumCDKB008, L. plantarumCDKB001, L. plantarumCDKB002, and L. plantarumKCTC 3108 were not affected by streptomycin treatment at concentrations below 400 mg / L, but when streptomycin was treated at concentrations above 500 mg / L, the number of bacteria decreased, confirming that they were inhibited by streptomycin. On the other hand, similar to vancomycin, L. fermentumSRK414, L. rhamnosusCKDB009, and L. paracaseiCKDB005 strains were not affected by streptomycin treatment at concentrations below 400 mg / L, but when streptomycin was treated at concentrations above 500 mg / L, the culture was inhibited, and the number of lactic acid bacteria was 10 8 CFU / g The remaining 12 strains of lactic acid bacteria were not cultured at 10 8 CFU / g It was not cultured beyond this point.

[0176] From the above results, when streptomycin was treated, a greater number of lactic acid bacteria strains and inhibition results were distinguished than when vancomycin was treated, so the efficiency of selecting L. plantarum alone was high. However, at concentrations exceeding 500 mg / L, the number of L. plantarum bacteria also decreased due to the effect of the antibiotic, so it was confirmed that the optimal concentration for L. plantarum selective media was 400 mg / L. In addition, streptomycin also could not select L. plantarum alone for some lactic acid bacteria, so it was found that there are limitations in using streptomycin alone in L. plantarum selective media.

[0177]

[0178] Example 3-3. Evaluation of combined treatment conditions for vancomycin and streptomycin.

[0179] As described above, it was confirmed that the species and degree of inhibition differed depending on the treatment with vancomycin and streptomycin, respectively. It was also confirmed that when vancomycin or streptomycin was treated alone, L. plantarum could not be selectively isolated from lactic acid bacteria strains. Therefore, it was decided to perform a combined treatment with vancomycin and streptomycin as follows for the L. plantarum selective medium.

[0180] To prevent L. plantarum strains from being inhibited, a mixture of vancomycin at a concentration of 1,600 mg / L and streptomycin at a concentration of 400 mg / L was applied to CKDB MRS medium, and cultured for 48 hours under aerobic conditions at pH 5.6 and 30°C, and the number of lactic acid bacteria was confirmed. As a control group, CKDB MRS medium pH 6.5, 37°C, and anaerobic conditions were cultured. The number of lactic acid bacteria was confirmed in the same manner as described in Example 2, and the results are shown in Table 9 below.

[0181] Lactic acid bacteria count according to combined treatment with vancomycin and streptomycin (48-hour culture, CFU / g) Classification Control group Experimental group Culture conditions Anaerobic, pH 6.5, 37℃ Aerobic, pH 5.6, 30℃ Vancomycin - 1,600 mg / L Streptomycin - 400 mg / LLP 15.1 x 10 11 5.2x10 11 LP25.5x10 11 5.5x10 11 LP33.5x10 11 3.5x10 11 LP44.2x10 11 4.2x10 11 LP53.8x10 11 3.8x10 11 LF3.0x10 11 3.1x10 10 LR3.2x10 11 < 107 LPC4.7x10 11 < 10 7 LC1.1x10 12 < 10 7 LA3.0x10 11 < 10 7 LH2.1x10 11 < 10 7 LRT3.3x10 11 < 10 7 LB2.0x10 11 < 10 7 ST5.5x10 11 < 10 7 LcL4.0x10 11 < 10 7 LM2.1x10 11 < 10 7 BL1.0x10 12 < 10 7 BB1.5x10 11 < 10 7 BLG2.1x10 11 < 10 7 BR3.9x10 11 < 10 7

[0182] As shown in Table 9 above, when treated with vancomycin and streptomycin in combination, L. plantarum Q180, L. plantarum CDKB008, L. plantarum CDKB001, L. plantarum CDKB002, and L. plantarum KCTC 3108 were not affected by vancomycin and streptomycin. On the other hand, culture was inhibited in all lactic acid bacteria strains except L. fermentum SRK414, and 10 7 CFU / g It was confirmed that the above did not result in culture. The survival rate of the lactic acid bacteria that inhibited the above culture was less than approximately 0.01%. In other words, it was confirmed that L. plantarum could be selected when treated with a combination of vancomycin and streptomycin.

[0183] However, in the case of L. fermentumSRK414, although the culture was somewhat inhibited when treated with a combination of vancomycin and streptomycin, it was still cultured with a survival rate of around 10%.

[0184] Therefore, the present inventors planned to evaluate the sugar utilization of each lactic acid bacteria strain in order to more efficiently select L. plantarum.

[0185]

[0186] Example 4: Evaluation of sugar availability for culturing lactic acid bacteria strains

[0187] As described above, even when combined treatment with vancomycin and streptomycin under culture conditions of pH 5.6, 30°C, and aerobic conditions was performed, it was difficult to completely isolate only the L. plantarum strain from the 20 lactic acid bacteria strains used in the experiment.

[0188] Therefore, if there is a sugar that only L. plantarum can utilize, in order to utilize it in the L. plantarum selective medium, the sugar utilization of the 20 strains used in the experiment was confirmed using the API 50 CH kit (BioMerieux, France). The strain cultures cultured in CKDB MRS liquid medium at 37°C for 16 hours were centrifuged at 3,500 rpm for 10 minutes to remove the supernatant, and 1 × 10 strains were incubated in API CHL medium. 8 The fungal solution was prepared at a concentration equal to CFU / mL. 120 μL of the diluted fungal solution was inoculated into each well and cultured at 37°C for 24 to 48 hours. Color changes were then observed visually. The sugar utilization of the strains was evaluated using API web, and the results were expressed as + (active sugar utilization), w (slight sugar utilization), and - (no sugar utilization) depending on the sugar utilization, which are shown in Figures 1a and 1b.

[0189] As shown in Figures 1a and 1b, D-Arabitol was found to be the only sugar utilized by L. plantarum.

[0190] Since most microorganisms use glucose as their main energy source for growth, D-arabitol was added instead of glucose in the CKDB MRS medium components, and the number of lactic acid bacteria according to the D-arabitol utilization ability of the 20 strains used in the experiment was confirmed.

[0191] For each lactic acid bacteria strain, the experimental group was divided into three groups and cultured for 48 hours each under the same CKDB MRS medium, pH 5.6, 30℃, aerobic conditions, under conditions excluding glucose, conditions using 2 g / L of D-arabitol instead of glucose, and conditions using 20 g / L of D-arabitol instead of glucose. The control group was cultured for each lactic acid bacteria strain in CKDB MRS medium, pH 6.5, 37℃, anaerobic conditions. The number of lactic acid bacteria was confirmed using the same method as described in Example 2. The results of confirming the number of lactic acid bacteria and the survival rate of the strain calculated based on the results are shown in Table 10 below. The survival rate of the lactic acid bacteria strain was calculated using the following equation.

[0192] Viability (%) = Number of viable cells in samples under various culture conditions / Number of viable cells in the control group for each sample Х 100

[0193] Lactic acid bacteria count according to D-Arabitol availability (48-hour culture) Classification CKDB MRS CKDB MRS (Without Glucose) Culture conditions Anaerobic, pH 6.5, 37℃ Aerobic, pH 5.6, 30℃ D-Arabitol--2 g / L 20 g / LCFU / g, % Viable cell count Viable cell count Survival rate Viable cell count Survival rate Viable cell count Survival rate LP 15.1x10 11 1.1x10 10 23.1x10 11 614.9x10 11 98LP25.5x10 11 2.0x10 10 43.5x1011 645.5x10 11 100LP33.5x10 11 1.7x10 10 52.0x10 11 573.5x10 11 100LP44.2x10 11 2.2x10 10 52.0x10 11 484.2x10 11 100LP53.8x10 11 1.3x10 10 31.7x10 11 453.7x10 11 97LF3.0x10 11 5.0x10 9 25.1x10 9 26.0x10 9 2LR3.2x10 11 1.2x10 10 41.2x10 10 41.2x10 10 4LPC4.7x10 11 5.2x10 9 15.4x10 9 14.7x10 9 1LC9.8x10 11 1.2x10 10 11.4x10 10 19.9x10 9 1LA3.0x10 11 7.0x10 9 27.2x10 9 27.3x10 9 2LH2.1x10 11 8.5x10 9 48.0x10 9 48.3x10 9 4LRT3.3x10 11 2.3x10 9 12.0x10 9 12.7x10 9 1LB2.0x10 11 4.0x10 9 23.7x10 9 24.2x10 9 2ST5.5x10 11 2.5x10 10 52.2x10 1042.1x10 10 4LcL4.0x10 11 1.0x10 10 31.1x10 10 31.4x10 10 4LM2.1x10 11 8.1x10 9 48.4x10 9 41.0x10 10 5BL1.0x10 12 3.0x10 9 03.4x10 9 05.0x10 9 1BB1.5x10 11 4.1x10 9 34.4x10 9 34.3x10 9 3BLG2.1x10 11 3.1x10 9 13.0x10 9 12.1x10 9 1BR3.9x10 11 4.7x10 9 12.5x10 9 12.3x10 9 1

[0194] As a result, as shown in Table 10 above, when glucose was excluded from the medium, all 20 types of lactic acid bacteria showed a survival rate of less than 5% compared to the control group, confirming that the strains were not cultured normally.

[0195] In the case of media using D-arabitol instead of glucose, all lactic acid bacteria strains except L. plantarumQ180, L. plantarumCDKB008, L. plantarumCDKB001, L. plantarumCDKB002, and L. plantarumKCTC 3108 showed a similar level of lactic acid bacteria count to that in media excluding glucose. This indicates that all lactic acid bacteria strains except L. plantarum cannot utilize D-arabitol as a carbon source.

[0196] On the other hand, in the case of L. plantarumQ180, L. plantarumCDKB008, L. plantarumCDKB001, L. plantarumCDKB002, L. plantarumKCTC 3108, the survival rate in the medium to which 2 g / L of D-arabitol was applied was 45% to 64% compared to the medium to which glucose was applied, and the survival rate in the medium to which 20 g / L of D-arabitol was applied was 100% compared to the medium to which glucose was applied. In other words, it was found that L. plantarumQ180, L. plantarumCDKB008, L. plantarumCDKB001, L. plantarumCDKB002, L. plantarumKCTC 3108 could grow using D-arabitol, which is consistent with the results confirmed with the API 50 CH kit.

[0197]

[0198] Example 5: Evaluation of culture conditions, antibiotic treatment conditions, and D-arabitol utilization complex application for culturing lactic acid bacteria strains.

[0199] Through Example 4, it was confirmed that selective isolation of L. plantarum was possible through the D-arabitol utilization of L. plantarumQ180, L. plantarumCDKB008, L. plantarumCDKB001, L. plantarumCDKB002, and L. plantarumKCTC 3108. However, since the CKDB MRS medium uses raw materials containing sugar components such as soy peptone and yeast extract in addition to D-arabitol, it was difficult to efficiently and selectively isolate only L. plantarum. Therefore, the present inventors, in order to further improve the sensitivity in selective culture and isolation of L. plantarum, applied the D-arabitol utilization confirmed in Example 4 in combination with the lactic acid bacteria culture conditions and antibiotic complex treatment conditions confirmed in Examples 2 and 3, respectively, to conduct an experiment to selectively isolate only L. plantarum as follows.

[0200] The experimental group was cultured in a CKDB MRS medium supplemented with 20 g / L of D-arabitol except for glucose, at pH 5.6, 30°C, for 48 hours under aerobic conditions, with a combination of 1,600 mg / L vancomycin and 400 mg / L streptomycin. The control group was cultured in CKDB MRS medium, pH 6.5, at a culture temperature of 37°C, for 48 hours under anaerobic conditions. The number of lactic acid bacteria was confirmed by the same method as described in Example 2, and the results are shown in Table 11 below.

[0201] Culture conditions, antibiotics, and sugar availability according to the number of cultured lactic acid bacteria (48 hours of culture) Classification Control group Experimental group Medium CKDB MRS CKDB MRS (Without Glucose) D-Arabitol-20 g / L Antibiotic-Vancomycin (1,600 mg / L) Streptomycin (400 mg / L) Culture conditions Anaerobic, pH 6.5, 37℃ Aerobic, pH 5.6, 30℃ LP 15.1 x 10 11 5.0x10 11 LP25.5x10 11 5.5x10 11 LP33.5x10 11 3.5x10 11 LP44.2x10 11 4.2x10 11 LP53.8x10 11 3.8x10 11 LF3.0x10 11 < 10 5 LR3.2x10 11 < 10 5 LPC4.7x10 11 < 10 5 LC9.8x10 11 < 10 5 LA3.0x10 11 < 10 5 LH2.1x10 11 < 10 5 LRT3.3x10 11 < 10 5 LB2.0x10 11 < 10 5 ST5.5x10 11 < 10 5LcL4.0x10 11 < 10 5 LM2.1x10 11 < 10 5 BL1.0x10 12 < 10 5 BB1.5x10 11 < 10 5 BLG2.1x10 11 < 10 5 BR3.9x10 11 < 10 5

[0202] As a result, as shown in Table 11, it was confirmed that only L. plantarum could be successfully selectively isolated and cultured under the above complex conditions. Except for L. plantarumQ180, L. plantarumCDKB008, L. plantarumCDKB001, L. plantarumCDKB002, and L. plantarumKCTC 3108, 15 types of lactic acid bacteria were inhibited from being cultured under the above complex conditions, and the number of lactic acid bacteria was less than 10 5 CFU / g It was not cultured beyond this point.

[0203] Thus, when all conditions of pH 5.6, 30℃, aerobic culture conditions, combined treatment with 1,600 mg / L vancomycin and 400 mg / L streptomycin antibiotics, and addition of 20 g / L D-arabitol were applied in combination and cultured for 48 hours, it was confirmed that only L. plantarum could be selectively cultured and isolated at a level of less than about 0.0001% among the 20 lactic acid bacteria strains used in this experiment, and the number of L. plantarum lactic acid bacteria could be quantitatively evaluated.

[0204]

[0205] In this way, the present inventors established the composition of a medium and culture conditions that can successfully perform selective isolation and culture of L. plantarum and quantitative evaluation of the number of lactic acid bacteria.

Claims

1. A medium composition for selective cultivation of Lactobacillus plantarum containing D-arabitol as a carbon source.

2. A medium composition for selective culture of Lactobacillus plantarum, wherein the concentration of D-arabitol in the medium composition in the first paragraph is 0.5 g / L to 50 g / L.

3. A medium composition for selective culture of Lactobacillus plantarum, further comprising a glycopeptide antibiotic, an aminoglycoside antibiotic, or a combination thereof, in accordance with claim 1.

4. A medium composition for selective culture of Lactobacillus plantarum, wherein the glycopeptide antibiotic in paragraph 3 is vancomycin or teicoplanin.

5. A medium composition for selective culture of Lactobacillus plantarum, wherein the aminoglycoside antibiotic in the third paragraph is at least one antibiotic selected from the group consisting of streptomycin, gentamycin, tobramycin, and amikacin.

6. A medium composition for selective culture of Lactobacillus plantarum, wherein the concentration of the glycopeptide antibiotic in the medium composition in the third paragraph is 10 mg / L to 2,000 mg / L.

7. A medium composition for selective culture of Lactobacillus plantarum, wherein the concentration of the aminoglycoside antibiotic in the medium composition in the third paragraph is 50 mg / L to 1,200 mg / L.

8. A medium composition for selective culture of Lactobacillus plantarum, further comprising soy peptone, yeast extract, ammonium citrate, dipotassium phosphate, and magnesium sulfate according to claim 1.

9. A medium composition for selective culture of Lactobacillus plantarum, wherein the pH of the composition in paragraph 1 is 5.0 to 7.

0.

10. A medium composition for selective culture of Lactobacillus plantarum, wherein the composition does not contain glucose in the first paragraph. 11.(a) A method for selectively isolating Lactobacillus plantarum, comprising a step of culturing a lactic acid bacteria sample in a medium composition for selectively culturing Lactobacillus plantarum according to any one of claims 1 to 10.

12. A method for selectively isolating Lactobacillus plantarum, comprising the step of (b) confirming whether a colony is formed in the medium composition in the 11th paragraph.

13. A method for selectively isolating Lactobacillus plantarum, wherein the culturing in step (a) is performed under aerobic conditions.

14. A method for selectively isolating Lactobacillus plantarum, wherein the culturing in step (a) is performed under conditions of pH 5.0 to pH 7.0 in the 11th paragraph.

15. A method for selectively isolating Lactobacillus plantarum, wherein the culturing in step (a) is performed at a temperature of 20°C to 45°C in the 11th paragraph.

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