WO2020171256A1 - Lactobacillus strain having growth-inhibitory effect on clostridium difficile - Google Patents

Abstract

The present invention relates to a Lactobacillus plantarum KBL396 (KCTC 13278 BP) strain characterized by having a growth-inhibitory effect on Clostridium difficile.

Description

Lactobacillus strain having the effect of inhibiting the growth of Clostridium difficile

The present invention relates to a CDI (Clostridium difficile infection) treatment pharmaceutical composition comprising the Lactobacillus plantarum strain or a culture thereof having a Clostridium difficile growth inhibitory effect, and a culture thereof. .

CDI (Clostridium difficile infection) is an antibiotic-related diarrhea disease that occurs when the composition of the normal flora changes in the intestinal tract of a patient receiving antibiotics, and Clostridium difficile proliferates and simultaneously secretes toxins. antibiotics-associated diarrhea, AAD).

C. difficile shows relatively high resistance to disinfectant treatment such as alcohol, and it is difficult to properly manage and remove because it can build spores that can survive in extreme environments for several years or more. According to a recent report by the US CDC, the number of CDI patients per year is around 500,000, and the death toll is 14,000 a year, a serious disease. In general, recurrence is frequent even after treatment. In particular, recurrence patients are classified as a major disease because it is difficult to see great efficacy even with general antibiotic treatment.

Currently, the standard treatment used for CDI treatment is antibiotic prescription, which is known to show relatively low treatment success rate and high recurrence rate. Briefly, Metronidazole and vancomycin both have a limited treatment success rate at the time of primary treatment and show a recurrence rate of 20 to 30%, the treatment success rate at the first recurrence is 70%, and at more recurrences it decreases to 35%, but due to highly toxic strains. Inadequate for use in refractory severe CDIs and recurrent CDIs. In addition, Fidaxomicin inhibits the synthesis of C. difficle toxins A and B and sporulation, and the recurrence rate is also low compared to other antibiotics, but the price is high, and nausea, vomiting, fever, dizziness, and increased liver enzyme levels are still problematic. The possibility of Nitazoxanide as a treatment has also been suggested, but related studies are still insufficient. In recent years, a non-antibiotic approach using fecal microbiota transplantation (FMT), vaccines, and administration of key microbiota as the prevention and treatment of CDIs has been suggested. FMT, in which stool from a healthy donor is administered to the patient's intestine, is one of the methods of treatment for refractory or recurrent CDIs. As a result of a summary of 28 studies of 317 CDI patients, it showed a recovery rate of 92%. In addition, it has a high recurrence prevention effect. However, despite the high treatment success rate and low recurrence rate, the FMT treatment has several constraints, such as a procedure that can be unpleasant for the general public to accept, non-standard treatment, and transmission of pathogens. C. difficile vaccines, including degenerative toxins A and B, are also being studied to be effective in patients with recurrent CDI, but commercialization is expected to take considerable time.

Useful microorganisms isolated in the intestine can cause changes in the intestinal ecosystem and interaction with the immune system, and as a result, maintain space and resource competition with C. difficile. When these are treated, it has been reported that they actually show the same level of therapeutic effect as metronidazole or vancomycin, and that the recurrence rate is also significantly lower (Ollech et al., 2016. Best Practice & Research Clinical Gastroenterology). Therefore, the process of securing and mass-producing functional beneficial strains that can make a big contribution to the prevention/treatment of CDI can be seen as having very important significance as a method that can supplement/replace chemical antibiotic therapy in the future.

Intestinal microbes play a role of protecting the intestinal epithelium from pathogens such as C. difficile, and when an imbalance of intestinal microbes is caused by certain causes such as antibiotics (dysbiosis), the pathogen penetrates the intestinal wall and prevents disease. cause. According to a recent study, IL-25 produced by intestinal microbes increased the tight junction of the intestinal wall to prevent CDI (Buonomo et al., 2016. Cell Reports). It has also been reported that there is a high correlation with the degree of diversity of (Milani et al., 2016. Scientific Reports). As a result of confirming the diversity of intestinal microflora by obtaining clinical samples of CDI patients, it was confirmed that the intestinal microbial diversity of CDI patients was significantly lower than that of normal people, and secondary bile acids produced by beneficial strains in the intestinal microflora were C. It has been reported that difficile inhibits the growth of C. difficile by inhibiting TCA and DCA, which are essential for sporulation (Winston et al., 2016. Anaerobe.).

An object of the present invention is to provide a Lactobacillus plantarum (Lactobacillus plantarum) KBL396 (KCTC 13278 BP) strain, characterized in that it has a Clostridium difficile growth inhibitory effect.

Another object of the present invention is to provide a pharmaceutical composition for the treatment of Clostridium difficile infection (CDI), comprising a Lactobacillus plantarum KBL396 (KCTC 13278 BP) strain or a culture therefor. .

Another object of the present invention is to provide a functional food for alleviation or improvement of Clostridium difficile infection (CDI), characterized in that it comprises a Lactobacillus plantarum KBL396 (KCTC 13278 BP) strain or a culture therefor. Is to do.

In order to achieve the above object, the present invention provides a Lactobacillus plantarum KBL396 (KCTC 13278 BP) strain, characterized in that it has a Clostridium difficile growth inhibitory effect.

The growth inhibitory effect of Clostridium difficile may be bile acid independent.

In addition, the present invention provides a pharmaceutical composition for treating or preventing CDI (Clostridium difficile infection), characterized in that it comprises a Lactobacillus plantarum KBL396 (KCTC 13278 BP) strain or a culture therefor.

In addition, the present invention provides a health functional food for alleviating or improving Clostridium difficile infection (CDI), characterized in that it comprises a Lactobacillus plantarum KBL396 (KCTC 13278 BP) strain or a culture therefor.

The Clostridium difficile infection (CDI) may be a recurrent CDI.

The Clostridium difficile infection (CDI) may be caused by an intestinal microbial imbalance.

In addition, the present invention provides a method for preventing, improving or treating CDI by administering an effective amount of Lactobacillus plantarum KBL396 (KCTC 13278 BP) strain to an individual in need thereof.

In addition, the present invention provides the use of the Lactobacillus plantarum KBL396 (KCTC 13278 BP) strain to inhibit the growth of Clostridium difficile in an individual.

In addition, the present invention provides the use of the Lactobacillus plantarum KBL396 (KCTC 13278 BP) strain for the manufacture of a pharmaceutical formulation for preventing, improving or treating CDI.

The present invention relates to a strain of Lactobacillus plantarum KBL396 (KCTC 13278 BP) characterized by having a Clostridium difficile growth inhibitory effect, and is effective in CDI treatment and symptom improvement, In particular, it is very effective in treating recurrent CDI and improving symptoms.

1 is a result of a comparative experiment on the growth inhibitory effect of Clostridium difficile (C.difficile).

Figure 2 is a result of the inhibitory effect of Lactobacillus plantarum (Lactobacillus plantarum) KBL396 (KCTC 13278 BP) strain (LP) on C. difficile growth experiments.

3 is an experimental process for establishing a C.difficile infection prevention model.

Figure 4 is a result of the weight loss prevention effect experiment by the Lactobacillus plantarum (Lactobacillus plantarum) KBL396 (KCTC 13278 BP) strain in the C.difficile infection prevention model.

Figure 5 is a result of the survival rate increase experiment by Lactobacillus plantarum (Lactobacillus plantarum) KBL396 (KCTC 13278 BP) strain in the C.difficile infection prevention model.

6 is a profile analysis result of bile acid, DCA and LCA in feces before/after C. difficile infection (FIG. 6A is a result of DCA content in feces 24 hours after C. difficile infection, and FIG. 6B is C. Results of LCA content in feces 24 hours after difficile infection).

7 shows the results of microbial analysis between the LP administration group and the infection control group.

8 is an experimental result for understanding the correlation between C. difficile infection and associated microbial species.

9 is a functional microbiom analysis (microbiom metabolic pathway analysis) between the LP administration group and the infection control experimental results.

Fig. 10 is a result of analysis of genes related to the biosynthetic pathway of secondary bile acids among fecal microvims of the LP administration group and the infection control.

11 is an experimental result of correlation between the amount of bile acids in feces and functional microbial analysis.

Fig. 12 is a result of experimentation of changes in bile acid composition according to LP culture (in vitro).

13 is an experimental result of the conversion effect of TCA and TDCA according to LP culture (in vitro) (FIG. 13A is an experimental result for Cholic acid, and FIG. 13B is an experimental result for Deoxycholic acid).

14 is an experimental procedure for establishing a model for preventing recurrence of C. difficile infection.

Figure 15 is a result of the weight recovery effect test caused by LP administration in the C. difficile infection recurrence prevention model.

The present invention provides a Lactobacillus plantarum (Lactobacillus plantarum) KBL396 (KCTC 13278 BP) strain, characterized in that it has a Clostridium difficile (C.difficile) growth inhibitory effect.

Lactobacillus plantarum KBL396 (KCTC 13278 BP) strain was isolated from feces provided by healthy adult volunteers. Since the strain is an absolute anaerobic strain, the separation process was performed under an anaerobic system.

The Clostridium difficile (C.difficile) is a gram-positive bacterium, an anaerobic bacterium, is a relatively large bacterium with a length of 2-17 μm, has a structure such as flagella or projections, and is a bacterium living in human intestine. Clostridium difficile is likely to occur even after administration of antibiotics such as Clindamycin, Lincomycin, Amoxicillin, Cephalotin, and Cefazoline. After the destruction of the normal intestinal flora due to antibiotics, colonization may occur due to toxic C. difficile. Clostridium difficile produces toxin A (enterotoxin) with a molecular weight of about 310,000 and toxin B (cytotoxin) with a molecular weight of about 270,000. Toxin A first injures the intestinal epithelial cells and then invades the intestinal membrane at the site of toxin B stage and exerts a poison effect.

The growth inhibitory effect on the Clostridium difficile strain can be confirmed through an inhibition assay, but is not limited thereto.

Growth inhibition of C.difficile may be due to inhibition of spore germination, but is not limited thereto.

The Lactobacillus plantarum KBL396 (KCTC 13278 BP) strain is a deposited strain and was deposited with the Biological Resource Center on May 29, 2017, and the accession number is KCTC 13278 BP.

The growth inhibitory effect of Clostridium difficile may be bile acid independent.

The present invention provides a pharmaceutical composition for the treatment or prevention of Clostridium difficile infection (CDI), characterized in that it comprises a Lactobacillus plantarum KBL396 (KCTC 13278 BP) strain or a culture therefor.

CDI may be caused by spore-forming bacteria, and specifically, may be caused by a Clostridium difficile strain. CDI can also be accompanied by diarrhea, high temperatures, and unusual pain.

The CDI may occur in patients whose bile secretion is not normally performed in the liver.

The CDI may be a type of antibiotic-associated diarrhea.

In addition, CDI may have occurred in patients receiving broad spectrum antibiotics (especially clindamycin) that alter the ecological balance of the intestinal flora.

In addition, the CDI may be mild CDI, severe CDI, complex CDI, or recurrent CDI, and more preferably, may be recurrent CDI. The severity of the CDI can be determined based on age, whether a systemic antitussive agent is used, the number of white blood cells, albumin, and serum creatine.

More specifically, in mild CDI, the number of leukocytes may be less than 15,000 cells/ml, and the serum creatine may be less than 1.5 times premorbid level, and in severe CDI, the number of white blood cells is 15,000 cells/ml or more, and serum creatine is 1.5 times premorbid level. It may be abnormal, and complex (complicated CDI) may involve hypotension, shock, rainy season, megacolon, and recurrent CDI may mean recurrence within 8 weeks after successful treatment for CDI.

The pharmaceutical composition according to the present invention can be administered to mammals including humans by various routes. The mode of administration may be any method commonly used, for example, may be administered by oral, dermal, intravenous, intramuscular, subcutaneous, and the like, preferably orally. The pharmaceutical compositions of the present invention are each parenterally in the form of oral dosage forms such as powders, granules, tablets, capsules, ointments, suspensions, emulsions, syrups, aerosols, or transdermal preparations, suppositories, and sterile injectable solutions according to conventional methods. It can be formulated and used as an old formulation. The pharmaceutical composition of the present invention may further contain adjuvants such as a pharmaceutically suitable and physiologically acceptable carrier, excipient, and diluent.

Carriers, excipients and diluents that may be included in the pharmaceutical composition of the present invention include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum acacia, alginate, gelatin, calcium phosphate, calcium. Silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, and mineral oils. In the case of formulation, diluents or excipients such as commonly used fillers, extenders, binders, wetting agents, disintegrants, and surfactants can be used.

In an embodiment in which the pharmaceutical composition of the present invention is applied to humans, the pharmaceutical composition of the present invention may be administered alone, but generally, a pharmaceutical composition selected in consideration of the mode of administration and standard pharmaceutical practice. It can be administered in admixture with a carrier. For example, the composition containing the Clostridium syndense strain of the present invention is in the form of a tablet containing starch or lactose, alone or in the form of a capsule containing an excipient, or contains a chemical agent to taste or color It can be administered orally, intraorally, or sublingually in the form of an elixir or suspension.

The dosage of the pharmaceutical composition containing the strain of Lactobacillus plantarum KBL396 (KCTC 13278 BP) of the present invention may vary depending on the patient's age, weight, sex, dosage form, health condition, and degree of disease. Alternatively, according to the judgment of the pharmacist, it may be dividedly administered once or several times a day at regular time intervals. For example, the daily dosage may be 0.1 to 500 mg/kg, preferably 0.5 to 300 mg/kg, based on the content of the active ingredient. The above dosage is an example of an average case, and the dosage may be high or low depending on individual differences. If the daily dosage of the composition containing the mixed extract of the present invention is less than the above dosage, a significant effect cannot be obtained, and if it exceeds that, it is not only uneconomical, but also outside the range of the normal dosage, there is a concern that undesirable side effects may occur. It may occur, so it is better to set it within the above range.

Another example of the present invention is a health functional food for alleviating or improving Clostridium difficile infection (CDI), characterized in that it comprises a Lactobacillus plantarum KBL396 (KCTC 13278 BP) strain or a culture therefor. to provide.

The health functional food may be various beverages, fermented milk, food additives, and the like.

The content of the Clostridium syndense strain as an active ingredient contained in the health functional food is not particularly limited appropriately depending on the form of the food, the desired use, etc., and may be added in an amount of 0.01 to 15% by weight of the total food ,

The healthy beverage composition may be added in an amount of 0.02 to 10 g, preferably 0.3 to 1 g, based on 100 ml.

Among the health functional foods of the present invention, there is no particular limitation on liquid ingredients other than those containing the Clostridium syndense strain as an essential ingredient in the indicated ratio as an essential ingredient in beverages, and various flavoring agents or natural carbohydrates, etc. May contain as an additional component.

Examples of the above-described natural carbohydrates are monosaccharides such as disaccharides such as glucose and fructose, such as maltose, sucrose, and the like, and polysaccharides such as dextrin, cyclodextrin, and the like. Sugar and sugar alcohols such as xylitol, sorbitol, and erythritol. As flavoring agents other than those described above, natural flavoring agents (taumatin, stevia extract (for example, rebaudioside A, glycyrrhizin, etc.)) and synthetic flavoring agents (saccharin, aspartame, etc.) can be advantageously used. The ratio of the natural carbohydrate is generally about 1 to 20 g, preferably about 5 to 12 g per 100 ml of the composition of the present invention.

In addition to the above, the health functional food of the present invention includes various nutrients, vitamins, minerals (electrolytes), flavors such as synthetic flavors and natural flavors, coloring agents and enhancers (cheese, chocolate, etc.), pectic acid and salts thereof, alginic acid, and It may contain salts, organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohols, carbonates used in carbonated beverages, and the like.

In addition, the health functional food of the present invention may contain flesh for the manufacture of natural fruit juice and fruit juice beverage and vegetable beverage. These components may be used independently or in combination. The proportion of these additives is not so important, but is generally selected from 0 to about 20 parts by weight per 100 parts by weight of the health functional food of the present invention.

Another example of the present invention is Lactobacillus plantarum (Lactobacillus plantarum) KBL396 (KCTC 13278 BP) using the Clostridium difficile growth inhibitory effect of the strain Lactobacillus plantarum (Lactobacillus plantarum) KBL396 ( KCTC 13278 BP) provides a method of inhibiting or enhancing CDI (Clostridium difficile infection) inhibition or curative ability of an individual, comprising administering a strain to an individual in need thereof, and a method of preventing, improving, or treating CDI.

Another example of the present invention is the use of the Lactobacillus plantarum KBL396 (KCTC 13278 BP) strain for inhibiting the growth of Clostridium difficile. Use of the Lactobacillus plantarum KBL396 (KCTC 13278 BP) strain for enhancing the CDI inhibitory or healing ability of an individual, and Lactobacillus plantarum KBL396 (Lactobacillus plantarum) for the prevention, improvement or treatment of CDI ( KCTC 13278 BP) strain is provided.

The'individual' refers to an animal that is expected to exhibit an effect according to the Lactobacillus plantarum KBL396 (KCTC 13278 BP) strain by administering a composition containing the strain or strain according to the present invention as an active ingredient, The animals include, for example, dogs, monkeys, goats, pigs, mice, and the like, and are preferably humans.

The'prevention' refers to any action of inhibiting or delaying the progress of CDI by administration of a composition comprising the strain or strain according to the present invention as an active ingredient.

The'treatment' or'improvement' refers to any action in which symptoms of CDI are improved or beneficially changed by administration of a composition comprising the strain or strain according to the present invention as an active ingredient.

The'administration' means providing a pharmaceutical composition comprising a predetermined strain or strain according to the present invention as an active ingredient to an individual by any suitable method.

Hereinafter, the present invention will be described in more detail by the following examples or experimental examples. However, the following examples are for illustrative purposes only, and the scope of the present invention is not limited thereto.

Experimental Example 1 . For CDI prevention and treatment Candidate strain secure

The research team intensively isolated candidate strains from feces provided by more than 40 healthy adult volunteers. For the comparison of efficacy, several commercial strains and 12 strains of a total of 5 species were cultured to conduct C. difficile inhibition experiments. Particularly, considering that most of the candidate strains are obligate anaerobes, all processes were performed under the anaerobic system of Go Bio Lab. The strains used for the C. difficile inhibitory effect comparison experiment were C. difficile KCTC 5009 strain and C. difficile ATCC43255 strain, and the growth inhibitory effect on the strain was compared. After diluting the OD of the C. difficile strain cultured in the liquid medium for 4 hours to 0.3 and re-inoculating the liquid medium at a concentration of 2%, the CD culture solution and the prepared test strain supernatant were mixed at a ratio of 1:1 at 37°C. Incubated for 24 hours, OD was measured to evaluate the CD growth rate.

As a result, Lactobacillus plantarum (Lactobacillus plantarum) KBL396 (KCTC 13278 BP) strain could be selected as a strain excellent in growth inhibition against C. difficile KCTC 5009 strain and C. difficile ATCC43255 strain. The Lactobacillus plantarum KBL396 (KCTC 13278 BP) strain showed superior growth inhibitory ability of C. difficile strains compared to other strains, Bacteroides caccae strain, Bacteroides ovatus strain, Faecalibacterium prausnitzi, and Roseburia intestinalis strain (Figure 1) Reference).

Experimental Example 2 . Lactobacillus Plantarum (Lactobacillus plantarum ) KBL396 ( KCTC 13278 BP)( Lp ) Strain of C. difficile Inhibitory effect on growth

In the C. difficile growth inhibition experiment, the inhibitory ability of C. difficile was confirmed using the supernatant obtained after culturing each isolated strain for 24 hours. Considering that the metabolism of bile affects spore germination and growth of C. difficile, the same experiment was conducted by adding bile of an appropriate concentration to the C. difficile culture medium so that candidate strains can metabolize bile. Observed. The concentration of bile acids in the small intestine of a living body was 0.2 to 2.0% (wt/vol) (Kristoffersen et al., 2007. Journal of Bacteriology). By adjusting the concentration of bile acids from 0 to 2.0% (wt/vol.), the inhibitory effect of C. difficile of the candidate strain was confirmed in the in vitro environment. Specifically, to perform the C. difficile viable bacteria inhibition assay, the test strain was inoculated into a liquid medium containing bile acids at concentrations of 0, 0.5, 1%, and 2%, cultured at 37°C for 24 hours, and then the supernatant was used. I did. After diluting the OD of the C. difficile strain cultured in the liquid medium for 4 hours to 0.3 and re-inoculating the liquid medium at a concentration of 2%, the CD culture solution and the prepared test strain supernatant were mixed at a ratio of 1:1 at 37°C. Incubated for 24 hours, OD was measured to evaluate the CD growth rate.

As a result, when the bile acid concentration increases, the CD inhibitory ability of LP increases, but it was confirmed that CD can be inhibited even in the absence of bile acids (Fig. 2). In other words, it can be seen that LP has both a bile acid concentration-dependent inhibitory effect and an independent CD inhibitory effect.

Experimental Example 3 . Mouse model to confirm CDI prevention (prevention model)

In the CDI prophylaxis animal model, C57BL/6 mice that were negatively administered antibiotics for 3 days to disturb intestinal microbial strains were orally administered the candidate strain for 2 days, followed by intraperitoneal administration of clindamycin and infection with C. difficile, resulting in CDI symptoms of the candidate strain ( Body weight change) and the efficacy on survival were measured every day for 3 weeks (see Fig. 3).

In the case of LP, compared to the control group, the effect of significantly preventing weight loss was exhibited, and this phenomenon continued to appear even in the recovery phase (see Fig. 4).

In addition, the survival rate also showed an effect of significantly increasing the survival rate in the case of the resulting LP compared to the control group (see FIG. 5).

Experimental Example 4 . Candidate strain Mechanism research experiment on efficacy

To study the mechanism of the efficacy of the candidate strain, profile analysis of representative bile acids (DCA and LCA) among feces before/after C. difficile infection was performed using LC-MS/MS.

After 24 hours of C. difficile infection in the group that ate LP in vivo, the concentrations of DCA (deoxycholic acid) and LCA (lithocholic acid) in feces were measured, and the concentration of DCA and LCA in feces was significantly increased. (See Fig. 6).

In addition, microbial analysis was performed that significantly changed between the LP administration group and the infection control group, and for this purpose, bacterial DNA was extracted from feces obtained on the 7th day of the animal experiment (24 hours after C. difficile infection) using the Qiagen FastStool DNA isolation kit. Then, PCR (polymerized chain reaction) was performed using primers corresponding to the V4-V5 region of the bacterial 16S rRNA gene. The amplified 16S rRNA gene profile was NGS sequenced using Illumina Miseq, followed by OTU analysis and taxon analysis using the Qiime 1.8 pipeline.

As a result, it was confirmed that after 24 hours of C. difficile infection in mice, Enterobacteriaceae were significantly increased in the infected control group, whereas Clostridium sp., Coprococcus ssp., Ruminococcaceae, etc., known as SCFA-producing strains, were significantly increased in the LP administration group. . The LefSe method was used for the above statistical analysis (see Fig. 7).

In addition, in order to identify the microbial species associated with C. difficile infection, microbial species significantly associated with the abundance of C. difficile were analyzed through fecal microbial analysis 24 hours after infection. Spearman correlation was used for the correlation analysis. As a result of the analysis, it was confirmed that C. difficile and Clostridium sp., Coprococcus ssp., Ruminococcaceae, etc. showed a significant negative correlation (see Fig. 8).

Following the above analysis, a functional microbial analysis was performed that significantly changed between the LP administration group and the infection control group. To this end, the OTU and biom files picked using Greengenes DB (gg _13.5.fasta) and Qiime pipeline were collected through PICRUSt. After switching to a functional metagenome, the statistical significance of the changed metabolic pathway was analyzed using LefSe analysis.

Compared to the infection control group, significant increased microbial metabolic pathways, including vitamin B6 metabolism, amino acid metabolism, nitrogen metabolism, glycoxylate and carboxylate metabolism, energy metabolism, and methanol metabolism, were identified in the LP group. (See Fig. 9).

As a result of analyzing the changes in the secondary bile acid biosynthesis pathway among fecal microbial microbial in the LP administration group and the infection control group, it was found that genes related to the secondary bile acid biosynthesis were increased in the LP administration group compared to the infection control group (see FIG. 10).

In order to analyze the correlation between the amount of bile acids in feces and functional microbial analysis, based on the change in the function of bile acids-related microorganisms in feces during LP administration, five of the bile acids in feces were directly quantified, and the concentration of bile acids and various The correlation between microbial metabolic pathways was analyzed.

As a result of the analysis, it was confirmed that DCA and LCA, which were significantly increased in the LP administration group, showed a very high correlation with the changed microbial metabolic pathways due to LP administration (see FIG. 11).

In vitro, the changes in the composition of bile acids according to Lp culture were examined, and for this purpose, mixed bile acids containing CA, CDCA (chenodeoxycholic acid), LCA, DCA (deoxycholic acid), and UDCA (Ursodeoxycholic acid) were used in 1% ( wt/vol) Lp was inoculated and cultured in MRS culture medium, and changes in bile acid composition before and after culture were quantitatively analyzed using HPLC-MS/MS.

As a result, by the LP culture, CA decreased and the concentration of CDCA and LCA increased (see Fig. 12). In addition, when LP was cultured in a medium containing TCA (taurocholic acid), TDCA (taurodeoxycholic acid), and TCA/TDCA mixture) in MRS, it was confirmed that deconjugation of TCA and TDCA occurred to generate CA and DCA (see FIG. 13). ). It was confirmed that LP's BSH (bile salt hydrolase) was present and was an effect.

Experimental Example 5 . Mouse model to confirm CDI recurrence (relapse model)

Confirmation of recurrence of CDI The animal model was confirmed by oral administration of C. difficile 2 days after ingesting the candidate strain. After 21 days, drinking water of antibiotics was re-dosed, and recurrence was confirmed through weight change of C. difficile (see Fig. 14).

As a result, in the case of LP showed the effect of significantly recovering the body weight compared to the control (see Fig. 15).

[Consignment institution]

Name of donated institution: Korea Research Institute of Bioscience and Biotechnology

Accession number: KCTC13278BP

Consignment Date: 2017529

Claims (11)

1. Clostridium difficile (Clostridium difficile) Lactobacillus plantarum (Lactobacillus plantarum) KBL396 (KCTC 13278 BP) strain, characterized in that it has a growth inhibitory effect.
2. The Lactobacillus plantarum KBL396 (KCTC 13278 BP) strain according to claim 1, wherein the Clostridium difficile growth inhibitory effect is bile acid independent.
3. Lactobacillus plantarum (Lactobacillus plantarum) KBL396 (KCTC 13278 BP) strain or CDI (Clostridium difficile infection) treatment or prevention pharmaceutical composition comprising a strain or a culture thereof.
4. Lactobacillus plantarum (Lactobacillus plantarum) KBL396 (KCTC 13278 BP) strain or CDI (Clostridium difficile infection) alleviation or improvement health functional food comprising a strain or a culture therefor.
5. The pharmaceutical composition for the treatment or prevention of Clostridium difficile infection (CDI) according to claim 3, wherein the Clostridium difficile infection (CDI) is a recurrent CDI.
6. The health functional food for alleviating or improving CDI (Clostridium difficile infection) according to claim 4, wherein the Clostridium difficile infection (CDI) is a recurrent CDI.
7. The pharmaceutical composition for the treatment or prevention of Clostridium difficile infection (CDI) according to claim 3, wherein the Clostridium difficile infection (CDI) is caused by an intestinal microbial imbalance.
8. The health functional food for alleviating or improving CDI (Clostridium difficile infection) according to claim 4, wherein the Clostridium difficile infection (CDI) is caused by an intestinal microbial imbalance.
9. A method of preventing, improving or treating CDI by administering an effective amount of Lactobacillus plantarum KBL396 (KCTC 13278 BP) strain to an individual in need thereof.
10. Use of Lactobacillus plantarum KBL396 (KCTC 13278 BP) strain to inhibit the growth of Clostridium difficile in an individual.
11. The use of Lactobacillus plantarum KBL396 (KCTC 13278 BP) strain for the manufacture of a pharmaceutical preparation for preventing, improving or treating CDI.

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