Lacticaseibacillus paracasei DG (registered trademark) CNCM I-1572 DSM 34154 for use in a method for treating dysbiosis in patients with IBS

Abstract

The object of the present invention is to use a bacterial strain named Lactobacillus paracasei DG (registered trademark) CNCM I-1572 DSM 34154 for the treatment of dysbiosis, in particular for use in a method for treating dysbiosis in patients with IBS (irritable bowel syndrome), preferably patients without constipation and with a high level of Collinsella aerofaciens in the feces.

Description

[Technical Field]

[0001] The object of the present invention is to use a bacterial strain named Lacticaseibacillus paracasei DG (registered trademark) CNCM I-1572 DSM 34154 for use in a method for treating dysbiosis (an imbalance in the intestinal microbiota, dysbiosis), particularly in patients with IBS (irritable bowel syndrome), preferably IBS patients without constipation and with high levels of Collinsella aerofaciens in their feces. [Background technology]

[0002] Technology background Irritable bowel syndrome (IBS) is one of the most common gastrointestinal disorders, affecting approximately 11% of the population in the United States and Europe. Recurrent abdominal pain in IBS is associated with changes in bowel habits. Traditionally, IBS has been considered a disorder involving bowel motor abnormalities and a psychosomatic component related to visceral hyperalgesia. Despite the absence of apparent physiological irregularities at the gastrointestinal level, quantitative morphological and molecular techniques have made it possible to demonstrate changes in the gastrointestinal mucosa and / or lumen at the tissue, cellular, and molecular levels in the majority of patients with IBS.

[0003] Furthermore, there is some experimental evidence suggesting that the qualitative and quantitative composition of the gut microbiota contributes to the pathophysiology of IBS. Firstly, prospective studies have shown that 3%–36% of intestinal infections cause significant disruption of the intestinal microbial ecosystem, resulting in a new diagnosis of IBS called post-infection IBS. Secondly, antibodies against flagellin (a globular protein found in the flagella of commensal bacteria inhabiting the human gut), toxin B and vinculin (related to changes in the gut microbiota), as well as elevated levels of human β-defensin-2 (an inducible antimicrobial protein), have been identified in at least one subgroup of patients with IBS. These data suggest the presence of a host immune response to components of the gut microbiota. In addition, many studies have reported that the composition and stability of the gut microbiota in subjects with IBS change over time. These studies are not entirely comprehensive and cannot draw definitive conclusions, but they still indicate that the gut microbiome of patients with IBS is significantly different from that of controls.

[0004] Further evidence suggests that the composition of the gut microbiota may have some influence on the pathophysiology of IBS syndrome, and that the modification of the gut microbiota with probiotics and non-absorbable antibiotics may, in some cases, improve patients' symptoms. This indicates that the interaction between gut bacteria and the host is involved in the progression and development of symptoms in patients with IBS.

[0005] Probiotics are defined as live microorganisms that, when administered in appropriate amounts, provide health benefits to the host. Their functions include, for example, preventing the overgrowth of potentially harmful bacteria in the gut, increasing the resistance of this anatomical region to pathogen invasion, inducing the secretion of soluble factors such as cytokines and antimicrobial peptides, and strengthening epithelial barrier function.

[0006] Reviews and meta-analyses of scientific literature generally indicate that microorganisms belonging to the probiotic class can provide therapeutic benefits for IBS symptoms. However, a general beneficial effect on symptoms was only observed after integrating data from various studies, regardless of the probiotic used. In contrast, analyses of individual subgroups of patients responding to specific treatments depending on the type of probiotic used did not show a statistically significant benefit from administration. The variety of probiotic species, strains, and dosages used in various studies, and in some cases even involving very limited case reports, makes it difficult to draw specific conclusions about what the optimal probiotic strategy, if any, is for treating IBS.

[0007] Furthermore, the mechanisms by which probiotics exert beneficial effects on humans are largely insufficient. The few available clinical trials have reported that some strains possess anti-inflammatory properties and / or may influence the qualitative / quantitative composition of the microbiome. In fact, certain strains show elevated dysbiosis levels compared to other strains and are present at high concentrations in IBS patients, including Ruminococcus bromii and Ruminococcus spp.

[0008] In clinical trials, O'Mahony L. et al. (Gastroenterology 2005, 128) (Non-patent Literature 1) reported that Bifidobacterium longum subsp. Infantis 35624, rather than Lactobacillus salivarius, normalized the IL-10 / IL-12 interleukin ratio, an indicator of the pro-inflammatory helper T1 immune response, in patients with IBS. In a study of healthy volunteers, Ferrario C. et al. (J Nutr 2014, 144) (Non-patent Literature 2) reported that L. casei DG Ingestion of L. casei DG (CNCM I-1572) has been reported to significantly regulate the levels of Clostridiales (now reclassified into Eubacteriales) bacteria and butyrate in feces, potentially offering benefits to host health. Furthermore, D'Inca et al. (Dig Dis Sci 2011, 56) (Non-Patent Literature 3) demonstrated that rectal administration of L. casei DG (CNCM I-1572) significantly reduced the levels of mRNA encoding TLR-4 and IL-1β, and significantly increased IL-10 in the colonic mucosa of patients with mild left-sided ulcerative colitis.

[0009] Khlinov et al. (Experimental & clinical gastroenterology, vol.1 (6), 2021-08-31, pages 57-62) (Non-patent document 4) reported a study of patients with IBS-C treated with L. paracasei DG containing mebeveline hydrochloride and fructooligosaccharides compared to placebo.

[0010] It should be noted that, following the reclassification of the Lactobacillus genus published by Zheng et al. in the scientific journal Int. J. Syst. Evol. Microbiol., 70(4):2782-2858, 2020 (Non-Patent Literature 5), the strain L. casei DG® (CNCM I-1572) or L. paracasei DG® (CNCM I-1572) was re-deposited on February 2, 2022, as Lacticaseibacillus paracasei DG I-1572 DSM 34154. The two names mentioned above are interchangeable as they always refer to the same strain (bacterial strain).

[0011] A study by Cremon C. et al. (UEG Journal, 2018, 6) (Non-patent Literature 6) reports on a randomized pilot clinical trial investigating the effects of lacticaseibacillus paracasei DG I-1572 DSM 34154 on clinical factors and microbiome composition in patients with diarrheal, constipated, mixed, and unclassifiable intestinal IBS. Specifically, administration of this strain significantly reduced Ruminococcus bacteria, increased short-chain fatty acids acetate and butyrate, and decreased IL-15. However, a statistically significant reduction in IBS symptoms could not be demonstrated.

[0012] Therefore, there is still a need for specific probiotic therapies that can act at the level of the gut microbiota and / or inflammatory factors in patients with IBS, alleviate their symptoms, be effective and safe to use, and overcome the shortcomings associated with known treatments. [Prior art documents] [Non-patent literature]

[0013] [Non-Patent Document 1] O'Mahony L. et al. (Gastroenterology 2005, 128) [Non-Patent Document 2] Ferrario C. et al. (J Nutr 2014, 144) [Non-Patent Document 3] D'Inca et al. (Dig Dis Sci 2011, 56) [Non-Patent Document 4] Khlinov et al. (Experimental & clinical gastroenterology, vol.1 (6), 2021-08-31, pages 57-62) [Non-Patent Document 5] Zheng et al. (Int. J. Syst. Evol. Microbiol., 70(4):2782-2858, 2020) [Non-Patent Document 6] Cremon C. et al. (UEG Journal, 2018, 6) [Overview of the project]

[0014] overview The object of the present invention is to provide a probiotic for use in a method of treating dysbiosis in patients with IBS, preferably in patients with non-constipation-type IBS, and in particular to reduce or decrease the presence of Cholinthera aerofaciens in the intestinal microbiota of said patients. Reducing dysbiosis also means reducing the amount of intestinal pathobiont bacteria such as Bacteroides plebeius, Dorea spp., Ruminococcus bromii, Ruminococcus, Blautia spp., and Cholinthera aerofaciens.

[0015] Another object of the present invention is to provide a probiotic that effectively and safely alleviates intestinal symptoms in patients with IBS, preferably those with non-constipation-type IBS.

[0016] These and other objects are achieved by the object of the present invention to provide an effective probiotic that treats the dysbiosis of the gut microbiota in patients with IBS and as a result reduces the symptoms of the disease.

Brief Description of the Drawings

[0017] [Figure 1A] Figure 1A: Study design with a total treatment period of 18 weeks. [Figure 1B] Figure 1B: Summary of IBS patients concluded to be per protocol in the study scheme (A) and the probiotic group (arm) included in this study that met the trial implementation plan of the PROBE-IBS / 2 trial (B). NRS: numeric rating scale. [Figure 2]Figure 2: Fecal bacterial taxa that distinguish responder (R) and non-responder (NR) patients with non-constipation IBS. P-values were by Mann-Whitney tests performed using CLR-transformed bacterial abundances. P-values with red background indicate taxa increased in the R group. Heatmaps represent the mean CLR-transformed abundances of reported taxonomic units. The taxonomic lineage of each taxon is shown below. p: phylum, c: class, o: order, f: family, g: genus, s: species. Modifications / updates to the taxonomy regarding the GreenGenes database nomenclature are shown in purple and are based on the NCBI Taxonomy database. Taxonomic names written in blue were determined by manual BLASTN searches in GenBank using the corresponding read sequences. The black and green bar histograms on the right show the contribution of each bacterial taxon to the first two components of the PLS discriminant analysis (PLSDA) biplot shown in Figure 3. Roman numerals are shown to associate bacterial taxa with the PLSDA loading plots in Figure 3. Eleven bacterial taxa that significantly contributed to the explanation of variation in the PLSDA analysis are shown in bold. [Figure 3] Figure 3: Biplots of PLS discriminant analysis (PLSDA) by prediction background for responder (R) and non-responder (NR) patients with non-constipation IBS (panel A) and for R, NR, and healthy control (HC) groups (panel B). Roman numerals in panel A refer to the bacterial taxa in Figure 2. Percentages indicate the explained variation on each axis. [Figure 4]Figure 4: Dot plot of the most abundant bacterial taxa found to better differentiate responder and non-responder patients. HC: healthy control group (n=100), R and NR: responder NC-IBS patients and non-responder NC-IBS patients, other NC-IBS: other non-constipation-type IBS patients recruited at baseline (n=161). Statistics follow the Mann-Whitney test. *: P<0.05, **: P<0.01. [Figure 5] Figure 5: Graph of the linear discriminant analysis (LDA) effect size (LEfSe) of responder(R) NC-IBS patients in the probiotic group of the clinical trial. The LDA score indicates a significantly higher (P<0.05) classification group before (V2; LDA negative) or after (V4; LDA positive) probiotic intake. The names of the classification groups are abbreviated as follows: p: phylum, c: class, o: order, f: family, g: genus, s: species. [Figure 6] Figure 6: Correlation analysis between bacterial taxa and serum markers for permeability (Panel A) and liver and kidney function (Panel B). In Panel A, V1 / V2 shows the correlation analysis performed using data from blood samples collected before the start of the clinical trial (visit V1) and stool samples collected before the probiotic intervention (visit V2). V4 shows the correlation analysis performed using data from blood and stool samples collected at the end of the probiotic intervention (visit V4). Only taxa with the same significant correlation in V1 / V2 and V4 are shown. The heatmap represents the τ coefficient of Kendall's rank correlation. Asterisks indicate significance in Kendall's rank correlation. *P<0.05, **P<0.01, ***P<0.001. The taxonomic phylogenetic relationships of each taxa are shown below. p: phylum, c: class, o: order, f: family, g: genus, s: species. ALT: Alanine aminotransferase, AST: Aspartate aminotransferase, Bil: Bilirubin, ALK: Alkaline phosphatase, BUN: Blood urea nitrogen, Crea: Creatinine. [Modes for carrying out the invention]

[0018] Description of the Invention The present invention includes the following embodiments.

[0019] Embodiment [1]: A bacterial strain belonging to the species Lacticaseibacillus paracasei, deposited as Lacticaseibacillus paracasei DG I-1572 DSM 34154, for use in a method of treating dysbiosis of the intestinal microbiota of a subject having IBS, wherein the subject is classified as non-constipated.

[0020] Embodiment [2]: Bacteria (or bacterial strains) for use according to Embodiment [1], wherein the subject having IBS has elevated levels of Collinsella aerofaciens, Bacteroides plebeius, Dorea spp., Ruminococcus bromii, Ruminococcus spp., and Blautia spp.

[0021] Embodiment [3]: Bacteria (or bacterial strains) for use according to Embodiment [1] or [2], wherein the above-mentioned elevated levels of Cholinthera aerofasiens, Bacteroides plebius, Dorea, Ruminococcus bromii, Ruminococcus, and Blautia are present in the feces of subjects having IBS as required.

[0022] Embodiment [4]: ​​A bacterium (or bacterial strain) for use according to any one of Embodiments [1] to [3], wherein the subject is an IBS patient, preferably a non-constipation type IBS patient.

[0023] Embodiment [5]: The bacteria (or bacterial strain) for use according to any one of Embodiments [1] to [4], wherein the bacteria (or bacterial strain) is administered orally, preferably in the form of an oral capsule [Enterolactis (R) Plus capsule] which is a human supplement.

[0024] Embodiment [6]: A bacterium (or bacterial strain) for use according to any of Embodiments [1] to [5], wherein the human supplement is preferably administered twice daily, preferably for 4 to 24 weeks, more preferably for 8 to 12 weeks.

[0025] Embodiment [7]: Each of the capsules is 1 × 10 6 ~1×10 12 CFU / capsule, more comfortable 1x10 8 ~1×10 10 CFU / capsule, even more preferably 10x10 9 ~50×10 9 Bacteria (or bacterial strains) for use according to any of embodiments [1] to [6], comprising CFU / capsules.

[0026] Embodiment [8]: The bacteria (or bacterial strain) for use according to any one of Embodiments [1] to [7], wherein the bacteria (or bacterial strain) are present in each of the capsules in a solid form, preferably in a powder, dried or freeze-dried form.

[0027] Embodiment [9]: A bacterium (or bacterial strain) for use according to any of Embodiments [1] to [8], wherein the daily dose may consist of 1 to 4 capsules / day, preferably 2 to 3 capsules / day.

[0028] Embodiment

[10] : The bacteria (or bacterial strain) for use according to any of Embodiments [1] to [9], wherein the bacteria (or bacterial strain) is used in a method for treating abdominal symptoms in patients with IBS, preferably in patients with non-constipation type IBS.

[0029] According to one aspect of the present invention, the present invention relates to a bacterial strain belonging to the species Lacticaseibacillus paracasei, deposited as Lacticaseibacillus paracasei DG I-1572 DSM 34154, for use in a method of treating dysbiosis of the intestinal microbiota of a subject having IBS, wherein the subject is classified as non-constipated.

[0030] Preferably, the subjects having IBS show elevated levels (abundances) of Collinsella aerofaciens, Bacteroides plebeius, Dorea spp., Ruminococcus bromii, Ruminococcus spp., and Blautia spp.

[0031] Preferably, the following species, with elevated levels (abundances), are present in the feces of subjects having in need IBS: Cholinthera aerofasciens, Bacteroides plebius, Drea, Ruminococcus bromii, Ruminococcus, and Brautia.

[0032] Preferably, the subject is an IBS sufferer, and preferably, the subject is a non-constipation type IBS sufferer.

[0033] Preferably, the bacteria are administered orally, preferably in the form of Enterolactis® Plus capsules, which are a human supplement.

[0034] Preferably, the human supplement is administered twice a day, preferably for 4 to 24 weeks, more preferably for 8 to 12 weeks.

[0035] Preferably, each of the capsules contains 1×10 6 ~1×10 12 CFU / capsule, more preferably 1×10 8 ~1×10 10 CFU / capsule, even more preferably 10×10 9 ~50×10 9 CFU / capsule.

[0036] Preferably, the bacteria are present in each of the capsules in a solid form, preferably in a powder, dried or lyophilized form.

[0037] Preferably, the daily dose may include 1 to 4 capsules / day, preferably 2 to 3 capsules / day.

[0038] Preferably, the bacteria are used in a method for treating abdominal symptoms of IBS patients, preferably non-constipated IBS patients.

[0039] According to one aspect of the present invention, it relates to a probiotic based on Lactiplantibacillus paracasei DG I-1572 DSM 34154 for use in a method for treating dysbiosis of the gut microbiota in patients with IBS, particularly when the patient is classified as non-constipated.

[0040] The present invention relates, in one aspect, to a probiotic based on Lacticaseibacillus paracasei DG® I-1572 DSM 34154 for use in a method of treating subjects with high levels of Cholinthera aerofaciens, Bacteroides plebius, Dorea species, Ruminococcus bromii, Ruminococcus species, and Brautia species in their feces. Preferably, the Lacticaseibacillus paracasei DG® I-1572 DSM 34154 is used in subjects suffering from IBS, preferably non-constipation type IBS. Preferably, the Lacticaseibacillus paracasei DG® I-1572 DSM 34154 is administered orally, preferably in the form of Enterolactis® Plus capsules. Preferably, the probiotic, Lacticaseibacillus paracasei DG® I-1572 DSM 34154, is administered preferably twice daily for 4 to 24 weeks. Preferably, Lacticaseibacillus paracasei DG® I-1572 DSM 34154 is used for the treatment of abdominal symptoms in patients with IBS, preferably in patients with non-constipation-predominant IBS.

[0041] In particular, Lacticaseibacillus paracasei DG(registered trademark) I-1572 DSM 34154 reduces abdominal pain in NC-IBS patients by 40% to 60%. It also improves the fecal type in the same patients.

[0042] In this specification, intestinal dysbiosis refers to a condition in which the quantity and types of microorganisms present in the gastrointestinal tract are altered compared to a normal physiological state, and this alteration is causally related to a pathological or dysfunctional state.

[0043] The group of IBS patients considered to be non-constipation-type mainly consists of IBS-D patients who primarily experience diarrhea and IBS-M patients who have mixed bowel movements.

[0044] Lacticaseibacillus paracasei DG I-1572 DSM 34154 is currently included in dietary supplements registered as Enterolactis® Plus, and is also currently known as registered trademark L.casei DG (CNCM I-1572) or L.paracasei DG (CNCM I-1572). It should be noted that, following the reclassification of the Lactobacillus genus published by Zheng et al. in the scientific journal Int. J. Syst. Evol. Microbiol., 70(4):2782-2858, 2020 (Non-Patent Literature 5), the strain L. casei DG (registered trademark) (CNCM I-1572) or L. paracasei DG (registered trademark) (CNCM I-1572) was re-deposited on February 2, 2022, as Lacticaseibacillus paracasei DG I-1572 DSM 34154. The two names mentioned above are interchangeable as they always refer to the same strain (bacterial strain).

[0045] The probiotic for use according to the present invention is Enterolactis® Plus, an oral capsule containing 24 billion or more L. casei DG® (Lactobacillus paracasei CNCM I-1572) (Lactobacillus paracasei DG I-1572 DSM 34154) per capsule, administered at a rate of 1 to 3 capsules per day, preferably 2 capsules per day, for 8 to 24 weeks, for example, 12 weeks.

[0046] Preferably, the capsule is administered on an empty stomach, for example, one hour before or two hours after a main meal.

[0047] As fully demonstrated in the following experimental part, the probiotics for use according to the present invention can improve abdominal symptoms in patients with non-constipation-predominant IBS. This improvement is related to the ability of the probiotics for use according to the present invention to intervene in intestinal dysbiosis afflicting a specific subgroup of patients with non-constipation-predominant IBS, particularly its ability to reduce the presence of Cholinthera aerofasciens in the gut microbiota of said patients.

[0048] As previously reported, the probiotic for use according to the present invention is lacticaseibacillus paracasei DG I-1572DSM 34154), which is administered orally and has been shown to be effective in treating intestinal dysbiosis in patients with non-constipation-type IBS, particularly when high levels of Cholinella aerofaciens are present in the gut microbiota.

[0049] In another embodiment, the object of the present invention is a method for reducing or decreasing abdominal symptoms in a subject having IBS, the method comprising administering one or more capsules of Enterolactis® Plus for use according to the present invention to the subject in need, one or more capsules per day, for example, two capsules per day, preferably for 4 to 24 weeks, and more preferably for 8 to 12 weeks. Herein, subject is understood to mean human.

[0050] The probiotics for use according to the present invention may be used alone or, if desired or necessary, in combination with other substances, provided that such other substances do not interfere with or limit the effects of the present invention.

[0051] The following experimental section presents the results of a clinical trial concerning the use of probiotics according to the present invention, comparing their use with a placebo.

[0052] Experiment section Objective: One of the objectives of this study is to identify markers for recognizing non-constipation type (NC) IBS patients who may show significant clinical improvement with treatment using the probiotic strain lacticase ibacillus paracasei DG (LDG).

[0053] Design: Post-hoc analysis of samples collected during a multi-center, randomized, double-blind, parallel-group, placebo-controlled trial. In this trial, NC-IBS patients were randomized to receive at least 24 billion CFU of LDG capsules or placebo capsules twice daily (bid) for 12 weeks. The primary endpoint was a composite response based on improvement in abdominal pain and stool type. Fecal microbiome and intestinal serum markers such as PV1, liver function, and kidney function were investigated.

[0054] Results: Responders (R) (25%) in the probiotic group differed from non-responders (NR) in the abundance of 18 bacterial taxa, including Coriobacteriaceae, Drea, and Cholinthera aerofaciens, which are excessively present in R patients. These taxa also distinguished R (not NR) from the healthy control group. Probiotic intervention significantly reduced the abundance of these bacteria in R patients, but not significantly in NR patients. Similar results were obtained for C. aerofaciens from data analysis of previous clinical trials conducted for IBS using the same probiotic. Ultimately, C. aerofaciens was positively correlated with PV-1 and liver function markers.

[0055] Advantageously, the bacterial strain L. casei DG® (Lacticaseibacillus paracasei DG I-1572 DSM 34154)-Enterolactis® Plus is effective in NC-IBS patients who have a higher proportion of potentially pathogenic locustodial bacteria. Of these, C. aerofasiens has emerged as a potential predictor of probiotic efficacy.

[0056] Purpose of the exam Primary objective: To evaluate the effect of L. casei DG® on abdominal symptoms in non-constipated patients with irritable bowel syndrome (IBS) and patients with symptoms meeting Rome IV criteria for the diagnosis of IBS without constipation (i.e., patients with IBS-D and IBS-M).

[0057] Secondary objective: To evaluate the following parameters: • Presence or absence of IBS symptoms, • Daily stool consistency • Patient's overall satisfaction, • Overall quality of life ·Psychological disorders, • Taking rescue medications, • Composition of gut microbiota and metabolites, ·Intestinal permeability, • Recall of L. casei DG (registered trademark) strain from feces.

[0058] Experimental Design Multicenter, randomized, double-blind, placebo-controlled, parallel-group comparative trial This study consisted of a two-week initial (induction) phase during which data on abdominal and alveolar pain and discomfort were collected, and these data were useful in establishing baseline levels used to evaluate therapeutic efficacy. A 12-week phase (treatment) followed, during which each patient took two oral capsules of Enterolactis® Plus (a single-strain probiotic formula containing at least 24 billion CFUs of L. paracasei DG) or Enterolactis®. Participants took two placebo capsules that were physically indistinguishable from Lactis® Plus capsules. Finally, an early (follow-up) condition lasted for 4 weeks, during which no capsules were taken. Thus, according to the study design in Figures 1A and 1B, the total treatment period for each cycle was 18 weeks.

[0059] For each patient, three stool samples (at visits at the end of weeks 2, 14, and 18) and three blood samples (at the initial visit (at zero) and at visits at the end of weeks 14 and 18) were collected. These samples were subjected to analysis for various markers. In particular, for fecal samples, taxonomic characterization of bacterial populations and quantification of short-chain fatty acids (acetate, butyrate, propionate, valerate, isovalerate, lactate, and succinate) were performed. For blood samples, the following were quantified: PV-1, liver [alanine aminotransferase (ALT), aspartate aminotransferase (AST), bilirubin (Bil), alkaline phosphatase (ALK)], and kidney [blood urea nitrogen (BUN) and creatinine (Crea)], which are markers of permeability function.

[0060] patient The patients selected for this study were men or women aged 18 or older diagnosed with IBS without constipation according to the Rome IV criteria.

[0061] The diagnostic criteria for IBS are that symptoms must have started at least 6 months prior to diagnosis, the criteria must have been met for 3 months prior to the test, there must be recurrent abdominal pain at least one day a week, and the patient must meet two or more of the following criteria: ○ Related to defecation, ○ Accompanied by changes in the frequency of bowel movements, ○ This involves a change in the shape (appearance) of the stool.

[0062] IBS patients without constipation include the following: • IBS-D, where diarrhea is the main symptom: More than a quarter (25%) of bowel movements are of stool type 6 or 7 (Bristol Stool). • IBS with mixed bowel movements (IBS-M): More than one-quarter (25%) of bowel movements are of stool type 1 or 2, and more than one-quarter (25%) of bowel movements are of stool type 6 or 7.

[0063] Inclusion criteria • Between 18 and 65 years old, • A positive diagnosis of IBS without constipation (IBS-D and IBS-M, both in men and women) according to the Rome IV criteria. • If the patient is under 50 years of age, or if the patient has any of the following warning signs, the colonoscopy results from the five years prior to the screening visit must be negative: • Significant weight loss has been recorded in the past 6 months, or • Symptoms occur at night, or • Family history of colon cancer, • There is blood in the stool (excluding blood from hemorrhoids). • If necessary, further relevant screenings or examinations must be negative. • Ability to adhere to the test protocol.

[0064] Exclusion criteria Patients with IBS-C or IBS-U according to the Rome IV criteria, • The presence of any relevant organic, systemic, or metabolic disease (particularly a significant history of cardiac, renal, nervous, psychiatric, neoplastic, endocrine, metabolic, or hepatic disease) or abnormal laboratory values ​​detected during the induction period that are considered clinically significant based on predefined values ​​(e.g., kidney or hepatic function levels more than twice the upper limit). • The presence of an established organic bowel disease, including celiac disease, food allergy, or inflammatory bowel disease (Crohn's disease, ulcerative colitis, diverticular disease, infectious colitis, ischemic colitis, microscopic colitis). • He had previously undergone major abdominal surgery. • Having any type of active malignant tumor, or a history of malignant tumor (patients with a history of other surgically resected malignant tumors and no evidence of recurrence for at least 5 years prior to trial enrollment are acceptable). Defined by a medical history assessment or, if appropriate, a lactose breath test. Untreated food intolerances, including established or suspected lactose intolerance, • Having used probiotics or topical / systemic antibiotics in the past month. • Frequent or regular use of contact laxatives. • Being a pregnant woman or a woman of reproductive age without effective contraception. • Failure to comply with the protocol, • Have you received treatment with an experimental drug within the past 30 days? • A recent history of or suspected history of alcohol abuse or drug addiction. • The presence of a red or white flag according to the criteria of the Rome IV Psychosocial Alarm Questionnaire for Functional Gastrointestinal Disorders.

[0065] Randomization Eligible patients entered a two-week induction period, after which they were randomly assigned in a 1:1 ratio to receive either Enterolactis® Plus treatment or an equivalent sterile product (placebo) with similar color, texture, and taste, twice daily for 12 weeks.

[0066] Effectiveness assessment Primary endpoint: Percentage of patients showing a combined response over 12 weeks: Patients who recorded a reduction of 30% or more from the baseline mean score of the most severe abdominal pain on 50% or more days, and who also had a stool consistency of 5 or less.

[0067] Abdominal pain was assessed using a standard 11-point numerical rating scale (0 = no pain to 10 = the most severe pain imaginable), and for abnormal bowel movements, the frequency and consistency of stools were measured using the Bristol Stool Scale (BSFS).

[0068] Secondary outcome items • Reduction of IBS symptoms. This is evaluated as follows: • Pain reduction: For more than 50% of the duration, the score indicating the most severe abdominal pain decreased by more than 30% from baseline. • Combined response at 4-week intervals, • Improvement in overall symptom score: Improvement of 0 or 1, or 2 or more points from baseline, over 50% or more of the duration. • IBS symptoms were sufficiently reduced for more than 50% of the past few weeks (answered "yes" to the question "Were your IBS symptoms sufficiently reduced in the past week?" for more than 50% of the weeks). • IBS-SSS score assessed at zero and at the end of treatment after 12 weeks (a decrease of at least 50 points is considered clinically significant). • Improvement of stool consistency: Stool consistency score is 5 or less. • Overall satisfaction with treatment as assessed by the VAS scale. • Assessment of quality of life on a 0-100 scale using the validated Short-Form 12 Items Health Survey (SF-12). • Taking rescue medication, • Composition of the gut microbiota and its metabolites (SCFAs, free amino acids, and bioamines), • Intestinal permeability based on evaluation of serum levels of zonulin, citrulline, and PV-1 (using blood samples) • L. casei DG® strain was recovered from feces according to the method described by Arioli et al. (Front. Microbiol 2018. 9:1720).

[0069] Safety evaluation Vital parameters including blood pressure, heart rate, and respiratory rate. A physical examination including assessment of hair and skin condition, lymph nodes, eyes, ENT, chest, respiratory system, cardiovascular system, abdomen, genitourinary system, pelvis, and rectum. • Clinical laboratory tests: • Blood chemistry tests: glucose, blood urea nitrogen (BUN), aspartate aminotransferase (AST), alanine aminotransferase (ALT), bilirubin, alkaline phosphatase, creatinine, Hematology tests: complete blood count and blood composition, platelets. • Adverse events. These are defined as the occurrence of undesirable medical signs, symptoms, or conditions that occur after being continuously monitored during the study and after informed consent has been obtained from the patient.

[0070] Consideration Of the 300 patients initially recruited, 72 patients completed the trial in the probiotic group and 70 patients completed the trial in the placebo group. The 300 recruited patients represent only the PP (per protocol) population and do not represent the ITT (intention to treat) population, which is typically considered in primary analyses. Furthermore, not all PP patients were considered (approximately 235 patients); only patients who deviated from the protocol were considered. This selection was due to the fact that this method only included patients who took the probiotic product, completed the trial, and for whom samples could be collected at various visits.

[0071] As anticipated, the primary endpoint of this trial was the percentage of patients who had a combined response over the 12-week probiotic or placebo period, i.e., the percentage of patients who experienced a 30% or greater reduction in their most severe abdominal pain from the baseline mean score for at least 50% of the duration, and whose stool consistency score was less than 5 for the same duration.

[0072] According to the primary endpoint, 16 patients (23.5%) in the probiotic group and 19 patients (29.2%) in the placebo group were found to have shown a positive response (R, responder) to the probiotic for use according to the present invention.

[0073] Table 1 shows the abdominal pain and fecal type of patients who received treatment at visit 2 (second visit) and visit 4 (fourth visit).

[0074] [Table 1]

[0075] The results obtained are explained below.

[0076] (i) Patients who had no pain at baseline or whose pain level was too low (IBS-SSS total score ≤ 175 and NRS ≤ 3) and who did not show significant improvement after treatment were also included.

[0077] (ii) The diary card states that the severity of pain / annoyance must be indicated daily (because annoyance can be reported interchangeably with pain, it becomes unclear which symptom the patient wanted to report, and it is unclear whether a comparison was made between baseline pain / annoyance and / or pain / annoyance at follow-up visits). (It will become certain.)

[0078] (iii) Analysis of the daily diaries kept by patients during the two-week induction period revealed that while compliance with the eligibility criteria was confirmed at baseline, many patients did not meet the Rome IV criteria for diagnosing IBS-D or IBS-M.

[0079] (iv) As a result, 183 patients were excluded from the ITT population of 264, which decisively reduced the valid sample size for statistical analysis of the results.

[0080] (v) Furthermore, regarding treatment compliance, microbiome analysis showed that L. casei DG® (lacticaseibacillus paracasei DG I-1572 DSM 34154) (Enterolactis® Plus) was not detected in 25 patients in the Enterolactis group (meaning that the patients may not have taken the product sufficiently or, in any case, not taken it correctly). On the other hand, L. casei DG® was detected in 5 patients in the placebo group (it is an ingredient present in other products freely available at pharmacies, and the patients may have found and taken it freely).

[0081] Significant changes in the quantity and types of bacterial populations in the microbiome of subjects administered placebo were far smaller than those observed in the group of subjects taking Enterolactis® Plus. This suggests that the improvement observed in the placebo group of subjects (R) may not be related to changes at the microbiome level, as in patients treated with Enterolactis® Plus, but rather to psychological factors, i.e., the known psychosomatic elements of IBS.

[0082] To determine whether patients who benefited from probiotic treatment possessed distinguishing features from those who did not show significant improvement (R vs. NR (non-responder)), the levels of bacteria and other markers were compared between the two patient groups. This analysis showed that R patients had significantly higher abundances of several microbial groups that cause dysbiosis in IBS patients, including Bacteroides plebius, Dorea, Ruminococcus bromii, Ruminococcus, Blautia, and Cholinthera aerofaciens, compared to healthy subjects. Specifically, principal component analysis based on the abundance of 46 bacterial taxa that were found to differ significantly between R and NR patients indicated that Cholinthera aerofaciens was the most important taxonomic group for distinguishing R from NR patients.

[0083] Similar analyses were performed between R patients and NR patients in the placebo group. In this case, fewer significantly different classification groups were observed. Notably, no significant difference in cholinethera aerofasciens was found between R patients and NR patients.

[0084] Subsequent statistical analysis showed that the probiotic contained in the Enterolactis® Plus formulation for use according to the present invention resulted in a decrease in cholinethera aerofasciens (significant trend, P=0.0681) in responder® patients. In contrast, cholinethera aerofasciens concentrations were not altered by administration of the probiotic for use according to the present invention in either NR patients or placebo patients.

[0085] Further investigation revealed that non-constipation-type IBS patients, particularly responder subjects treated with Enterolactis® Plus for use according to the present invention, had significantly higher initial levels of cholinethera aerofascens in their feces than a control group of 100 healthy adults.

[0086] Furthermore, it was shown that patients whose levels of Cholinella aerofaciens decreased with the use of the probiotic according to the present invention simultaneously showed a significant reduction in abdominal pain (P=0.0497) and a tendency toward a decrease in fecal type (P=0.0709). This suggests that this bacterial strain may be mechanistically related to IBS symptoms.

[0087] In addition to demonstrating the efficacy of the probiotic for use according to the present invention in reducing abdominal symptoms in patients with non-constipation-predominant IBS by lowering the concentration of Cholinthera aerofasciens, experimental data from the described clinical protocol highlight the potential of measuring the concentration of this microorganism as a predictive test to determine whether a particular patient will respond to the probiotic for use according to the present invention, L. casei DG®.

[0088] Quantification of organic acids As previously described by Gargari G, et al. (Environ Microbiol 2018;20:3201-13), organic acids (acetic acid, butyric acid, propionic acid, valeric acid, isovaleric acid, lactic acid, and succinic acid) in fecal samples were detected and quantified by ultrafast liquid chromatography-high-resolution mass spectrometry (UPLC-HR-MS) using an electrospray ionization HESI-II probe (Thermo Scientific, San Jose, California) combined with Exactive Orbitrap MS on an Acquity UPLC separation module (Waters, Milford, Massachusetts).

[0089] Table 2 shows the quantification of organic acids measured at visits V2 and V4.

[0090] [Table 2]

[0091] Quantification of PV-1 Plasmalemma vesicle-associated protein (PLVAP) / PV-1, an endothelial permeability marker, was measured in serum samples using the Human PVLAP ELISA Kit (Fine test, China). Samples were processed according to the manufacturer's instructions, and absorbance at 450 nm was measured using an Eon plate reader. The absorbance data were then interpolated using logarithmic standard curves calculated for each plate analyzed.

[0092] [Table 3]

[0093] Analysis of liver and kidney function markers The following liver and kidney function markers were evaluated in serum samples: alanine aminotransferase (ALT), aspartate aminotransferase (AST), bilirubin (Bil), alkaline phosphatase (ALK), blood urea nitrogen (BUN), and creatinine (Crea). AST and ALT were evaluated by enzyme administration without activation with pyridoxal phosphate (Cobas 8000 - Roche Diagnostics). Bil and ALK were administered by enzyme colorimetric testing (Cobas 8000 - Roche Diagnostics). (as 8000 - Roche Diagnostics). A dynamic enzyme test was performed to evaluate urea, and creatinine was evaluated by dynamic staining (Jaffe method) (Cobas 8000 - Roche Diagnostics).

[0094] [Table 4]

Claims

1. A bacterial strain belonging to the species Lacticaseibacillus paracasei, deposited as Lacticaseibacillus paracasei DG I-1572 DSM 34154, for use in a method of treating dysbiosis of the intestinal microbiota of subjects with IBS, wherein the subject is classified as non-constipated.

2. The bacterial strain for use according to claim 1, wherein the subject having IBS has elevated levels of Collinsella aerofaciens, Bacteroides plebeius, Dorea spp., Ruminococcus bromii, Ruminococcus spp., and Blautia spp.

3. The bacterial strains for use according to claim 2, wherein the aforementioned levels of Cholinthera aerofasiens, Bacteroides plebius, Dorea, Ruminococcus bromii, Ruminococcus, and Brautia are present in the feces of subjects having IBS as required.

4. The bacterial strain for use according to claim 1 or 2, wherein the bacterial strain is administered orally.

5. The bacterial strain for use according to claim 4, wherein the bacterial strain is administered in the form of Enterolactis (R) Plus capsules, which are a human supplement.

6. The bacterial strain for use according to claim 5, wherein the human supplement is administered twice daily.

7. Each of the aforementioned capsules is 1 x 10 ６ ~1 x 10 １２ A bacterial strain for use according to claim 5, comprising CFU / capsule.

8. The bacterial strain for use according to claim 5, wherein the bacterial strain is present in a solid form in each of the capsules.

9. A bacterial strain for use according to claim 5, wherein the daily dose comprises 1 to 4 capsules / day.

10. The bacterial strain for use according to claim 1 or 2, wherein the bacterial strain is used in a method for treating abdominal symptoms in patients with non-constipation-type IBS.

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