Lactobacillus rhamnosus DSM 33960 and its use in a method for preventing and treating urogenital dysbiosis

Abstract

The present invention relates to the strain Lactobacillus rhamnosus DSM 33960 and its use in a method for preventing and treating conditions of female urogenital dysbiosis, preferably vaginal dysbiosis.

Description

Technical Field

[0003] Although characterized by a small number of microbial species, the vaginal microbiome is a dynamic ecosystem that changes throughout a woman's life (from birth to the pre- and post-menopausal stages). In addition, intrinsic factors (such as ethnicity, immune system imbalances, and genetic susceptibility, etc.) and extrinsic factors (such as a vitamin- and folic acid-rich diet, oral contraceptive use, sexual behavior, hygiene habits, and the use of antibiotics or immunosuppressive therapy) can affect the balance of the vaginal microbiome, thereby changing the balance of the vaginal microbiome.

[0004] Alterations in the vaginal microbiota (vaginal dysbiosis) are associated with a number of diseases, such as urogenital tract infections (e.g., Candida infections), reproductive dysfunction, obstetric complications, and cancer. Traditionally, vaginal dysbiosis has been treated with antimicrobial drugs, such as metronidazole and clindamycin. However, antimicrobial drugs have some undesirable effects, such as short-term relapses and the promotion of antibiotic resistance. For these reasons, alternative treatment strategies that can modulate the vaginal microbiota and correct its imbalance have been studied over time. In this context, the administration of probiotics, whether alone or in addition to antibiotic therapy, has received increasing attention. However, the probiotics currently known in the art often have host specificity issues and are therefore not effective in combating vaginal dysbiosis and related diseases. In addition, the probiotics currently known in the art are generally found to be unsafe and therefore unsuitable for use in the medical field, such as for the prevention or treatment of vaginal dysbiosis. In fact, although most probiotics currently known in the art are generally considered safe, both safety and probiotic characteristics are specific to each strain and cannot be considered representative of the entire species to which the strain belongs.

[0005] Furthermore, known probiotic strains often cannot be used in combination with antibiotic therapy.

[0006] International patent application WO 2022 / 009170 A1, and scientific literature publication Pino, A., Rapisarda, AMC, Vitale, SG et al. "A clinical pilot study on the effect of the probiotic Lacticaseibacillus rhamnosus TOM 22.8 strain in women with vaginal dysbiosis," and related "Supplementary information," Sci Rep 11, 2592 (2021), https: / / doi.org / 10.1038 / s41598-021-81931-z, disclose a Lactobacillus rhamnosus (strain TOM 22.8-DSM 33500) for women with vaginal dysbiosis.

[0007] Therefore, there remains a need for bacterial strains, particularly probiotic strains, that can prevent and / or combat vaginal dysbiosis.

[0008] In particular, there is now a need to have available bacterial strains capable of acting at the level of the vaginal microbiota (probiotics) in order to prevent and / or combat imbalances of this microbiota and the associated pathologies, such as urogenital tract infections.

[0009] Purpose of the invention

[0010] It is an object of the present invention to provide isolated bacterial strains of human origin.

[0011] Another object of the present invention is to provide an isolated bacterial strain for use in a method for preventing and treating urogenital dysbiosis, preferably vaginal dysbiosis.

[0012] Another object of the present invention is to provide an isolated bacterial strain effective in preventing and / or treating diseases or conditions associated with urogenital dysbiosis, such as urogenital tract infections, in particular vaginal infections.

[0013] It is a further object of the present invention to provide a probiotic strain which can be used, for example, effectively and safely to rebalance the vaginal microbiota and which is host specific.

[0014] These and other objects are achieved by the object of the present invention, which is to provide an isolated bacterial strain suitable for the prevention and / or treatment of urogenital dysbiosis, in particular vaginal dysbiosis.

[0015] Description of the Invention

[0016] The object of the present invention is to provide a novel isolated microorganism, in particular a novel, genetically unmodified bacterial strain belonging to the genus Lacticaseibacillus, named Lactobacillus rhamnosus CA15, which was deposited by ProBioEtna Srl in the German Collection of Microorganisms (Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSMZ)) on July 28, 2021, with the deposit number DSM 33960.

[0017] The microorganism (strain) according to the present invention, Lactobacillus rhamnosus CA15 DSM 33960 (also referred to as "Lactobacillus rhamnosus DSM 33960" or more simply "DSM33960" in this specification) was isolated from the vaginal ecosystem of healthy asymptomatic Italian women attending the AOU Department of Obstetrics and Gynecology, Policlinico-Vittorio Emanuele POGRodolico, University of Catania. To isolate the strains of the present invention, vaginal secretion samples were processed as previously reported (Pino et al., 2021; 2019). Vaginal secretions were collected using suitable sterile pads provided with the transport medium and analyzed as follows. After removing the cells, serial dilutions were prepared using sterile phosphate-buffered saline (PBS) and plated in deMan Rogosa and Sharp growth medium (MRS, Oxoid, Milan, Italy). The plates were incubated at 35-37°C for 40-48h. In the context of the present invention, "h" can refer to one or more hours, depending on the context, and is intended to be a time.

[0018] The strain Lactobacillus rhamnosus CA15 DSM 33960 has a bacillary morphology and is Gram-positive. The microorganism according to the invention is catalase-negative, ie the microorganism according to the invention does not have catalase which can hydrolyze hydrogen peroxide.

[0019] Additionally, the DSM 33960 strain was unable to produce spores and showed no motility.

[0020] Advantageously, the strain of the invention can be used as a medicament.The object of the present invention is therefore the Lactobacillus rhamnosus DSM 33960 strain for use as a medicament.

[0021] Surprisingly, it was observed that the Lactobacillus rhamnosus CA15 DSM 33960 strain according to the invention was effective against a broad spectrum of potentially pathogenic bacteria and yeasts, such as Gardnerella vaginalis

[0022] It has high antagonistic activity against Gardnerella vaginalis, Escherichia coli, Staphylococcus aureus, Candida albicans, Candida krusei, Candida glabrata, Candida parapsilosis, Candida tropicalis, Listeria monocytogenes, Pseudomonas aeruginosa, Pseudomonas monteilii and Streptococcus agalactiae.

[0023] In addition to the above-mentioned antagonistic activity, the Applicant was able to observe other advantageous properties of the strains of the present invention, such as high anti-inflammatory and antioxidant activity, the ability to adhere to the intestinal mucosa, the ability to produce exopolysaccharides (EPS), the ability to produce biofilms and the ability to antagonize biofilm formation by different Candida species, as well as the ability to inhibit the adhesion of various pathogens and to resist the development of various adherent pathogens.

[0024] These characteristics make the strains of the present invention particularly suitable for preventing and / or treating dysbiosis of the urogenital tract microbiota, in particular dysbiosis of the female urogenital tract microbiota, preferably dysbiosis of the vaginal microbiota. In other words, the strains of the present invention can be advantageously used to prevent and / or treat changes in the urogenital tract microbiota, preferably the vaginal microbiota.

[0025] Thus, according to one of its aspects, the present invention has as its object the Lactobacillus rhamnosus CA15 DSM 33960 strain for use in preventing and / or treating dysbiosis of the urogenital microbiota, preferably vaginal dysbiosis.

[0026] In this specification, the term "dysbiosis" refers to a condition in which the homeostasis of the microbiota associated with a specific microbial ecosystem of the human body is altered. The terms "urogenital dysbiosis" and "vaginal dysbiosis" refer to a condition in which the microbiota of the urogenital tract and vaginal microbiota, respectively, are altered compared to their normal physiological conditions.

[0027] According to another aspect, the present invention is directed to a method for the use of the strain Lactobacillus rhamnosus CA15 DSM 33960 in the prevention and / or treatment of diseases associated with dysbiosis of the urogenital tract microbiota, in particular of the female urogenital tract microbiota, preferably of the vaginal microbiota.

[0028] Examples of pathologies associated with dysbiosis of the vaginal microbiota are vaginal infections, vaginal inflammation, bacterial vaginosis (BV) and its recurrence, vulvovaginal candidiasis (VVC) and sexually transmitted bacterial infections caused by, for example, Chlamydia trachomatis, Neisseria gonorrhoeae and Trichomonas vaginalis.

[0029] In particular, the Lactobacillus rhamnosus CA15 DSM 33960 strain can be used for preventing and / or treating urogenital tract infections, in particular female urogenital tract infections, preferably vaginal infections.

[0030] In an embodiment, urogenital tract infections, in particular vaginal infections, can be selected from infections caused by one or more microorganisms, such as Gardnerella vaginalis, Escherichia coli, Staphylococcus aureus, Candida species, Listeria monocytogenes, Pseudomonas aeruginosa, Pseudomonas montersii and Streptococcus agalactiae and combinations thereof. Preferably, the Lactobacillus rhamnosus CA15 DSM 33960 strain can be used to prevent and / or treat urogenital tract infections, urogenital tract inflammation, bacterial vaginosis (BV) and its recurrence, vulvovaginal candidiasis (VVC), mixed dysbiosis and group B streptococcal infection.

[0031] In an embodiment, the strains of the present invention can be used to prevent and / or treat bacterial vaginosis (BV) and its recurrence, and / or vulvovaginal candidiasis (VVC), Group B Streptococcus (GBS) infection, urinary tract infection caused by Pseudomonas aeruginosa and Escherichia coli.

[0032] The Lactobacillus rhamnosus CA15 DSM 33960 strain can be advantageously used for preventing and / or treating inflammation of the urogenital tract, in particular inflammation of the female urogenital tract, preferably vaginal inflammation, such as inflammation caused by Gardnerella vaginalis, Escherichia coli, Candida species, Pseudomonas aeruginosa and Streptococcus agalactiae.

[0033] In embodiments, the strains of the invention can be administered orally (ie, enterally) and topically.

[0034] Preferably, the DSM 33960 strain of the present invention is administered orally.

[0035] In fact, it was surprisingly observed that the strains of the present invention exhibited a high ability to survive stress conditions in the gastrointestinal tract (GI). In particular, it was observed that the strains of the present invention exhibited a high resistance to lysozyme (e.g. present in saliva), bile salts, gastric juice and acidic pH.

[0036] When administered orally, the Lactobacillus rhamnosus CA15 DSM 33960 strain may be administered as a powder or granules, as contained in a tablet, or as a capsule that can be swallowed.

[0037] In embodiments, the strains of the invention may be administered in a dried or lyophilized form.

[0038] Advantageously, the Lactobacillus rhamnosus DSM 33960 strain did not show resistance to antibiotics recommended by EFSA (European Food Safety Authority), while resistance had been detected to metronidazole, clotrimazole and boric acid, antibacterial agents routinely used to treat urogenital tract infections, particularly vaginal infections such as bacterial vaginosis (BV) and vulvovaginal candidiasis (VVC).

[0039] Furthermore, the Lactobacillus rhamnosus DSM 33960 strain also showed resistance to fluconazole, an antifungal agent used to treat fungal infections that can also proliferate in the vagina.

[0040] Therefore, the DSM 33960 strain can be advantageously used as an adjuvant in antibacterial therapy, in particular antibiotic / antifungal therapy, of urogenital tract infections, in particular female urogenital tract infections, preferably vaginal infections.

[0041] In this specification, the term "adjuvant" refers to a substance that has the ability to enhance or supplement the therapeutic effect of a chosen therapy.

[0042] In an embodiment, the strain of the present invention can be administered in combination with one or more antibacterial agents. Preferably, the strain of the present invention can be administered in combination with one or more antibacterial agents selected from metronidazole, clotrimazole, boric acid, fluconazole and mixtures thereof.

[0043] According to the description, the phrase "administered in combination with one or more antimicrobial agents" refers to the combined use of the strains of the present invention with one or more antimicrobial agents, particularly for preventive and / or therapeutic purposes. The strains of the present invention can be administered together with the antimicrobial agent or separately from the antimicrobial agent. For example, the strains of the present invention can be administered before or after the administration of the antimicrobial agent, or simultaneously with the antimicrobial agent.

[0044] Compared to the probiotic microorganisms known in the art, the strains of the present invention have various advantages.

[0045] In fact, the strain of the present invention can be used to effectively and safely prevent and / or treat dysbiosis of the urogenital tract, especially the female urogenital tract, preferably vaginal dysbiosis, in a host.

[0046] The strain of the present invention produces H2O2 and lactic acid. This feature makes it particularly effective as an antagonist against many pathogenic microorganisms of the urogenital tract, especially vaginal pathogens.

[0047] In addition, the DSM 33960 strain showed the ability to adhere to Caco-2 (intestinal epithelial model cells) HT29 (grade II colon adenocarcinoma cells) and VK2 / E6E7 cell lines (vaginal epithelial cell lines). In addition, the strains of the present invention exhibit hydrophobicity, self-aggregation ability and the ability to co-aggregate with pathogens. In order to be able to have a beneficial health effect on the host, microbial strains with probiotic potential should be able to adhere to and colonize the gastrointestinal tract to interact with resident bacteria. The bacterial adhesion process involves a variety of mechanisms, which depend on intracellular and extracellular properties. It is known that the inherent self-aggregation of specific microbial strains increases the possibility of colonization, while the hydrophobicity associated with the presence of hydrophobic molecules on the surface of the strain cells enhances its ability to adhere to the intestinal mucosa of the host. In addition, the ability to co-aggregate with pathogens is considered to be a hallmark of specific probiotic strains because the ability to co-aggregate with pathogens promotes the formation of a physicochemical barrier to prevent the colonization of pathogenic microorganisms. Without wishing to be combined with a specific explanation, it has been assumed that these abilities enable the strains of the present invention to inhibit the growth of pathogens and adhesion to the vaginal epithelium, making them particularly effective in protecting the urogenital tract, especially the vagina.

[0048] The DSM 33960 strain was also observed to produce exopolysaccharides (EPS) that are associated with various health-promoting functions.

[0049] Furthermore, the Lactobacillus rhamnosus DSM 33960 strain exhibits important antioxidant and anti-inflammatory capabilities to prevent tissue damage and the development of related diseases due to oxidative stress and stimulates the immune system to respond in an anti-inflammatory manner as a response to pro-inflammatory stimuli.

[0050] The DSM 33960 strain also shows high survival rates at low pH in the presence of lysozyme, bile salts and during simulated gastrointestinal digestion in vitro. These characteristics make the strains of the invention particularly suitable for oral administration.

[0051] The invention will now be illustrated by the following experimental section which is to be regarded as purely illustrative and not limiting. BRIEF DESCRIPTION OF THE DRAWINGS

[0052] Figure 1 : Gene fingerprint generated by PFGE ("Pulsed Field Gel Electrophoresis"). PFGE pattern of genomic DNA digested with AscI restriction enzyme of Lactobacillus rhamnosus CA15 DSM33960 strain. M: lambda PFG marker (NEB, US).

[0053] Figure 2 : Results of ABTS assay, aimed at measuring the antioxidant activity of Lactobacillus rhamnosus CA15 DSM 33960 strain using 2,2-azinobis-(3-ethylbenzothiazoline sulfonate) (ABTS) substrate. The results are expressed as absorbance values ​​at 500 nm.

[0054] Figure 3 : Adhesion of Lactobacillus rhamnosus CA15 DSM 33960 and Lactobacillus rhamnosus GG strains to human intestinal mucus glycoproteins. Results are expressed as the mean percentage of adhesion and standard deviation of three replicates, each consisting of six technical replicates.

[0055] Experimental Section

[0056] Example 1 - Isolation, phenotypic and genotypic characterization of Lactobacillus rhamnosus CA15 DSM 33960 strain

[0057] The Lactobacillus rhamnosus CA15 DSM 33960 strain was isolated from the vaginal ecosystem of healthy patients attending the AOU Obstetrics and Gynecology Clinic of the Policlinico-Vittorio Emanuele POGRodolico of the University of Catania using De Man, Rogosa and Sharpe (MRS) growth medium. Pure cultures of the strain obtained by purification smears were phenotypically characterized by microscopic observation, catalase test, Gram staining, spore production ability and motility assessment. The strain was stored at -80°C in pure culture using 20% ​​glycerol (v / v) solution.

[0058] The phenotypic characteristics of Lactobacillus rhamnosus CA15 DSM 33960 strain are shown in Table 1.

[0059]

[0060] According to the FAO / WHO 2002 guidelines, the strain was genotypically characterized by PCR of species-specific tuf gene, species-level characterization by 16S rDNA gene sequencing, and strain-level typing by pulsed-field gel electrophoresis (PFGE) analysis.

[0061] Genomic DNA extraction was performed on cultures of Lactobacillus rhamnosus DSM 33960 strains in the stationary growth phase using a commercial kit (Qiagen GmbH, Hilden, Germany). Genomic DNA was subsequently amplified by conventional PCR using P1-P4 primers targeting the V1-V3 region of the 16S rDNA gene. The resulting amplicon was sequenced and species were identified by the BLAST database "Nucleotide collection (nt / nt)" (http: / / blast.ncbi.nlm.nih.gov / Blast.cgi). At >99% sequence homology, identification was considered to be unambiguous. Pulsed field electrophoresis (PFGE) was used for strain level typing. Plug preparation, DNA extraction, and enzymatic restriction were performed using the CHEF Bacterial Genomic DNA Plug Kit (Bio-Rad, UK) according to the manufacturer's protocol for Gram-positive bacteria. After digestion with AscI restriction enzyme at 37°C for 18 h, electrophoresis was performed in 1% macrobase agarose (Bio-Rad, Milan, Italy) in 0.5X TBE buffer using CHEF DR II (Bio-Rad, Milan, Italy). The gel was then stained with 1X Atlas ClearSight DNA stain (BIOATLAS Estonia) for 1 h and observed under UV light.

[0062] The results of PFGE analysis were as follows Figure 1 shown.

[0063] Example 2 - Safety Performance

[0064] The safety claims of Lactobacillus rhamnosus CA15DSM 33960 strain were evaluated according to the guidelines for probiotics and prebiotics established by the Ministry of Health. Specifically, the following aspects were studied: hemolytic activity, the ability to produce DNAse and gelatinase, the ability to degrade mucins, the ability to produce biogenic amines and the ability to develop antibiotic resistance to a number of antibiotics, the breakpoint values ​​of which have been set by the European Food Safety Authority (EFSA). In addition, resistance / susceptibility to antimicrobial agents used in common clinical practice (metronidazole, clotrimazole and boric acid) was tested.

[0065] The characterization of the studied strains is described in detail below.

[0066] 2.1. Hemolytic activity

[0067] Lactobacillus rhamnosus CA15 DSM 33960 strain was revived in MRS broth medium and incubated at 37°C for 16-18 hours. Cell cultures in exponential growth phase were transferred to blood agar plates (Biolife, Milan, Italy) containing 5% defibrinated sheep blood by smear and incubated at 37°C for 24-48 hours. Visual hemolytic activity was determined and distinguished as β-hemolysis, α-hemolysis or γ-hemolysis based on the presence or absence of transparent areas, green haloes around the colonies. β-hemolytic strain Streptococcus pyogenes ATCC 19615 and α-hemolytic strain Streptococcus pneumoniae ATCC 6303 were used as positive controls. The results shown in Table 2 are expressed as "+" (hemolytic activity is present) and "-" (hemolytic activity is not present). The strains tested did not show hemolytic activity.

[0068]

[0069] 2.2. Ability to produce DNase and gelatinase

[0070] DNase production was tested by transferring 5 μL of cell culture in exponential growth phase onto DNase agar plates (Oxoid). After incubation at 37°C for 48 hours, the plates were treated with HCl 1N for 5 minutes. The presence of clear zones around the colonies was considered an indicator of positive DNase production.

[0071] Gelatinase production was assessed using gelatin agar plates (30 g / L gelatin, 5 g / L peptone, 3 g / L yeast extract, and 17 g / L agar). After incubation at 37°C for 48 hours, the plate surface was treated with saturated ammonium sulfate (Merck, Milan). The presence of clear areas around the colonies was considered an indicator of positive gelatinase activity.

[0072] For these two tests, the results shown in Table 3 are expressed as: "-" (no activity), "+" (active). The strains tested did not show the ability to produce DNase and gelatinase.

[0073]

[0074] 2.3. Ability to degrade mucin

[0075] The ability of Lactobacillus rhamnosus CA15 DSM 33960 strain to degrade mucin was evaluated using an appropriate growth medium modified by the addition of 0.5% (w / v) porcine stomach type III (Sigma-Aldrich, St. Louis, MO, USA). Cell cultures in the exponential growth phase were transferred to the above growth medium and the plates were incubated at 37°C for 72 hours. Next, the plates were stained with 0.1% (w / v) starch black (Merck, Milan) in 3.5 M acetic acid for 30 minutes and then washed with 1.2 M acetic acid.

[0076] (Merck, Milan) and washed. The presence of a halo around the colonies was considered a positive result.

[0077] The results shown in Table 4 are expressed as: "-" (no activity), "+" (activity). The tested strains did not show the ability to degrade mucin.

[0078]

[0079] 2.4. Production of biogenic amines

[0080] The Lactobacillus rhamnosus CA15 DSM 33960 strain was reactivated by 2% inoculation in de Man Rogosa and Sharp broth medium (MRS, Oxoid, Milan, Italy) and incubated overnight at 37°C. The resulting biomass was subcultured five times in liquid medium containing 0.005% pyridoxal-5'-phosphate (Sigma Aldrich St. Louis, MO, USA) and stimulated to produce biogenic amines by adding 0.1% histidine, 0.1% lysine, 0.1% tyrosine or 0.1% ornithine (Sigma Aldrich, St. Louis, MO, USA). The biogenic amine extraction and estimation protocol was performed as follows. Specifically, the extracted and derivatized samples were separated by HPLC chromatography (Shimadzu NexeraXL UHPLC system equipped with SPD M30A diode array detector) using a Discovery HS C18 250 mm x 46 mm x 5 μm column (Sigma Aldrich, St. Louis, MO, USA) using a low pressure gradient of acetonitrile and water. The separation temperature was kept constant at 26°C to allow for better reproducibility of the results. A six-point standard curve was generated and if their correlation values ​​R 2 >0.99, they were considered suitable. The samples were analyzed in duplicate and the results are shown in Table 5 and expressed as mean and standard deviation.

[0081]

[0082] Quantification was performed according to the procedure described in ISO 19343:2017 and it was found that the tested strains did not produce relevant amounts of the measured biogenic amines.

[0083] 2.5. Antibiotic susceptibility and MIC determination

[0084] According to the European Food Safety Authority (EFSA), the Lactobacillus rhamnosus CA15DSM 33960 strain was tested for resistance or susceptibility to certain antibiotics by minimum inhibitory concentration (MIC) assessment using the cut-off values ​​recommended by EFSA (2018) as reference. For each antibiotic tested, the strain was classified as resistant (R) or susceptible (S). In addition to the antibiotics recommended by EFSA, the strain was tested for resistance / susceptibility to some antimicrobial agents used in common clinical practice (metronidazole, clotrimazole and boric acid).

[0085] From the results shown in Table 6, it can be seen that the Lactobacillus rhamnosus CA15 DSM 33960 strain is susceptible to all antibiotics recommended by EFSA.

[0086] As can be seen from the results shown in Table 7, the Lactobacillus rhamnosus CA15 DSM 33960 strain showed resistance to metronidazole, clotrimazole and boric acid. The strain also did not show genome-wide antibiotic resistance genes. In Table 6, the MIC and breakpoint values ​​(thresholds that allow strains to be classified as susceptible or resistant) for each tested antibiotic are shown in μg / ml.

[0087]

[0088]

[0089] In a survival test during gastrointestinal transit using the dynamic SHIME system, the Lactobacillus rhamnosus DSM 33960 strain also showed resistance to fluconazole, an antifungal agent used to treat fungal infections that can also proliferate in the vagina. See Section 3.17-C below for details.

[0090] Example 3 - Functional Performance

[0091] Antimicrobial activity

[0092] The strain Lactobacillus rhamnosus CA15 DSM 33960 was tested against the potential pathogens Enterobacter cloacae DSM 30054, Enterococcus faecalis DSM 2570, Escherichia coli ATCC 25922, Escherichia coli ATCC 700414, Escherichia coli DSM105393, Candida albicans ATCC 10231, Candida glabrata ATCC 90030, Candida krusei ATCC 14243, Candida parapsilosis ATCC 90018, Candida tropicalis ATCC 13803, Gardnerella vaginalis ATCC 14019, Gardnerella vaginalis ATCC 14018, Listeria monocytogenes DSM 12464, Proteus mirabilis DSM Antagonistic activity against Pseudomonas aeruginosa DSM 30116, Pseudomonas aeruginosa DSM 1117, Pseudomonas aeruginosa DSM 3227, Pseudomonas monteri ATCC 700476, Staphylococcus aureus ATCC 6538 and Streptococcus agalactiae DSM 2134. The antibacterial activity was evaluated by using cells and metabolites produced during cell growth. Cell testing was performed by agar spot test. After incubation, the antibacterial activity was evaluated by measuring the inhibition halo obtained. The results shown in Table 8 are expressed as "-" (inactive); "+" (inhibition halo diameter <10 mm); "++" (inhibition halo diameter between 11 and 20 mm); "+++" (inhibition halo diameter>20 mm). In order to evaluate the antibacterial activity produced by metabolites produced during cell growth, the supernatant obtained after centrifugation and filtration of cell cultures in broth medium ("CFS", "cell-free supernatant") was tested before and after neutralization, heat inactivation, treatment with catalase and proteolytic enzymes.

[0093] All experiments were performed three times.

[0094] The strains studied showed a broad spectrum of antimicrobial activity. Based on the treatments performed on CFS, the antagonistic activity was in all cases attributed to the production of organic acids.

[0095] Specifically, the highest antibacterial activity was shown against Candida albicans ATCC 10231, Candida krusei ATCC 14243, Gardnerella vaginalis ATCC 14018 and Streptococcus agalactiae DSM 2134 (inhibition zones greater than 20 mm). Inhibition zones with diameters between 11 and 20 mm were observed against Candida glabrata ATCC 90030, Candida tropicalis ATCC 13803, Gardnerella vaginalis ATCC 14019, Listeria monocytogenes DSM 12464, Pseudomonas monteridium ATCC 700476 and Staphylococcus aureus ATCC 6538. Inhibition zones with diameters less than 10 mm were found against: Escherichia coli ATCC 700414, Candida parapsilosis ATCC 90018, Pseudomonas aeruginosa DSM 1117 and Pseudomonas aeruginosa DSM 3227.

[0096]

[0097]

[0098] 3.2. Production of hydrogen peroxide

[0099] The ability to produce hydrogen peroxide (H2O2) was assessed by culturing strains previously revived in broth medium on MRS agar containing 0.25 mg / mL of 3,3′,5,5′-tetramethylbenzidine and 0.01 mg / mL of horseradish peroxidase. After 72 hours of incubation, the plates were exposed to air and the production of H2O2 was assessed by the appearance of blue staining. The ability to produce hydrogen peroxide (H2O2) was assessed based on the time required for the appearance of blue staining, as follows: absent (score 0, no blue staining); low (score 1, time>20 minutes), medium (score 2, time 10-20 minutes) and high (score 3, time<10 minutes). The results are shown in Table 9.

[0100] The Lactobacillus rhamnosus CA15 DSM 33960 strain showed the ability to produce H2O2. In particular, the strain was classified as a high producer of H2O2 (score 3) because the time required for the appearance of blue staining was less than 10 minutes.

[0101]

[0102] 3.3. Production of organic acids and lactic acid

[0103] The ability of Lactobacillus rhamnosus CA15 DSM33960 to produce organic acids and lactic acid was quantitatively evaluated by HPLC chromatography according to the protocol suggested by Chenoll et al. The test was performed three times and the results are expressed as mean values ​​(mmol / L) and standard deviations, as shown in Table 10.

[0104] The Lactobacillus rhamnosus CA15 DSM 33960 strain showed the ability to produce large amounts of acetic acid, butyric acid, lactic acid, propionic acid, and succinic acid.

[0105]

[0106] 3.4. Anti-inflammatory activity of macrophages

[0107] U937 cell line (human premonocyte cell line) is used as an in vitro model of inflammation.U937 cells are suspended in Dulbecco's modified Eagle medium (DMEM) containing 1g / LD-glucose (Gibco, Life Technologies, Milan, Italy), and the culture medium is supplemented with 10% v / v bovine serum (FBS) (Invitrogen, Carlsbad, California, USA), 1% penicillin / streptomycin (Carlo Erba, Milan, Italy) and 60mg / mL gentamicin (Gibco).U937 cells are differentiated into macrophages by treating them with 200nM PMA (phorbol 12-myristate 13-acetate) for 72 hours.In order to simulate the inflammatory process, cells are pretreated with lipopolysaccharide (LPS) at a concentration of 100ng / mL for 2 hours.The anti-inflammatory effect of Lactobacillus rhamnosus CA15 DSM 33960 strain is evaluated by treating differentiated cells with a concentration of 10μg / mL of the research strain for 6 hours. At the end of the treatment, the cells were washed with PBS, harvested by trypsin digestion, and then lysed to extract RNA. COX-1 (cyclooxygenase-1), COX-2, IL-8 and IL-10 genes were quantified by real-time qRT-PCR. Untreated cells were used as controls. The analysis was performed three times, and the results are shown in Table 11 with mean values ​​and standard deviations.

[0108] The analysis showed that Lactobacillus rhamnosus CA15 DSM 33960 strain exhibited anti-inflammatory activity, resulting in a decrease in the gene expression of COX-2 and the pro-inflammatory interleukin IL-8 and an increase in the anti-inflammatory interleukin IL-10 (Table 11).

[0109]

[0110] Antioxidant activity

[0111] The antioxidant activity of Lactobacillus rhamnosus CA15 DSM 33960 strain was tested by ABTS assay using substrate 2,2-azinodi-(3-ethylbenzothiazoline sulfonate). Standard curves were obtained using Trolox at different concentrations (20-1000M). Results were expressed as molar concentrations (M) of Trolox equivalents per liter (mol TE / L). In addition, antioxidant activity was estimated by testing the oxidation of oleic acid. Specifically, 1 mL of cell-free supernatant was added to 1 mL of PBS (0.1M, pH 7.0) and 1 mL of linoleic acid (50 mM) in ethanol (99.5%). Oxidation was measured by ferric thiocyanate assay. Butylated hydroxytoluene (BHT) and α-tocopherol (1 mg / mL) were used as positive controls. Uninoculated MRS broth without antioxidants was used as a negative control. The analysis was performed three times.

[0112] The results of antioxidant activity were as follows Figure 2 As shown, and expressed as absorbance value at 500nm.

[0113] The tested strains showed antioxidant activity against linoleic acid peroxidation. The antioxidant activity of Lactobacillus rhamnosus CA15 DSM33960 strain was similar to that of α-tocopherol (P<0.05).

[0114] 3.6. Adhesion to cell lines

[0115] The adhesion ability of Lactobacillus rhamnosus CA15 DSM33960 strain was tested using intestinal cell lines Caco-2 (ATCC HTB-37) and HT29 (Caucasian colon adenocarcinoma grade II, Merck, Milan, Italy) and human vaginal epithelial cell line (VK2 / E6E7 ATCC-CRL-2616). Each test was repeated three times in three independent experiments, and the results are shown in Table 12, expressed as average adhesion percentage (%) and standard deviation. The adhesion ability is expressed as a percentage and is calculated by comparing the number of viable cells tested (8log ufc / ml) and the number of adherent cells in the cell line.

[0116] The Lactobacillus rhamnosus CA15 DSM 33960 strain showed adhesion ability to all cell lines tested.

[0117]

[0118] 3.7. Adhesion to intestinal mucus

[0119] The strains under investigation were labeled by adding 10 μl / ml tritiated thymidine (5-3Htymidine, 120 Cimmol21; Amersham Biosciences, Little Chalfont, UK) to the growth medium (MRS broth). After incubation at 37°C for 18 h, the cells were washed with PBS and normalized to OD 600 It is 0.25±0.05, corresponding to a cell number of about 8 log ufc / ml.

[0120] Intestinal mucus solution was prepared by dissolving mucin (mucin from porcine stomach type III, Merck, Milan, Italy) in 1M NaOH and diluting the solution in 1X PBS at a ratio of 1:20. 150 μl of intestinal mucus solution was fixed in the wells of a polystyrene microplate (Maxisorp, Nunc, Denmark) by incubation overnight at 4°C. The plate was washed three times with PBS. 100 μL of radiolabeled and standardized bacterial suspension was transferred to the wells and incubated at 37°C for 1 hour. Subsequently, the wells were washed three times with 200 μL of PBS to remove non-adherent cells. Adherent cells were released and lysed with sodium dodecyl sulfate (SDS) (0.1 mol / liter) (200 μl per well) in 1% (w / v) NaOH by incubation overnight at 37°C. The contents of the wells were transferred to microcentrifuge tubes containing scintillation fluid (OptiPhase "HiSafe 3", Wallac, Turku, Finland) and radioactivity was measured by liquid scintillation. Adhesion was expressed as the percentage of radioactivity recovered after adhesion relative to the radioactivity of the bacterial suspension added to the fixed mucus. Adhesion was determined in three independent experiments, and each test was repeated six times to calculate intra-assay variation.

[0121] The ability of Lactobacillus rhamnosus CA15 DSM 33960 to adhere to human intestinal mucus Figure 3 As shown. The Lactobacillus rhamnosus LGG strain was used as a reference strain. Specifically, the adhesion percentages of CA15 and LGG strains were 11.99±0.27 and 11.61±0.76, respectively.

[0122] Biofilm formation

[0123] The ability of Lactobacillus rhamnosus CA15 DSM 33960 to produce biofilm was tested according to the following method. For the test, 96-well polystyrene microtiter plates were used. Cell cultures standardized to 9 log ufc / ml were inoculated in 5% (v / v) MRS broth and MRS broth supplemented with Tween 80 (1% v / v) and incubated at 37 °C for 24 hours and 72 hours. After incubation, the wells were washed three times with PBS (pH 7.0) and treated with 200 μL of crystal violet (0.1% w / v) prepared in an isopropanol-methanol-PBS solution (1:1:18 v / v) for 30 minutes. The excess dye was washed with sterile water. After drying for 2 - 3 hours, the wells were treated with 200 μL of 30% glacial acetic acid and the biofilm produced by the strain was quantified by spectrophotometric readings at 595 nm. An enzyme-linked immunosorbent assay reader iMark TM (Biorad) was used to measure the optical density (OD 595nm ). A sterile medium was used as a negative control (ODc). The biofilm-producing ability was reported as follows: no biofilm production (OD ≤ ODc); weak biofilm production (ODc < OD ≤ 2xODc); moderate biofilm production (2xODc < OD ≤ 4xODc); high biofilm production (4xODc < OD ≤ 8xODc); very high biofilm production (8xODc < OD). The test was carried out three times and the results are shown in Table 13.

[0124] The Lactobacillus rhamnosus CA15 DSM 33960 strain showed a high ability to produce biofilm in MRS after 24 hours and 72 hours of incubation. After growing in MRS with Tween 80 for 72 hours, a very high ability to produce biofilm was found.

[0125]

[0126] 3.9. Ability to inhibit adhesion of Candida species

[0127] Using 96-well polystyrene microtiter plates, the ability of Lactobacillus rhamnosus CA15 DSM 33960 to resist the adhesion of Candida albicans ATCC 10231, Candida glabrata ATCC 90030, Candida krusei ATCC 14243, Candida parapsilosis ATCC 90018, and Candida tropicalis ATCC 13803 was evaluated by the "precoating" and "co-incubation" methods. In both methods, the cell-free supernatant (CFS) of the Lactobacillus rhamnosus CA15 DSM 33960 strain was obtained by centrifuging a standardized cell culture of 9 log ufc / ml and filtering the supernatant through a 0.22 μm filter.

[0128] Pre-coating: CFS (200 μL) was transferred to the wells of a polystyrene microplate and incubated at 37°C for 24 hours. After incubation, the CFS was removed and the wells were washed twice with 100 μL of PBS (pH 7.2). A cell suspension (150 μL) of each Candida species tested was standardized to 7 log ufc / ml, added to separate wells, and incubated at 37°C for 24 hours. After incubation, the wells were gently washed with PBS (pH 7.2), and 99% methanol (100 μL) was added to fix the biofilm. After 15 minutes, the residual methanol was removed by washing, and 2% crystal violet (100 μL) was added. After 20 minutes, the excess dye was removed and 100 μL of 33% glacial acetic acid was added. Use the microplate reader iMark TM The optical density (ODc) was measured at 595 nm by (Biorad). A suspension of Candida species without CFS was used as a negative control (OD0). The percentage of Candida species adhesion in the presence of CFS of Lactobacillus rhamnosus CA15 DSM 33960 strain was calculated as follows:

[0129] Adhesion % = 1-ODc / OD0×100

[0130] Co-incubation: 100 μL of CFS of Lactobacillus rhamnosus CA15 DSM 33960 strain and 150 μL of cell suspension of each Candida species tested (standardized to 7 log ufc / ml) were transferred to a 96-well polystyrene microplate and incubated at 37°C for 24 hours. Washing and staining steps were performed as reported for the pre-coating test. A suspension of Candida species without CFS of Lactobacillus rhamnosus CA15 DSM 33960 strain was used as a negative control. The optical density was measured at 650 nm. The percentage reduction of biofilm formation was calculated as follows:

[0131] Reduction % = 1-OD A / OD B ×100OD A The optical density and OD of the samples (CFS of Lactobacillus rhamnosus CA15 DSM 33960 strain and cell suspension of each Candida species) are shown in Table 1. B Represents the optical density of the negative control.

[0132] The results for the ability of the Lactobacillus rhamnosus CA15 DSM 33960 strain to resist the adhesion of Candida species are reported in Table 14 as the mean and standard deviation of three determinations.

[0133] The Lactobacillus rhamnosus CA15 DSM 33960 strain was found to inhibit the adhesion of all Candida species tested, showing more than 50% inhibition in the pre-coating assay and more than 20% inhibition in the co-incubation assay.

[0134]

[0135] 3.10. Inhibition of pathogen adhesion

[0136] The ability of the Lactobacillus rhamnosus CA15 DSM 33960 strain to inhibit pathogen adhesion was tested on polystyrene plates by transferring 100 μl of a cell suspension of the strain standardized to 8 log cfu / ml to each well. After incubation at 37°C for 1 hour, unbound cells were removed by washing twice with PBS buffer. The pathogen strains tested were previously grown in liquid culture medium and radioactively labeled. 100 μl of each pathogen standardized to 8 log cfu / ml was added to the wells and incubated at 37°C for 1 hour. Unbound labeled bacteria were then removed by washing, and bound bacterial cells were released and lysed with SDS (0.1M) in 1% (w / v) NaOH. After incubation overnight at 37°C, radioactivity was assessed by liquid scintillation. The results are shown in Table 15 and are expressed as the percentage of radioactivity recovered after adhesion compared to the radioactivity of the bacterial suspension added to the wells. The ability to inhibit pathogen adhesion was expressed as a percentage and calculated as the difference between pathogen adhesion in the presence and absence of Lactobacillus rhamnosus CA15 DSM 33960. The inhibition was determined in three independent experiments with six replicates for each test.

[0137]

[0138] The Lactobacillus rhamnosus CA15 DSM 33960 strain showed the ability to inhibit the adhesion of all pathogens tested with an inhibition percentage of more than 50%.

[0139] 3.11. Inhibition of the development of adherent pathogens

[0140] The ability of the rhamnosus Lactobacillus CA15 DSM 33960 strain to resist previously adhered pathogens was determined as follows. Specifically, as previously described, radioactively labeled and standardized pathogens were added to the wells and incubated for 1 hour at 37°C. Unbound pathogens were removed by washing three times with PBS buffer, and non-radioactively labeled rhamnosus Lactobacillus CA15 DSM 33960 strains (100 μl, 8 log cfu / ml) were added to each well. The plate was incubated for 1 hour at 37°C. Then, the wells were washed again, and the bound bacteria were released and lysed as described above. Radioactivity was measured as described above, and the results obtained are shown in Table 16. The ability to resist previously adhered pathogens was calculated as the difference between pathogen adhesion before and after adding the rhamnosus Lactobacillus CA15 DSM 33960 strain. Three independent experiments were carried out. Each test was repeated six times to calculate the variation within the test.

[0141]

[0142] The Lactobacillus rhamnosus CA15 DSM 33960 strain showed the ability to inhibit all pathogens tested with a percentage of more than 50%.

[0143] 3.12. Production of extracellular polysaccharides (EPS)

[0144] The ability of Lactobacillus rhamnosus CA15 DSM 33960 strain to produce exopolysaccharides (EPS) was evaluated using the phenol / sulfuric acid method, and the amount of EPS produced was estimated using glucose (50-500 mg / L) as a standard.

[0145] The determination was performed three times and the results were expressed in mg / L. The results are shown in Table 17.

[0146]

[0147] 3.13. Hydrophobicity, self-aggregation and co-aggregation

[0148] The hydrophobicity (H%) was determined by mixing equal volumes of a cell suspension of Lactobacillus rhamnosus CA15 DSM 33960 strain (9 log cfu / ml) in xylene and PBS. After incubation at 37°C for 10 minutes, the suspension was kept at 37°C for 5 hours to achieve phase separation. The aqueous phase was taken and the absorbance was determined at 600 nm. The hydrophobicity was calculated as the percentage decrease in the absorbance of the aqueous phase after incubation (ΔAbs x 100) compared to the absorbance of the base suspension according to the following formula:

[0149] H%=ΔAbs / Abs t0 x 100

[0150] The self-aggregation (Auto-A%) was determined by using a cell suspension (9 log ufc / ml) of Lactobacillus rhamnosus CA15 DSM 33960 strain in 1 ml PBS buffer, vortexing for 10 seconds and incubating at room temperature for 5 hours. Then, an aliquot (0.1 ml) of the upper suspension layer was mixed with 500 μl of PBS buffer and the absorbance at 600 nm was determined. The self-aggregation (Auto-A%) was expressed as the percentage (ΔAbs x 100) of the decrease in absorbance after 5 hours compared to the original suspension (Abst0), as follows:

[0151] Auto-A%=ΔAbs / Abs t0 x 100

[0152] Coaggregation (Co-A%) was evaluated using Escherichia coli 555, Gardnerella vaginalis ATCC 14018, Gardnerella vaginalis ATCC 14019, Candida albicans ATCC 10231, Candida glabrata ATCC 90030, Candida krusei ATCC 14243, Candida parapsilosis ATCC 90018, and Candida tropicalis ATCC 13803 strains as coaggregation partners. Specifically, an equal volume (2 mL) of Lactobacillus rhamnosus CA15 DSM 33960 strain and a cell suspension of each pathogenic strain were mixed and vortexed for 30 seconds and then kept at room temperature for 5 hours. The DSM 33960 + pathogen mixture (Abs (CA15+病原体) ) and DSM 33960 strain alone (Abs CA15 ) at 600 nm. Coaggregation was calculated as follows:

[0153] Co-A%=((Abs CA15 +Abs 病原体 ) / 2-Abs (CA15+病原体) / (Abs CA15 +Abs 病原体 / 2)x 100.

[0154] The results reported in Table 18 are means and standard deviations of three determinations.

[0155] Lactobacillus rhamnosus CA15 DSM 33960 strain showed good self-aggregation, hydrophobicity, and co-aggregation with all tested pathogens, indirectly demonstrating the ability to adhere to the intestinal mucosa and prevent the development of pathogens.

[0156]

[0157]

[0158] 3.14. Lysozyme resistance

[0159] The ability of Lactobacillus rhamnosus CA15 DSM 33960 strain to survive in the presence of lysozyme (100 mg / L) was assessed in MRS broth after incubation at 37°C for 0, 30 and 120 minutes. Lysozyme resistance was determined by in-plate counting of viable cells. Survival in the presence of lysozyme was assessed by measuring the survival rate (SR%), i.e. the survival percentage, which was calculated by considering the final (cfuF) and initial (cfuI) population density (cfuF / cfuI*100). A value of 80% was considered to be the minimum survival limit. In the absence of lysozyme, a cell culture of Lactobacillus rhamnosus CA15 DSM 33960 strain was used as a control. The analysis was performed three times.

[0160] The results are expressed as the average survival rate (SR%) and are shown in Table 19.

[0161] The strains tested showed the ability to survive in the presence of lysozyme, with survival rates of 95% and 93% after 30 and 120 minutes of incubation at 37°C, respectively.

[0162]

[0163] 3.15. Tolerance to low pH

[0164] The ability of Lactobacillus rhamnosus CA15DSM 33960 strain to tolerate low pH was tested in MRS broth acidified to pH 2.0 and pH 3.0 by adding 1M HCL. MRS broth at pH 6.2 was used as a control. The strain under study was revived in MRS broth, and a cell suspension (9log ufc / ml) in the exponential growth phase was inoculated into the acidified medium. Aliquots were taken immediately after inoculation (0 hour) and after incubation at 37°C for 2 hours and 4 hours. Tolerance to low pH was determined by counting the plate of living cells. The survival percentage (%) calculated by considering the final (cfuF) and initial (cfuI) population density (cfuF / cfuI*100) was evaluated, and a value of 80% was considered to be the minimum survival limit.

[0165] The results are shown in Table 20 and expressed as survival rate % (SR %).

[0166] The strains tested showed the ability to survive at pH 3.0 and 2.0, with survival rates greater than 80% after 2 and 4 hours of incubation at 37°C (Table 20). The survival percentage after 4 hours at pH 3.0 was 86%. The survival percentage after 4 hours at pH 2.0 was 83%.

[0167]

[0168] 3.16. Tolerance to bile salts

[0169] The ability of the Lactobacillus rhamnosus CA15 DSM 33960 strain to survive in the presence of bile salts (cow bile salts, Oxgall; Sigma-Aldrich) at a final concentration of 0.5% and 1.0% was evaluated as follows. The strain under study was revived in MRS broth, and a cell suspension (9log ufc / ml) in the exponential growth phase was inoculated into MRS broth containing the above-mentioned percentage of bile salts. MRS without bile salts was used as a control. Aliquots were taken immediately after inoculation (0 hours) and after incubation at 37°C for 2 hours and 4 hours, and viable cells were counted in the plate. The analysis was performed three times, and the results are shown in Table 21, expressed as survival percentage (SR%), which is calculated by considering the final (cfuF) and initial (cfuI) population density (cfuF / cfuI*100), and a value of 80% is considered to be the minimum survival limit.

[0170] The strains tested showed the ability to survive in the presence of bile salts at concentrations of 0.5% and 1%. For 0.5% bile salts, the survival rate was 87% after 4 hours of incubation. For 1% bile salts, the survival rate was 82% after 4 hours.

[0171]

[0172]

[0173] 3.17. Survival during simulated gastrointestinal (GI) digestion

[0174] The ability of Lactobacillus rhamnosus CA15 DSM 33960 strain to survive gastrointestinal (GI) transit was determined in vitro using both static (SGJ / SIF) and dynamic systems (SHIME), the latter accurately reproducing the physiological, chemical, and microbiological conditions of the GI tract in vitro.

[0175] 3.17-A. Survival during simulated gastrointestinal (GI) digestion in the SGJ / SIF system

[0176] The ability of Lactobacillus rhamnosus CA15 DSM33960 strain to survive gastrointestinal (GI) transit was determined in vitro using simulated gastric fluid ("SGJ") and simulated intestinal fluid ("SIF"). Specifically, SGJ (0.3% pepsin, 0.5% NaCl, adjusted to pH 2 by adding 1 M HCl) and SIF (0.1% pancreatin, 0.5% bile salts, 0.5% NaCl, 0.4% phenol, adjusted to pH 8 by adding 1 M NaOH) were prepared immediately before use and filtered using 0.22 μM cellulose acetate filters (Minisartfilters, Sartorius, Germany) sterilization. All chemicals were purchased from Sigma Aldrich (St.Louis, MO). Cell cultures (9log cfu / ml) in the exponential growth phase were precipitated by centrifugation and resuspended in phosphate buffered saline (PBS). The resulting cell suspension was mixed with SGJ and incubated at 37°C for 2 hours under stirring (200 rpm). The cells precipitated by centrifugation were resuspended in SIF and incubated at 37°C for 3 hours. The cells treated with SGJ and SGJ-SIF were counted on the plate for viable cells. The analysis was performed three times, and the results are shown in Table 19, and the results are expressed as survival rate%.

[0177] The strains showed excellent survival during simulated gastrointestinal transit under static conditions. During simulated gastrointestinal digestion, survival rates of greater than 90% were observed. Specifically, SR% was found to be 99.7% after treatment with simulated gastric fluid (SGJ), and SR% was found to be 95.7% after treatment with simulated intestinal fluid (SIF) (Table 22).

[0178]

[0179] 3.17-B. Using dynamic Survival of the system during gastrointestinal transit

[0180] Using a simulator of the human gut microbial ecosystem ( ProDigest, Belgium) to determine the ability of Lactobacillus rhamnosus CA15 DSM 33960 strain to survive gastrointestinal (GI) transit under fasting and full conditions. This system accurately simulates the entire human GI tract, including different regions of the stomach, small intestine and colon. As it is a dynamic system, the physiological, chemical and microbiological conditions of the GI tract are accurately reproduced.

[0181] A dynamic SHIME system was set up using a double-walled reactor, maintained at 37°C under constant stirring and anaerobic conditions with nitrogen sparging to simulate the entire gastrointestinal tract. The Lactobacillus rhamnosus CA15 DSM 33960 strain was tested in capsule form with a population density of 10 log cfu / g and maintained in a specific capsule content in the SHIME system.

[0182] Survival under fasting conditions

[0183] Gastric fluid at pH 2 (composed of KCl 0.66 g / L, NaCl 3.63 g / L, mucin 3.95 g / L, lecithin 3.4 g / L, and pepsin 10 g / L) was used to reproduce the gastric microenvironment. Gastric horizontal digestion was simulated for 45 minutes. Next, the gastric digesta were subjected to intestinal digestion, and then pancreatic juice (composed of NaHCO3 2.6 g / L, bile salts 4.8 g / L, pancreatic enzymes 1.9 g / L, trypsin 10 g / L, and chymotrypsin 10 g / L) was added. Thus, the duodenum (incubation for 27 minutes with a controlled pH increase from 2.0 to 6.5), jejunum (incubation for 63 minutes with a controlled pH increase from 6.5 to 7.5), and ileum (incubation for 90 minutes at a constant pH of 7.5) parts were simulated. During intestinal digestion, aliquots of NaHCO3 were added after 60 minutes, 90 minutes, and 120 minutes.

[0184] (8.4g / L) was added to determine that the pH increased to the above value. The pH was continuously monitored using a Senseline F410 pH meter (ProSense, Oosterhout, the Netherlands). A pump system was used to automatically administer HCl (0.5M) and NaOH (0.5M) solutions to determine that the pH was maintained constant within the above range. At the same time, a negative control (digested as above, but without the encapsulated Lactobacillus rhamnosus CA15DSM 33960 strain) was set up to exclude the presence of contamination and to monitor process parameters. All determinations were performed twice.

[0185] To determine the survival of Lactobacillus rhamnosus CA15 DSM 33960 strain during gastrointestinal transit, gastric, duodenal, jejunal and ileal digesta were aseptically collected and subjected to culture-dependent analysis. Specifically, 5 ml of the sample was serially diluted using anaerobic PBS (K2HPO4, 8.8 g / L; KH2PO4, 6.4 g / L; NaCl, 8.5 g / L; L-cysteine ​​HCl, 0.5 g / L) and incubated at 37°C for 48 hours under anaerobic conditions. The culture was then cultured using Lactobacillus MRS agar medium (BD Difco). TM ) The viability of Lactobacillus rhamnosus CA15 DSM 33960 strain was determined by counting live cells in the plate. The survival percentage (%) was evaluated and calculated by considering the final (cfuF) and initial (cfuI) population density (cfuF / cfuI*100). The percentage of capsule degradation was determined visually.

[0186] Table 23 shows the survival results of Lactobacillus rhamnosus CA15 DSM 33960 strain, expressed as log cfu / ml and survival rate % (SR %), and capsule degradation rate.

[0187] Specifically, considering that the initial population density of Lactobacillus rhamnosus CA15 DSM 33960 strain was 10 logcfu / g, the SR% values ​​in the duodenum, jejunum and ileum were found to be greater than 50%, indicating a high ability to survive the stress conditions of the gastrointestinal tract. The small presence of viable cells in the stomach can be attributed to the viability and non-culturable conditions of the strain, given the extremely acidic conditions of the stomach (pH 2) and the presence of pepsin. Regarding the degradation percentage of the capsule, it was found that the capsule gradually dissolved in various regions of the gastrointestinal system, reaching a degradation percentage of 100% in the jejunum.

[0188]

[0189] Survival on a full stomach

[0190] Use containing The gastric microenvironment was reproduced by gastric juice at pH 4.5 containing nutrient medium (PDNM001B 20.53 g / L, ProDigest, Ghent, Belgium), NaCl 3.63 g / L, KCl 0.65 g / L, lecithin 13.5 g / L and pepsin 40 g / L. Digestion at the gastric level was simulated for 120 minutes. Next, the gastric digesta were subjected to intestinal digestion and then pancreatic juice consisting of: NaHCO3 7.7 g / L, bile salts 15 g / L, pancreatic enzymes 10 g / L, trypsin 10 g / L and chymotrypsin 10 g / L was added. Thus, the duodenum (incubation for 27 minutes with a controlled pH increase from 2.0 to 6.5), the jejunum (incubation for 63 minutes with a controlled pH increase from 6.5 to 7.5) and the ileum (incubation for 90 minutes at a constant pH of 7.5) parts were simulated. During intestinal digestion, NaHCO (8.4 g / L) was added in equal parts after 60 minutes, 90 minutes and 120 minutes to determine that the pH value was increased to the above value. Use Senseline F410 pH meter (ProSense, Oosterhout, the Netherlands) to continuously monitor pH. Use pump system to automatically give HCl (0.5 M) and NaOH (0.5 M) solution so that the pH value remains within the above range. Meanwhile, negative control (digested as above, but not in the presence of encapsulated Lactobacillus rhamnosus CA15 DSM 33960 bacterial strain) was set up, and the negative control was intended to exclude the existence of pollution and the monitoring process parameters. All determinations were carried out twice.

[0191] To determine the survival rate of Lactobacillus rhamnosus CA15 DSM 33960 strain during gastrointestinal transit, digesta were collected aseptically at the end of the stomach, duodenum, jejunum, and ileum and culture-dependent analysis was performed. Specifically, 5 ml of the sample was serially diluted using anaerobic PBS (K2HPO4, 8.8 g / L; KH2PO4, 6.4 g / L; NaCl, 8.5 g / L; L-cysteine ​​HCl, 0.5 g / L) and incubated at 37°C for 48 h under anaerobic conditions. The culture was then cultured using Lactobacillus MRS agar medium (BD Difco TM ) The viability of Lactobacillus rhamnosus CA15 DSM 33960 strain was determined by counting live cells in the plate. The survival percentage (%) was evaluated and calculated by taking into account the final (cfuF) and initial (cfuI) population densities (cfuF / cfuI*100).

[0192] The survival results of Lactobacillus rhamnosus CA15 DSM 33960 strain, expressed as log cfu / ml and survival rate % (SR %), and the percentage of capsule degradation are shown in Table 24.

[0193] Specifically, considering that the initial population density of Lactobacillus rhamnosus CA15 DSM 33960 strain was 10 logufc / g, this strain showed SR% values ​​higher than 50% in all tested gastrointestinal system parts. Regarding the capsule degradation rate, it was found that the capsule completely disintegrated in the duodenum (100% degradation percentage).

[0194]

[0195] 3.17-C. Under fasting conditions and in the presence of antibiotics (fluconazole 200 mg), the dynamic SHIME system Survival of the system during gastrointestinal transit

[0196] By using a human gut microbial ecosystem simulator ( ProDigest, Belgium) determined the ability of the Lactobacillus rhamnosus CA15 DSM 33960 strain to survive gastrointestinal (GI) transit under fasting conditions and in the presence of the antibiotic fluconazole (200 mg). A dynamic SHIME system was established using a double-walled reactor, maintained at a temperature of 37°C under anaerobic conditions with constant stirring and nitrogen sparging to simulate the entire gastrointestinal tract. The Lactobacillus rhamnosus CA15 DSM 33960 strain and the antibiotic were introduced into the reactor simultaneously. Specifically, the Lactobacillus rhamnosus CA15 DSM 33960 strain was tested in capsule form with a population density of 10 log cfu / g and maintained in a specific capsule content in the SHIME system. According to the dosage instructions, the antibiotic fluconazole (200 mg) pre-dissolved in 50 ml of sterile water was aseptically introduced through an additional port.

[0197] The gastric microenvironment was reproduced by using gastric juice (consisting of KCl 0.66 g / L, NaCl 3.63 g / L, mucin 3.95 g / L, lecithin 3.4 g / L, and pepsin 10 g / L) at pH 2. The gastric horizontal digestion was simulated for 45 minutes. Next, the gastric digesta were subjected to intestinal digestion, and then pancreatic juice (consisting of NaHCO3 2.6 g / L, bile salts 4.8 g / L, pancreatic enzymes 1.9 g / L, trypsin 10 g / L, and chymotrypsin 10 g / L) was added. Thus, the duodenum (incubated for 27 minutes with a controlled pH increase from 2.0 to 6.5), the jejunum (incubated for 63 minutes with a controlled pH increase from 6.5 to 7.5), and the ileum (incubated for 90 minutes at a constant pH of 7.5) parts were simulated. During the intestinal tract, after 60 minutes, 90 minutes and 120 minutes, add the NaHCO of 8.4g / L to determine that the pH value increases to the above-mentioned value. Use Senseline F410 pH meter (ProSense, Oosterhout, the Netherlands) to continuously monitor pH. Use pump system to automatically give HCl (0.5M) and NaOH (0.5M) solution, so that the pH value remains within the above-mentioned range. Meanwhile, only set up negative control with antibiotics, and digest as above, but do not have the Lactobacillus rhamnosus CA15 DSM 33960 bacterial strain of sealing. This contrast is intended to exclude the existence of pollution and the monitoring process parameters. All mensuration are carried out twice.

[0198] To determine the survival rate of Lactobacillus rhamnosus CA15 DSM 33960 strain in the presence of fluconazole, digestive samples were collected at the end of the stomach, duodenum, jejunum and ileum and culture-dependent analysis was performed. Specifically, 5 ml of the sample was serially diluted using anaerobic PBS (K2HPO4, 8.8 g / L; KH2PO4, 6.4 g / L; NaCl, 8.5 g / L; L-cysteine ​​HCl, 0.5 g / L) and incubated at 37°C for 48 hours under anaerobic conditions. The culture medium was then diluted using Lactobacillus MRS agar medium (BD Difco). TM ) The viability of Lactobacillus rhamnosus CA15 DSM 33960 strain was determined by counting live cells in the plate. The survival percentage (%) was evaluated and calculated by considering the final (cfuF) and initial (cfuI) population density (cfuF / cfuI*100). The percentage of capsule degradation was determined visually.

[0199] The survival results of Lactobacillus rhamnosus CA15 DSM 33960 strain in the presence of fluconazole (200 mg), expressed as log cfu / ml and survival rate % (SR%), and the degradation percentage of Lactobacillus rhamnosus CA15 DSM 33960 strain capsule are shown in Table 25.

[0200] Specifically, considering that the initial population density of Lactobacillus rhamnosus CA15 DSM 33960 strain was 10 logcfu / g, the SR% values ​​of this strain in the duodenum, jejunum and ileum were higher than 60%, indicating its high viability under the stress conditions of the gastrointestinal tract. The absence of viable cells in the stomach may be attributed to the viable but non-culturable conditions of the strain, given the extremely acidic conditions of the stomach (pH 2) and the presence of pepsin. Regarding the degradation percentage of the capsule, it was found that the capsule gradually dissolved in various regions of the gastrointestinal system, reaching a degradation percentage of 100% in the jejunum.

[0201]

[0202] 3.18. Acidification capacity in MRS medium

[0203] The acidification capacity of Lactobacillus rhamnosus CA15 DSM 33960 strain was tested in de Man Rogosa and Sharp broth medium (MRS, Oxoid, Milan, Italy) and monitored for 24 hours by pH determination. Specifically, an overnight culture of Lactobacillus rhamnosus CA15 DSM 33960 strain with a population density of 8.60 cfu / ml was transferred to 30 ml of MRS broth medium and incubated at a temperature of 37° C. under constant stirring. The pH of the culture medium was regularly measured by a pH meter (XS, pH 60VIO LAB).

[0204] Table 26 shows the pH values ​​measured at different sampling times.

[0205] In particular, the Lactobacillus rhamnosus CA15 DSM 33960 strain caused a gradual decrease in the pH of the medium used, reaching a pH value of 3.82 after 24 hours of incubation at 37°C.

[0206]

[0207]

Claims

1. A Lactobacillus rhamnosus CA15 bacterial strain, deposited in the German Collection of Microorganisms (Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH-DSMZ) with the deposit number DSM 33960.

2. The Lactobacillus rhamnosus CA15 bacterial strain with the deposit number DSM 33960 according to claim 1, which is used as a medicine.

3. The Lactobacillus rhamnosus CA15 bacterial strain with the deposit number DSM 33960 according to claim 1 or 2, for use in a method for preventing and / or treating diseases associated with dysbiosis of the microbiota of the urogenital tract, preferably diseases associated with dysbiosis of the microbiota of the female urogenital tract.

4. The Lactobacillus rhamnosus CA15 bacterial strain with the deposit number DSM 33960 used according to claim 3, wherein the dysbiosis refers to the dysbiosis of the vaginal microbiota.

5. The Lactobacillus rhamnosus CA15 bacterial strain with the deposit number DSM 33960 for use according to claim 3 or 4, wherein the disease associated with dysbiosis of the microbiota of the female urogenital tract is selected from the group comprising or alternatively consisting of: vaginal infection, urogenital tract infection, vaginal inflammation, urogenital tract inflammation, bacterial vaginosis (BV) and its recurrence, vulvovaginal candidiasis (VVC), sexually transmitted bacterial infections, preferably caused by Chlamydia trachomatis, Neisseria gonorrhoeae and Trichomonas vaginalis, mixed dysbiosis, group B streptococcus (GBS) infection.

6. The Lactobacillus rhamnosus CA15 bacterial strain with the deposit number DSM 33960 for use according to claim 5, wherein the urogenital tract infection is selected from infections / inflammations caused by one or more microorganisms, such as Gardnerella vaginalis, Escherichia coli, Staphylococcus aureus, Candida spp., Candida albicans, Candida krusei, Candida glabrata, Candida parapsilosis, Candida tropicali, Listeria monocytogenes, Pseudomonas aeruginosa, Pseudomonas monteilii and Streptococcus agalactiae and / or combinations thereof.

7. The Lactobacillus rhamnosus CA15 bacterial strain with the deposit number DSM 33960 for use according to any one of claims 1 to 6, wherein the bacterial strain is administered orally.

8. The Lactobacillus rhamnosus CA15 bacterial strain with the deposit number DSM 33960 for use according to any one of claims 1 to 7, wherein the bacterial strain is in a freeze-dried form.

9. The Lactobacillus rhamnosus CA15 bacterial strain with the deposit number DSM 33960 for use according to any one of claims 1 to 8, wherein the bacterial strain is administered in combination with one or more antimicrobial agents.

10. The Lactobacillus rhamnosus CA15 bacterial strain with the deposit number DSM 33960 for use according to claim 9, wherein the one or more antibacterial agents are selected from metronidazole, clotrimazole, boric acid, fluconazole and mixtures thereof.

11. The Lactobacillus rhamnosus CA15 bacterial strain with the deposit number DSM 33960 for use according to any one of claims 1 to 9, wherein the bacterial strain is administered in the form of capsules.

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