Composition for promoting physiological microbiota growth especially in a pregnant woman or newborns and preventing dysbiosis and related diseases

Abstract

The present invention relates to selected strains of Lactiplantibacillus plantarum and compositions containing the strains for promoting the growth of physiological microbiota and preventing dysbiosis, infections and inflammatory diseases. The composition is for topical or oral administration.

Description

[0001] COMPOSITION FOR PROMOTING PHYSIOLOGICAL MICROBIOTA GROWTH ESPECIALLY IN A PREGNANT WOMAN OR NEWBORNS AND PREVENTING DYSBIOSIS AND RELATED DISEASES

[0002] DESCRIPTION

[0003] FIELD OF THE INVENTION

[0004] The present invention relates to a composition for promoting physiological microbiota growth especially in women and newborns and preventing dysbiosis, related infections and inflammatory conditions.

[0005] The present invention origins in the field of products or nutritional compositions containing selected lactobacilli species suitable for preventing and treating dysbiosis. Suitable compositions are for oral administration or for topical application in the form of a gel for application on the abdominal orfemale genital region of body of a woman to promote the growth of physiological microbiota which is transferred and delivered to the newborn during partum. The gel contains lactobacilli species, which colonize the skin of the newborn immediately after birth and continue developing with the host into a highly diversified ecosystem.

[0006] BACKGROUND

[0007] Recent studies demonstrated that the early-life microbiota of new-borns is affected by mode of delivery during partum. Specifically, the human microbiota differs between caesarean section bom and vaginally delivered infants especially over the first year of life.

[0008] The term “microbiota” refers to the total collection of microorganisms of a specific region or a period, while the term “microbiome” refers to the collection of genomes of all the microbes that are part of a specific ecosystem (Schwiertz 2016).

[0009] The human body hosts a huge number of microorganisms that are particularly abundant in certain organs, such as the oral cavity, the gastro-intestinal tract, the skin, and the vagina. Our microbiota does not only include bacteria, but also viruses, archaea, and eukaryotes, such as protozoa, fungi, and nematodes (Schwiertz 2016). Commensal bacteria colonize the human host and develop into a highly diversified ecosystem. Over time, host-bacterial associations have developed into beneficial relationships (Wang et al. 2017). A microbial balanced ecosystem, defined as “eubiosis”, is characterized by the presence and dominance of beneficial species that keep potential pathogenic species in very low percentage. Differently, a condition of “dysbiosis” is established when “good bacteria” no longer control the “bad / pathogen bacteria”, which take over. The balance of bacteria defining the human microbiota can be disturbed by different factors, such as antibiotic treatments, pathogen infection but also lifestyle, such as diet, sport, and stress (Lebba et al. 2016).

[0010] In addition, recent studies have shown that the microbiota composition at one week of life is associated with the development of infections, inflammatory and immunological diseases, especially in the female genital tract (FGT). This is a complex and dynamic system of organs, which goes through numerous and significant changes during a woman’s lifecycle. The FGT mucosal surfaces are coated with cervicovaginal secretion (CVS), a mixture of cervical mucus and vaginal transudate, and colonized by a diverse microbial population, the vaginal microbiota. This microbiota consists of a diversity of anaerobic and aerobic microorganisms, with a predominance of lactobacilli species (Pekmezovic et al. 2019). Most of the members of the vaginal microbiota exist in a mutualistic relationship with the host and they play a fundamental role in maintaining a gynaecological healthy status (Barrientos-dur et al., 2020).

[0011] A dysbiosis of the vaginal microbiota is defined as “a polymicrobial condition characterized by a low prevalence of Lactobacillus spp. and by an increase in anaerobic microorganisms” (Barrientos-Duran et al. 2020). This condition can be caused by several factors such as the prolonged use of antibiotics, an unbalanced diet, stress, hygiene practices, sexual habits, smoking cigarettes, etc. (Barrientos- Duran et al. 2020). A dysbiotic condition can lead to genital infection, which has been associated with increased risk for several reproductive and obstetric disorders. Most of the microorganisms causing FGT infections are part of the vaginal microbiota, therefore are considered opportunistic pathogens or commensals (Barrientos-Duran et al. 2020).

[0012] Accordingly, vaginal microbiota is of fundamental importance for the maintenance of a gynaecological healthy status. Several factors may lead to a condition of dysbiosis in the FGT. This condition increases the risk of pathogen infections and consequent adverse reproductive and obstetric health outcomes (Barrientos-Duran et al. 2020).

[0013] Vaginal infections are extremely common, and they can be symptomatic or asymptomatic. Depending on the agent causing it, we distinguish bacterial vaginosis, aerobic vaginitis, or vulvovaginal candidiasis (WC). The therapies used to treat these conditions have changed little in the last 40 years (Macklaim et al. 2015) and they are often unsuccessful. The antimicrobial failures have been attributed to different factors, such as drug resistance, failure of the drug to penetrate biofilms of the aetiological agent, elimination of beneficial microorganisms by the therapeutic antimicrobial, and inability to restore a eubiotic status, that also leads to infection recurrence (Cribby et al. 2008; Macklaim et al. 2015).

[0014] Epidemiological studies have reported associations between C-section delivered babies and an increased risk of developing metabolic and chronic disorders including celiac disease, diabetes mellitus, obesity, asthma, allergies, and immune deficiencies (Stennett et al. 2020).

[0015] Accordingly, at present there is a need for new products for maintaining microbiota eubiosis especially in pregnant women, independently by the mode of delivery.

[0016] One of the general aims of the present invention is to provide a product, which maintains the microbiota in physiological conditions when administered either by the topical or oral route of administration.

[0017] A further aim resides in providing a composition for topical application in the female genital tract to maintain vaginal microbiota eubiosis to prevent urogenital infections and inflammatory or allergic diseases.

[0018] SUMMARY OF THE INVENTION

[0019] The present invention origins from the finding that selected bacterial strains belonging to Lactobacillus species isolated from food sources and which are not endogenous vaginal lactobacilli, are suitable to maintain microbiota eubiosis and to prevent related infections and inflammatory or allergic diseases.

[0020] In addition, the inventors investigated the activities and effects of nomadic lifestyle lactobacilli on vaginal microbiota and their inhibition on the growth of Candida albicans. The inventors also investigated the effects of certain Lactiplantibacillus plantarum strains on Candida albicans infections of human mucosae such as vaginal mucosae.

[0021] In a first aspect, the present invention relates to composition comprising a strain of

[0022] Lactiplantibacillus plantarum selected from the group consisting of:

[0023] Lactiplantibacillus plantarum having the accession number DSM 34000,

[0024] Lactiplantibacillus plantarum having the accession number DSM 33995,

[0025] Lactiplantibacillus plantarum having the accession number DSM 33999

[0026] Lactiplantibacillus plantarum having the accession number DSM 33996

[0027] Lactiplantibacillus plantarum having the accession number DSM 33997

[0028] Lactiplantibacillus plantarum having the accession number DSM 33998, and

[0029] Lactiplantibacillus plantarum having the accession number DSM 34001 , or mixtures thereof and a physiologically acceptable carrier, for use in the prevention and / or treatment of skin or mucosa dysbiosis of a new-born or of a pregnant woman.

[0030] In a second aspect, the invention relates to lactobacilli belonging to Lactiplantibacillus plantarum species which are strains selected from the group comprising:

[0031] Lactiplantibacillus plantarum having the accession number DSM 33995, filed on 4 August 2021 at Leibniz Institute DSMZ-German Collection of Microorganisms and Cell Cultures (also referred as DSMZ hereinbelow), Inhoffenstr. 7B D-38124 Braunschweig;

[0032] Lactiplantibacillus plantarum having the accession number DSM 33996, filed on 4 August 2021 at Leibniz Institute DSMZ-German Collection of Microorganisms and Cell Cultures, Inhoffenstr. 7B D-38124 Braunschweig;

[0033] Lactiplantibacillus plantarum having the accession number DSM 33997, filed on 4 August 2021 at Leibniz Institute DSMZ-German Collection of Microorganisms and Cell Cultures, Inhoffenstr. 7B D-38124 Braunschweig;

[0034] Lactiplantibacillus plantarum having the accession number DSM 33998, filed on 4 August 2021 at Leibniz Institute DSMZ-German Collection of Microorganisms and Cell Cultures, Inhoffenstr. 7B D-38124 Braunschweig;

[0035] Lactiplantibacillus plantarum having the accession number DSM 33999, filed on 4 August 2021 at Leibniz Institute DSMZ-German Collection of Microorganisms and Cell Cultures, Inhoffenstr. 7B D-38124 Braunschweig; Lactiplantibacillus plantarum having the accession number DSM 34000, filed on 4 August 2021 at Leibniz Institute DSMZ-German Collection of Microorganisms and Cell Cultures, Inhoffenstr. 7B D-38124 Braunschweig;

[0036] Lactiplantibacillus plantarum having the accession number DSM 34001 filed on 4 August 2021 at Leibniz Institute DSMZ-German Collection of Microorganisms and Cell Cultures, Inhoffenstr. 7B D-38124 Braunschweig, and mixtures thereof. The above strains were filed at DSMZ as International

[0037] Depositary Authority under the Budapest Treaty for the purposes of patent procedure. Certificates of deposit are submitted on filing.

[0038] The inventors find out that the herein disclosed species and strains are effective in restoring the eubiosis and in promoting the physiological growth of microbiota. In addition, the selected strains of the invention form biofilms on the skin that prevent the adhesion of pathogen microorganisms thereon.

[0039] The selected strains of this invention may be formulated as a composition either for oral administration or topical application.

[0040] The composition for topical application is suitable and intended to prevent and treat skin diseases to dysbiosis such as skin infections or inflammation.

[0041] In accordance with a third aspect, a composition is provided comprising lactobacilli belonging to Lactiplantibacillus plantarum species which are strains selected from

[0042] Lactiplantibacillus plantarum having the accession number DSM 33995,

[0043] Lactiplantibacillus plantarum having the accession number DSM 33996,

[0044] Lactiplantibacillus plantarum having the accession number DSM 33997,

[0045] Lactiplantibacillus plantarum having the accession number DSM 33998,

[0046] Lactiplantibacillus plantarum having the accession number DSM 33999,

[0047] Lactiplantibacillus plantarum having the accession number DSM 34000,

[0048] Lactiplantibacillus plantarum having the accession number DSM 34001 and mixtures thereof and a physiologically acceptable carrier.

[0049] In certain embodiments the composition is an oral composition, for example a nutritional composition.

[0050] In accordance with certain aspects of the invention, the herein disclosed strains of Lactiplantibacillus plantarum or the compositions containing these strains are for use in the prevention or / and treatment of dysbiosis or in maintaining a physiological microbiota of the new-born.

[0051] According to further embodiments, the composition is a topical composition suitable for application on the skin.

[0052] The topical compositions containing the above referred selected strains reduce inflammation on the area or portion of the skin on which are applied.

[0053] The topical compositions are suitable to treat diseases of the skin, especially inflammatory and / or allergic and / or immunologic skin diseases.

[0054] The composition containing the selected strains of Lactiplantibacillus plantarum of the invention may be applied on the skin of parturient abdominal area or mucosae of the genital tract either before and / or during partum.

[0055] The composition containing the lactobacilli of the selected strains may be applied on the skin in any forms suitable for topical application.

[0056] Advantageously, the composition is applied on the abdominal or female genital region of body of a woman to promote the growth of physiological microbiota which is transferred and delivered to the newborn during partum.

[0057] The composition contains the selected Lactobacilli strains, which colonize the skin of the newborn immediately after birth and continue developing with the host into a highly diversified ecosystem. The host-bacterial association provides beneficial relationships and plays an important role in the health of the newborn via several mechanisms. The microbiota transferred to the newborn plays an important role in human health preventing and treating diseases such as inflammatory conditions, infections and immunological diseases via several mechanisms.

[0058] Preferably, the composition is an anhydrous gel, or an aqueous gel containing lactobacilli belonging to the L plantarum strains as disclosed herein. The gel may be a water-based gel extemporaneously prepared.

[0059] The inventors have also discovered that the composition containing the selected Lactiplantibacillus plantarum strains maintains vaginal microbiota eubiosis. This effect reduces the risks of colonization by pathogens and the correlated risks of developing vaginal infections and inflammatory diseases.

[0060] Thus, in accordance with an additional aspect, the invention concerns with a topical composition for maintaining vaginal microbiota eubiosis containing the above selected strains of Lactiplantibacillus plantarum for use in the prevention and treatment of urogenital infections or inflammatory diseases or immune disorders. Preferably, for the uses according to this aspect, the composition is applied on mucosae of the female genital tract.

[0061] In accordance with other aspects, the inventors have also observed that the selected Lactiplantibacillus plantarum strains as described herein, when applied onto the skin, provide an inhibitory effect on pathogens of the skin and regulate the biological mechanism of the local immune response blocking or slowing the inflammatory cascade.

[0062] Accordingly, in a further aspect the invention concerns with uses of the herein disclosed strains in the gynaecological field, especially in the treatment of immune- related skin diseases and in skin infections, in particular with inflammation.

[0063] Preferably, the composition according to an aspect of the invention finds application in the gynaecological field, for example in the treatment of mucosal inflammation or infections from bacteria, fungi or protozoa.

[0064] BRIEF DESCRIPTION OF THE FIGURES

[0065] The invention will now be described in detail and in reference to the attached Figures wherein:

[0066] Figure 1 is a picture showing the operations of scraping the biofilm colony for ECM characterisation according to Example 1

[0067] Figure 2 illustrates the steps followed for the cell-free supernatants (CFSs) preparation and the high-throughput C. albicans inhibition screening according to Example 1 ,

[0068] Figure 3 shows the plate set-up for the gene expression analysis of HaCat cell lines with Candida albicans and four lactobacilli strains of Example 1 ,

[0069] Figure 4 shows the biofilm colony of the seven selected strains used in the study, grown on membrane-filters placed on MRS agar plates. The images show their appearance after 24 h and 48 h from the starting of the incubation of Example 1 , Figure 5 shows bar graphs illustrating the cell densities of the seven strains expressed in Log [CFU / mL], calculated from duplicate serial dilutions and plate count of two different biofilm colonies of Example 1 ,

[0070] Figure 6 shows bar graphs illustrating the average weight of the biomass produced by the seven strains used in the study. The result is obtained by calculating the mean value and standard deviation of the duplicates.

[0071] Figure 7 shows bar graphs illustrating the percentage of the ECM components, calculated as component concentration (mg / mL) divided by the average biomass (mg), divided by the total weight of the ECM fractions of each strain;

[0072] Figures 8 a, b, c, d, e, f, g, illustrate biofilm images obtained with a confocal laser scanning microscope with 488 and 552 nm lasers, and 40x lens. Texas red-labeled ConA marked the polysaccharides (in red) and SYTO9 the cells (in green).

[0073] Figure 9 shows the pathogen inhibition using well diffusion method and WSE. The first well on the top contains cycloheximide as positive control (red circle), the second on the right contains distilled water as negative control (blue circle), the other 2 contain the WSE. On the left petri plate with Borlotti beans WSE, on the right petri plate with buckwheat WSE. Positive result, meaning pathogen inhibition, was shown with a halo of inhibition around the well after 24 h of incubation at 25 °C,

[0074] Figures 10 a, b, c, d show bar graphs illustrating the Expression of Interleukin 8, Interleukin 6, Tumor necrosis factor a, and Interleukin 1 / 3 by HaCat cell lines, determined by qRT-PCR. The control sample represents the gene expression in untreated cells, and it is standardized as equal to 1. Under-expression or overexpression of the genes are represented by lower or higher values, respectively. Expression rates were calculated as the relative quantification data. The data represent mean and standard error of the mean of duplicate analysis,

[0075] Figure 11 shows bar graphs illustrating the cell densities of the gel containing lactobacilli expressed in Log [CFU / mL], calculated from duplicate plate count, after 24 h from the preparation and before the application on HVE,

[0076] Figure 12 shows bar graphs illustrating the percentage of viability of Human Vaginal Model. Control sample was the untreated epithelium, blank sample was the epithelium model treated with blank gel, C. albicans is the epithelium infected with the pathogen (positive control), L plantarum C5, L plantarum POM1 and C5 + POM1 are the epithelium models infected with the pathogen and then treated with the gel containing the respective strain or combination. Data relative to Control, Blank, and C. albicans represent mean and standard error of the mean of technical duplicate analysis. Data relative to L plantarum C5, L plantarum POM1 and the combination represent mean and standard error of the mean of duplicate biological and technical analysis.

[0077] Figure 13 shows the MTT results. On the left side the inserts treated with MTT reagent and isopropanol. On the right side the two aliquots taken from each well. Control sample in black circle, blank sample in white circle, sample infected with Candida albicans in red circle, samples infected and treated with Lactiplantibacillus plantarum C5, Lactiplantibacillus plantarum POM1 and the combination of the two in light blue, blue and green circles, respectively,

[0078] Figures 14 a, b illustrates bar graphs showing the expression of Interleukin 8 and E- cadherine by Human Vaginal Epithelium, determined by qRT-PCR. The control sample represent the gene expression in untreated epithelium model, and it is standardized as equal to 1 . Under-expression or over-expression of the genes are represented by lower or higher values, respectively. Blank sample was the epithelium model treated with blank gel, C. albicans is the epithelium infected with the pathogen (positive control), L plantarum C5, L plantarum P0M1 , and C5 + P0M1 are the epithelium models infected with the pathogen and then treated with the gel containing the respective strain or combination. Expression rates were calculated as the relative quantification data. The data represent mean and standard error of the mean of duplicate analysis.

[0079] DETAILED DESCRIPTION OF THE INVENTION

[0080] In one aspect, the invention origins from the observation that the vaginal microbiota during pregnancy is an important factor for the health of the infant. This is the first source of microorganisms for the new-born that will colonize his gut and skin. In addition, in women, the intestinal and genital microbiota are biological ecosystems in communication and their interaction exerts effects on the immunological homeostasis of the host and vice versa.

[0081] The vaginal microbiota in physiological conditions at the time of partum prevents diseases, especially skin diseases or infections, in the new-born.

[0082] In addition, it has been observed that vaginal delivered babies harbour bacterial communities resembling those of the maternal vagina, while C-section delivered infants do not. This fact is especially relevant because the microorganisms that colonize the body and especially the gut of the newborn have a determinant role in the immune system development and in the development of immune disorders ranging from allergies to inflammatory skin and / or bowel diseases.

[0083] Starting from these observations, the inventors found out that either the oral administration and the topical application on skin or mucosae of selected strains or compositions containing selected Lactobacilli strains of the species Lactiplantibacillus plantarum prevent dysbiosis in the human organism and also promote the growth of lactobacilli-dominated vaginal microbiota in pregnant women which can be transferred to the newborn either during natural and caesarean partum. According to a main aspect, the invention concerns a composition comprising a strain of Lactiplantibacillus plantarum selected from the group consisting of:

[0084] Lactiplantibacillus plantarum having the accession number DSM 34000,

[0085] Lactiplantibacillus plantarum having the accession number DSM 33995,

[0086] Lactiplantibacillus plantarum having the accession number DSM 33999,

[0087] Lactiplantibacillus plantarum having the accession number DSM 33996,

[0088] Lactiplantibacillus plantarum having the accession number DSM 33997,

[0089] Lactiplantibacillus plantarum having the accession number DSM 33998, and

[0090] Lactiplantibacillus plantarum having the accession number DSM 34001, or mixtures thereof and a physiologically acceptable carrier, for use in the prevention and / or treatment of skin or mucosa dysbiosis of a new-born or of a pregnant woman.

[0091] In a second aspect the invention relates to the selected Lactiplantibacillus plantarum species defined in claim 7 and uses thereof as defined herein.

[0092] In a preferred aspect, the composition comprises a strain of Lactiplantibacillus plantarum selected from the group consisting of:

[0093] Lactiplantibacillus plantarum having the accession number DSM 34000, Lactiplantibacillus plantarum having the accession number DSM 33995, and Lactiplantibacillus plantarum having the accession number DSM 33999, or mixtures thereof.

[0094] More preferably the composition comprises the strain of Lactiplantibacillus plantarum: Lactiplantibacillus plantarum strain having the accession number DSM 34000.

[0095] The Lactiplantibacillus plantarum strains herein described can be viable, tyndallized or lyophilized. According to another aspect, the invention provides a composition comprising a Lactiplantibacillus plantarum strain of claim 1 and mixtures thereof. Advantageously, the composition of this invention is intended for use in preventing or treating dysbiosis or maintaining a physiological microbiota of an individual, especially a new-born.

[0096] In certain embodiments the composition is for oral administration and contain a Lactiplantibacillus plantarum strain of claim 1 and mixtures thereof and a physiologically acceptable carrier.

[0097] For example, the composition for oral administration is contained in or formulated as a nutritional or dietetic product for oral administration.

[0098] In accordance with additional aspects, the composition is for topical application and may be applied on the skin of abdominal area and / or mucosae of the female genital tract of a pregnant woman or of a woman in labour.

[0099] Advantageously, the lactobacilli of the selected Lactiplantibacillus plantarum strains contained in the composition applied either on the abdominal area or on female genital tract, depending by the delivery way, are transferred during partum from the parturient to the new-born.

[0100] The inventors have also observed that maintaining a vaginal microbiota eubiosis is a key factor to prevent urogenital infections and inflammatory diseases and to avoid the increased risk of preterm delivery and preterm rupture of the fetal membrane in pregnant women.

[0101] Preferably the urogenital infections are vaginal infections, in particular bacterial or fungal infections.

[0102] In addition, it is estimated that up to 50% of preterm birth are caused by infections and the aetiological agents often ascend from the vagina along the mucosal surface. Moreover, pregnant women with bacterial vaginosis, have an increased risk of late miscarriage too.

[0103] The inventors observed that application of the topical composition as described herein on the vaginal area, especially of pregnant women, prevents dysbiosis and related urogenital infections and inflammatory diseases.

[0104] Accordingly, a further aspect the invention concerns with a topical composition for maintaining vaginal microbiota eubiosis comprising lactobacilli of the Lactiplantibacillus plantarum strains defined in the present invention for use in the prevention and treatment of urogenital infections or inflammatory diseases or immune disorders.

[0105] The composition of the invention contains selected strains of lactobacilli of the species Lactiplantibacillus plantarum. The inventors found out that these strains colonize the new-born immediately after birth and continue to developing with the host into ecosystem which prevents dysbiosis. In addition, the selected strains of Lactiplantibacillus plantarum form a biofilm on the skin of the new-born and pregnant women.

[0106] Advantageously, the herein disclosed Lactiplantibacillus plantarum species when formulated as a topical composition, provide either immunomodulation and antiinflammatory actions enabling the physician to treat a wide range of diseases especially allergic, autoimmune, infectious disease of the skin or mucosa of human beings.

[0107] In addition, the composition containing the selected strains of the invention when applied on the skin or / and mucous of an individual triggers the formation of a biofilm, which improves the resistance to hostile environments and provides for the activation of human cells inflammatory response.

[0108] These effects make the disclosed lactobacilli species and strains useful in the treatment of infections, especially skin and mucosal infections.

[0109] In particular, the herein described association is effective in the treatment of most of common bacterial infections, that mainly involve gram-positive bacteria, such as Staphylococcus aureus or Escherichia coli and pathogenic yeasts such as those of the genus Candida, for example Candida albicans.

[0110] Amongst lactobacilli belonging to Lactiplantibacillus plantarum, the strains as herein disclosed were isolated from fruits, vegetables, sourdough.

[0111] The composition herein disclosed may contain one or more of the following selected strains :

[0112] Strains Code Source of isolation

[0113] Lactiplantibacillus plantarum DSM 33995 C5 Fruits / vegetables

[0114] Lactiplantibacillus plantarum DSM 34000 P0M1 Fruits / vegetables

[0115] Lactiplantibacillus plantarum DSM 34001 1 LS 16 Fruits / vegetables Lactiplantibacillus plantarum DSM 33997 D3.15 Sourdough

[0116] Lactiplantibacillus plantarum DSM 33999 0E1 Fruits / vegetables

[0117] Lactiplantibacillus plantarum DSM 33996 D2.6 Sourdough

[0118] Lactiplantibacillus plantarum DSM 33998 E3.8 Sourdough

[0119] The herein described Lactiplantibacillus plantarum strains find industrial application in the preparation of compositions, such as pharmaceutical compositions, especially for topical application.

[0120] In accordance with an aspect, the present invention relates to a composition comprising a Lactiplantibacillus plantarum strains as defined herein and a pharmaceutically or physiologically acceptable carrier.

[0121] The compositions of the present invention encompass any compositions made by mixing a Lactiplantibacillus plantarum spp such as the one or more of the strains as herein defined and a physiologically acceptable carrier. Such compositions are suitable for use in humans especially in a female and newborn.

[0122] In certain aspects a method to modulate the skin microbiome composition or to promote the physiological growth of microbiota especially in a pregnant woman or in a new-born is hereby provided, wherein the method comprises the oral administration or topical application of a composition comprising a Lactiplantibacillus plantarum strain as defined in claim 1 or a mixture thereof.

[0123] In some aspects, a composition is provided containing a composition comprising a Lactiplantibacillus plantarum strain as defined in claim 1 or a mixture thereof for use in the treatment of a skin disease especially an inflammatory disease of the skin.

[0124] The composition of the present invention comprises a therapeutically effective amount of one or more of Lactiplantibacillus plantarum strains as herein disclosed and a pharmaceutically acceptable carrier.

[0125] In certain embodiments, the strain of the present invention can be combined as an active ingredient in intimate admixture with a suitable pharmaceutical carrier and / or excipient according to conventional pharmaceutical compounding techniques.

[0126] Suitable route of administration in any given case will depend in part on the nature and seventy of the conditions being treated.

[0127] The composition of the invention can be intended for external use and for oral administration. The composition of the invention can be authorized for sale as a product for local application, or as a drug, a dietary or nutritional supplement intended for oral administration.

[0128] Typically, the composition of the invention comprises a carrier, diluent or physiologically acceptable excipient.

[0129] Typically, the physiologically acceptable carrier of the composition of the invention is an excipient, or diluent suitable for topical application and / or oral administration. Within the scope of the present invention the term “carrier” refers to an excipient, carrier, diluent or coadjuvant that may be present in the composition of the invention. Any carrier and / or excipient suitable for the desired form of preparation for administration is contemplated in the uses of the plant extract or active ingredients present therein described here.

[0130] The carrier, diluent or physiologically or pharmaceutically acceptable excipient may be selected on the basis of the administration route for which the resulting composition is intended.

[0131] According to some preferred embodiments the composition may be in the form for oral administration.

[0132] The compositions for oral administration may be in solid or liquid form wherein the strains are suspended.

[0133] Typical compositions in solid form comprise tablets, capsules, powders, granules and pills.

[0134] Typical compositions in liquid form comprise solutions, emulsions, suspensions and syrups.

[0135] According to certain embodiments, the composition of the invention further comprises one or more active ingredients such as vitamins, minerals, micronutrients and or other active ingredients.

[0136] According to some embodiments the composition for oral administration is a functional food, a nutraceutical composition, a dietary product, a supplement or nutritional product or a medical device.

[0137] Functional food means any modified food or food ingredient that can provide a benefit or protection against a drawback or physiological affection, in addition to the traditional nutrients that it contains. Nutraceutical product means an isolated or purified product from edible substances. A nutraceutical is such when it is demonstrated that it has a physiological benefit or that it provides protection against a drawback or physiological disorder.

[0138] Dietary or food supplement means a product that contains a vitamin, mineral, plant extract, amino acid, metabolite, extract, concentrate or mixture of these ingredients. The amount administered and the frequency of administration of the composition will depend on the nature and severeness of the dysbiosis to be treated.

[0139] In some embodiments the administration route of the composition of the invention is the topical one. According to these embodiments the composition is for topical application and is applied to the skin and mucous membranes, especially in the vaginal area.

[0140] The composition for topical application may be in solid, semi-solid or fluid form. Suitable formulations in semi-solid form include creams, balms, emulsions, pastes, ointments.

[0141] In some embodiments the formulation for local administration is in fluid form, e.g. in the form of lotions, shampoos, suspensions, in the oily form or anhydrous such as oleolites or semi-fluid e.g. in the form of gels and emulsions either of the o / w and w / o type and liposomes. For example, the bacterial strain can be diluted in a carrier in physiologically acceptable liquid form such as water or glyceryl solution or mixed with other liquids suitable for local application.

[0142] In some embodiments the compositions of the invention may comprise excipients commonly used in the formulation of cosmetic or pharmaceutical preparations, for local use, such as preservatives, stabilizers, emulsifiers, buffers, wetting agents, dyes and other excipients commonly used in cosmetic / pharmaceutical preparation techniques. Preferably the excipients do not interfere with the local microbiota.

[0143] In one embodiment the formulation for local application is in emulsion form containing the extract carried in a suitable excipient. In some embodiments the composition for topical application comprises an excipient of the hydroxymethyl cellulose type and / or gelling agents suitable for the formulation and for the substances.

[0144] By way of an example, the compositions of the invention in liquid form can be prepared by dissolving or dispersing the bacterial species and a gelling or thickening agent in a liquid such as water. The liquid composition can be buffered to reach a pH range conveniently selected from 4 to 7,5 to be compatible with the pH of the skin or mucosae.

[0145] Preferably, the composition is an anhydrous gel.

[0146] Suitable gel contains water, preferably deionized water.

[0147] Preferably, the composition is a gel containing one or more active strains of the invention, which colonizes the skin of the newborn immediately after birth and continues developing with the host into a highly diversified ecosystem.

[0148] The amount of active strain in such therapeutically useful compositions is such that therapeutically active dosage will be obtained. The compositions may be prepared by any of the methods well-known in the art of pharmacy.

[0149] In some embodiments, in the pharmaceutical compositions of the present invention the active principle or active principles are generally formulated in dosage units and prepared by any of the methods well-known in the art of pharmacy.

[0150] When used in combination with one or more other active ingredients, the strain of the present invention and the other active ingredient may be used in lower doses than when each is used singly.

[0151] In certain embodiments, a dosage unit is provided containing a composition as defined in claim 2.

[0152] In certain embodiments the unit of dosage of a composition includes a lyophilized strain of lactobacilli as herein disclosed with a bacterial load in the range of 1 * 1O10- 9* 1011unit forming colonies (ufc) per gr.

[0153] Preferably, the lyophilized Lactoplantibacillus plantarum strains are diluted in a carrier for the preparation of a suitable form of administration.

[0154] Typically, in the composition, the vitality of the Lactoplantibacillus plantarum strains as defined herein is maintained at a concentration expressed as cfu / g or ufc / g, or cfu / dose or ufc / dose which promote the physiological growth of microbiota and prevent dysbiosis especially in pregnant women or new-born.

[0155] In certain aspects a composition as defined in claim 2 is hereby provided for use in the treatment of dysbiosis. The treatment may be carried out for a period of 1 to 3 months or at time of needs for example for treating urogenital tract infections. In certain aspects the use of the composition of claim 2 is provided for promoting (preserving and maintaining) the physiological microbiota growth in a pregnant woman. The composition may be administered through the oral route or by topical application in the body area surrounding the vagina of a pregnant women. For example, the treatment may be started 6 months before partum.

[0156] The treatment with the composition may be carried out either by the topical or oral administration or a combination thereof.

[0157] The composition containing the Lactobacilli of the Lactoplantibacillus plantarum strains as defined herein may be applied on the skin of human beings.

[0158] Typically, the composition may contain an amount of the above identified bacterial species from 0.00001 % to 10%, from 0.0001 to 3%, from 0,01 to 2% weight with respect to the total weight of the composition. Typically, the percentages (%) are by weight and expressed as a lyophilised product with a titre of 1-2* 1011ufc.

[0159] According to certain embodiments, the topical composition is a gel comprising deionized water preferably from 65 to 90% by weight, a glycerol suspension containing lactobacilli of claim 1 , preferably from 15 to 35%by weight, a thickening agent preferably from 0, 5 to 10%by weight, and optionally a preservative, preferably from 0,01 to 3%by weight.

[0160] In some embodiments, the amounts effective of lactobacilli for topical formulation will depend on the seventy of the disease, disorder or condition, previous therapy, the individual’s health status and response to the drug. In some embodiments, the dose is in the range from 0.001 % by weight to about 60% by weight of the formulation.

[0161] The composition may be conveniently presented in unit dosage form. The dosage unit may contain from 0.00001 to 1000 mg of a strain per dosage unit for daily administration.

[0162] In certain embodiments the invention provides a supernatant of a strain or strains herein disclosed. The term supernatant means non-viable bacterial products including metabolic by-products secreted by the strains of the invention and which have biological activity in the host, especially when orally administered or topically applied on the skin of human body. In certain embodiments the supernatant also comprises any materials of the selected strains of the invention, advantageously including its wall, cytoplasm, cytoplasmatic membrane, genetic material (nucleoid), ribosomes which may be released or obtained after bacterial lysis.

[0163] The supernatants provide physiological benefits to the host and may be used to formulate a composition for oral and / or topical administration as herein described. A suitable supernatant is obtained as described hereinbelow in Example 1 , par. 3.3.1.

[0164] The invention is further disclosed by way of the following examples.

[0165] EXAMPLE 1

[0166] This example provides experimental evidence of the effects / activity of compositions containing the selected Lactiplantibacillus plantarum strains.

[0167] 1. Gel

[0168] The tested gel contains 77% of deionized water, 20% vegetal glycerol suspension containing the following lactobacilli of the 2% of Solagum tara (Seppic, France), and 1 % of EUXYL K712, a liquid cosmetic preservative, that contains sodium benzoate and potassium sorbate.

[0169] 2. Aim of the study

[0170] The aim of this study was to investigate and select nomadic lifestyle lactobacilli, to be used in the formulation of a probiotic gel, to promote lactobacilli-dominated vaginal microbiota in pregnant women. This probiotic gel would be used to prevent urogenital infections, obstetrical complications and to help the establishment of a proper gut microbiota on the newborn.

[0171] The screening activities presented in this study are part of a broader project, which started from a collection of 109 lactobacilli isolated from different sources (fruits, vegetables, dairy products, and sourdough). These candidates were first screened for their functional characteristics (production of hydrogen peroxide, production of L- and D- lactic acid isomers, co-aggregation capacity, hydrophobicity characteristics, and peptide hydrolysis activity) and then for their inhibition ability against three main vaginal pathogens (Streptococcus agalactiae, Staphylococcus aureus, and Candida albicans).

[0172] From this first screening, seven best performing strains were selected and subjected to the analysis presented in this study, to further investigate their potential fitness in vaginal microbiota. The present study included an investigation of the ability of the candidate strains to form biofilms. This bacterial property is thought to be associated to improved surface adhesion capabilities and to human cells and niches colonization, also by pathogen exclusion. In addition, it seems to improve the resistance to hostile environments, such as the gastro-intestinal tract.

[0173] 3. Materials and methods

[0174] 3. 1 Candidate strains

[0175] Seven strains (Lactobacillus spp.) previously isolated from various sources (fruits and vegetables, sourdough, and milk) and belonging to the “Micro4Food” culture collection, were tested (NOI TECHPARK, Free University of Bolzano-Bozen) (Table 1 ). The strains were stored in cryo vials (20% glycerol) at -20°C.

[0176] Table 1. Candidate strains

[0177] Lactiplantibacillus plantarum DSM 33995 is referred as C5

[0178] Lactiplantibacillus plantarum DSM 34000 is referred as POM 1

[0179] Lactiplantibacillus plantarum DSM 34001 is referred as 1 LS 16

[0180] Lactiplantibacillus plantarum DSM 33997 is referred as D3.15

[0181] Lactiplantibacillus plantarum DSM 33999 is referred as OE1

[0182] Lactiplantibacillus plantarum DSM 33996 is referred as D2.6

[0183] Lactiplantibacillus plantarum DSM 33998 is referred as E3.8

[0184] 3.2 Biofilm formation on membrane-filters The candidate strains were refreshed from cryo-vials and cultivated in MRS broth medium at 37 °C until they reached the stationary phase of growth (~ 24 h). Then, the cultures were centrifuged (7500 rpm, 10 min) washed and resuspended in physiological solution. The optical density of each suspension was adjusted to ca. 109CFU / mL (at a wavelength of 620nm), using the UV-1800 Spectrophotometer (SHIMADZU). Meanwhile, membrane-filters (0.22 pm, , Whatman) were placed on aluminum foil and sterilized in a laminar hood, with UV light for 15 min on each side. The membrane-filters were then placed on MRS agar plates. Finally, 5 pL of the cell suspensions, adjusted to 109CFU / mL, were inoculated on the sterile membranefilters, placed on MRS agar plates, and incubated at 37°C for 48 h. Each strain was inoculated on 5 different membrane-filters, which were subjected to different analyses. Every 8-10 hours the membrane-supported biofilms were placed on a fresh agar plates to supply new nutrients.

[0185] 3.2. 1 Cell density e valuation

[0186] Two membrane-filters, for each strain, with the biofilm colonies were placed in 15 mL falcon tubes containing 9 mL of physiological solution and vortexed vigorously to suspend the biofilm. Serial dilutions, of each falcon tube, were prepared and inoculated by inclusion in MRS agar plates. The plates were incubated for 48 h and the colonies were then counted to calculate the cell density.

[0187] 3.2.2 Biomass quantification

[0188] To quantify the biomass, we first weighed 3 membrane-filters and calculated the average weight Then, we filled falcon tubes with 9 mL of physiological solution, and we weighed them. Finally, we inserted the membrane-supported biofilm into the falcon tube, and we weighed it. The biomass weight was calculated by subtracting the average weight of the membrane-filters and the initial weight of the falcon tube containing physiological solution from the final weight of falcon tube containing the membrane-supported biofilm. The experiment was performed in duplicate.

[0189] 3.2.3 Collection and characterization of extracellular matrix ( ECM)

[0190] To collect and characterize the ECM components of the biofilm colonies, we followed the method described by Chiba et al. 2015. Two membrane-supported biofilms were used for this experiment. Briefly, from the membrane-filters the biofilm colonies were scraped with a scraper (as shown in Fig. 1) and suspended with 1 mL of 1.5 M NaCI solution. These suspensions were centrifuged (5000 g, 10 min) at 25 °C, without any incubation period. The supernatants were harvested as ECM fractions and analyzed for their proteins, DNA, and total saccharides content.

[0191] Proteins in the isolated ECM fractions were quantified using the Bradford method. Bovine serum albumin (BSA) was used as a standard for the calibration curve. The calibration curve was built up by measuring different BSA concentrations (from 5 mg / mL to 0.001 mg / mL). Blank sample contained 800 pL Milli-Q water and 200 pL of Bradford reagent. Then, ten microliters of ECM fractions were mixed with 790 pL of Milli-Q water and 200 pL of Bradford reagent inside cuvettes. The concentration was measured at 590 nm using UV-1800 Spectrophotometer, SHIMADZU.

[0192] The total saccharide concentrations in the ECMs were measured by phenol sulfuric acid method. Glucose was used as a standard for the calibration curve. The calibration curve was built up by measuring different glucose concentrations (from 10 mg / mL to 0.05 mg / mL). Twenty microliters of the isolated ECM were mixed with 20 pL of 5% phenol in the 96 wells plate. Then, 100 pL of sulfuric acid was added and the plate was incubated for 10 min at 25°C. The concentration was measured at 492 nm with Infinite M Nano+ Spectrophotometer (TECAN, Austria).

[0193] The concentration of the DNA was measured with NanoDrop 2000 (Thermo Fisher Scientific, Waltham, MA, USA).

[0194] 3.2.4 Confocal Laser Scanning Microscopy

[0195] The last membrane of each strain was characterized using confocal laser scanning microscopy. The membrane-filter with the biofilm was carefully mounted on a glass slide, covered with 50 pL of a staining solution, and incubated for 1 h in darkness, inside a petri plate, to avoid the drying up of the biofilm. The staining solution contained 200 pg / mL texas red-labeled Concanavalin A (ConA) e 5 pM SYTO9, which respectively marked the polysaccharides (in red) and the cells (in green). After the incubation, the biofilm was carefully covered with a cover glass slide, paying attention to preserve the biofilm colony structure. Biofilm images were obtained with a confocal laser scanning microscope (Leica SP8LIA, Leica Microsystems, Germany) with 488 and 552 nm lasers, and 40x lens using immersion oil. Images were analyzed with the software LAS X. 3.3 High-throughput C. albicans inhibition screening

[0196] Figure 2 reports all the steps that were followed for the cell-free supernatants (CFSs) preparation and the high-throughput C. albicans inhibition screening.

[0197] 3.3. 1 Cell free supernatant preparation

[0198] The strains were cultivated in falcon tubes containing MRS (de Man Rogosa Sharp) broth medium for 24 h at 37°C. Then, they were centrifuged (7500 rpm, 10 min), and the CFS was collected for each strain. One aliquot of the CFS was kept at the initial acidic pH (non-neutralized pH), while another aliquot of CFSs was adjusted at pH 7 (neutralized pH), using NaOH. The CFSs were then sterilized by filtration using 0.22 pm filters and stored at -20°C.

[0199] The CFS, obtained from the seven different strain cultures, were then mixed, in equal amount, in all the possible combinations, each one containing three supernatants, obtaining 56 combinations at neutralized pH and 56 combinations at non-neutralized pH.

[0200] 3.3.2 C. albicans inhibition using combinations of CFSs

[0201] C. albicans was cultivated in Sabouraud broth with 5% Tween® 80 (SIGMA- ALDRICH), for 24h at room temperature (~25 °C). Tween® 80 (SIGMA-ALDRICH) was used to reduce C. albicans aggregation and flocculation, which would make difficult to follow its growth by spectrophotometric detection. After the incubation period, the cells were centrifuged (7500 rpm, 10 min), washed, and resuspended in physiological solution. The optical density (O.D.) was adjusted to 107CFU / mL (at a wavelength of 600 nm), using UV-1800 Spectrophotometer (SHIMADZU), diluting with Sabouraud broth medium.

[0202] Then, 96-well plates were set using 150 pL of C. albicans, previously adjusted in the OD, and 50 pL of the CFSs of the strains in single or in combination at neutralized and non-neutralized pH. The experiment was performed in triplicate. In addition, each 96-wellplate, was set with two blank wells, containing 150 pL of Sabouraud broth medium either with neutralized pH (50 pL) or non-neutralized pH (50 pL), to check the media sterility. Two control wells were also set containing 50 pL of MRS broth medium at neutralized pH or at non-neutralized pH and 150 pL of C. albicans cell suspension, previously adjusted in the OD, to check the pathogen growth. The growth kinetics were monitored every 15 minutes for 24 h at 37 °C, by measuring the absorbance at 600 nm, using Infinite M Nano+ Spectrophotometer (TECAN, Austria). Growth parameters, which are lag phase (A), growth rate (p), and maximum absorbance (A), were determined using grofit R package (Kahm et al., 2010) for each replicate of CFSs and controls (one way). Inhibition effect was assessed through non-parametric one-way Kruskal-Wallis followed by Dunn Control test provided by PMCMR R package (Pohlert, 2016).

[0203] 3.4 Pathogen inhibition using well diffusion method and water-soluble vegetable extracts

[0204] The inhibition of C. albicans was evaluated using water-soluble extract (WSE) obtained from buckwheat flour (Fagopyrum esculentum) and borlotti beans (Phaseolus vulgaris). WSE of whole-meal buckwheat flour and dry borlotti beans, found in a local supermarket, were prepared as described by Coda et al. 2008. Briefly, 50 g of each vegetable was homogenized with 50 mL of distilled water in a blender (Kinematica, Microtron, MB 550, Switzerland). The ground material was added to 50 mL of boiling distilled water, incubated for 10 min at room temperature while being stirred, then centrifuged (14000 g, 20 min) to recover the supernatant, which was then sterilized by filtration using 0.22 pm filters. C. albicans was cultivated in Sabouraud broth, at 25°C for 24h. Then, the cell suspension was centrifuged (7500 rpm, 10 min), washed, and resuspended in physiological solution. The O.D. was adjusted to ca. 107CFU / mL (at a wavelength of 600 nm), using UV-1800 Spectrophotometer (SHIMADZU). The cell suspension was inoculated by inclusion in two petri plates with Sabouraud agar, in 1 :10 dilution. After agar solidification, four wells (3 mm diameter) were obtained in each plate. One plate was used for the buckwheat WSE testing and the other one for the borlotti beans WSE. In each plate the following suspensions were added: one well was filled with 100 pL of negative control (distilled water), a second well was filled with 100 pL of positive control (cycloheximide 1 %), and the last two were filled with 100 pL of the two WSEs respectively. The plates were incubated at 4°C for 1 h, to allow diffusion of the extracts in the agar, and then the plates were incubated at 25°C for 24 h.

[0205] 3.5 Cell culture

[0206] A human immortalized HaCaT keratinocyte cell line (BS CL 168) was obtained from Istituto Zooprof ilattico (Brescia, Italy). It was grown in 75 cm2tissue culture flask, in RPMI-1640 medium (Sigma Aldrich, Milan, Italy) containing 10% fetal bovine serum (FBS) (Sigma Aldrich, Milan, Italy) and 100 U / mL penicillin / streptomycin mixture (Gibco, Milan, Italy). The cells were incubated in a humidified, 5% CO2 atmosphere at 37°C. After the cell monolayers reached the confluency, circa two days, they were reseeded in a 1 :2 split ratio.

[0207] 3.5. 1 Gene expression analysis of Ha Cat cell lines treated with C. albicans and lactobacilli

[0208] HaCat keratinocyte cell lines were seeded in 12-well culture plates and incubated in the same medium and conditions described before, until reaching the confluency. L plantarum C5, L plantarum POM1 and L plantarum D2.6 were grown in MRS broth medium at 37 °C for 24 h, then they were centrifuged (7500 rpm, 10 min) washed and resuspended in physiological solution. The optical density of each suspension was adjusted to ca. 108CFU / mL (at a wavelength of 620 nm) using the 6715 UVA / IS Spectrophotometer (Jenway, UK). C. albicans was grown in Sabouraud broth at 25 °C for 24 h, then it was centrifuged (7500 rpm, 10 min) washed and resuspended in physiological solution. Its optical density was adjusted to ca. 107CFU / mL (at a wavelength of 600 nm).

[0209] Then, 500 pL of the suspension of 108CFU / mL of lactobacilli and 500 pL of the suspension of 107CFU / mL of C. albicans were placed individually in wells with confluent monolayer cells. A combination of the four lactobacilli were also tested, in this case 125 pL of each cell suspension was added in the well with confluent monolayer cells. In addition, we tested combination of 250 pL of the suspension of 108CFU / mL of lactobacilli and 250 pL of the suspension of 107CFU / mL of C. albicans, in wells with confluent monolayer cells, and 400 pL of the combination of the four lactobacilli cell suspensions and 100 pL of C. albicans cell suspension. The plate also contained a control well, with HaCat cells alone. The 12-well plate was then incubated for 2 h, in a humidified, 5% CO2 atmosphere at 37 °C. The plate setup is shown in Figure 3.

[0210] After the incubation period, TRI Reagent (Sigma Aldrich, Italy) method, as described by Chomczynski and Mackey (1995), was used to isolate and purify RNA from HaCat cell lines. Briefly, the supernatant was removed from each well and cells were lysed with 1 mL of TRI reagent, which was added in each well and passed through the pipette several times to form a homogeneous lysate.

[0211] Then the lysate was transferred in an Eppendorf tube and 200 pL of Chloroform per tube were added and vortexed for 15 sec. After 15 minutes resting at room temperature, the resulting mixture was centrifuged (12000 g, 15 min at 4 °C). Thereafter, we obtained the separation in three phases and the upper aqueous phase contained the RNA. This aqueous phase was transferred in a new Eppendorf tube and 200 pL of isopropanol for each tube was added for the precipitation of the RNA. After overnight resting, the samples were centrifuged (12000 g, 10 min at 4 °C) to collect the RNA pellet. The supernatant was removed, and the RNA pellet was washed by adding 1 ml of sterile filtered 75% ethanol. The sample was vortexed and then centrifuged (7500 g, 5 min at 4 °C), the supernatant was removed, and the pellet was let drying for 15 min. Finally, the pellet was resuspended in 20 pL of DEPC-water and its concentration was determined using the spectrophotometer (Spettrofotometro UVA / IS Jenway 6715, Cole-Parmer, Staffordshire, ST15 OSA, UK). Then, 2 pg RNA template were used for cDNA synthesis in a 20pL reaction volume, using the PrimeScriptTM RT Reagent Kit (TakaraBioInc., Japan). The cDNA was amplified and detected by the Stratagene Mx3000P Real-Time PCR System (Agilent Technologies Italia S.p.A., Milan, Italy). PCR conditions were the following: 37 °C for 15 minutes, 85 °C for 5 seconds and 25°C for 2 minutes. Afterwards, the following Taqman gene expression assays were used for quantitative Real-time PCR (qRT-PCR): GADPH Hs99999905_m1 (human glyceraldehyde-3-phosphate dehydrogenase, GAPDH), IL-8 Hs00174103_m1 (Interleukin 8, IL-8), IL-6 Hs00985639_m1 (Interleukin 6, IL-6), TNF-a Hs00174128_m1 (Tumor necrosis factor alpha, TNF-a) and IL-1 [3 Hs01555410_m1 (Interleukin 1 (3, IL-1 (3). GAPDH was used as the housekeeping gene. PCR amplifications were carried out in a 20pL of total volume. The mixture of reaction contained 10 pL of 2X Premix Ex Taq (Takara, Japan), 1 pL of 20* TaqMan gene expression assay, 0.4 pL of RoX Reference Dye II (Takara, Japan), 4.6 pL of water, and 4 pL of cDNA. PCR reactions were performed in duplicate using Stratagene Mx3000P Real-Time PCR System (Agilent Technologies Italia S.p.A., Milan, Italy). PCR conditions were the following: 95°C for 30 sec followed by 40 cycles of 95°C for 5 sec, 60°C for 20 sec. The average value of the target gene was normalized using GAPDH gene, and the relative quantification of the levels of gene expression was determined by comparing the A cycle threshold (ACt) value (Vigetti et al. 2008).

[0212] The statistical analysis was performed using Graphpad Prism 6 (GraphPad Software Inc). Data were assessed for normality using the D’Agostino & Pearson omnibus normality test. Subsequently, datasets assumed to follow a normal distribution were compared with an unpaired t-test (2 groups being compared). Data are expressed as the mean, mean ± SEM, or mean fold change ± SEM. p-values <0.05 were regarded as statistically significant.

[0213] 3.6 Human Vaginal Epithelium model

[0214] SkinEthicTM Human Vaginal Epithelium (HVE) was obtained from EpiSkin (Lyon, France). The HVE model is composed of A431 cells (derived from a vulval epidermoid carcinoma) cultivated on an inert polycarbonate filter at the air liquid interface in a chemically defined medium After receiving the HVE, 15 inserts were placed in 24-wells plate, containing 1 mL of Maintenance medium (SkinEthic, Episkin), and re-equilibrated for 24 h at 37 °C, in a humidified, 5% CO2 atmosphere. In this study we present analyses performed on the inserts, while the culture media were conserved at -80 °C for following protein analyses.

[0215] 3.6. 1 Formulation of gel containing lactobacilli

[0216] L plantarum C5 and L plantarum POM1 were selected to be inserted in gel formulations, for the treatment of HVE. The lactobacilli-containing gel was developed in collaboration with the R&D Innovation center of Giuliani SpA (Milan, Italy). The selected lactobacilli were cultivated in MRS broth medium for 24 h at 37 °C. The cell density of each culture was calculated by measuring the optical density (at a wavelength of 620 nm) using the 6715 UV / VIS Spectrophotometer (Jenway, UK). With this value of optical density, we estimated the necessary amount of culture to be centrifuged, to obtain a pellet, containing ca. 1 -2 * 1011CFU. The pellet was obtained by centrifuging the culture (10000 rpm, 5 min) and by washing it with physiological solution. The pellet was then resuspended in 10 mL of vegetal glycerol (Acef s.p.a., Italy), to a final cell density of ~ 1 -2 * 1010CFU / mL. Finally, the glycerol suspension containing lactobacilli was inserted in an aqueous-based gel formulation.

[0217] The tested composition contained 77% of deionized water, 20% vegetal glycerol suspension containing lactobacilli, 2% of Solagum tara (Seppic, France), and 1 % of EUXYL K712, a liquid cosmetic preservative, that contains sodium benzoate and potassium sorbate. The final cell density of the lactobacilli in the probiotic gel was about 2-4 * 109CFU / mL. The viability of the lactobacilli in the gel formulation was assessed by preparing serial dilutions and by plating these on MRS agar medium, before applying them to the HVE, which was 24 h after the gel preparation. Four gels were prepared, one contained cell suspension of L plantarum C5, one L plantarum POM1 , one the combination of the two strains and the last one did not contain any cell suspension (blank gel).

[0218] 3.6.2 Infection and treatment

[0219] C. albicans was cultivated in Sabouraud broth medium for 24 h at 25 °C, cells were washed and resuspended in physiological solution and optical density was adjusted to ca. 5 * 106CFU / mL (at a wavelength of 600 nm) by diluting in Maintenance medium (SkinEthic, Episkin). Then, 30 pL of this cell suspension was added to eleven HVE inserts, after their re-equilibration period, and they were incubated in a humidified, 5% CO2 atmosphere at 37°C for 24 h, to allow the pathogen adhesion and infection. Four inserts were not infected and kept for control. After the incubation, the gel containing lactobacilli (24 h from the preparation) were applied on the so infected HVE, while the blank gel was applied in two not infected inserts. Finally, we obtained: two (negative) control inserts (not infected and not treated with gel), two blank inserts (not infected and treated with blank gel), two (positive) inserts infected with C. albicans (untreated), three inserts infected with C. albicans and treated with L plantarum C5 -containing gel, three inserts infected with C. albicans and treated with L plantarum P0M1 -containing gel, three inserts infected with C. albicans and treated with L plantarum C5 + L plantarum P0M1 -containing gel. After gel application the inserts were incubated in the same condition described before for 24 h.

[0220] 3.6.3 MTT assay

[0221] For MTT assay we used inserts: one control insert, one blank insert, one insert infected with C. albicans, and two inserts for each infected and treated with lactobacilli containing gel.

[0222] For the preparation of MTT solution, a stock solution of MTT (5 mg / mL in PBS) was diluted 1 :10 in the cell culture medium.

[0223] After 24 h of incubation with gel containing lactobacilli, the inserts were rinsed with PBS and placed in new 12-well plate containing 300 pL per well of the MTT solution. After 2 h of incubation in a humidified, 5% CO2 atmosphere at 37°C, 800 pL of isopropanol were added in each well. The plate was incubated again for 1 .5 h, in the same conditions above mentioned. Thereafter, three aliquots (200 pL) were taken from each well and placed in 96-well plates. The optical density was measured at 570 nm with a BioTek Micro-volume Plate Reader (BioTek Instruments Inc., Bad Friedrichshall, Germany) and elaborated with the ELX808 software (BioTek Instruments Inc., Bad Friedrichshall, Germanyrif) (reference filter: 690 nm).

[0224] Results are expressed as percentage of viability compared to negative control (mean ± standard error of duplicate cultures).

[0225] % of viability = [OD(57o nm - 630 nm) test product I OD(57o nm - 630 nm) negative control] x 100

[0226] 3.6.4 Gene expression analysis ofHVE infected with C. albicans and treated with lactobacilli-containing gel

[0227] The remaining six inserts were used for the gene expression analysis. Total RNA was extracted from the HVE using the RNeasy mini kit according to De Vuyst et al. (2014). Briefly, we dissected the circumference of the polycarbonate filter, using a sharp surgical blade, from the bottom of the insert. Then, the dissected disc was transferred into a 12-well culture plate containing 600 pL of RLT buffer.

[0228] After 1 or 2 min, stratum corneum detaches from the epidermis. Remove it with a pair of tweezers and discard.

[0229] For disrupting keratinocytes, we scratched the epidermis with a micropipette tip in lysis buffer provided by the kit. The lysate was homogenized by several passages in the pipette and transferred into a 2 ml collection tube. From this point, we followed the instructions of the manufacturer.

[0230] To obtain cDNA, we followed the same procedure described for “Gene expression analysis of HaCat cell lines” (3.5.1 ).

[0231] Afterwards, the following Taqman gene expression assays were used for qRT-PCR: GAPDH Hs99999905_m1 (human glyceraldehyde-3-phosphate dehydrogenase, GAPDH), IL-8 Hs00174103_m1 (Interleukin 8, IL-8), CDH1 Hs01023894_m1 (E- caderin, CDH1). GAPDH was used as the housekeeping gene. PCR amplifications were carried out in a 20pL of total volume. The mixture of reaction contained 10 pL of 2X Premix Ex Taq (Takara, Japan), 1 pL of 20* TaqMan gene expression assay, 0.4 pL of RoX Reference Dye II (Takara, Japan), 4.6 pL of water, and 4 pL of cDNA. PCR reactions and data elaboration were performed as described for “Gene expression analysis of HaCat cell lines” (3.5.1 ).

[0232] 4. Results

[0233] 4. 1 Biofilm formation on membrane-filters

[0234] All the seven strains were able to produce biofilm in the tested conditions. The positive result was shown through the visualization of a biofilm colony on the membrane-filter, placed on the MRS agar medium. The appearance of the biofilm colony at 24 and 48 h from the starting of the incubation is shown in Figure 4. No remarkable differences were observed in the color or dimension of the biofilm colonies.

[0235] 4. 1. 1 Cell density evaluation

[0236] In Figure 5 it is shown the cell density of the biofilm from the seven strains, grown on the membrane-filter. In general, the cell density of the different biofilm colonies ranged from 9.22 Log CFU / mL, (L plantarum C5), to 10.13 Log CFU / mL L plantarum POM1.

[0237] 4.1.2 Biomass quantification

[0238] The biomass of the seven strains used in the study in Figure 6. The average weights ranged from a minimum of 11 .30 mg (L plantarum C5) to a maximum of 36.20 of (L plantarum POM1 ).

[0239] 4. 1.3 Characterization of the ECM

[0240] After extracting the extracellular matrix components, the suspension was characterized for DNA, proteins, and exopolysaccharide concentration. In table 2. the results are reported.

[0241] Table 2. Characterization of the ECM: DNA, proteins, and exopolysaccharide concentration, expressed as pg / mg of the total biomass, is reported as mean ± standard deviation of the duplicate analysis.

[0242] Figure 7 shows the relative abundance of the components in the corresponding ECM fractions. The concentrations were comparable among strains. The main constituents of the ECM fraction were the EPS, followed by proteins and DNA.

[0243] 4. 1.4 Confocal Laser Scanning Microscopy CLSM allowed to obtain 3D images of the biofilm colonies grown on membranefilters and to visualize their structures. Biofilm width was similar among samples and ranged from 10 to 20 pm. In each sample was possible to identify both cells and polysaccharides as shown in Fig. 8 (a-g).

[0244] 4.2 High-throughput C. albicans inhibition screening

[0245] Tables 3 and 4. Growth kinetics of Candida albicans grown in presence of CFS of the candidate strains in single, at non-neutralized pH (above) and at neutralized pH (below). In the tables 3, 4, 5, and 6 are shown parameters about the growth kinetics of C. albicans, grown in presence of single or combinations of CFS obtained from the candidate strains. The statistical analysis did not show any significant difference between C. albicans inhibition with single supernatants and inhibition with combination of supernatants, therefore no synergistic effect was observed.

[0246]

[0247] Table 5. Growth kinetics of Candida albicans grown wit combinations of CFSs of the candidate strains, at non-neutralized pH.

[0248]

[0249] Table 6. Growth kinetics of Candida albicans grown with combinations of CFSs of the candidate strains, at neutralized pH.

[0250] 4.3 Pathogen inhibition using well diffusion method and water-soluble vegetable extracts

[0251] None of the WSE tested in this study were able to inhibit the growth of C. albicans, as shown in Figure 9, since no inhibition halo can be observed around the wells containing WSEs, but only around the well containing the antibiotic (positive control).

[0252] 4.4 Gene expression analysis of HaCat cell lines treated with C. albicans and lactobacilli

[0253] Figure 10 reports the results of Taqman gene expression assays, performed with qRT-PCR. C. albicans increased the expression of all the genes considered in this study. Regarding IL-8 and IL-6 expression, all the strains in single were able to decrease the expression of these genes, the only exception is L plantarum D2.6 which did not seem to have any effect. When they were applied with C. albicans, only L plantarum C5 and POM1 seemed to have a positive effect, but only the gene expression of cell treated with L plantarum C5 was significantly different (p<0.05) , from that of cells treated only with C. albicans. On the other hand, L. plantarum D2.6 seemed to increase the expression of the genes. Regarding the combinations of the four strains, we did not observe positive effects when they were applied alone, while a decrease in the gene expression was observed, when applied together with C. albicans, compared to C. albicans alone, with a statistical significant difference (p<0.05), compared to cells treated only with C. albicans. Under-expression of TNF-o was observed when L. plantarum C5 was present in single (with statistical significant difference, p<0.05), but none of the strains in combination with C. albicans had a positive effect on the expression of this gene and not even the combination of the four strains.

[0254] Regarding IL-1 / 3 expression only L. plantarum C5 seemed to have a positive effect when applied in single (but without statistical significance), and L. plantarum C5 and POM1 showed under-expression of the gene when applied in combination with C. albicans (but without statistical significance), compared to the gene expressed by cells infected with C. albicans alone. Similarly, to what is observed with IL-8 and IL- 6 the combination of the four strains did not show positive effect when they were applied alone, while a decrease in the gene expression was observed, when applied together with C. albicans, compared to C. albicans alone.

[0255] 4.5 Human Vaginal Epithelium model infection and treatment with probiotic gel

[0256] 4.5. 1 Formulation of gel containing lactobacilli

[0257] Figure 11 shows the cell densities of the three gel containing lactobacilli after 24 h from the preparation. The lactobacilli cell number was about 8 Log CFU / mL, for both the strains in single and in combination. Specifically in Figure 11 the cell densities of the gel containing lactobacilli expressed in Log [CFU / mL], are calculated from duplicate plate count, after 24 h from the preparation and before the application on HVE.

[0258] 4.5.2 MTT assay

[0259] The results regarding cell viability of HVE, evaluated with MTT assay, are shown in Figure 12. As can be observed, C. albicans sharply decreased the viability of the cells in this model (25.9%), while by treating the infected epithelium with the probiotic gel the cells’ viability improved (from 56.8 to 64.9%).

[0260] In Figure 13 it is possible to see the result of the MTT assay. Higher purple color intensity is associated to higher cell viability. It can be clearly observed that wells associated to C. albicans developed a less intense color than the control, the blank and the samples treated with gel containing lactobacilli.

[0261] 4.5.3 Gene expression analysis of HVE infected with C. albicans and treated with lactobacilli-containing gel

[0262] The results of the gene expression analyses of HVE, infected with C. albicans and treated with gel containing lactobacilli, can be observed in Figure 14. The epithelium models, treated with gels containing lactobacilli, showed a marked decrease in IL-8 expression compared to the positive control. In all the three cases, the difference was significant (p<0.05), according to two sample t-test. In addition, a synergistic effect was observed when the epithelium model was treated with the gel containing both lactobacilli strains.

[0263] Positive effects were observed also in the case of E-cadherin expression, which sharply decreased in the epithelium model infected with C. albicans, while it was significantly higher (p<0.05) when the epithelium model was treated with gel containing lactobacilli.

[0264] Discussion

[0265] In the present study it is investigated the potential fitness of nomadic lifestyle lactobacilli in vaginal microbiota and their ability to inhibit the growth of C. albicans, or to contrast the negative effect on human mucosa associated to its infection. These bacteria were isolated from food sources, and they were not endogenous vaginal lactobacilli. Nevertheless, nomadic bacteria are characterized by large genomes, which reflect high metabolic flexibility, and ability to migrate and thrive on different environments (Duar et al. 2017), therefore we hypothesized that they could be able to establish themselves also in the vaginal environment. These bacteria should then be used in a vaginal probiotic gel, for topical use, to prevent urogenital infections, obstetrical complications and to help the establishment of a proper gut microbiota on the newborn.

[0266] Biofilm is involved in the adhesion and colonization of surfaces and human cells (Caggianiello, Kleerebezem, and Spano 2016). Furthermore, biofilm, produced after adhesion to vaginal epithelial mucosa, might result in the formation of a barrier that masks the epithelial cell receptors and prevents pathogen attachment (Martin et al. 2008).

[0267] In the research, to evaluate the ability to form biofilm, the strains were inoculated and grown on a membrane-filter, placed on MRS agar medium. Positive result was shown by visualization of a biofilm colony, grown on the membrane-filter. To further confirm that the visualized biomass on the membrane-filter was a biofilm colony, we extracted the ECM components with a solution of NaCI, following a method optimized by Chiba et al. (2015), and we analyzed the composition with biochemical assays. The principle of the extraction method is the following: generally, Grampositive bacterial surface is considered as negatively charged, due to outer components of the cell wall (peptidoglycans and teichoic acids). However, some positively charged molecules (membrane embedded proteins and cell wall- anchored proteins) are also present on bacterial surfaces. ECM components (proteins, polysaccharides and eDNAs) can be positively or negatively charged too. Hence, ionic interactions seem to be the most important forces that keep ECM constituents attached to bacterial surfaces. Therefore, using higher ionic strength of Na+and Cl’ ions, we triggered the release of ECM components from bacterial cells, and analyzed them separately. Finally, confocal laser scanning microscopy was used to further evaluate the tridimensional structure of the biofilm and the distribution of the EPS among the cells. This microscope type is a very valuable tool for biofilm visualization (Swope and Flickinger 1996), since it has some advantages over conventional light and fluorescent microscopes. First of all, thick objects (up to 0.25 mm) and opaque objects such as animal tissue or biofilm colonies can be examined, image blurring caused by light scattering is reduced, the effective resolution is increased, and inclusion of a scan in the Z direction is possible, which in combination with computer image analysis can reconstruct 3D images (Swope and Flickinger 1996).

[0268] All the strains tested in this study were able to form biofilm in the tested conditions and in a relatively short time, since they were incubated for 48 hours, but already after 24 hours the colonies were visible. Biofilm colonies were similar in color and appearance. Differences were found among biomass weight and cell densities of the different biofilm colonies. In particular, L plantarum POM1 distinguished itself for the highest cell density and for the highest biomass weigth, that reflects in the CLSM image (Fig. 8.b) where cells appear very abundant and tightly packed. Instead, L plantarum C5 had the lowest cell density and the lowest biomass average weigth.

[0269] The analyses of the ECM components could show that the biofilm colony were formed mainly by EPS (82-91 %), followed by proteins and eDNA. Here, no noteworthy differences were observed among the compositions of the different ECM components. Our results suggest that these strains may be good candidate for the fitness in vaginal environment and for their use in a vaginal probiotic gel. Forming biofilm can help them to establish themselves in this niche, at least for a period and prevent the attachment of other microorganisms, including pathogens. In addition, biofilm is associated with higher survival during gastrointestinal transit (Caggianiello et al. 2016), and this is also fundamental for the colonization and establishment of the probiotic in the intestine of the newborn.

[0270] In the first screening activities of this project, which are not presented in this study, 109 nomadic lifestyle lactobacilli were tested for the inhibition against three main vaginal pathogens (Streptococcus agalactiae, Staphylococcus aureus and Candida albicans) using CFSs. The analyses gave satisfactory results regarding growth inhibition of S. agalactiae and S. aureus, while C. albicans resulted to be the most resistant pathogen and no, or very slight, growth inhibition was observed by growing it with the CFSs of the strains in single. So, in this study we decided to further investigate possible synergistic effects in C. albicans inhibition, by growing this yeast in presence of combinations of CFSs, obtained from our seven candidate strains. The growth kinetics of C. albicans were monitored by measuring the absorbance at 600 nm, then absorbance data were modelled using Gompertz equation. Gompertz is one of the most widely used sigmoid model to describe the growth of bacteria and yeast. By fitting the data obtained from absorbance measurements with this model, we can obtain three parameters, that allow us to describe and compare growth curves more easily. These are lag phase (A), growth rate (p), and maximum absorbance (A). The A is the period during which the microorganisms are adapting themselves to the environment and they are not dividing, so the absorbance data will remain stable. In our study, longer lag phases, in C. albicans growth kinetics, corresponded to a better inhibition ability of the lactobacilli CFSs. Growth rate describes the speed of C. albicans multiplication, in this case lower values of p corresponded to improved inhibition. Finally, A is the maximum registered absorbance value, that corresponds to the highest cell density reached. In this case, lower values corresponded to improved inhibition. In our study no significant differences were observed among growth kinetics of C. albicans cultivated with the single supernatants and with the combinations, both at neutralized and nonneutralized pH, thus, no synergisms were observed. In general, none of the CFSs obtained from our strains’ cultures were able to inhibit the growth of the pathogen. Moreover, cultivating C. albicans in presence of acidic CFSs seemed to have no effect on its growth kinetics. This suggests that the CFSs do not contain any inhibitory compounds for C. albicans. Nevertheless, these results do not exclude the possibility that our strains can inhibit C. albicans growth in vivo. Further research is necessary, in fact, we must consider that the supernatants used in our study were collected starting from a pure culture of lactobacilli in MRS medium, an optimal and sterile environment for lactobacilli growth. It is possible that in this environment lactobacilli do not express genes related to the production of bacteriocins. Our results are aligned with those of Allonsius et al. (2017) whose study showed, through a well-diffusion assay, that CFS of a Lacticaseibacillus rhamnosus strain could not inhibit C. albicans growth, but they demonstrated that by replacing the CFS with the cell culture (cell density of 108CFU / mL) the inhibition was possible. In addition, it would be interesting to investigate the possible inhibitory activity of ECM components collected from biofilm colonies. In fact, it is known that bacteria express different genes in biofilm growth compared to planktonic growth (Aoudia et al. 2015), and we cannot exclude that in this condition more genes related to the inhibition of pathogens are expressed.

[0271] Another interesting way to investigate fungal inhibition is through the study of plant extracts. It is widely known that plants produce antifungal compounds, which are thought to play a role in diseases resistance. Some studies already demonstrated the presence of compounds with antifungal activity in buckwheat (Fagopyrum esculentum) and borlotti beans (Phaseolus vulgaris) (Coda et al. 2008; Koval et al. 2020) and investigated their potential in improving shelf life of various foods, by inhibition of fungi responsible for their spoilage. In this study, we used agar well diffusion method to evaluate the possible antifungal activity of buckwheat and borlotti beans WSEs against one of the main vaginal pathogens. None of the two tested WSEs were able to inhibit the growth of C. albicans in our tested conditions.

[0272] Conclusion

[0273] The tested seven strains were all able to produce biofilm, a characteristic that can be associated to adhesion and pathogen exclusion, which may give them a competitive advantage in human associated niches. It is also demonstrated that at least two of the candidate strains were able to reduce the inflammatory response of host cells infected with C. albicans and contrast the process of human cells invasion. EXAMPLE 2

[0274] DIETETIC PRODUCT - OROSOLUBLE GRANULATE Each sachet contains: . amounts u.m.

[0275] Lyophilisate of Lactiplantibacillus plantarum mix: POM1 , OE1 - 2*1011cfu / g for single strain (carrier: com maltodextrin) . 20 mg

[0276] Xylo-oligosaccharides . 100 mg

[0277] D-mannose . 50-500 mg

[0278] Inulin . 5-100 mg

[0279] Vitamin D3 100 CWS I AM [composition: gum arabic (acacia gum), sucrose, corn starch, medium chain triglycerides, cholecalciferol (Vitamin D3)] for a total of 15 pg / dose . 7.2 mg

[0280] Maltodextrin . 0.5-2.0 g

[0281] Malic acid . 1-10 mg

[0282] Blueberry flavor . 10.0-50.0 mg

[0283] Silicon dioxide . 20-70 mg

[0284] Sucralose . 0.005 mg

[0285] EXAMPLE 3

[0286] TOPICAL PREPARATION - OILY SUSPENSION

[0287] Oily suspension in amber glass container with measuring cap; 10 drops / dose. Composition . amounts u.m.

[0288] Lyophilisate of Lactiplantibacillus plantarum mix: POM1 , C5 and OE1 - 2.5*1010cfu / g for single strain (microencapsulated) . 10 mg

[0289] Silicon dioxide . 100 mg

[0290] Mineral oil . to reach 5 mL

[0291] EXAMPLE 4

[0292] TOPICAL VAGINAL PREPARATION IN GEL Composition: . % w / w

[0293] Cell-free supernatant spray-dry mix C5 -1 LS15 - POM1 (carrier: com maltodextrin, silicon dioxide) 0.01-5

[0294] Carboxymethylcellulose, sodium salt 0.1 -4.0

[0295] Carrageenan 0.1 -4.0

[0296] Carob gum (Ceratonia siliqua) gel 0.1 -4.0

[0297] Glucose 0.1 -4.0

[0298] Glycerin 1.0-10.0

[0299] Trimethylglycine (glycine-betaine) 0.5-1 .0

[0300] Trehalose 0.5-1 .0

[0301] Hyaluronic acid 0.01-0.5

[0302] 2-ethylhexylanol ether with glycerin 0.5-1.0

[0303] Potassium sorbate 0.08-0.10

[0304] Sodium benzoate 0.08-0.10

[0305] Edetic acid, disodium salt 0.05-0.10

[0306] Sodium citrate buffer (citric acid, sodium hydroxide) to reach pH 4.7

[0307] Demineralized water to reach 100

[0308] Example 5

[0309] DIETETIC PRODUCT - SOFTGEL CAPSULES

[0310] Each softgel capsule contains amounts u.m.

[0311] Lyophilisate of Lactiplantibacillus plantarum mix: POM1 , C5 e OE1 - 3*1010ufc / g for single strain (carrier: com maltodextrin) 10 mg

[0312] Vitamin D3 in vegetable oil stabilized with dl-alpha tocopherol, for a total amount of

[0313] 2,5 pg of cholecalciferol / dose 0,04-0,2 mg

[0314] Soy oil > . 250 mg

[0315] Soy lecithin 5 mg

[0316] Mono e diglycerides of fatty acids 30 mg

[0317] Capsule constituents:

[0318] Gelatin 145 mg

[0319] Glycerol 67 mg

[0320] Example 6

[0321] DIETETIC PRODUCT - OILY SUSPENSION Oily suspension in amber glass container with measuring cap; 10 drops / dose.

[0322] Composition: amount u.m.

[0323] Lyophilisate of Lactiplantibacillus plantarum mix: POM1 , C5 - 2,5*1010ufc / g for single strain (microencapsulated) 10 mg Medium chain triglycerides 2,5 g

[0324] Delta alpha Tocopherol (vitamin E, natural) > 0,6 g

[0325] Com oil to reach 5 mL

Claims

CLAIMS1. A composition comprising a strain of Lactiplantibacillus plantarum selected from the group consisting of:Lactiplantibacillus plantarum having the accession number DSM 34000,Lactiplantibacillus plantarum having the accession number DSM 33995,Lactiplantibacillus plantarum having the accession number DSM 33999,Lactiplantibacillus plantarum having the accession number DSM 33996,Lactiplantibacillus plantarum having the accession number DSM 33997,Lactiplantibacillus plantarum having the accession number DSM 33998, andLactiplantibacillus plantarum having the accession number DSM 34001, or mixtures thereof and a physiologically acceptable carrier, for use in the prevention and / or treatment of skin or mucosa dysbiosis of a new-born or of a pregnant woman.

2. The composition for use according to claim 1 , wherein said strain of Lactiplantibacillus plantarum is selected from the group consisting of:Lactiplantibacillus plantarum having the accession number DSM 34000, Lactiplantibacillus plantarum having the accession number DSM 33995, and Lactiplantibacillus plantarum having the accession number DSM 33999, or mixtures thereof.

3. The composition for use according to claim 1 , wherein said strain of Lactiplantibacillus plantarum is the Lactiplantibacillus plantarum strain having the accession number DSM 34000.

4. The composition for use according to any one of claims 1 to 3, wherein the composition is a topical composition which is applied before and / or during partum to the skin of the abdominal area or on the mucosae of the genital tract of a pregnant woman.

5. The composition for use according to any one of claims 1 to 4, wherein the treatment of skin or mucosa dysbiosis is carried out by maintaining or preserving the physiological microbiota.

6. The composition for use according to any one of claims 1 to 4, wherein the treatment of skin or mucosa dysbiosis is carried out by promoting the physiological growth of microbiota of skin or mucosa, bowel or urogenital tract.

7. A strain of Lactiplantibacillus plantarum selected from the group consisting ofLactiplantibacillus plantarum having the accession number DSM 34000,Lactiplantibacillus plantarum having the accession number DSM 33995,Lactiplantibacillus plantarum having the accession number DSM 33999,Lactiplantibacillus plantarum having the accession number DSM 33996,Lactiplantibacillus plantarum having the accession number DSM 33997,Lactiplantibacillus plantarum having the accession number DSM 33998, andLactiplantibacillus plantarum having the accession number DSM 34001, or mixtures thereof.

8. A composition comprising a strain of Lactiplantibacillus plantarum according to claim 7 or a mixture thereof, and a physiologically acceptable carrier.

9. The composition according to claim 8, wherein the Lactiplantibacillus plantarum strains are viable, tyndallized or lyophilized.

10. The composition according to any one of claims 8 or 9, for oral or topical administration.11 . A composition comprising a strain of Lactiplantibacillus plantarum according to any one of claims 8 to 10, for use in the prevention and treatment of urogenital infections or inflammatory diseases or immune disorders of the skin or mucosa.

12. The composition for use according to claim 11 , wherein said urogenital infections are vaginal infections, in particular bacterial or fungal infections.

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