Use of lactobacillus paracasei bacterial strains to treat neonates

Abstract

The present invention relates to at least one bacterial strain belonging to the species of Lactobacillus paracasei, preferably Lactobacillus paracasei # imgabs0 # CNCM I-1572 and / or Lactobacillus paracasei LPC-01DSM 26760, at least one bacterial strain belonging to the species of Lactobacillus paracasei, preferably Lactobacillus paracasei # imgabs0 # CNCM I-1572 and / or Lactobacillus paracasei LPC-01DSM 26760; and compositions thereof for the prophylactic and / or curative treatment of inflammatory and / or functional gastrointestinal diseases, gastrointestinal infections caused by pathogenic microorganisms, gastrointestinal infections caused by parasites, allergies, immune-mediated diseases or autoimmune diseases in neonates and / or subjects of 1 month age to < = 12 months age; and to support the growth of the subject.

Description

[0001] The present invention relates to at least one bacterial strain belonging to the species Lactobacillus paracasei, preferably Lactobacillus paracasei CNCM 1-1572 and / or Lactobacillus paracasei LPC-S01 DSM 26760 and compositions thereof are used in neonatal subjects in the first 4 weeks of life for the prophylactic and / or curative treatment of gastrointestinal diseases of an inflammatory and / or functional nature, preferably neonatal colic or chronic enterocolitis (e.g. necrotizing enterocolitis) in premature neonates, for the prophylactic and / or curative treatment of gastrointestinal infections caused by pathogenic microorganisms (e.g. viruses or bacteria), gastrointestinal infections caused by parasites, allergies, immune-mediated diseases or autoimmune diseases, and supportive treatment of the growth of subjects.

[0002] Microbial colonization of the neonatal intestine begins immediately after birth and is essential for the development of mucosal barrier function, intestinal homeostasis, and maturation of the immune system.

[0003] In the context of the present invention, the term "neonatal" or "neonatal subject" refers to a mammal (human subject or animal subject) between the moment of birth and the first 4 weeks (28 days) of life.

[0004] In the first few days of an infant's life, multiple factors influence the composition of the intestinal microbiome: the mother's vaginal and / or skin microbiome, vaginal birth or caesarean section, milk (breastfeeding) or artificial milk feeding, and the administration of antibiotics and / or other drugs. In particular, caesarean section, artificial milk, premature birth, and the use of antibiotics reduce the abundance and diversity of beneficial bacterial species in the microbiome, thereby promoting a state of dysbiosis. This in turn may increase the risk of colic attacks, premature neonatal necrotizing enterocolitis, and the development of immune-mediated diseases such as allergies or inflammatory or functional bowel diseases and / or related symptoms later in life.

[0005] Currently, approximately 12% of premature newborns weighing less than 1500 g develop necrotizing enterocolitis and approximately one third die from sepsis or other complications.

[0006] Neonates are not physiologically comparable to "fragile" adults, or even to young children, because during the first weeks of life, maturation of the internal organs is not complete and their consequent functions are not comparable to those of children or adults, however weak or sick they may be.

[0007] "Gut microbiota" refers to the totality of microorganisms (all bacteria, archaea, eukaryotes, and viruses) present in the gastrointestinal (or gastrointestinal tract) environment.

[0008] Microbial communities perform many useful activities of the host organism. Indeed, the microorganisms that make up the microbial community degrade polysaccharides, assist digestive functions, synthesize vitamins, hinder the colonization of pathogenic species, provide signals necessary for intestinal development, and help regulate inflammatory and immune responses. It is believed that the etiology of many gastrointestinal diseases and those that affect other systems is an abnormal immune response to intestinal microorganisms. In addition, the intestinal microbial community, together with the metabolites and intestinal cells it produces, constitutes a barrier whose dysfunction is involved in the pathophysiology of many immune-mediated diseases and infections by pathogenic microorganisms.

[0009] In order to maintain a good state of health, it is necessary to have a balance between the immune system and the microorganisms that reside in the intestine. It has also been shown that this state of balance depends on the composition of the intestinal microbiota: an imbalance of the microbiota, known under the term "dysbiosis", can lead to immune system diseases, which cause harmful effects in the intestine and other systems.

[0010] The beneficial effects of probiotic bacterial strains (ie live microorganisms that confer health benefits when administered in adequate amounts) are therefore mainly attributed to normalizing the permeability of the intestinal barrier, modulating the intestinal microbiota and restoring the balance between the microbiota and the immune response.

[0011] The technical problem that the present invention is directed to and solves is to provide an effective solution for treating gastrointestinal diseases or symptoms of an inflammatory or functional nature (e.g., colic, chronic enterocolitis) and gastrointestinal infections caused by pathogenic microorganisms or gastrointestinal infections caused by parasites in newborn subjects and / or mammalian subjects in the first few months of life (1 month to ≤12 months of age; one month can have 28, or 29, or 30 or 31 days). In addition, the present invention is directed to and solves the technical problem of the prophylactic treatment of allergic reactions and / or immune-mediated diseases or autoimmune diseases (e.g., coeliac disease) related to the gastrointestinal system in the later years of life of the treated subjects. Finally, an object of the present invention is to provide an effective solution to support the body growth of newborns and / or mammalian subjects in the first few months of life.

[0012] After research and development activities, the applicant aimed at and solved the aforementioned technical problem by providing at least one strain of bacteria belonging to the species Lactobacillus paracasei, such as Lactobacillus paracasei CNCM 1-1572 and / or Lactobacillus paracasei LPC-S01 DSM 26760 and compositions thereof (in short, compositions of the present invention) which effectively and efficiently promote the formation of an appropriate and lactobacillus-rich intestinal microflora, especially Lactobacillus paracasei, in newborns and / or subjects in the first few months of life.

[0013] Among the Lactobacillus bacterial strains, Lactobacillus paracasei species (such as Lactobacillus paracasei strains CNCM 1-1572 and / or Lactobacillus paracasei strain LPC-S01 (DSM 26760) belong to species commonly used as probiotic bacterial strains. In particular, Lactobacillus paracasei strains have been extensively studied. The beneficial properties of CNCM I-1572. However, Lactobacillus paracasei has never been studied so far. Effects of CNCM I-1572 on neonatal subjects and / or subjects aged 1 month to ≤12 months.

[0014] It should be noted that after the reclassification of the genus Lactobacillus published by Zheng et al. in the scientific journal Int. J. Syst. Evol. Microbiol., 70(4): 2782-2858, 2020, the bacterial strain Lactobacillus casei (CNCM I-1572) or Lactobacillus paracasei (CNCM I-1572) was redeposited on February 2, 2022 as Lacticaseibacillus paracasei DG I-1572 DSM 34154. The two aforementioned names are interchangeable with each other as they always refer to the same bacterial strain.

[0015] Administration of the Lactobacillus paracasei strain of the present invention to subjects of neonatal age (first 4 weeks of life) CNCM I-1572 and Lactobacillus paracasei strain LPC-S01 DSM 26760, mixtures and compositions thereof appear to be able to effectively and efficiently colonize the intestine during the administration phase and / or the first months of life, leading to a balance between the intestinal microbiota and the immune system that is beneficial to the host and to strengthening of the intestinal barrier permeability.

[0016] Finally, the bacterial strains, mixtures and compositions of the present invention are well tolerated, have no associated side effects, are easy to prepare and are cost-effective.

[0017] These and other objects, the latter of which will become apparent from the following detailed description of the invention, are achieved by means of the compositions and mixtures according to the invention, thanks to the technical features claimed in the appended claims. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 .Schematic diagram of clinical research;

[0020] FIG. 2A to FIG. 2ELactobacillus casei in stool samples collected during V1-V5 control visits in Group 1 (analyzed composition; Tx) and Group 2 (placebo) Quantitative;

[0021] Figure 3 A to 3D. Bar graphs of the relative abundance of Lactobacillus spp. at time points during the V1-V5 control visits;

[0022] Figure 4 . Metabolome dominance showing partial least squares discriminant analysis (PLS-DA) of identified metabolites, including butyrate and propionate.

[0023] Figure 5 .Flowchart of the study design. 60 participants were enrolled in the clinical trial and randomized to one of the administration groups in a 1:1 ratio. 20 subjects in the placebo group and 26 subjects in the treatment group completed the study, respectively. 20 subjects in the placebo group and 26 subjects in the treatment group completed the study, respectively.

[0024] Figure 6. Lactobacillus casei DG (LCDG) was detected in the intestine from 10 days after the first administration and remained until the last visit (84 days).

[0025] A) Box plot showing LCDG concentrations that differentiate between placebo and treatment at each follow-up time point; B) Data are stratified according to birth weight of subjects: VLW (very low weight), LW (low weight), NW (normal weight). The median, 25th percentile, 75th percentile, minimum and maximum values ​​of Lactobacillus casei DG concentration (CFU / mL) of each group are shown. The upper bars represent statistically significant comparisons (p value < 0.05).

[0026] Figure 8. LCDG treatment can protect the heterogeneity of the intestinal microbiome. (A) Alpha diversity analysis between the placebo group and the treatment group at days 10, 28, 56 and 84 (V2, V3, V4 and V5, respectively). B) β-diversity analysis. The diversity matrix obtained using the non-weighted UniFrac algorithm (reproduced as PCoA at various time points between the placebo group and the treatment group) is shown. C) Histogram of OTU distribution at the phylum level and histogram of the Kruskall Wallis test suitable for comparison at various time points. D) Histogram showing the relative abundance of Lactobacillus at all time points.

[0027] Figure 9. Administration of LCDG increases short-chain fatty acid (SCFA) concentrations. Metabolite analysis is shown as partial least squares discriminant analysis (PLS-DA; left: score plot; right: VIP score) comparing placebo to treatment at each time point: A) V1; B) V2; C) V3; D) V4; E) V5.

[0028] Figure 10. The administration of LCDG affects the ecology and function of the microbiota, thereby adjusting the ecological niche of the bacteria that usually live in the digestive tract and the corresponding functional characteristics. Correlation analysis between Lactobacillus paracasei, metabolites and OTUs at 28 days (V3; end of treatment). A) Heat map of the correlation between metabolites and Lactobacillus paracasei (CFU / mL); B) Heat map of the correlation between OTUs and Lactobacillus paracasei (CFU / mL); C) Heat map of the correlation between OTUs and metabolites. DETAILED DESCRIPTION OF THE INVENTION

[0030] An object of the present invention is at least one strain of bacteria belonging to the species Lactobacillus paracasei as defined herein below, preferably a strain of Lactobacillus paracasei CNCM I-1572 and / or Lactobacillus paracasei strain LPC-S01DSM26760, mixtures or compositions thereof (in short, mixtures or compositions of the invention) are used in neonatal subjects (first 4 weeks of life) and / or subjects aged 1 month to ≤12 months for the preventive and / or curative treatment of gastrointestinal diseases of an inflammatory and / or functional nature, such as neonatal colic or intestinal colic, chronic enterocolitis or chronic enterocolitis of premature neonates, necrotizing enterocolitis, sepsis, irritable bowel syndrome (abbreviated as IBS), IBS with generalized constipation or constipation-type bowel, IBS with generalized diarrhea or diarrhea-type bowel, IBS with alternating bowel, unclassified IBS or chronic inflammatory bowel disease (IBD) such as Crohn's disease, ulcerative colitis, asthma, obesity, type 1 diabetes, atopic dermatitis, multiple sclerosis, cancer and autism.

[0031] Preferably, the strains, mixtures or compositions of the invention are used for the prophylactic and / or curative treatment of neonatal colic or chronic enterocolitis or necrotizing enterocolitis in premature neonates in neonatal subjects and / or in subjects aged 1 month to ≤12 months.

[0032] An object of the present invention is at least one strain of bacteria belonging to the species Lactobacillus paracasei, preferably a strain of Lactobacillus paracasei as defined herein below CNCM I-1572 and / or Lactobacillus paracasei strain LPC-S01DSM26760, mixtures or compositions thereof (in short, mixtures or compositions of the invention) are used for the prophylactic and / or curative treatment of pathogenic microorganisms (e.g., viruses (e.g., rotavirus, enteroadenovirus, calicivirus, astrovirus, influenza virus) or bacteria such as Salmonella, Shigella, Staphylococcus, Campylobacter, Escherichia coli) in neonatal subjects (first 4 weeks of life) and / or subjects aged 1 month to ≤12 months coli), gastrointestinal infections caused by parasites (preferably helminths or intestinal helminths (e.g., Ossiuri, Giardia, etc.), allergies, immune-mediated diseases or autoimmune diseases (e.g., celiac disease, Graves' disease, rheumatoid arthritis, Hashimoto's thyroiditis, type 1 diabetes, systemic lupus erythematosus (lupus), vasculitis, Addison's disease, polymyositis, Sjögren's syndrome, progressive systemic sclerosis, glomerulonephritis (inflammation of the kidney)), infertility, asthma, obesity, type I diabetes, atopic dermatitis, multiple sclerosis, cancer, and autism.

[0033] An object of the present invention is at least one strain of bacteria belonging to the species Lactobacillus paracasei as defined herein below, preferably a strain of Lactobacillus paracasei CNCM 1-1572 and / or Lactobacillus paracasei strain LPC-S01DSM26760, a mixture or composition thereof (in short, a mixture or composition of the present invention) is used in neonatal subjects (first 4 weeks of life) and / or subjects aged 1 month to ≤12 months for the supportive (adjuvant) treatment of the growth of the subject, which is understood as the weight gain of the administered subject evaluated as g / week.

[0034] Lactobacillus paracasei The bacterial strain of ANTIMICRO-OIL was deposited by SOFAR SpA on May 5, 1995 under the accession number CNCM I-1572 at the Institut Pasteur of Paris, 28, rue du Dr., 75724 Paris 15, France (in short, Lactobacillus paracasei CNCM I-1572); originally the strain was named Lactobacillus casei Lactobacillus casei sub.casei); later classified as Lactobacillus paracasei CNCM I-1572. It is clear that no matter whether it is named Lactobacillus casei Lactobacillus paracasei It is always and only the same bacterial strain.

[0035] The bacterial strain identified as Lactobacillus paracasei LPC-S01 (alternatively named Lactobacillus paracasei S01) was deposited by SOFARS.pA on November 20, 2012 with the deposit number DSM 26760 (in short, LPC-S01 or Lactobacillus paracasei LPC-S01 DSM 26760) at the German Collection of Microorganisms (Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH) (DSMZ) at 7B Inhofenstrasse D-38124 Braunschweig. It is clear that no matter it is named as Lactobacillus paracasei S01 DSM 26760 or Lactobacillus paracasei LPC-S01 DSM 26760 adopted by the applicant, it is always and only the same bacterial strain.

[0036] The composition of the invention for use in the method or treatment of the invention comprises: (i) the mixture M of the invention comprising Lactobacillus paracasei CNCM 1-1572 and / or Lactobacillus paracasei LPC-S01 DSM 26760 or alternatively consisting thereof; and, optionally, (ii) at least one food grade or pharmaceutical grade additive and / or excipient.

[0037] According to one aspect of the present invention, the mixture M included in the composition of the present invention may comprise or alternatively consist of:

[0038] - Lactobacillus paracasei CNCM I-1572 and / or Lactobacillus paracasei LPC-S01 DSM 26760; and

[0039] - at least one other bacterial strain selected from group (I) consisting of:

[0040] (a) Bifidobacterium breve BbIBS01 DSM 33231,

[0041] (b) Bifidobacterium breve BbIBS02 DSM 33232,

[0042] (c) Bifidobacterium animalis subsp. lactis BlIBS01 DSM 33233,

[0043] (d) Lactobacillus plantarum LpIBS01 DSM 33234,

[0044] (e) Bifidobacterium bifidum BbfIBS01 DSM 32708; and

[0045] A mixture thereof.

[0046] The bacterial strain belonging to the species Bifidobacterium breve identified as Bifidobacterium breve BbIBS01 was deposited by SOFAR SpA on July 31, 2019 under the deposit number DSM 33231 (in short, BbIBS01 or Bifidobacterium breve BbIBS01 DSM 33231) at the German Collection of Microorganisms (DSMZ), Inhofenstraße 7B, D-38124 Braunschweig.

[0047] The bacterial strain belonging to the species Bifidobacterium breve identified as Bifidobacterium breve BbIBS02 was deposited by SOFAR SpA on July 31, 2019 under the deposit number DSM 33232 (in short, BbIBS02 or Bifidobacterium breve BbIBS01 DSM 33232) at the German Collection of Microorganisms (DSMZ), Inhofenstraße 7B, D-38124 Braunschweig.

[0048] The bacterial strain belonging to the species Bifidobacterium animalis identified as Bifidobacterium animalis subsp. lactis B1IBS01 was deposited by SOFAR SpA on July 31, 2019 under the deposit number DSM 33233 (in short, B1IBS01 or Bifidobacterium animalis subsp. lactis B1IBS01 DSM 33233) at the German Collection of Microorganisms (DSMZ), Inhofenstraße 7B, D-38124 Braunschweig.

[0049] The bacterial strain belonging to the species Lactobacillus plantarum identified as Lactobacillus plantarum LpIBS01 was deposited by SOFAR SpA on July 31, 2019 under the deposit number DSM 33234 (in short, LpIBS01 or Lactobacillus plantarum LpIBS01) at the German Collection of Microorganisms (DSMZ), Inhofenstraße 7B, D-38124 Braunschweig.

[0050] A bacterial strain belonging to the species Bifidobacterium bifidum identified as Bifidobacterium bifidum MIMBb23sg = BbfIBS01, or a derivative thereof, wherein the bacterial strain was deposited by Sofar SpA on December 4, 2017 under the deposit number DSM 32708 (in short, BbfIBS01 or Bifidobacterium bifidum BbfIBS01 DSM 32708) at the German Collection of Microorganisms (DSMZ), Inhofenstrasse 7B D-38124 Braunschweig. It is clear that whether it is named Bifidobacterium bifidum BbfIBS01 DSM 32708 or Bifidobacterium bifidum MIMBb23sg DSM 32708 adopted by the applicant, it is always and only the same bacterial strain.

[0051] All bacterial strains mentioned in the present invention (i.e., The depositor of the bacterial strains described and / or claimed in this patent application and the patent application owner hereby represent that they agree that all such strains will be available for the entire duration of this patent.

[0052] Advantageously, the bacterial strain included in the composition of the present invention (i.e. LPC-S01, BbIBS01, BbIBS02, BlIBS01, LpIBS01 and BbfIBS01) are live bacterial strains (probiotics). Alternatively, the bacterial strains of the present invention may be derivatives of live strains as described in the present invention.

[0053] In the context of the present invention, the term "derivative" of a bacterial strain (or "derivative" of a live bacterial strain) is understood to mean a postbiotic or parabiotic known to those skilled in the art, for example, a tyndallized, sonicated, irradiated (preferably gamma-irradiated) inactivated bacterial strain, a bacterial strain lysate or homogenate, a bacterial strain extract or wall fraction, a metabolite or metabolic bioproduct produced by a bacterial strain or an exopolysaccharide (EPS) and / or any other derivative product of a bacterial strain. The derivative is obtained according to a methodology known to those skilled in the art. Preferably, the term "derivative" of a bacterial strain means a tyndallized, sonicated, irradiated (preferably gamma-irradiated) inactivated strain, a bacterial strain lysate or homogenate, an extract or parietal fraction of a bacterial strain; more preferably a tyndallized strain.

[0054] Examples of mixtures of bacterial strains (En) contained in a mixture M, optionally together with at least one probiotic selected from group (II) and / or an additional active ingredient selected from group (III), are given below, wherein the mixture M is included in the composition according to the invention:

[0055] E1: and LPC-S01;

[0056] E2: and at least one strain selected from group (I);

[0057] E3: LPC-S01 and at least one strain selected from group (I);

[0058] E4: and / or LPC-S01, and BbfIBS01 and at least one strain selected from group (I);

[0059] E5: and / or LPC-S01, and a mixture of BbIBS01, BbIBS02, BlIBS01 and LpIBS01;

[0060] E6: and / or a mixture of LPC-S01, BbfIBS01, and BbIBS01, BbIBS02, BlIBS01 and LpIBS01.

[0061] Lactobacillus paracasei In addition to CNCM I-1572 and / or Lactobacillus paracasei LPC-S01DSM 26760 and optionally at least one or more bacterial strains selected from group (I) and / or at least one other active ingredient selected from group (III) described below, the mixture M included in the composition of the present invention may further comprise at least one prebiotic, which is preferably selected from the group (II) consisting of: inulin, fructooligosaccharides (FOS), galacto-oligosaccharides (GOS), xylitol-oligosaccharides (XOS), guar gum, lactoferrin, and mixtures thereof; preferably inulin.

[0062] Advantageously, the mixture M included in the composition of the invention comprises or alternatively consists of: Lactobacillus paracasei CNCM I-1572 and / or Lactobacillus paracasei LPC-S01DSM 26760 and inulin; preferably Lactobacillus paracasei CNCM I-1572 and inulin.

[0063] Lactobacillus paracasei strains In addition to CNCM I-1572 and / or Lactobacillus paracasei LPC-S01DSM 26760 and optionally at least one or more bacterial strains selected from group (I) and / or at least one prebiotic selected from group (II), the mixture M included in the composition of the present invention may further comprise at least one other active ingredient selected from group (III) consisting of:

[0064] - vitamins of group A, B, C, D, E and / or K, preferably vitamins of group B and / or vitamin D;

[0065] - Antioxidants such as glutathione, polyphenols such as resveratrol and trans-resveratrol, coenzyme Q10, astaxanthin, lycopene;

[0066] - plant substances (botanicals) or extracts thereof which have a relaxing effect on the intestine, such as valerian, passion flower, lemon balm, hawthorn and camomile;

[0067] - Minerals or their salts, such as zinc, selenium, magnesium, potassium;

[0068] - monounsaturated fatty acids, such as ω9, and / or polyunsaturated fatty acids, such as ω3 and ω6;

[0069] - immunostimulant substances, antidiarrheal substances and / or nutrients; and

[0070] A mixture thereof.

[0071] Advantageously, the mixture M included in the composition of the invention comprises or alternatively consists of: Lactobacillus paracasei CNCM I-1572 and / or Lactobacillus paracasei LPC-S01DSM 26760 and B vitamins and vitamin D; preferably Lactobacillus paracasei CNCM I-1572 and B vitamins and vitamin D.

[0072] In the context of the present invention, food grade or pharmaceutical grade acceptable "additives and / or excipients" optionally included in the composition of the present invention together with the strain bacteria include all auxiliary substances known to those skilled in the art for preparing solid form, semi-solid or liquid form compositions, such as carriers, diluents, solvents, solubilizers, acidulants, thickeners, sweeteners, flavoring agents, colorants, sweeteners, lubricants, surfactants, preservatives, stabilizers, pH stabilizing buffers and mixtures thereof. For example, in the case where the composition is in the form of drops for oral administration to a newborn or a 1-month to 12-month-old subject, seed oil can be used as a diluent.

[0073] According to any of the described embodiments, the mixture M or composition of the present invention can be a pharmaceutical composition (or a living cell biotherapeutic product), a composition for a medical device, a composition for a food supplement, a food (or a new food or a food for special medical purposes (FSMP)), a composition for a food supplement or a food.

[0074] According to any of the described embodiments, the bacterial strains of the invention, mixtures thereof and compositions thereof can be formulated for oral or nasal administration, preferably for oral use, in solid form or alternatively in liquid form, for example in the form of water-based or oil (e.g., various seed oils or sunflower oil) based drops.

[0075] Advantageously, with regard to the daily dose of the M mixture or composition according to the invention, the at least one bacterial strain or each bacterial strain is present in an amount of 10×10 6 CFU to 10x10 12 CFU, preferably 10x10 8 CFU to 10x10 10 The concentration of CFU is preferably about 10x10 8 CFU or 10x10 9 The concentration of CFU (CFU: Colony Forming Unit) is present in the mixture M or composition according to the invention.

[0076] For example, a daily dose of a mixture or composition according to the invention (eg, oral administration of drops) comprises: > 1x10 9 Lactobacillus paracasei CNCM I-1572 and seed oil.

[0077] The above daily doses can be administered to a subject in need thereof in separate doses (single administration) or in repeated doses, such as two, three or four daily doses. For example, 15-5 drops (oil-based), preferably 12 to 8 drops, such as about 9-10 drops of the composition of the present invention twice a day is equivalent to about 2 billion bacterial strains, preferably Lactobacillus paracasei CNCM I-1572.

[0078] In order to assess the number of viable bacterial strains in the compositions or mixtures M of the invention, these compositions or mixtures M can be analyzed by the plate count method to determine the CFU value.

[0079] For the sake of clarity, with the aim of achieving one of the objects of the present invention, the bacterial strains, prebiotics and / or other (optional) active ingredients included in the mixture (M) of the present invention can also be administered separately (preferably at intervals of 30 to 60 minutes) and in any order, but preferably at the same time, even more preferably in a single composition, to the subject, in order to achieve a faster effect and easy administration. When the strains and (optional) active ingredients are administered in a single composition, the single composition corresponds to the composition of the present invention.

[0080] Unless otherwise stated, stating that a composition or mixture or otherwise comprises a component in an amount ranging from "x to y" means that the component may be present in the composition or otherwise in all amounts within that range, including the extremes of that range even if not explicitly stated.

[0081] Unless otherwise stated, the statement that a composition "comprises" one or more components or materials means that other components or materials may be present in addition to that or those specifically stated.

[0082] In the context of the present invention, "treatment method" means an intervention on a subject in need thereof, comprising administering to the subject a bacterial strain or composition of the present invention in a therapeutically effective amount, with the aim of eliminating, reducing / lowering or preventing pathology or disease and related symptoms or disorders.

[0083] In the context of the present invention, the term "subject" refers to mammals (animals and humans), preferably human subjects of neonatal age (0 to 4 weeks old) and / or 1 month to ≤12 months of life (starting from the 2nd month until the end of the 12th month).

[0084] Advantageously, the subject treated or administered with the strain or mixture or composition of the present invention may be a neonatal subject or a subject from 1 month to ≤12 months of life born by caesarean section and / or born prematurely.

[0085] The term "therapeutically effective amount" refers to that amount of active compound and / or bacterial strain that elicits the biological or medicinal response in a tissue, system, mammal or human that is sought and defined by the individual, researcher, veterinarian, physician or other clinician or health care practitioner.

[0086] In the context of the present invention, the term "medical device" is used in the meaning according to Legislative Decree No. 46 of 24 February 1997 or according to the new Medical Devices Regulation (EU) 2017 / 745 (MDR).

[0087] In the context of the present invention the term "novel food" is used in the sense according to EC Regulation 258 of 1997.

[0088] Implementation

[0089] Preferred embodiments FRn of the present invention are as follows.

[0090] FR1. A bacterial strain or composition for use in neonatal subjects in the first 4 weeks of life and / or in subjects aged 1 month to ≤ 12 months for the prophylactic and / or curative treatment of subjects suffering from a gastrointestinal disease of an inflammatory and / or functional nature, such disease being selected from the group comprising or alternatively consisting of: neonatal colic, intestinal colic, chronic enterocolitis or chronic enterocolitis of premature neonates, necrotizing enterocolitis, sepsis, irritable bowel syndrome (IBS), IBS with generalized constipation or constipation-type bowel, IBS with generalized diarrhea or diarrhea-type bowel, IBS with alternating IBS of the intestine, unclassified IBS, chronic inflammatory bowel disease (IBD), Crohn's disease, ulcerative colitis, asthma, obesity, type 1 diabetes, atopic dermatitis, multiple sclerosis, cancer, autism, allergies, immune-mediated diseases, autoimmune diseases selected from celiac disease, Graves' disease, rheumatoid arthritis, Hashimoto's thyroiditis, type 1 diabetes, systemic lupus erythematosus (lupus), vasculitis, Addison's disease, polymyositis, Sjögren's syndrome, progressive systemic sclerosis, glomerulonephritis (kidney inflammation), infertility; and supporting weight gain in the subject;

[0091] wherein the bacterial strain belongs to the species Lactobacillus paracasei;

[0092] Wherein the composition comprises:

[0093] (i) a mixture M comprising or alternatively consisting of at least one strain belonging to the species Lactobacillus paracasei; and

[0094] (ii) at least one food grade or pharmacological grade additive and / or excipient.

[0095] FR2. A strain for use according to FR1 or a composition for use therefor,

[0096] Wherein the bacterial strain is selected from Lactobacillus paracasei CNCM I-1572, Lactobacillus paracasei LPC-S01DSM 26760 and mixtures thereof;

[0097] Wherein the composition comprises:

[0098] (i) a mixture M comprising or alternatively consisting of: Lactobacillus paracasei CNCM I-1572 and / or Lactobacillus paracasei LPC-S01 DSM 26760; and

[0099] (ii) at least one food grade or pharmacological grade additive and / or excipient.

[0100] FR3. A strain for use according to FR1 or FR2 or a composition for use therefor, wherein the strain or composition is for use in the prophylactic and / or curative treatment of neonatal colic or intestinal colic or chronic enterocolitis or chronic enterocolitis in premature neonates in neonatal subjects and / or subjects aged 1 month to <12 months.

[0101] FR4. A strain for use according to FR1 or FR2 or a composition for use therefor, wherein the strain or composition is used for the preventive and / or curative treatment of gastrointestinal infections caused by pathogenic microorganisms or gastrointestinal infections caused by parasites in neonatal subjects and / or subjects aged 1 month to ≤12 months.

[0102] FR5. A strain for use according to FR1 or FR2 or a composition for use therefor, wherein the strain or the composition is for use in the preventive and / or curative treatment of allergies in neonatal subjects and / or subjects aged 1 month to ≤12 months.

[0103] FR6. A strain for use according to FR1 or FR2 or a composition for use therefor, wherein the strain or composition is used for the prophylactic and / or curative treatment of immune-mediated diseases, autoimmune diseases such as celiac disease, Graves' disease, rheumatoid arthritis, Hashimoto's thyroiditis, type 1 diabetes, systemic lupus erythematosus (lupus), vasculitis, Addison's disease, polymyositis, Sjögren's syndrome, progressive systemic sclerosis, glomerulonephritis (kidney inflammation) and infertility in neonatal subjects and / or subjects aged 1 month to ≤12 months.

[0104] FR7. A strain for use according to FR1 or FR2 or a composition for use therefor, wherein the strain or composition is used for preventive and / or curative treatment in neonatal subjects and / or subjects aged 1 month to ≤12 months to support weight gain in subjects.

[0105] FR8. A composition for use according to any one of FR1-7, wherein the (i) mixture M comprises or alternatively consists of:

[0106] - Lactobacillus paracasei CNCM I-1572 and / or Lactobacillus paracasei LPC-S01 DSM 26760; and

[0107] - at least one other bacterial strain selected from the group consisting of:

[0108] - Bifidobacterium breve BbIBS01 DSM 33231,

[0109] - Bifidobacterium breve BbIBS02 DSM 33232,

[0110] - Bifidobacterium animalis subsp. lactis BlIBS01 DSM 33233,

[0111] - Lactobacillus plantarum LpIBS01 DSM 33234,

[0112] - Bifidobacterium bifidum BbfIBS01 DSM 32708; and

[0113] A mixture thereof.

[0114] FR9. A composition for use according to any one of FR1-8, wherein the (i) mixture M further comprises at least one other active ingredient selected from the group consisting of:

[0115] - at least one vitamin from the A, B, C, D, E and / or K groups, preferably B vitamins and / or vitamin D;

[0116] - Antioxidants such as glutathione, polyphenols such as resveratrol and trans-resveratrol, coenzyme Q10, astaxanthin, lycopene;

[0117] - a plant substance, a botanical drug or an extract thereof having an intestinal relaxant effect, preferably selected from valerian, passionflower, lemon mint, hawthorn and chamomile;

[0118] - Minerals or their salts, such as zinc, selenium, magnesium, potassium;

[0119] - monounsaturated fatty acids, such as ω9, and / or polyunsaturated fatty acids, such as ω3 and ω6;

[0120] - immunostimulant substances, antidiarrheal substances and / or nutrients;

[0121] - at least one prebiotic, preferably selected from the group (II) consisting of inulin, fructooligosaccharides (FOS), galacto-oligosaccharides (GOS), xylitol-oligosaccharides (XOS), guar gum, lactoferrin, and mixtures thereof; more preferably inulin; and

[0122] A mixture thereof.

[0123] FR10. A strain for use according to any one of FR1-9 or a composition for use according to the same,

[0124] The strain is a live bacterial strain or a bacterial strain derivative, wherein the bacterial strain derivative is selected from: intermittently sterilized, sonicated, irradiated (preferably gamma-irradiated) inactivated bacterial strains, bacterial strain lysates or homogenates, extracts or wall fractions of bacterial strains; preferably intermittently sterilized bacterial strains.

[0125] Experimental Section

[0126] The applicant conducted a clinical study in neonates to evaluate the administration of Lactobacillus paracasei Effects of CNCM I-1572 probiotics (live cells) on the intestinal microbiota of hosts in the first 3 months of life.

[0127] 1. Objectives of clinical research

[0128] Main research objectives:

[0129] It was evaluated whether supplementation for 28 days with a composition of the invention in the form of drops for oral administration (oil base) (in short, the composition analyzed) comprising the Lactobacillus paracasei strain CNCM I-1572 modifies the composition of the fecal intestinal microbiota in terms of the concentration of Lactobacillus paracasei CNCM I-1572 in the study population.

[0130] Compositions analyzed:

[0131] Sunflower oil (Heliantus annuus L.), DL-α-tocopherol, Lactobacillus paracasei CNCM I-1572 (not less than 14 billion viable cells per 8 ml).

[0132] Secondary study objectives:

[0133] The ingestion of the analyzed compositions for 28 days was evaluated with respect to:

[0134] - Changes in the composition of the fecal intestinal microbiota (real-time PCR), while predicting the ecology of the microbiota system 56 and 84 days after the intake of the analyzed composition;

[0135] - Changes in the functional activity of the fecal gut microbiome (metabolome), while predicting the ecology of the microbiome system at 28, 56 and 84 days after ingestion;

[0136] - changes in stool quantity and quality (frequency and consistency);

[0137] - Occurrence of neonatal colic as defined by the Rome IV criteria;

[0138] - Safety and tolerability of the product;

[0139] - Patient growth.

[0140] 2. Organization of this study

[0141] Study schedule Figure 1 )

[0142] - Enrollment period: 24 weeks

[0143] - Duration of treatment: 4 weeks;

[0144] - Follow-up duration: 8 weeks;

[0145] - Duration of study per patient: 12 weeks (84 days);

[0146] - Total duration of study: 36 weeks.

[0147] 3. Study Design

[0148] This is a randomized, placebo-controlled, double-blind, single-center clinical study. Newborns were stratified into three groups according to birth weight:

[0149] - Normal birth weight (>2500g, NBW),

[0150] - Low birth weight (1500-2500 g, LBW),

[0151] - Very low birth weight (1000-1500g, VLBW).

[0152] The trial was conducted at the UO Department of Neonatology at the Policlinico Casilino in Rome as the only participating and coordinating center.

[0153] Study population

[0154] The study included 60 preterm and term neonates divided into the following categories: 20 NBW, 20 LBW, and 20 VLBW.

[0155] 3.2 Types of intervention

[0156] The 60 enrolled subjects were randomized into two groups at a 1:1 ratio in each of the three groups:

[0157] Group 1

[0158] The test composition in the form of drops, 9 drops once, twice a day, is equivalent to 2 billion CFU (colony forming units) of Lactobacillus paracasei (CNCM I-1572), for 4 weeks (28 days).

[0159] Group 2

[0160] Control: Placebo, 9 drops once, twice a day, for 4 weeks (28 days).

[0161] 3.3 Duration of this study

[0162] A 12-week period allowed for the growth of Lactobacillus casei based on the intake of the analyzed composition. (Lactobacillus paracasei CNCM I-1572) persistence, assessing changes in the intestinal microbiome. This 12-week phase is divided into 4 weeks of treatment and 8 weeks of follow-up. Including an enrollment phase of approximately 24 weeks, the total duration of the study will be 36 weeks.

[0163] 4. Selection of research subjects

[0164] 4.1 Inclusion criteria

[0165] - Life 0-48 hours;

[0166] - Newborns of either sex with a birth weight ≥ 1000 g;

[0167] - Newborns delivered by caesarean section.

[0168] 4.2 Exclusion criteria (only major items included)

[0169] - Vaginal delivery;

[0170] - Extremely low birth weight (ELBW) (<1000g);

[0171] - Established or suspected systemic infection;

[0172] - Any serious lesions that may interfere with treatment in the opinion of the investigator;

[0173] - Neonates or lactating mothers taking probiotics other than the trial product since the start of the study.

[0174] - Systemic antibiotic treatment or prophylaxis (NBW only) since birth and throughout the study duration;

[0175] - Lactating mothers receiving systemic antibiotic treatment or prophylaxis within 30 days prior to birth and throughout the study duration (for NBW newborns).

[0176] A newborn will be considered eligible for the protocol population if he / she has received at least 80% and no more than 120% of the prescribed treatment.

[0177] 5. Procedure and Methods

[0178] If possible, control visits and collect stool samples in context:

[0179] V1 Day 1 (life 0-48 hours); V2 Day 10 (+ / - 2 days); V3 Day 28 (+ / - 3 days); V4 Day 56 (+ / - 3 days); V5 Day 84 (+ / - 3 days) ( Figure 1 ).

[0180] In addition, parent-collected stool samples were delivered during the study period according to the following schedule (1 sample was collected within 24 hours prior to each visit):

[0181] Before treatment

[0182] - Stools within the first 48 hours of life and, when available, meconium.

[0183] During the treatment phase:

[0184] - stool 10 days after starting intake;

[0185] - stool after 28 days from the start of ingestion;

[0186] During the follow-up phase:

[0187] - stool after 56 days from the start of ingestion;

[0188] - stool after 84 days from the start of ingestion;

[0189] Physicochemical analysis of fecal samples:

[0190] -Evaluation of Lactobacillus casei strains Real-time PCR analysis of bacterial counts of (Lactobacillus paracasei CNCM 1-1572);

[0191] - Metagenomic and metabolomic analyses characterizing the gut microbiota before and after probiotic administration.

[0192] Specifically, the microbiome was analyzed by nucleotide sequence analysis of a portion of the gene encoding the bacterial ribosomal subunit 16S rRNA. Specifically, a metagenomic strategy consisting of the following steps was adopted:

[0193] - Extraction, quantification and normalization of metagenomic DNA from fecal samples;

[0194] - PCR amplification of the V3-V4 hypervariable region of the bacterial gene encoding 16S rRNA;

[0195] -PCR product quantification;

[0196] - Sequencing using Illumina MiSeq technology;

[0197] - Bioinformatics sequence analysis (characterization of microbial communities, hierarchical clustering, taxonomic analysis, construction of phylogenetic trees with heatmaps).

[0198] For metabolome analysis, microbial metabolites produced as volatile organic compounds (VOCs) were identified and quantified. These compounds were extracted using a GC-MS / SPME (gas chromatography coupled mass spectrometry with solid phase microextraction) system. For VOC extraction, carboxyl-polydimethylsiloxane (CAR-PDMS) coated fibers (85 μm) were used in the SPME method. From each sample analyzed in triplicate, an average of 100-500 mg of sample was placed in a 10 ml glass vial, while 4-methyl-2-pentanol was added as an internal standard (IS). Subsequently, fecal samples were balanced at 45 ° C for 10 minutes. The fiber was exposed to each sample for 45 minutes, and then loaded onto a GC-MS (Hewlett Packard 6890 GC) coupled to a 5973C mass selective detector and equipped with a Supelcowax 10 capillary column. Metabolites were identified using the retention time (Rt) of metabolites relative to pure compounds. Chromatograms were integrated and identified by comparison of fragmentation patterns with those from the NIST library followed by manual visual inspection. Quantitative metabolite data were obtained by area interpolation relative to the IS area.

[0199] 6. Effectiveness and safety evaluation

[0200] 6.1. Evaluating effectiveness

[0201] Primary End Point

[0202] - Lactobacillus casei by real-time PCR on day 28 Bacterial genome counts.

[0203] Secondary End Points

[0204] - indicators of dysbiosis of the gut microbiota (α-diversity and β-diversity) at days 28, 56 and 84;

[0205] - Bacteria Lactobacillus casei on days 56 and 84 Genome count;

[0206] - the number, consistency, amount and colour of stool passages on the day before the visit (days 1, 10, 28, 56, 84) using the Amsterdam Neonatal Stool Scale;

[0207] - Physicochemical analysis of stool (day 1, day 10, day 28, day 56, day 84);

[0208] - Quantitative assessment of volatile substances, i.e., fecal volatile metabolites (chemical categories: short-chain fatty acids (SCFA), alcohols, ketones, aldehydes, thiols, acids, esters, pyrazines, pyridines, phenols, furans, terpenes, alkanes, alkenes, etc.) by evaluating changes in the overall metabolic profile of the microbiota on days 28, 56, and 84;

[0209] - the presence of colic assessed according to the "Rome IV" diagnostic criteria;

[0210] - Evaluate adverse events.

[0211] 7. Statistical Analysis

[0212] Statistical methods

[0213] Categorical data were reproduced in terms of counts and percentages, whereas continuous data were reproduced as mean and standard deviation or median and range.

[0214] Categorical data were compared by chi-square test or Fisher's exact test, as appropriate. Continuous data were compared by Student's t-test (for independent or dependent data), analysis of variance (Anova), and repeated measures Anova, if data normality was determined by appropriate statistical tests; in the case of nonnormal distribution of data, nonparametric tests such as the rank-sum test (Mann Whitney rank-sum test) or the signed-rank test (Wilkoxon signed-rank test), Kruskal Wallis test, or Friedman test were used. Multivariate regression techniques were used if indicated to assess the simultaneous effects of several independent variables on the response variable or to identify possible confounders and / or to analyze temporal data. Results were considered statistically significant when the P value was <0.05.

[0215] All analyses were performed using Stata 15 software (StatCorp).

[0216] 8. Microbiological Results of This Study

[0217] 8.1 Primary endpoint: Lactobacillus casei by real-time PCR at day 28 Bacterial genome counting

[0218] Results from stool samples collected during control visits V1-V5 are illustrated in Figures 2A to 2E .

[0219] These graphs (box plots) show for each follow-up time point the presence of Lactobacillus casei in the placebo group and in the "Tx" group administered with the analyzed composition. For each group, the concentration of Lactobacillus casei is shown Median, 25th percentile, 75th percentile, minimum and maximum values ​​of concentration (CFU / ml).

[0220] Probiotic Lactobacillus casei detected in the intestines of treatment group subjects starting 10 days after first administration It persisted for up to 84 days after ingestion and began to decrease slightly at the follow-up time point V3 (56th treatment day).

[0221] 8.2 Metagenomic Analysis

[0222] Figure 3 A to Figure 3 D shows a bar graph of the relative abundance of Lactobacillus species at time points of control visits V2-V5.

[0223] From the ecological analysis of the microbiota, it can be inferred that the administration of the analyzed composition (Tx group) caused a statistically significant increase (pFDR≤0.05) in the relative abundance of Lactobacillus species until ten days after the start of administration (time point V2) and that this increase continued until time points V4 and V5, with a trend at p<0.05.

[0224] 8.3 Metabolome analysis

[0225] Figure 4 A graph showing the results of PLS-DA metabolome analysis: the vertical axis lists the identified metabolites, and the horizontal axis shows the variable importance in projection (VIP).

[0226] PLS-DA analysis identified butyrate and propionate metabolites (SCFA) as the main players in the clustering between the placebo and Tx groups, with elevated concentrations of these metabolites in Tx samples.

[0227] Considering the time points separately according to PLS-DA, it can be seen that the maximum separation of the samples occurred between time V2 and V4, and the SCFA concentration increased in the TX sample.

[0228] This trend is consistent with the higher relative abundance of Lactobacillus in the subjects of the Tx group who were administered the analyzed composition (Section 8.2).

[0229] 8.4 Correlation between all microbiological data obtained

[0230] The results obtained from the three different methods discussed in the previous paragraphs (real-time PCR, metagenomic analysis, metabolomic analysis) were correlated with each other according to Spearman correlation.

[0231] For correlation analysis, time points V2 and V3 (V2: 10 days; V3: 28 days) corresponding to the administration of the analyzed composition were selected as metagenomic analysis, metabolomic analysis and real-time PCR analysis, showing the greatest statistically significant differences between the placebo group and the Tx group treated with the analyzed composition.

[0232] By integrating the data obtained from the three methods, it can be shown that the probiotic Lactobacillus casei The administration of these drugs affects the ecology and function of the microbiota, thereby adjusting the ecological niches of intestinal resident bacteria and the corresponding functional characteristics.

[0233] Specifically, certain short-chain fatty acids (SCFAs)—such as butyrate and propionate—showed positive correlations with certain beneficial bacteria from the genera Faecalibactarium, Oscillospira, Eggerthella, Bacteroides, and Bifidobacterium.

[0234] 9. Clinical outcomes of this study: stool frequency, consistency, quantity and color

[0235] The number of stools observed per study visit was substantially higher in subjects treated with the analyzed compositions than in subjects treated with placebo.

[0236] With regard to the amount of feces excreted, there were no statistically significant differences between the two study groups at 10, 56 and 84 days after the start of treatment (P = 0.12; 0.61 and 0.25, respectively).

[0237] Statistically significant differences in the amount of excreta were observed only at the end of the treatment period (day 28, V3) in favor of the subjects treated with the analyzed composition (P=0.03).

[0238] With regard to the color and consistency of the stool (parameters assessed according to the "Amsterdam Infant Stool Scale"), statistically significant differences in favor of the subjects treated with the analyzed composition were observed 10 days (P<0.0001) and 56 days (P=0.007) after the start of treatment in terms of stool color; and 10 days (P=0.0016), 56 days (P<0.0001) and 84 days (P=0.05) after the start of treatment in terms of stool consistency.

[0239] 10. Safety Results of This Study

[0240] The results of the safety analysis are summarized in Table 1 below.

[0241]

[0242] Table 1

[0243] *AE = adverse event

[0244] A total of 37 adverse events (AEs) have been recorded, 21 in the placebo-treated group and 16 in the analyzed composition-treated subject population (Tx).

[0245] Of the 59 subjects enrolled and randomized, 27 experienced one or more adverse events (15 subjects in the placebo group; 12 subjects in the Tx group).

[0246] Only two serious adverse events (necrotizing colitis and suspected sepsis) were reported in one subject treated with the analyzed composition. However, none of the serious adverse events appeared to be related to the use of the analyzed composition.

[0247] The same judgment could be made for the serious adverse events observed in the placebo group (mild gastroesophageal reflux requiring hospitalization).

[0248] The investigators classified deaths due to disseminated intravascular coagulation and septic shock recorded in patients treated with the analyzed combination as “unlikely related to study treatment.”

[0249] All remaining severe and non-severe adverse events were deemed either unrelated (N=29) or unlikely related (N=3) to the administered study treatment (placebo or analyzed combination).

[0250] No eczema or skin disorders were recorded in either group during the study period.

[0251] 11. Conclusion:

[0252] Based on the above, we can conclude that:

[0253] - Regarding the primary endpoint, the probiotic Lactobacillus casei in the Tx group treated with the analyzed composition In the intestine, it was detected from the 10th day after the start of treatment (V2; Figure 2B ) and persists until the end of observation phase V5 ( Figure 2E );

[0254] - With regard to the relative abundance of Lactobacilli, the analyzed compositions showed a statistically significant increase in the relative abundance of Lactobacilli until the end of observation period V5 ( Figure 3 D)

[0255] - In terms of PLS-DA metabolome analysis, the metabolites butyrate and propionate were identified, which showed higher concentrations in Tx samples ( Figure 4 );

[0256] - From the correlations of previous microbiological analyses, it can be inferred that the presence of butyrate and propionate showed a positive correlation with certain bacterial species that are beneficial to the microbiota of the treated subjects;

[0257] - Subjects treated with the analyzed composition had statistically significantly higher number of stools and better stool color and consistency - assessed according to the Amsterdam Neonatal Stool Scale - than the placebo-treated group;

[0258] - Finally, based on the safety and tolerability profile analyzed above and the evaluation of severe adverse events recorded in both study groups, the analyzed composition was demonstrated to be safe and well tolerated, with a safety profile comparable to that of placebo.

[0259] The results obtained are far from obvious, since, as mentioned above, in the first weeks of life, mammals and in particular humans are not even comparable to children, since they have not yet completed the maturation of their internal organs. It is therefore necessary to carry out the clinical trials described above in order to determine the efficacy and safety of the strains described here, since it is not foreseeable in any way that these strains will be able to provide the pharmacological responses obtained, nor that they will be well tolerated by newborns.

[0260] Experimental Section

[0261] To date, no studies have been conducted to evaluate the effects of LCDG on vulnerable groups such as healthy newborns (in preterm and term newborns).

[0262] One objective of this double-blind, placebo-controlled study was to confirm the ability of a pediatric formulation of LCDG to survive passage through the gastrointestinal tract of neonates (stratified into three groups based on birth weight: normal birth weight, low birth weight, and very low birth weight) during and after the administration period by evaluating the ability of LCDG to positively influence the composition of the microbiota. Product safety (weight, length, and head circumference parameters were also monitored), bowel movement frequency, stool consistency, and the occurrence of colic were also evaluated.

[0263] Materials and methods

[0264] Experimental Products

[0265] Lactobacillus paracasei (Lactobacillus casei -CNCM I-1572; LCDG) supplied by SOFAR SpA.

[0266] 9 drops of Lactobacillus paracasei (corresponding to 1 billion colony forming units (CFU) of LCDG) were applied twice a day for 28 days. The drops were applied directly on the tongue or mixed with a cold or lukewarm liquid.

[0267] Participants of this study

[0268] This study was conducted from September 2018 to March 2021. Patients participating in this study were selected from the group of preterm and term newborns in the Neonatal Department of Policlinico Casilino Hospital, Rome, Italy. The inclusion criteria for this study were determined as follows: newborns of both sexes aged between 0 and 48 hours, birth weight ≥ 1000 g, newborns delivered by caesarean section, and written informed consent from the newborn's parents / guardians. Exclusion criteria were: vaginal delivery, extremely low birth weight (ELBW) (<1000 g), established or suspected systemic infection, known severe neurological disease, known severe metabolic disease, known genetic and chromosomal disease, severe malformations (e.g., short bowel syndrome; intestinal obstruction; including patent ductus arteriosus if asymptomatic and not requiring treatment), known severe primary or secondary maternal immunodeficiency, known maternal food allergy, maternal diabetes (including gestational diabetes), maternal history of recent or suspected alcohol or drug abuse, any severe condition that the investigator believed could interfere with treatment, insufficient reliability, or the presence of a condition that could lead to patient noncompliance or nonadherence to the study protocol. In addition, neonates were prohibited from receiving probiotics other than the test product throughout the study duration, and lactating mothers were prohibited from receiving any probiotics throughout the study duration. For normal weight (NBW) neonates only, systemic antibiotic therapy or prophylaxis was not allowed from birth and for the entire study duration, and systemic antibiotic therapy or prophylaxis was not allowed for lactating mothers (of NBW infants) within 30 days before the study and for the entire study duration.

[0269] The parents / guardians of the newborns each signed an informed consent to participate in the study and received an instruction sheet with the conditions of use of the test product. Based on the inclusion / exclusion criteria, 60 preterm and term newborns were selected.

[0270] Study Design

[0271] The goal of this 12-week, randomized, double-blind, single-center, placebo-controlled clinical trial was to evaluate whether administration of LCDG to neonates in the first 48 hours after delivery alters the composition of the fecal gut microbiota after 28 days of product supplementation. The 12-week period was divided into 4 weeks of treatment and 8 weeks of follow-up.

[0272] Children attended 5 on-site visits: Visit 1 (V1; 0-48 hours after birth), Visit 2 (V2; 10 days + / - 2 days), Visit 3 (V3; 28 days + / - 3 days; end of product ingestion), Visit 4 (V4; 56 days + / - 3 days), and Visit 5 (V5; 84 days + / - 3; end of study).

[0273] At V1, newborns will be stratified into three groups according to birth weight: normal birth weight (>2500g, NBW), low birth weight (1500-2500g, LBW), and very low birth weight (1000-1500g, VLBW).

[0274] Eligible patients so stratified will be randomized in a ratio of 1:1 to one of the following groups: Group 1: Treatment group: 9 drops, twice a day of LCDG corresponding to 2 billion CFU (colony forming units), for 4 weeks (28 days), starting at 48 hours of life; Group 2: Placebo group: 9 drops, twice a day of placebo (product indistinguishable from the test product), for 4 weeks (28 days), starting at 48 hours of life.

[0275] During the same visits, infant and maternal demographic data and stool samples from the child were collected. The primary outcome was the composition of the fecal gut microbiota in terms of bacterial genome counts by real-time PCR at day 28 (V3) of LCDG treatment. Secondary outcomes assessed during the study were: modifications in the functional activity of the fecal gut microbiota (metabolome analysis - alpha- and beta-diversity) along with microbiota ecological predictions at 28, 56, and 84 days after the start of administration (V3, V4, and V5, respectively); changes in the composition of the fecal gut microbiota (real-time PCR) along with microbiota ecological predictions at 56 and 84 days after the start of administration (V4 and V5, respectively); changes in stool quality and quantity (frequency and consistency); the occurrence of neonatal colic as defined by the Rome IV criteria [Castelluzzo]; product safety and tolerability; and patient well-being.

[0276] Ethics

[0277] This study adhered to the ethical principles derived from or derived from the Declaration of Helsinki and was conducted in accordance with the Good Clinical Practice guidelines. The investigators submitted all study documents to the relevant ethics committee and sent the sponsor a copy of the approval / supportive opinion received from the ethics committee (ASL Roma 2: Prot.n°01220672 / 2018). The parents / guardians of the newborns provided signed informed consent.

[0278] Results Evaluation

[0279] Stool samples were collected within 24 hours before each visit.

[0280] For each newborn, the number of stools was recorded and the color and consistency of stool samples were assessed using the Amsterdam Stool Scale (described below).

[0281] In addition, physicochemical analyses of stool samples were performed by the Clinical Pathology Laboratory of the Casilino Hospital on the day of follow-up, including stercobilinogen, neutral fats, fatty acids, soaps, amides, meat and vegetable fiber, mucus, and pH. To ensure optimal storage conditions, stool samples were stored in a hospital freezer at −20°C and sent once a month with dry ice to the Parasitology / Human Microbiome Laboratory of the Bambino Gesù Children's Hospital (São Paulo region) for molecular analyses by a courier controlled by the sponsor.

[0282] Real-time PCR analysis was performed to evaluate Lactobacillus casei The bacterial load of the strains (according to a validated and published method; Ferrario) and metagenomic and metabolomic analyses were performed to characterize the intestinal microbiome before and after probiotic administration. Specifically, the microbiome was assessed by nucleotide sequence analysis of a portion of the gene encoding the bacterial ribosomal subunit 16S rRNA. Metagenomic DNA was extracted from fecal samples, quantified and normalized. The V3-V4 hypervariable region of the bacterial gene encoding 16S rRNA was amplified by PCR and the PCR products were quantified and sequenced using Illumina MiSeq technology. Bioinformatic sequence analysis was performed to characterize the microbial community, hierarchical clustering, taxonomic analysis and construction of phylogenetic evolutionary tree with the help of heat maps.

[0283] For metabolome analysis, microbial metabolites produced as volatile organic compounds (VOC-chemical categories: short-chain fatty acids, alcohols, ketones, aldehydes, thiols, acids, esters, pyrazines, pyridines, phenols, furans, terpenes, alkanes, alkenes, etc.) were identified and quantified. These compounds were extracted by GC-MS / SPME (gas chromatography coupled mass spectrometry with solid phase microextraction) system. For VOC extraction, carboxyl-polydimethylsiloxane (CAR-PDMS) coated fibers (85 μm) were used in the SPME method. Each sample was analyzed in triplicate, and an average of 100-500 mg of sample was placed in a 10 ml glass bottle, while 4-methyl-2-pentanol was added as an internal standard (IS). Subsequently, fecal samples were equilibrated at 45 ° C for 10 minutes. The fiber was exposed to each sample for 45 minutes, and then loaded onto a GC-MS (Hewlett Packard 6890GC) coupled to a 5973C mass selective detector and equipped with a Supelcowax 10 capillary column. Metabolites were detected based on their characteristics. Metabolites were detected based on their retention time (Rt) relative to the pure compound. Chromatograms were integrated and identified by comparing the fragmentation patterns with those from the NIST library followed by manual visual inspection. Quantitative metabolite data were obtained by area interpolation relative to the IS area.

[0284] Safety was assessed by monitoring adverse events during the study and measuring neonatal weight, length, and head circumference on days 1 (V1), 10 (V2), 28 (V3), 56 (V4), and 84 (V5).

[0285] Finally, the assessment of the occurrence of colic was performed according to the "Rome IV" diagnostic criteria (described below) and the daily frequency and consistency of stools were monitored with the help of a dedicated diary given to the mothers at V1.

[0286] Amsterdam stool scale

[0287] The scale divides stool volume into four categories: 1: diffuse, 2: up to 25%, 3: 25-50% and 4: >50%.

[0288] Stool consistency is classified as: A: watery, B: soft, C: formed, and D: hard.

[0289] Finally, stool color was classified into six categories (I-VI).

[0290] "Rome IV" diagnostic criteria for infantile colic

[0291] For clinical purposes, the diagnosis of infantile colic must be based on the presence of all of the following criteria: the child is less than 5 months of age when symptoms occur and resolve; there are recurrent periods of prolonged crying, agitation, or irritability that occur for no apparent reason and cannot be prevented or eliminated by the parents; and there are no signs of growth failure, fever, or illness. The term "agitation" refers to intermittent vocalizations and is defined as 'behavior' that is not exactly crying, but is not the same as someone who is awake and happy. Children often alternate between crying and agitation, which can make it difficult to distinguish the two symptoms.

[0292] For study purposes, a diagnosis of infantile colic had to include the previous criteria plus both of the following: parental reporting of crying or agitation lasting at least 3 hours per day at least 3 days per week during a telephone interview or one-on-one interview with an investigator or physician; and prospective quantification of the duration of crying or agitation by completion of a daily diary reported for 24 consecutive hours in a selected group of children, confirmed as lasting at least 3 hours.

[0293] Statistical analysis

[0294] Lactobacillus casei (Lactobacillus paracasei CNCM I-1572; LCDG) behaves similarly to Lactobacillus salivaris, which showed a difference of 2.36 Log10 copies (from 0.04, SD 0.4 to 2.5, SD 2.5) in 200 mg stool after 28 days in the same study [Putignani et al., submitted]. In each group (NBW, LBW, VLBW), recognizing that 2 billion CFU / day was ingested in the treatment group, samples from 9 patients in each treatment group would give this study 80% power with a two-tailed t-test and 95% significance level in each stratum.

[0295] It should be noted that after the reclassification of the genus Lactobacillus published by Zheng et al. in the scientific journal Int. J. Syst. Evol. Microbiol., 70(4): 2782-2858, 2020, the bacterial strain Lactobacillus casei (CNCM I-1572) or Lactobacillus paracasei (CNCM I-1572) was redeposited on February 2, 2022 as Lactobacillus paracasei DG I-1572 DSM 34154 at the German Collection of Microorganisms, Inhofenstrasse 7B D-38124 Braunschweig. The two aforementioned nomenclatures are interchangeable with each other, as they always refer to the same bacterial strain.

[0296] Anticipating possible loss to follow-up (10% dropout), 20 neonates per weight stratum were enrolled (10 to receive active treatment and 10 to receive placebo), for a total of 60 neonates.

[0297] Categorical data were expressed as counts and percentages, and continuous data were expressed as mean and standard deviation or median and range.

[0298] Categorical data were compared using the chi-square test or Fisher's exact test, as appropriate. Continuous data were compared using Student's t-test (for independent or dependent data), analysis of variance, and repeated measures Anova if appropriate statistical tests determined data normality; in the case of nonnormal distribution, nonparametric tests such as the Mann Whitney rank-sum test or the Wilkoxon signed-rank test, the Kruskal Wallis test, or the Friedman test were applied. Multivariate regression methods were used if indicated to assess the simultaneous effects of a set of independent variables on the response variable or to identify potential confounders and / or to analyze temporal data. The results obtained were considered statistically significant when the P value was <0.05. All analyses were performed using Stata 15 software (StatCorp).

[0299] result

[0300] Patient disposition and basic characteristics

[0301] Sixty subjects, preterm and term neonates selected from the Neonatal Department of the Policlinico Casilino Hospital, were randomized in a 1:1 ratio into two treatment groups: 30 treated with LCDG and 30 treated with placebo ( Figure 5 ).

[0302] In the placebo group, 20 subjects (66.7%) completed the study and 10 (33.3%) did not: 2 (6.7%) subjects withdrew their consent, 1 (3.3%) patient discontinued study participation prematurely due to an adverse event (AE), 6 (20.0%) subjects were lost to follow-up (FUP) and 1 (3.3%) subject withdrew due to hypopharyngeal hamartoma requiring surgical treatment.

[0303] In the LCDG group, 26 subjects (86.7%) completed the study and 4 (13.3%) did not complete; of these subjects, 3 (10.0%) were lost to follow-up during FUP and 1 (3.3%) died due to "disseminated intravascular coagulation" and "septic shock".

[0304] Regarding demographic and baseline characteristics, the mean age of mothers in the placebo group was 35 ± 6 years and in the LCDG (treatment) group was 35 ± 5 years (P = 0.94), and there were no statistically significant differences between the two treatment groups with respect to maternal ethnicity, breastfeeding type, maternal weight and height, dietary type, smoking status, physical activity, presence of pets, and education level.

[0305] Regarding the characteristics of the newborns, they were divided into 3 groups according to their birth weight in this study: normal birth weight (NBW), low birth weight (LBW) and very low birth weight (VLBW). During the study period, 12 pairs of twins were born (a total of 24 newborns), especially 14 (46.7%) subjects were included in the LCDG group and 10 (34.5%) were included in the placebo group (P = 0.34). 18 (62.1%) and 17 (56.7%) newborns in the placebo group and LCDG group were female (P = 0.67) and the average birth weight of the newborns in the placebo group and LCDG group were 2050g ± 784g and 2005g ± 768g, respectively (P = 0.82). There was no statistically significant difference between the two treatment groups in terms of APGAR score, gestational age and PN / EG ratio (AGA and SGA).

[0306] Lactobacillus casei DG (LCDG) was detected in the intestine from 10 days after the first administration and remained until the last visit (84 days).

[0307] We analyzed 251 stool samples collected from 59 subjects stratified at birth into VLW (n=20), LW (n=20), and NW (n=19). LCDG strains were detected in stool samples starting 10 days after the first application (V2) and persisted until the last visit (84 days; end-of-study visit), with a slight decrease ( 56 days after the first follow-up time point) from the first follow-up time point (study day 58, 28 days since the last product application) to the last visit (day 84; end-of-study visit). Fig. 6A ). In samples collected at visits during the treatment phase (at 10 and 28 days) and follow-up (at 56 and 84 days), the presence of LCDG was significantly higher in the treatment group than in the control group. This evidence was also confirmed by stratifying the samples from each group according to birth weight (NW, LW and VLW). Specifically, the comparison between the three weight categories in the placebo group (intra-group analysis) showed a statistical difference in LCDG concentrations at V2 (10 days after the first administration) (p-value = 0.01), while in the treatment group, a statistical difference in LCDG concentrations between the three weight categories (p-value = 0.01) appeared at V3 (28 days after the last administration of the product). No statistically significant differences were observed between the weight categories at the other time points evaluated ( Figure 6B In addition, at day 56, the differences in LCDG concentrations between the treatment and placebo groups were statistically significant for NW infants and VLW infants ( Figure 6B ).

[0308] Wilcoxon signed rank test was performed to compare LCDG concentrations in each group at different time points. In the placebo group, comparisons between V1 and V2 (p value = 0.012), between V1 and V3 (p value = 0.016), and between V1 and V4 (p value = 0.012) were statistically significant. For the treatment group, comparisons between V1 and V2 (p value = 1.5x10 -12 ), between V1 and V3 (p value = 5.8x10 -9 ), between V1 and V4 (p value = 1.8x10 -8 ), between V1 and V5 (p value = 2.0x10 -6 ), between V2 and V4 (p value = 1.6x10 -3 ) and between V2 and V5 (p value = 9.7x10 -3 ) is statistically significant ( Figure 7 ).

[0309] Treatment with LCDG preserves the heterogeneity of the gut microbiome

[0310] To verify whether LCDG treatment could preserve the heterogeneity of the gut microbiome, fecal samples were analyzed for α- and β-diversity using a metagenomic approach targeting 16S-RNA.

[0311] Analysis of the α-diversity of subjects assigned to the placebo and treatment groups at each time point showed that at time points V2 (10 days) and V3 (28 days), the microbiota of the placebo group appeared to have a greater richness of microbial species than the treatment group (p ≥ 0.05). At time point V4, each α-diversity index seemed equivalent (p ≥ 0.05), while at time point V5, the treatment group had a greater richness than the placebo group (p ≥ 0.05) ( Fig. 8A ). However, there were no statistically significant differences between the placebo group and the treatment group at various time points (p≥0.05).

[0312] For the β-diversity analysis, the diversity matrix obtained with the unweighted UniFrac algorithm (expressed as PCoA at each time point between the placebo group and the treatment group) showed no statistically significant comparison between the two groups at each time point (p ≥ 0.05). This result was confirmed with the PERMANOVA statistical analysis, applied to the distance matrix calculated with the unweighted UniFrac algorithm ( Figure 8B ).

[0313] Analysis of the distribution of operational taxonomic units (OTUs) at the phylum level and the Kruskall-Wallis test for comparisons at each time point showed no statistically significant differences (p ≥ 0.05) in any placebo / treatment comparison ( Figure 8C ).

[0314] At the species level, statistically significant comparisons between placebo and treatment were observed only for time point V2 (p < 0.05); in particular, Lactobacillus species were more abundant in the treatment group ( Fig.8D ).

[0315] LCDG administration increases short-chain fatty acid (SCFA) concentrations

[0316] Metabolome analysis was performed on stool samples (n=85) collected from subjects who completed collection at all follow-up time points (V1 to V5).Stool samples were analyzed by gas chromatography coupled to mass spectrometry (GC-MS-SPME) using a solid phase microextraction system.

[0317] 492 metabolites were identified, quantified and divided into 15 chemical categories: acids, alcohols, aldehydes, alkanes, alkenes, amines, aromatic hydrocarbons, esters, furans, furanones, indoles, ketones, phenols, pyrazines and terpenes. As expected, the metabolic profile of each sample showed considerable variability between subjects. The original data matrix was compressed into a matrix of 98 metabolites, maintaining metabolites present in at least 10% of the entire sample set, aiming to make the analysis as robust as possible. A complete data analysis was developed for this compressed matrix. For each time point, a multivariate analysis was implemented using a supervised scheme, which was a partial least squares discriminant analysis (PLS-DA). The largest clustering of samples according to treatment (placebo / treatment) occurred between time points V2 (10 days) and V4 (56 days), with an increase in SCFA concentrations in treated samples (Figure 9).

[0318] Additionally, at each study visit, physicochemical evaluation of stool was performed to assess the presence of neutral fat, fatty acids, soaps, starch, meat and vegetable fiber, mucus, and pH. Chi-square test was applied to compare any differences between the two treatment groups and no significant differences were found for any parameter (P>0.05).

[0319] The administration of LCDG affects the ecology and function of the microbiota, thereby adjusting the ecological niche of intestinal resident bacteria and the corresponding functional characteristics.

[0320] The results obtained with the 3 different methods (real-time PCR, metagenomics and metabolomics) were correlated with each other according to Spearman correlation. Time points V2 and V3, which coincide with 10 and 28 days of probiotic administration, respectively, were selected for correlation analysis because metagenomics, metabolomics and real-time analysis showed the greatest statistically significant differences between placebo and treatment groups at these time points.

[0321] Specifically, at time point V2, Lactobacillus paracasei (CFU / mL) was positively correlated with the metabolites cyclohexanone, n-decanoic acid, and 3,4-dimethylheptane and with the OTUs Lactobacillus and Granulicatella (p≤0.05). In addition, several statistically significant positive correlations between OTUs and metabolites can be noted. The metabolites with the greatest significance after probiotic ingestion were SCFAs, including butyrate, which showed significant correlations with Ruminococcus, Prevotella, Collinsella, Faecalibactarium, and Oscillospira (p≤0.05). Valeric acid showed significant correlation with Streptococcus, Granulostomyces, and Enterococcus (p≤0.05), while propionic acid had significant correlation with Eggerthella, Bacteroides, and Bifidobacterium (p≤0.05).

[0322] At time point V3, Lactobacillus paracasei (CFU / mL) was positively correlated with 2,3-dimethylpentanal, 1-hexanol, decanal, and dodecanoic acid (p≤0.05) ( Fig. 10A ), while there was no significant correlation between Lactobacillus paracasei (CFU / mL) and various OTUs ( Fig. 10B Finally, several statistically significant positive correlations between OTUs and metabolites can be noted (p ≤ 0.05). In particular, butyrate and propionate showed significant correlations with Collinsella (p ≤ 0.05), while valeric acid was positively correlated with Propionibacterium, Faecalibacterium, Prevotella, and Coprococcus ( Fig. 10C ).

[0323] Administration of LCDG did not reduce the number of colic episodes.

[0324] According to the "Rome IV" diagnostic criteria, the investigators recorded colic episodes if crying lasted longer than 3 hours on at least 3 days a week and if there was at least 3 hours of agitation a day. During the study period, mothers were required to fill in a diary in which they recorded their infant's crying if crying lasted longer than 3 hours. At all study visits involved (10, 28, 56 and 84 days after randomization), there were no statistically significant differences between the two study groups (placebo and Enterolactis Baby). In fact, the number of patients in each study group who cried for more than 3 hours per day and the mean duration of crying were almost similar at each study visit, with a total of 8 cases of colic occurring during the study period (4 in the placebo group and 4 in the LCDG group).

[0325] Stool consistency, amount, and color

[0326] During the clinical trial, stool consistency, volume, and color were assessed using the Amsterdam Stool Scale. There were significant differences in color and consistency between the placebo and treatment groups on Day 10 (V2) and Day 56 (V4).

[0327] In V2, for stool color, the p-value was P<0.0001, and for stool consistency, the p-value was P=0.0016. Specifically, among the total 143 and 221 stools recorded for the PT of the placebo group and the treatment group, respectively, 37 (25.9%) stools of the placebo group and 18 (8.1%) stools of the treatment group were divided by color II, and 7 (4.9%) stools of the placebo group and zero stools of the treatment group were divided by color IV. Regarding consistency, 80 (55.9%) stools of the placebo group and 152 (68.8%) stools of the treatment group were divided by consistency B, and zero stools of the placebo group and 6 (2.7%) and 2 (0.9%) stools of the treatment group were divided by consistency C / D and D.

[0328] At visit 4, there were 119 stools in the placebo group and 183 stools in the treatment group, with statistically significant differences in color and consistency between treatment groups (P = 0.007 for stool color and P < 0.0001 for stool consistency using the Amsterdam scale).

[0329] Regarding the amount, it was significant only in the samples collected on day 28 (V3), with a total of 122 and 219 excretions recorded for the placebo and treatment groups, respectively.

[0330] Safety Assessment

[0331] To monitor the growth of enrolled newborns, weight, length and head circumference parameters were assessed at each study visit. Comparing the treatment and placebo groups, there were no statistically significant differences during the study. Finally, there were no differences in vital signs (the exception was RR, which was higher in the placebo group than in the treatment group).

[0332] Regarding adverse events (AEs), a total of 21 AEs were recorded for the placebo group and a total of 16 AEs were recorded for the LCDG group during the study. Among these adverse events, 10 SAEs were recorded for the placebo group and 6 SAEs were recorded for the treatment group, and 2 severe AEs were recorded for the placebo group and 4 severe AEs were recorded for the treatment group.

[0333] In the placebo group, 10 (47.6%) AEs were considered mild, 9 (42.9%) AEs were moderate in severity, and 2 (9.5%) AEs were severe in severity, while in the LCDG group, 8 (50.0%) AEs were considered mild, 4 (25.0%) AEs were moderate in severity, and 4 (25.0%) AEs were severe in severity.

[0334] Among the AEs occurring in patients (PTs) belonging to the placebo group, 3 (14.3%) AEs were considered unlikely to be related to treatment (1 "neonatal respiratory distress syndrome" and 2 "abdominal pain") and 1 (4.8%) AE was considered possibly related to treatment ("gastroesophageal reflux disease"). Among the AEs occurring in PTs belonging to the LCDG group, 2 (12.5%) AEs were considered unlikely to be related to treatment ("disseminated intravascular coagulation" and "septic shock", which resulted in the death of one patient 9 days after visit 2) and 2 (12.5%) AEs were considered possibly related to treatment ("necrotizing colitis" and "sepsis").

[0335] discuss

[0336] Some neonatal clinical trials have investigated the role of probiotics in the treatment of colic, showing that they were effective in reducing crying episodes and the duration of colic symptoms compared with placebo [Lundelin].

[0337] A long-term follow-up study of probiotic treatment during the neonatal period revealed that a lower percentage of subjects in the probiotic treatment group experienced allergic episodes compared with those assigned to the placebo group [Denkel].

[0338] Additionally, probiotic administration has been associated with less incidence of necrotizing enterocolitis and reduced mortality in premature neonates [Lundelin, Strunk, Shane, Costeloe].

[0339] According to these findings, probiotics are characterized by an excellent safety profile. Cases of bacteremia caused by bacterial strains contained in probiotics have been documented, although they are extremely rare and were determined in the presence of severe immunodeficiency [Lundelin, Salminen, Thomas].

[0340] However, more evidence is needed regarding the safety and potential efficacy of probiotic supplementation in neonates.

[0341] Considering the increasing use of Lactobacillus species in probiotic mixtures, correct identification and enumeration of LCDG cells in stool will improve studies on the actual stability and persistence of these probiotics in the human gut microbiome [Arioli].

[0342] In this study, the safety of LCDG and its ability to positively influence microbiome composition were tested for the first time in a specific population that included not only NMW infants but also LW subjects and VLW subjects.

[0343] LCDG was detected in the intestine from 10 days after the first administration and remained until the last visit (84 days; end of study), with a slight decrease from the first follow-up time point (study day 56, 28 days since the last administration of the product). For both NW infants and VLW infants, the presence of LCDG was significantly greater in the treatment group than in the control group at V3 (day 28; last day of treatment).

[0344] Probiotic strains were analyzed in fecal samples, and although changes in the overall microbiota spectrum appeared to be time-dependent at the species level and unaffected by treatment considering alpha and beta diversity analysis (suggesting that treatment can preserve the heterogeneity of the gut microbiome), according to this preliminary analysis, probiotic administration caused a statistically significant increase in Lactobacillus 10 days after the start of administration. This increase was also detected at later times, but became non-statistically significant.

[0345] As for the metabolome analysis, as expected, the metabolic profiles of each sample showed considerable variability between subjects. PLS-DA analysis identified butyrate and propionate metabolites (SCFA) as the main contributors to the clustering between the placebo and treatment groups, with elevated concentrations of these metabolites in samples collected from the treatment groups.

[0346] All these results emphasize and confirm the ability of LCDG to survive the gastrointestinal passage while preserving the heterogeneity of the intestinal microbiota. These results are consistent with the conclusions of studies conducted in healthy children (3-12 years) [Radicioni] or adults [Drago].

[0347] In addition, the results obtained with the three different methods (real-time PCR, metagenomics, and metabolomics) were correlated with each other based on Spearman correlation.

[0348] Integration of the data obtained from these protocols showed that the administration of probiotic LCDG affects the ecology and function of the microbiota, thereby adjusting the niche of the resident intestinal bacteria and the corresponding functional characteristics. Specifically, some SCFAs such as butyrate and propionate showed positive correlations with certain beneficial bacteria such as Faecalibacterium, Oscillatory Spirochete, Eggerthella, Bacteroides, and Bifidobacterium. These molecules have shown the ability to affect nutrient absorption [Heimann]. In addition, a positive correlation was found between Lactobacillus paracasei (CFU / mL) and cyclohexanone, n-decanoic acid, and 3,4-dimethylheptane on day 28 (p≤0.05).

[0349] Stool consistency, volume and color were also recorded during the clinical trial. Volume was significant only in samples collected on day 28, while color and consistency were significantly different on days 10 and 56 (4 weeks after the end of administration).

[0350] On the other hand, no differences were observed in the occurrence of angina according to the "Rome IV" diagnostic criteria, but considering that the duration of treatment and the total study duration might not have been sufficient to see an improvement in this parameter, perhaps these results should be analyzed. et al. Extended probiotic supplementation for up to 2 months (twice the duration of this study), with follow-up up to 1 year, demonstrated that it can reduce symptoms associated with crying and agitation in premature newborns.

[0351] Regarding safety, as mentioned above, there were no differences in vital signs (the exception was the RR, which was higher in the placebo group than in the treatment group).

[0352] During the study, weight, length, and head circumference parameters were also assessed at each study visit to monitor the growth of the enrolled neonates. Comparing the treatment and placebo groups, there were no statistically significant differences during the study. Although a previous article has shown that a commercially prepared symbiotic solution containing a combination of probiotics and prebiotics can improve weight and head circumference in preterm neonates [Guney], other studies have shown data consistent with our results (i.e., no differences between the treatment and placebo groups) [Vlieger], especially for weight parameters [Indrio, Underwood]. Weight gain in neonates is affected by many coexisting pathologies and the overall supplementation availability and / or milk fortification of the parents [Moni], which may have affected the weight trends in both groups of this study.

[0353] Recently, Matin et al. [Matin] published the results of a study involving oral administration of probiotics (1.5 × 10 9 CFU / g Lacticaseibacillus paracasei subsp. paracasei) to test the changes in total serum bilirubin (TSB) levels and weight gain of newborns. After the first week of intervention, the administration of the selected probiotics reduced the TSB levels of newborns, but did not significantly affect weight gain.

[0354] In addition, based on the analyzed safety and tolerability profiles and the evaluation of severe AEs recorded in both study groups, the test product was shown to be safe and well tolerated as placebo in this study.

[0355] Limitations and future plans

[0356] In conclusion, the results of this study confirm that the safety profile of the product is high even for vulnerable groups such as NBW, LBW and VLBW neonates. Although LDCG is confirmed to be able to survive in the gastrointestinal tract, affecting the gut microbiota and SCFA composition, it will be useful in the future to analyze the long-term effects of this product by considering a follow-up of at least one year, given the key role of the gut microbiota in nutrient absorption and therefore neonatal growth.

[0357] Another interesting aspect that has not been fully strengthened in this clinical trial is the potential impact of the product on the microbiome and serum metabolome under different nutritional conditions. In fact, nutrition during early childhood determines the formation of the intestinal microbiome. Unlike breastfeeding, artificial feeding has been shown to influence the intestinal microbiome and the serum metabolome towards a more unfavorable state. Recently, it has been shown that another strain of Lactobacillus paracasei, although not eliminating the effects of artificial feeding, affects the microbiome and serum metabolome, and this effect could mitigate some of the adverse metabolic effects of (artificial) feeding [Lee].

[0358] 1. A bacterial strain for use in a method for the prophylactic and / or curative treatment of a subject suffering from a gastrointestinal disease of an inflammatory and / or functional nature, the disease being selected from the group comprising or alternatively consisting of: neonatal colic, intestinal colic, chronic enterocolitis or chronic enterocolitis of premature neonates, necrotizing enterocolitis, sepsis, irritable bowel syndrome (IBS), IBS with generalized constipation or constipation-type bowel, IBS with generalized diarrhea or diarrhea-type bowel, IBS with alternating bowel, unclassified IBS, chronic Inflammatory bowel disease (IBD), Crohn's disease, ulcerative colitis, asthma, obesity, type 1 diabetes, atopic dermatitis, multiple sclerosis, cancer, autism, allergies, immune-mediated autoimmune diseases selected from celiac disease, Graves' disease, rheumatoid arthritis, Hashimoto's thyroiditis, type 1 diabetes, systemic lupus erythematosus (lupus), vasculitis, Addison's disease, polymyositis, Sjögren's syndrome, progressive systemic sclerosis, glomerulonephritis (inflammation of the kidney), infertility; and supporting weight gain in said subject,

[0359] - wherein the bacterial strain belongs to the species Lactobacillus paracasei and is selected from the group comprising or alternatively consisting of: Lactobacillus paracasei CNCM I-1572, Lactobacillus paracasei LPC-S01DSM 26760 and mixtures thereof;

[0360] - wherein the subject is a neonatal subject in the first four weeks of life.

[0361] 2. The bacterial strain for use according to claim 1, wherein the age of the subject is from 1 month to less than or equal to 12 months.

[0362] 3. The bacterial strain for use according to claim 1 or 2, wherein the strain is a live bacterial strain or a derivative of the bacterial strain, the latter being selected from the group comprising or alternatively consisting of: an intermittently sterilized bacterial strain, an ultrasonically treated bacterial strain, a bacterial strain inactivated by irradiation, preferably gamma irradiation, a lysed bacterial strain or a bacterial homogenate, an extract or a wall fraction of a bacterial strain; preferably an intermittently sterilized bacterial strain.

[0363] 4. The bacterial strain for use according to any one of claims 1 to 3, wherein the disease is selected from the group comprising or alternatively consisting of neonatal colic, intestinal colic, chronic enterocolitis or chronic enterocolitis of premature neonates, necrotizing enterocolitis, sepsis, irritable bowel syndrome (IBS), IBS with generalized constipation or constipation-type bowel, IBS with generalized diarrhea or diarrhea-type bowel, IBS with alternating-type bowel, unclassified IBS, chronic inflammatory bowel disease (IBD), Crohn's disease, ulcerative proctitis.

[0364] 5. A mixture M for use according to any one of claims 1 to 4, wherein the mixture comprises or alternatively consists of a bacterial strain belonging to the species Lactobacillus paracasei and at least one food grade or pharmaceutical grade additive and / or excipient, the bacterial strain being selected from the group comprising or alternatively consisting of: Lactobacillus paracasei CNCM 1-1572, Lactobacillus paracasei LPC-S01 DSM 26760 and mixtures thereof.

[0365] 6. A mixture M for use according to claim 5, wherein the mixture M comprises or alternatively consists of a bacterial strain belonging to the species Lactobacillus paracasei and at least one other bacterial strain, the bacterial strain of the species Lactobacillus paracasei being selected from the group comprising or alternatively consisting of: Lactobacillus paracasei CNCM 1-1572, Lactobacillus paracasei LPC-S01 DSM 26760 and mixtures thereof; said at least one other bacterial strain is selected from the group comprising or alternatively consisting of:

[0366] - Bifidobacterium breve BbIBS01 DSM 33231,

[0367] - Bifidobacterium breve BbIBS02 DSM 33232,

[0368] - Bifidobacterium animalis subsp. lactis BlIBS01 DSM 33233,

[0369] - Lactobacillus plantarum LpIBS01 DSM 33234,

[0370] - Bifidobacterium bifidum BbfIBS01 DSM 32708; and

[0371] A mixture thereof.

[0372] 7. The mixture M for use according to claim 5 or 6, wherein the mixture M further comprises at least one other active ingredient selected from the group comprising or alternatively consisting of:

[0373] - at least one vitamin from the A, B, C, D, E and / or K groups, preferably B vitamins and / or vitamin D;

[0374] - one or more antioxidant substances selected from the group comprising or alternatively consisting of: glutathione, polyphenols such as resveratrol and trans-resveratrol, coenzyme Q10, astaxanthin, lycopene;

[0375] - one or more botanical substances with intestinal relaxant effect selected from the group comprising or alternatively consisting of botanicals or extracts thereof, preferably selected from valerian, passionflower, lemon mint, hawthorn and chamomile;

[0376] - Minerals or their salts, such as zinc, selenium, magnesium, potassium;

[0377] - one or more monounsaturated fatty acids selected from the group comprising or alternatively consisting of: ω9, and / or polyunsaturated fatty acids, ω3 and ω6;

[0378] - one or more immunostimulant substances, antidiarrheal substances and / or nutrients;

[0379] - at least one prebiotic, preferably selected from the group (II) consisting of inulin, fructooligosaccharides (FOS), galacto-oligosaccharides (GOS), xylitol-oligosaccharides (XOS), guar gum, lactoferrin, and mixtures thereof; more preferably inulin; and

[0380] A mixture thereof.

[0381] The present invention relates to at least one bacterial strain belonging to the species Lactobacillus paracasei, preferably Lactobacillus paracasei CNCM I-1572 and / or Lactobacillus paracasei LPC-01DSM 26760; and compositions thereof for the preventive and / or curative treatment of gastrointestinal diseases of inflammatory and / or functional nature, gastrointestinal infections caused by pathogenic microorganisms, gastrointestinal infections caused by parasites, allergies, immune-mediated diseases or autoimmune diseases in newborns and / or subjects aged 1 month to ≤12 months; and for supporting the growth of subjects.

Claims

1. A bacterial strain for use in a method for the prophylactic and / or curative treatment of a subject suffering from a gastrointestinal disease of an inflammatory and / or functional nature, the disease being selected from the group comprising or alternatively consisting of: neonatal colic, intestinal colic, chronic enterocolitis or chronic enterocolitis of premature neonates, necrotizing enterocolitis, sepsis, irritable bowel syndrome (IBS), IBS with generalized constipation or constipation-type bowel, IBS with generalized diarrhea or diarrhea-type bowel, IBS with alternating bowel, unclassified IBS, chronic Inflammatory bowel disease (IBD), Crohn's disease, ulcerative colitis, asthma, obesity, type 1 diabetes, atopic dermatitis, multiple sclerosis, cancer, autism, allergies, immune-mediated autoimmune diseases selected from celiac disease, Graves' disease, rheumatoid arthritis, Hashimoto's thyroiditis, type 1 diabetes, systemic lupus erythematosus (lupus), vasculitis, Addison's disease, polymyositis, Sjögren's syndrome, progressive systemic sclerosis, glomerulonephritis (inflammation of the kidney), infertility; and supporting weight gain in said subject, - wherein the bacterial strain belongs to the species Lactobacillus paracasei and is selected from the group comprising or alternatively consisting of: Lactobacillus paracasei CNCM I-1572, Lactobacillus paracasei LPC-S01DSM 26760 and mixtures thereof; - wherein the subject is a neonatal subject in the first four weeks of life.

2. The bacterial strain for use according to claim 1, wherein the age of the subject is from 1 month to less than or equal to 12 months.

3. The bacterial strain for use according to claim 1 or 2, wherein the strain is a live bacterial strain or a derivative of the bacterial strain, the latter being selected from the group comprising or alternatively consisting of: an intermittently sterilized bacterial strain, an ultrasonically treated bacterial strain, a bacterial strain inactivated by irradiation, preferably gamma irradiation, a lysed bacterial strain or a bacterial homogenate, an extract or a wall fraction of a bacterial strain; preferably an intermittently sterilized bacterial strain.

4. The bacterial strain for use according to any one of claims 1 to 3, wherein the disease is selected from the group comprising or alternatively consisting of neonatal colic, intestinal colic, chronic enterocolitis or chronic enterocolitis of premature neonates, necrotizing enterocolitis, sepsis, irritable bowel syndrome (IBS), IBS with generalized constipation or constipation-type bowel, IBS with generalized diarrhea or diarrhea-type bowel, IBS with alternating-type bowel, unclassified IBS, chronic inflammatory bowel disease (IBD), Crohn's disease, ulcerative proctitis.

5. A mixture M for use according to any one of claims 1 to 4, wherein the mixture comprises or alternatively consists of a bacterial strain belonging to the species Lactobacillus paracasei and at least one food grade or pharmaceutical grade additive and / or excipient, the bacterial strain being selected from the group comprising or alternatively consisting of: Lactobacillus paracasei CNCM I-1572, Lactobacillus paracasei LPC-S01 DSM 26760 and mixtures thereof.

6. A mixture M for use according to claim 5, wherein the mixture M comprises or alternatively consists of a bacterial strain belonging to the species Lactobacillus paracasei and at least one other bacterial strain, the bacterial strain of the species Lactobacillus paracasei being selected from the group comprising or alternatively consisting of: Lactobacillus paracasei CNCM 1-1572, Lactobacillus paracasei LPC-S01 DSM 26760 and mixtures thereof; said at least one other bacterial strain is selected from the group comprising or alternatively consisting of: - Bifidobacterium breve BbIBS01 DSM 33231, - Bifidobacterium breve BbIBS02 DSM 33232, - Bifidobacterium animalis subsp. lactis BlIBS01 DSM33233, - Lactobacillus plantarum LpIBS01 DSM 33234, - Bifidobacterium bifidum BbfIBS01 DSM 32708; and A mixture thereof.

7. The mixture M for use according to claim 5 or 6, wherein the mixture M further comprises at least one other active ingredient selected from the group comprising or alternatively consisting of: - at least one vitamin from the A, B, C, D, E and / or K groups, preferably B vitamins and / or vitamin D; - one or more antioxidant substances selected from the group comprising or alternatively consisting of: glutathione, polyphenols such as resveratrol and trans-resveratrol, coenzyme Q10, astaxanthin, lycopene; - one or more botanical substances with intestinal relaxant effect selected from the group comprising or alternatively consisting of botanicals or extracts thereof, preferably selected from valerian, passionflower, lemon mint, hawthorn and chamomile; - Minerals or their salts, such as zinc, selenium, magnesium, potassium; - one or more monounsaturated fatty acids selected from the group comprising or alternatively consisting of: ω9, and / or polyunsaturated fatty acids, ω3 and ω6; - one or more immunostimulant substances, antidiarrheal substances and / or nutrients; - at least one prebiotic, preferably selected from the group (II) consisting of inulin, fructooligosaccharides (FOS), galacto-oligosaccharides (GOS), xylitol-oligosaccharides (XOS), guar gum, lactoferrin, and mixtures thereof; more preferably inulin; and A mixture thereof.

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