Use of Lactobacillus paracasei strains in the treatment of neonates

Abstract

The present invention relates to at least one strain belonging to the species Lactobacillus paracasei, preferably Lactobacillus paracasei DG® CNCM I-1572 and / or Lactobacillus paracasei LPC-01 DSM 26760, for use in newborns and / or subjects aged between 1 and 12 months, and compositions thereof, in the treatment, prevention and / or healing of inflammatory and / or functional gastrointestinal disorders, pathogenic microbial gastrointestinal infections, parasitic gastrointestinal infections, allergies, immune-mediated or autoimmune disorders, and in supporting the growth of the subject.

Description

Technical Field

[0001] The present invention relates to at least one strain belonging to the species Lactobacillus paracasei, preferably Lactobacillus paracasei DG (registered trademark) CNCM I-1572 and / or Lactobacillus paracasei LPC-S01 DSM 26760, and their compositions for use in the treatment, prevention and / or cure of inflammatory and / or functional gastrointestinal tract disorders, preferably neonatal colic or chronic enteritis (e.g., necrotizing enteritis) of premature neonates, in the treatment, prevention and / or cure of gastrointestinal infections by pathogenic microorganisms (e.g., viruses or bacteria), gastrointestinal infections by parasites, allergies, immune-mediated or autoimmune disorders, and in the supportive treatment of the growth of a subject, for neonatal subjects within 4 weeks after birth.

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

[0003] In the context of the present invention, the term "newborn" or "newborn subject" refers to a mammal (human or animal subject) during the period from the moment of birth to 4 weeks (28 days) after birth.

[0004] In the first few days after birth, a number of factors affect the composition of the gut microbiota: the mother's vaginal and / or skin microbiota, vaginal delivery or cesarean section, breastfeeding or formula feeding, and the administration of antibiotics and / or other drugs. In particular, cesarean section, formula feeding, prematurity, and the use of antibiotics reduce the abundance and diversity of beneficial bacterial species in the microbiota and promote a state of dysbiosis. This, in turn, may increase the risk of colic, the development of necrotizing enterocolitis in premature neonates, and the onset of immune-mediated diseases in later life (such as allergies or inflammatory or functional bowel diseases, and / or related symptoms).

[0005] Currently, approximately 12% of premature neonates weighing less than 1,500 g suffer from necrotizing enterocolitis, and approximately one-third die of sepsis or other complications.

[0006] In the first few weeks after birth, the internal organs are not fully mature, and as a result, their functions do not match those of a child or an adult (even a child or an adult who is frail or ill). Therefore, neonates are physiologically "fragile" compared to both "weak" adults and even young children.

[0007] The "gut microbiota" refers to all microorganisms (all bacteria, archaea, eukaryotes, and viruses) present in the gastro-enteric or gastro-intestinal environment.

[0008] The microbiota performs many useful activities for the host organism. In fact, the microorganisms that make up the microbiota break down polysaccharides to assist digestion, synthesize vitamins, inhibit the colonization of pathogenic species, provide signals necessary for intestinal development, and contribute to the regulation of inflammation and immune responses. The etiology of many gastrointestinal disorders is thought to be an abnormal immune response to the gut microbiota, similar to disorders that affect other organ systems. In addition, the gut microbiota, together with the metabolites produced by the microbiota and intestinal cells, forms a barrier, and its dysfunction is involved in the pathophysiology of not only pathogenic microbial infections but also many immune-mediated disorders.

[0009] To maintain good health, it is necessary to maintain a balance between the immune system and the microorganisms present in the intestine. Therefore, it has also been shown that this balance state depends on the composition of the gut microbiota. An imbalance in the microbiota, known as the term "dysbiosis", can lead to immune system disorders and cause harmful effects both in the intestine and in other organ systems.

[0010] Therefore, the beneficial effects of probiotic strains (i.e., live microorganisms that bring health benefits when administered in appropriate amounts) are mainly due to normalizing the permeability of the intestinal barrier, regularizing the intestinal microbiota, and restoring the balance between the microbiota and the immune response.

[0011] The technical problem addressed by the present invention is to provide an effective solution for the treatment of inflammatory or functional gastrointestinal diseases or symptoms (such as colic, chronic enteritis, etc.) and gastrointestinal infections of pathogenic microorganisms or gastrointestinal infections of parasites in neonatal subjects and / or mammalian subjects within several months after birth (within 1 to 12 months; 1 month may be 28 days or 29 days or 30 days or 31 days).

[0012] Furthermore, the present invention addresses and solves the technical problem of prophylactically treating the onset of allergies and / or immune-mediated or autoimmune disorders (e.g., celiac disease) related to the gastrointestinal system in the later years of a treated subject. Finally, an object of the present invention is to provide an effective solution for supporting the physical growth of neonatal and / or mammalian subjects within several months after birth.

[0013] Following research and development activities, the applicant addresses and solves the above-mentioned technical problem by providing at least one strain belonging to the species Lactobacillus paracasei (in particular, Lactobacillus paracasei DG (registered trademark) CNCM I-1572 and / or Lactobacillus paracasei LPC-S01 DSM 26760, etc.), which effectively and efficiently contributes to the development of an appropriate Lactobacillus-rich (especially Lactobacillus paracasei-rich) gut microbiota in neonatal and / or subjects within several months after birth, and compositions thereof (i.e., the compositions of the present invention).

[0014] Among the strains of the genus Lactobacillus (bacterial strains), the species Lactobacillus paracasei [Lactobacillus paracasei strain DG (registered trademark) CNCM I-1572 and / or Lactobacillus paracasei strain LPC-S01 DSM 26760, etc.] belongs to the species generally used as probiotic strains. In particular, L. paracasei strain DG (registered trademark) CNCM I-1572 has been widely studied regarding its beneficial properties. However, the effect of L. paracasei DG (registered trademark) CNCM I-1572 on neonatal subjects and / or subjects within 1 to 12 months has not been studied until now.

[0015] It should be noted that, in view of 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 strain L. casei DG (L. casei DG)(registered trademark)(CNCM I-1572) or L. paracasei DG (registered trademark)(CNCM I-1572) was redeposited as Lacticaseibacillus paracasei DG I-1572 DSM 34154 on February 2, 2022. Since the above two names always refer to the same strain (bacterial strain), they are interchangeable with each other.

[0016] The L. paracasei strain DG (registered trademark) CNCM I-1572, the L. paracasei strain LPC-S01 DSM 26760, their mixtures and compositions according to the present invention administered to subjects within the neonatal period (within 4 weeks after birth) seem to be able to effectively and efficiently colonize the intestinal tract during the administration period and / or for several months after birth, balance the intestinal microbiota and the immune system for the benefit of the host, and enhance the permeability of the intestinal barrier.

[0017] Finally, the strains, their mixtures and compositions of the present invention have good tolerance, no related side effects, are easily prepared, and are cost-effective.

[0018] These objectives and the like will become apparent from the following detailed description and are achieved by the compositions and mixtures of the present invention by the technical features according to the appended claims.

[0019] That is, the present invention includes the following aspects.

[0020] Aspect [1]: A strain for use in a method for treating, preventing, and / or curing a subject having an inflammatory and / or functional gastrointestinal disorder, wherein the disorder is selected from the group consisting of or including neonatal colic, intestinal colic, chronic enteritis or chronic enteritis in premature neonates, necrotizing enterocolitis, sepsis, irritable bowel syndrome (IBS), IBS with prevalent constipation or constipated bowel, IBS with prevalent diarrhoea or diarrhoeic bowel, IBS with alternating bowel, unclassified IBS, inflammatory bowel disease (IBD), Crohn's disease, and ulcerative proctocolitis. The strain belongs to the Lactobacillus paracasei species and is selected from the group consisting of or including Lactobacillus paracasei DG (registered trademark) CNCM I-1572, Lactobacillus paracasei LPC-S01 DSM 26760, and mixtures thereof. The subject is a neonatal subject within 4 weeks after birth and / or is 1 month to 12 months of age or younger. The strain.

[0021] Aspect [2]: The strain is a viable strain of bacteria or a derivative of the strain, and the derivative of the strain is selected from the group consisting of or including a tyndallized strain, an ultrasonically treated strain, a radiation-inactivated strain, a lysed strain, or a bacterial homogenate, the strain extract, or the cell wall fraction, for use as described in the above Aspect [1].

[0022] Aspect [3]: The strain is a viable strain of bacteria or a derivative of the strain, and the derivative of the strain is selected from the group consisting of or including a tyndallized strain, an ultrasonically treated strain, a gamma-radiation-inactivated strain, a lysed strain, or a bacterial homogenate, the strain extract, or the cell wall fraction, for use as described in the above Aspect [1] or [2].

[0023] Aspect [4]: The strain is a viable strain of bacteria or a derivative of the strain, and the derivative of the strain is a tyndallized strain, for use as described in any of the above Aspects [1] to [3].

[0024] Aspect [5]: The strain for use according to any of the aspects [1] to [4] above, wherein the disorder is selected from the group consisting of, or consists of, neonatal colic, intestinal colic, chronic enteritis or chronic enteritis in premature neonates, necrotizing enterocolitis, sepsis, irritable bowel syndrome (IBS), IBS mainly accompanied by constipation or constipation-type intestine, IBS mainly accompanied by diarrhea or diarrhea-type intestine, alternating-type intestine IBS, unclassified IBS, inflammatory bowel disease (IBD), Crohn's disease, ulcerative proctocolitis.

[0025] Aspect [6]: A mixture M for use according to any of the aspects [1] to [5] above, wherein the mixture M comprises, or consists of, strains belonging to the species Lactobacillus paracasei selected from the group consisting of Lactobacillus paracasei DG (registered trademark) CNCM I-1572, Lactobacillus paracasei LPC-S01 DSM 26760 and mixtures thereof, and at least one food-grade or pharmaceutical-grade additive and / or excipient, or consists of the same.

[0026] Aspect [7]: A mixture M for use according to the aspect [6] above, wherein the mixture M comprises, or consists of, strains belonging to the species Lactobacillus paracasei selected from the group consisting of Lactobacillus paracasei DG (registered trademark) CNCM I-1572, Lactobacillus paracasei LPC-S01 DSM 26760 and mixtures thereof, and (a) Bifidobacterium breve BbIBS01 DSM 33231, (b) Bifidobacterium breve BbIBS02 DSM 33232, (c) Bifidobacterium animalis subsp. lactis BlIBS01 DSM 33233, (d) Lactobacillus plantarum LpIBS01 DSM 33234, (e) Bifidobacterium bifidum BbfIBS01 DSM 32708, and mixtures thereof and comprises, or consists of, at least one additional strain selected from the group consisting of the same, or consists of the same.

[0027] Aspect [8]: A mixture M for use according to the aspect [6] or [7] above, (1) At least one vitamin from vitamin groups A, B, C, D, E and / or K, (2) One or more polyphenols selected from the group consisting of glutathione, resveratrol and trans-resveratrol, coenzyme Q10, astaxanthin, lycopene, or one or more antioxidants selected from the group consisting thereof, (3) One or more plant substances having a laxative effect, including or consisting of botanicals (crude plant products, botanicals) or extracts thereof, selected from the group consisting thereof, (4) One or more minerals or salts thereof selected from the group consisting of zinc, selenium, magnesium and potassium, (5) One or more monounsaturated fatty acids containing or selected from the group consisting of omega-9, and / or one or more polyunsaturated fatty acids containing or selected from the group consisting of omega-3 and / or omega-6, (6) One or more immunostimulants, antidiarrheal substances and / or nutrients, (7) At least one prebiotic, and mixtures thereof Mixture M further comprising at least one additional active ingredient selected from the group consisting of or containing the same.

[0028] Aspect [9]: (1-1) The vitamin is from vitamin group B and / or vitamin group D, and / or (3-1) The plant substance is selected from the group consisting of valerian, passion flower, lemon balm, hawthorn and chamomile, and / or (7-1) The prebiotic is selected from group (II) consisting of inulin, fructooligosaccharide (FOS), galactooligosaccharide (GOS), xylooligosaccharide (XOS), guar gum, lactoferrin and mixtures thereof, for use according to the mixture M described in the above aspect [8].

[0029] Aspect

[10] : The prebiotic is selected from group (II) consisting of inulin and mixtures thereof, for use according to the mixture M described in the above aspect [8] or [9].

[0030] Aspect

[11] : The disorder includes or consists of neonatal colic, intestinal colic, chronic enteritis or chronic enteritis of premature neonates, necrotizing enterocolitis, and is selected from the group consisting thereof, for use according to the strain described in any one of the above aspects [1] to [5].

[0031] Aspect

[12] : Mixture M for use according to any of the aspects [6] to

[10] above, wherein the disorder is selected from the group consisting of or comprising neonatal colic, infantile colic, chronic enteritis or chronic enteritis in premature neonates, necrotizing enterocolitis.

Brief Description of the Drawings

[0032]

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Mode for Carrying Out the Invention

[0033] Detailed Description of the Invention The object of the present invention is to provide at least one strain belonging to the species Lactobacillus paracasei, preferably Lactobacillus paracasei strain DG (registered trademark) CNCM I-1572 and / or Lactobacillus paracasei strain LPC-S01 DSM 26760, their mixtures or compositions as hereinafter shown in the present invention (that is, the mixtures or compositions of the present invention) for use in the treatment (treatment), prevention and / or cure of inflammatory and / or functional gastrointestinal disorders such as neonatal colic or infantile colic, chronic enteritis or chronic enteritis in premature neonates, necrotizing enterocolitis, sepsis, irritable bowel syndrome (abbreviated as IBS), IBS with mainly constipation (prevalent constipation) or constipated bowel, IBS with mainly diarrhoea (prevalent diarrhoea) or diarrhoeic bowel, alternating bowel IBS, unclassified IBS, or inflammatory bowel disease (IBD) (such as Crohn's disease), ulcerative proctocolitis, asthma, obesity, type 1 diabetes, atopic dermatitis, multiple sclerosis, cancer and autism in neonatal subjects (within 4 weeks after birth) and / or subjects within 1 to 12 months.

[0034] Preferably, the strains, their mixtures or compositions of the present invention are for use in neonatal subjects and / or subjects within 1 to 12 months in the treatment, prevention and / or cure of neonatal colic or chronic enteritis or necrotizing enterocolitis in premature neonates.

[0035] The object of the present invention is for use in neonatal subjects (within 4 weeks after birth) and / or subjects from 1 month to 12 months in the treatment, prevention and / or cure of gastrointestinal infections by pathogenic microorganisms [e.g., viruses (rotavirus, Adenovirus entericus, calicivirus, astrovirus, influenza virus, etc.) or bacteria (Salmonella, Shigella, Staphylococcus, Campylobacter, Escherichia coli, etc.)], gastrointestinal infections caused by parasites [preferably, helminths or roundworms (e.g., Oxyuris, Giardia, etc.)], allergies, immune-mediated or autoimmune disorders [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 kidneys), infertility, asthma, obesity, type 1 diabetes, atopic dermatitis, multiple sclerosis, cancer and autism], in at least one strain belonging to the species Lactobacillus paracasei, preferably, Lactobacillus paracasei strain DG® CNCM I-1572 and / or Lactobacillus paracasei strain LPC-S01 DSM 26760, their mixtures or compositions as shown below of the present invention (i.e., the mixtures or compositions of the present invention).

[0036] The object of the present invention is for use in neonatal subjects (within 4 weeks after birth) and / or subjects from 1 month to 12 months in the supportive (adjuvant) treatment of the growth of a subject, in at least one strain belonging to the species Lactobacillus paracasei, preferably, Lactobacillus paracasei strain DG® CNCM I-1572 and / or Lactobacillus paracasei strain LPC-S01 DSM 26760, their mixtures or compositions as shown below of the present invention (i.e., the mixtures or compositions of the present invention), wherein the growth of the subject is understood as an increase in the body weight of the administered subject and is evaluated as g / week.

[0037] The strain identified as Lactobacillus paracasei DG (registered trademark) (registered trademark of Sofar S.p.A., Italy) was deposited by Sofar S.p.A. on May 5, 1995, with the accession number CNCM I-1572 at the National Collection of Cultures of Microorganisms of the Institut Pasteur in Paris (that is, DG (registered trademark) or L. paracasei DG (registered trademark) CNCM I-1572). Initially, this strain was named Lactobacillus casei DG (R) sub. casei and was later reclassified as Lactobacillus paracasei DG (registered trademark) CNCM I-1572. It is clear that it is always the same unique strain, regardless of the name Lactobacillus casei DG (registered trademark) or Lactobacillus paracasei DG (registered trademark).

[0038] The strain identified as Lactobacillus paracasei LPC-S01 (or named Lactobacillus paracasei S01) was deposited by Sofar S.p.A. on November 20, 2012, with the accession number DSM 26760 at the Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSMZ) (that is, LPC-S01 or L. paracasei LPC-S01 DSM 26760). It is clear that it is always the same unique strain, regardless of the name Lactobacillus paracasei S01 DSM 26760 or Lactobacillus paracasei LPC-S01 DSM 26760 adopted by the applicant of this application.

[0039] The composition of the present invention for use in the method or treatment of the present invention comprises (i) the mixture M of the present invention comprising or consisting of Lactobacillus paracasei DG (registered trademark) CNCM I-1572 and / or Lactobacillus paracasei LPC-S01 DSM 26760, and optionally (ii) at least one additive and / or excipient of food grade or pharmaceutical grade.

[0040] According to one aspect of the present invention, the mixture M comprised in the composition of the present invention · comprises or consists of Lactobacillus paracasei DG (registered trademark) CNCM I-1572 and / or Lactobacillus paracasei LPC-S01 DSM 26760, and · at least one further strain selected from the group (I) consisting of: (a) Bifidobacterium breve BbIBS01 DSM 33231, (b) Bifidobacterium breve BbIBS02 DSM 33232, (c) Bifidobacterium animalis subsp. lactis BlIBS01 DSM 33233, (d) Lactobacillus plantarum LpIBS01 DSM 33234, (e) Bifidobacterium bifidum BbfIBS01 DSM 32708, and mixtures thereof and may comprise or consist of these.

[0041] Strains belonging to the species Bifidobacterium breve, identified as Bifidobacterium breve BbIBS01, were deposited with the German Collection of Microorganisms and Cell Cultures (DSMZ) on July 31, 2019, under the deposit number DSM 33231 by Sofar Essi P.A. (in short, BbIBS01 or B. breve BbIBS01 DSM 33231).

[0042] Strains belonging to the species Bifidobacterium breve, identified as Bifidobacterium breve BbIBS02, were deposited with the German Collection of Microorganisms and Cell Cultures (DSMZ) on July 31, 2019, under the deposit number DSM 33232 by Sofar Essi P.A. (in short, BbIBS02 or B. breve BbIBS02 DSM 33232).

[0043] Strains belonging to the species Bifidobacterium animalis, identified as Bifidobacterium animalis subsp. lactis BlIBS01, were deposited with the German Collection of Microorganisms and Cell Cultures (DSMZ) on July 31, 2019, under the deposit number DSM 33233 by Sofar Essi P.A. (in short, BlIBS01 or B. animalis subsp. lactis BlIBS01 DSM 33233).

[0044] Strains belonging to the species Lactobacillus plantarum, identified as Lactobacillus plantarum LpIBS01, were deposited with the German Collection of Microorganisms and Cell Cultures (DSMZ) on July 31, 2019, under the deposit number DSM 33234 by Sofar Essi P.A. (in short, LpIBS01 or L. plantarum LpIBS01).

[0045] A strain belonging to the species Bifidobacterium bifidum identified as Bifidobacterium bifidum MIMBb23sg = BbfIBS01 or a derivative thereof. Said strain was deposited with the German Collection of Microorganisms and Cell Cultures (DSMZ) on December 4, 2017 under the deposit number DSM 32708 by Sofar Essi P.A. (i.e., BbfIBS01 or Bifidobacterium bifidum BbfIBS01 DSM 32708). It is clear that, regardless of the name Bifidobacterium bifidum BbfIBS01 DSM 32708 or Bifidobacterium bifidum MIMBb23sg DSM 32708 adopted by the applicant, it is always the same unique strain.

[0046] All of the strains described in the present invention (i.e., DG®, LPC-S01, BbIBS01, BbIBS02, BlIBS01, LpIBS01, and BbfIBS01) have been deposited in accordance with the provisions of the Budapest Treaty. The depositors of the strains described in and / or related to the present patent application, as well as the owner of the present patent application, hereby declare their consent to make all such strains available during the term of this patent.

[0047] Advantageously, the strains described in the composition of the present invention (i.e., DG®, LPC-S01, BbIBS01, BbIBS02, BlIBS01, LpIBS01, and BbfIBS01) are viable bacterial strains (probiotics). Alternatively, the strains of the present invention may be derivatives of viable strains as defined in the present invention.

[0048] In the context of the present invention, the term "derivative" of a strain (or "derivative" of a viable strain) means, for example, a tyndallized strain, an ultrasonicated strain, an inactivated strain by radiation (preferably gamma radiation), a lysate (lysate, lysate, disintegration product or debris) or homogenate (crush) of the strain, a strain extract or wall fraction, a metabolite or metabolic bioproduct produced by the strain or exopolysaccharide (EPS) and / or any other derivative product of the strain known to those skilled in the art, such as postbiotics or parabiotics. The derivatives are obtained according to methodologies known to those skilled in the art. Preferably, the term "derivative" of a strain means a tyndallized strain, an ultrasonicated strain, an inactivated strain by radiation (preferably gamma radiation), a lysate or homogenate of the strain, a strain extract or parietal fraction, and more preferably, a tyndallized strain.

[0049] Examples of mixtures containing the strain (En) contained in mixture M and, optionally, at least one Prebiotic and / or further active ingredient selected from group (III) (wherein said mixture M is contained in the composition of the present invention) include the following: E1: DG® and LPC-S01, E2: DG® and at least one strain selected from group (I), E3: LPC-S01 and at least one strain selected from group (I), E4: DG® and / or LPC-S01, and BbfIBS01, and at least one strain selected from group (I), E5: DG® and / or LPC-S01, and a mixture of BbIBS01, BbIBS02, BlIBS01 and LpIBS01, E6: DG (Registered Trademark) and / or LPC-S01, BbfIBS01, and a mixture of BbIBS01, BbIBS02, BlIBS01, and LpIBS01.

[0050] The mixture M contained in the composition of the present invention is the strain L. paracasei DG (Registered Trademark) CNCM I-1572 and / or L. paracasei LPC-S01 DSM 26760, and optionally at least one or more strains selected from group (I) and / or at least one additional active ingredient selected from group (III) described below, and may further contain at least one prebiotic. The prebiotic is preferably inulin, fructooligosaccharide (FOS), galactooligosaccharide (GOS), xylitol-oligosaccharide (XOS), guar gum, lac Selected from the group (II) consisting of toferin and mixtures thereof, preferably inulin.

[0051] Advantageously, the mixture M contained in the composition of the present invention contains or consists of L. paracasei DG (Registered Trademark) CNCM I-1572 and / or L. paracasei LPC-S01 DSM 26760 and inulin, preferably contains or consists of L. paracasei DG (Registered Trademark) CNCM I-1572 and inulin.

[0052] The mixture M contained in the composition of the present invention is the strain L. paracasei DG (Registered Trademark) CNCM I-1572 and / or L. paracasei LPC-S01 DSM 26760, and optionally at least one or more strains selected from group (I) and / or at least one prebiotic selected from group (II), in addition to · Groups of vitamins A, B, C, D, E, and / or K, preferably groups of vitamins B and / or D, · Polyphenols such as glutathione, resveratrol, and trans-resveratrol, antioxidants such as coenzyme Q10, astaxanthin, and lycopene, · Botanical substances or extracts thereof having a laxative effect, such as valerian, passion flower, lemon balm, hawthorn (Hawthorne), chamomile, · Minerals or salts thereof, such as zinc, selenium, magnesium, potassium, · Monounsaturated fatty acids such as omega-9 (monounsaturated fatty acids), and / or polyunsaturated fatty acids such as omega-3 and omega-6 (polyunsaturated fatty acids), · Immunostimulants, antidiarrheal substances and / or nutrients, and Mixtures thereof Substance Or It may further contain at least one additional active ingredient selected from the group (III) consisting of.

[0053] Advantageously, the mixture M contained in the composition of the present invention comprises, or consists of, L. paracasei DG® CNCM I-1572 and / or L. paracasei LPC-S01 DSM 26760, and the vitamin B group and the vitamin D group, preferably, L. paracasei DG® CNCM I-1572 and the vitamin B group and the vitamin D group, or consists of them.

[0054] In the context of the present invention, together with the strain, the food-grade or pharmaceutical-grade acceptable "additives and / or excipients" optionally contained in the composition of the present invention include all auxiliary substances known to those skilled in the art for preparing solid, semi-solid or liquid compositions, and the auxiliary substances include, for example, carriers, diluents, solvents, solubilizers, acidifying agents, thickeners, sweeteners, flavorings, colorants, sweeteners, lubricants, surfactants, preservatives, stabilizers, pH-stabilizing buffers and mixtures thereof. For example, in the case of a drop-form composition for oral administration to a neonate or a subject aged 1 to 12 months, seed oil may be used as a diluent.

[0055] According to any one of the described embodiments, the mixture M or composition of the present invention may be a pharmaceutical composition (or Live Biotherapeutic Products), a composition for a medical device, a composition for a food supplement, a food (or novel food or Food for Special Medical Purposes (FSMP)), a food supplement, or a composition for food.

[0056] According to any one of the described embodiments, the strains, their mixtures and compositions of the present invention may be formulated for oral or nasal administration, preferably, for oral use, in solid form or in liquid form, for example, in the form of droplets based on aqueous or oily (e.g., various seed oils or sunflower seed oil).

[0057] Advantageously, said at least one strain or each strain is in the range of 10×10 6 CFU~10×10 12 CFU, preferably, 10×10 8 CFU~10×10 10 CFU, more preferably, at a concentration of about 10×10 8 CFU or 10×10 9 CFU (CFU: Colony Forming Unit), and is present in the mixture M or composition of the present invention.

[0058] For example, the daily dose of the mixture or composition of the present invention (e.g., droplets for oral administration) contains more than 1×10 9 live bacteria of L. paracasei DG® CNCM I-1572 and contains seed oil.

[0059] The above daily dosages may be administered to a subject in need thereof, in individual dosages (single dosages) or in repeated dosages, for example, 2, 3 or 4 times a day. For example, the composition of the present invention at 15 to 5 drops (oil-based), preferably 12 to 8 drops, for example, about 9 to 10 drops, twice a day corresponds to about 2 billion strains, preferably L. paracasei DG® CNCM I-1572.

[0060] To evaluate the number of live bacterial strains in the composition or mixture M of the present invention, these compositions or mixture M can be analyzed by the plate count method to measure the CFU value.

[0061] For the purpose of clarity, and to achieve the object of the present invention, the strain(s), prebiotics and / or further (optional) active ingredient(s) contained in the mixture (M) of the present invention may be administered separately (preferably at time intervals of 30 minutes to 60 minutes) and in any order, but are preferably administered to the subject simultaneously, and even more preferably, for obtaining a more rapid effect and for facilitating administration, are administered in a single composition. When the said strain and (optional) active ingredient(s) are administered in a single composition, the said single composition corresponds to the composition of the present invention.

[0062] Unless otherwise specified, an expression such as a composition or mixture containing an amount of a component "within the range of x to y" means that the said component may be present in the composition or the like in all amounts within the said range, and even if not explicitly stated, the values at both ends of the range are included.

[0063] Unless otherwise specified, a description that a composition "comprises" one or more components or substances means that other components or substances may be present in addition to the specifically described component or substance(s).

[0064] In the context of the present invention, "method of treatment" means an intervention to a subject in need thereof, including administering to the subject a therapeutically effective amount of a strain or composition of the present invention, with the aim of eliminating, reducing / decreasing, or preventing a pathology or disease and related symptoms or disorders.

[0065] In the context of the present invention, the term "subject (singular or plural)" refers to mammals (animals and humans), preferably human subjects in the neonatal period (0 - 4 weeks of age) and / or within 1 month to 12 months after birth (from the beginning of the second month to the end of the twelfth month).

[0066] Advantageously, the subject treated or administered with the strain or mixture or composition of the present invention may be a neonatal subject born by cesarean section and / or preterm birth or a subject within 1 month to 12 months after birth.

[0067] The term "therapeutically effective amount" refers to the amount of an active compound and / or strain that induces a biological or medical response (response) in a tissue, system (organ), mammal, or human, and is determined and defined by an individual, researcher, veterinarian, physician, or other clinician or healthcare provider.

[0068] In the context of the present invention, the term "medical device" is used in the sense based on Legislative Decree No. 46 of 24 February 1997 or based on the new Medical Device Regulation (EU) 2017 / 745 (MDR).

[0069] In the context of the present invention, the term "novel food" is used in the sense based on EC Regulation 258 of 1997.

[0070] Embodiment A preferred embodiment FRn of the present invention is as follows.

[0071] FR1. In the treatment, prevention and / or cure of a subject having an inflammatory and / or functional gastrointestinal disorder, A strain or composition for use in neonatal subjects, subjects within 4 weeks after birth, and / or subjects within 1 to 12 months of age, such disorders include, or consist of, neonatal colic, intestinal colic, chronic enteritis or chronic enteritis in premature neonates, necrotizing enterocolitis, sepsis, irritable bowel syndrome (IBS), IBS mainly accompanied by constipation or constipation-type intestine, IBS mainly accompanied by diarrhea or diarrhea-type intestine, alternating intestine IBS, unclassified IBS, inflammatory bowel disease (IBD), Crohn's disease, ulcerative proctocolitis, asthma, obesity, type 1 diabetes, atopic dermatitis, multiple sclerosis, cancer, autism, allergy, immune-mediated and autoimmune disorders, and the immune-mediated and autoimmune disorders are selected from celiac disease, Graves' disease, rheumatoid arthritis, Hashimoto's thyroiditis, type 1 diabetes, systemic lupus erythematosus (lupus), vasculitis, Addison's disease, polymyositis, Sjogren's syndrome, progressive systemic sclerosis, glomerulonephritis (inflammation of the kidneys), infertility and support for weight gain of the subject, the strain belongs to the Lactobacillus paracasei species, the composition is, (i) a mixture M comprising, or consisting of, at least one strain belonging to the Lactobacillus paracasei species, and (ii) containing at least one additive and / or excipient of food grade or pharmaceutical grade, a strain or composition.

[0072] FR2. The strain is selected from Lactobacillus paracasei DG (registered trademark) CNCM I-1572, Lactobacillus paracasei LPC-S01 DSM 26760 and mixtures thereof, the composition is, (i) A mixture M comprising or consisting of Lactobacillus paracasei DG (registered trademark) CNCM I-1572 and / or Lactobacillus paracasei LPC-S01 DSM 26760, and (ii) comprising at least one additive and / or excipient of food grade or pharmaceutical grade, A strain or composition for use according to the use described in FR1.

[0073] FR3. The strain or composition as described in FR1 or FR2, wherein the strain or composition is used in the treatment, prevention and / or cure of neonatal colic or intestinal colic, or chronic enteritis or chronic enteritis in premature neonates, in neonatal subjects and / or subjects from 1 month to 12 months Within of age.

[0074] FR4. The strain or composition as described in FR1 or FR2, wherein the strain or composition is used in the treatment, prevention and / or cure of gastrointestinal infections by pathogenic microorganisms or gastrointestinal infections by parasites, in neonatal subjects and / or subjects within 1 month to 12 months of age.

[0075] FR5. The strain or composition as described in FR1 or FR2, wherein the strain or composition is used in the treatment, prevention and / or cure of allergies, in neonatal subjects and / or subjects within 1 month to 12 months of age.

[0076] FR6. The strain or composition as described in FR1 or FR2, wherein the strain or composition is used in the treatment, prevention and / or cure of immune-mediated and autoimmune disorders such as celiac disease, Graves' disease, rheumatoid arthritis, Hashimoto's thyroiditis, type 1 diabetes, systemic lupus erythematosus (lupus), vasculitis, Addison's disease, polymyositis, Sjogren's syndrome, progressive systemic sclerosis, glomerulonephritis (inflammation of the kidneys) and infertility, in neonatal subjects and / or subjects within 1 month to 12 months of age.

[0077] FR7. A strain or composition for use according to FR1 or FR2, wherein the strain or composition is used in neonatal subjects and / or subjects within 1 to 12 months in the treatment, prevention and / or cure of supporting the weight gain of a subject.

[0078] FR8. The (i) mixture M is Lactobacillus paracasei DG (registered trademark) CNCM I-1572 and / or Lactobacillus paracasei LPC-S01 DSM 26760, and · Bifidobacterium breve BbIBS01 DSM 33231, · Bifidobacterium breve BbIBS02 DSM 33232, · Bifidobacterium animalis subsp. lactis BlIBS01 DSM 33233, · Lactobacillus plantarum LpIBS01 DSM 33234, · Bifidobacterium bifidum BbfIBS01 DSM 32708, and their mixtures at least one additional strain selected from the group consisting of A composition for use according to any one of FR1 to 7, comprising or consisting of them.

[0079] FR9. The (i) mixture M is · At least one vitamin selected from vitamin groups A, B, C, D, E and / or K, preferably vitamin group B and / or vitamin group D, · Antioxidants such as glutathione, polyphenols such as resveratrol and trans-resveratrol, coenzyme Q10, astaxanthin, lycopene, · Substances of plants, botanicals (crude plant products or plant-derived products) or their extracts having an intestinal relaxant effect, preferably substances of plants, botanicals or their extracts selected from valerian, passion flower, lemon balm, rose hip and chamomile, · Minerals or their salts, for example, zinc, selenium, magnesium, potassium, · Monounsaturated fatty acids such as omega 9, and / or polyunsaturated fatty acids such as omega 3 and omega 6, · Immunostimulants, antidiarrheal substances and / or nutrients, · At least one prebiotic, preferably at least one prebiotic selected from the group (II) consisting of inulin, fructooligosaccharide (FOS), galactooligosaccharide (GOS), xylooligosaccharide (XOS), guar gum, lactoferrin and mixtures thereof, more preferably inulin, and mixtures thereof A composition for use according to any one of FR1 to 8, further comprising at least one further active ingredient selected from

[0080] FR10. The strain is a viable strain or a derivative of the strain, and the derivative of the strain is selected from an intermittently sterilized strain, an ultrasonically treated strain, a radiation (preferably gamma radiation)-inactivated strain, a lysate or homogenized strain, a strain extract or a cell wall fraction, preferably an intermittently sterilized strain, a strain or composition for use according to any one of FR1 to 9.

[0081] EXPERIMENTAL PART The applicant conducted a clinical study in neonates to evaluate the effect of administration of Lactobacillus paracasei DG strain (registered trademark) CNCM I-1572 probiotic (viable cells) on the intestinal microbiota of the host within 3 months after birth.

[0082] 1. Purpose of the clinical trial Main purpose of the test: The composition of the present invention in the form of droplets (oil-based) for oral administration (i.e., the composition to be analyzed), containing Lactobacillus paracasei strain CNCM I-1572, was supplemented for 28 days to evaluate whether the composition of the faecal gut microbiota changes in terms of the concentration conversion of Lactobacillus paracasei CNCM I-1572 in the study population. To evaluate whether there is such a change.

[0083] Composition to be analyzed: Sunflower seed oil (Heliantus annuus L.), DL-α-tocopherol, Lactobacillus paracasei CNCM I-1572 (more than 14 billion viable cells per 8 ml).

[0084] Secondary objectives of the study: To evaluate the following by ingesting the composition to be analyzed for 28 days: · Changes in the composition of the faecal gut microbiota (real-time PCR) based on the ecological prediction of the microbiota system at 56 days and 84 days after ingestion, · Changes in the functional activity of the faecal gut microbiota (metabolomics) based on the ecological prediction of the microbiota system at 28 days, 56 days, and 84 days after ingestion, · Changes in the amount and quality of faeces (frequency and consistency), · Occurrence of neonatal colic defined according to the Rome IV criteria, · Safety and tolerance of the product, · Growth of the patient.

[0085] 2. Structure of the study Timing of the investigation (Figure 1) · Enrollment period: 24 weeks, · Treatment period: 4 weeks, · Follow-up period: 8 weeks, · Duration of the study per patient: 12 weeks (84 days), · Total study period: 36 weeks.

[0086] 3. Study design Randomized placebo-controlled double-blind single-center clinical trial. Neonates were stratified into three groups according to birth weight: · Normal birth weight (>2500 g, NBW), · Low birth weight (1500 - 2500 g, LBW), · Very low birth weight (1000 - 1500 g, VLBW).

[0087] The trial was conducted at the U.O. of Neonatology of the Policlinico Casilino in Rome as the only participating and coordinating centre.

[0088] 3.1. Study population Sixty preterm and term neonates were enrolled in the study and divided as follows: 20 NBW, 20 LBW, and 20 VLBW.

[0089] 3.2 Type of intervention The 60 enrolled subjects were randomized (randomly assigned) in a 1:1 ratio into two groups within each of the three groups.

[0090] Group 1: Droplet-shaped test composition, 9 drops twice a day, equivalent to 2 billion CFU (colony-forming units) of L. paracasei DG (registered trademark) (CNCM I - 1572), for 4 weeks (28 days).

[0091] Group 2: Control: placebo, 9 drops twice a day, for 4 weeks (28 days).

[0092] 3.3 Study period Over a 12-week period, changes in the gut microbiota can be evaluated based on the persistence of L. casei DG strain (registered trademark) (Lactobacillus paracasei CNCM I-1572) measured by the intake of the composition to be analyzed. This 12-week period is divided into a 4-week treatment period and an 8-week follow-up period. Including an approximately 24-week registration period, the total duration of the trial is 36 weeks.

[0093] 4. Selection of Subjects for the Trial 4.1 Eligibility Criteria · Newborns aged 0 to 48 hours after birth, · Newborns of both genders with a birth weight of 1000 g or more, · Newborns delivered by cesarean section.

[0094] 4.2 Exclusion Criteria (including only the main ones) · Vaginal delivery, · Extremely low birth weight (ELBW) (<1000 g), · Established or suspected systemic infection, · Severe medical conditions determined by the principal investigator to potentially interfere with treatment, · Intake of probiotics other than the test product by the newborn or lactating mother after the start of the trial, · Systemic therapy or prophylaxis with antibiotics from birth throughout the trial period (for NBW only), · Systemic therapy or prophylaxis with antibiotics by the lactating mother (of NBW newborns) during the 30 days before birth and throughout the trial period.

[0095] Newborns are considered eligible for the Per Protocol population that conforms to the trial implementation protocol if they do not exceed at least 80% and 120% of the prescribed treatment.

[0096] 5. Procedures and Methods Scheduled hospital visits, accompanied by contextual collection of stool samples if possible according to the situation of the stool samples: V1 Day 1 (0 - 48 hours after birth), V2 Day 10 (±2 days), V3 Day 28 (±3 days), V4 Day 56 (±3 days), V5 Day 84 (±3 days) (Figure 1).

[0097] Furthermore, during the test, fecal samples received from the parents (both parents) were provided according to the following scheme (1 sample collected within 24 hours before each visit):

[0098] Before treatment: · Feces within 48 hours after birth, and meconium if available.

[0099] During the treatment period: · Feces 10 days after the start of intake, · Feces 28 days after the start of intake, During the follow - up observation period: · Feces 56 days after the start of intake, · Feces 84 days after the start of intake,

[0100] Chemical - physical analysis of fecal samples: · Real - time PCR analysis to evaluate the bacterial count of L. casei strain DG® (Lactobacillus paracasei CNCM I - 1572), · Metagenomics and metabolomics analysis to characterize the gut microbiota before and after probiotic administration.

[0101] Specifically, the microbiota was analyzed by nucleotide sequence analysis of a part of the gene encoding the bacterial ribosomal subunit 16S rRNA. Specifically, a metagenomic strategy consisting of the following steps was adopted: · Extraction, quantification, and normalization of metagenomic DNA from fecal samples, · PCR amplification of the V3 - V4 hypervariable regions of the bacterial gene encoding 16S rRNA, · Quantification of PCR products, · Sequencing by Illumina MiSeq technology, · Bioinformatics array analysis (characterization of microbiota, hierarchical clustering, taxonomic analysis, phylogenetic dendrograms by heatmap construction).

[0102] For metabolome analysis, microbial-derived metabolites produced as volatile organic compounds (VOCs) were identified and quantified. These compounds were extracted using a GC-MS / SPME (gas chromatography-mass spectrometry by solid-phase microextraction) system. For VOC extraction, carboxy-polydimethylsiloxane (CAR-PDMS) coated fibers (85 μm) were used in the SPME process. From each sample analyzed three times, an average of 100 - 500 mg was placed in a 10 ml glass vial, and 4-methyl-2-pentanol was added as an internal standard (IS). Subsequently, the fecal samples were equilibrated at 45 °C for 10 minutes. The fibers were exposed to each sample for 45 minutes before injection into a GC-MS (Hewlett Packard 6890 GC) equipped with a 5973C mass selective detector and a Supelcowax 10 capillary column. Metabolites were identified using the retention time (Rt) of the metabolite relative to the pure compound. The chromatogram was integrated and identified by comparing the fragment pattern with the fragment pattern in the NIST library and then manually visually inspecting. Quantitative metabolite data was obtained by interpolating the relative area to the IS area.

[0103] 6. Evaluation of effectiveness and safety 6.1. Evaluation of effectiveness Primary evaluation items · Bacterial genome count of L. casei DG (registered trademark) on day 28 by real-time PCR.

[0104] Secondary evaluation items ·Indices of dysbiosis of the gut microbiota (α-diversity and β-diversity) on the 28th, 56th, and 84th days, ·Number of L. casei DG® bacteria genomes on the 56th and 84th days, ·Using the Amsterdam stool scale for newborns, the number, hardness, amount, and color of bowel movements on the day before each visit (1st, 10th, 28th, 56th, 84th days), ·Chemical and physical analysis of feces (1st, 10th, 28th, 56th, 84th days), ·Quantitative evaluation of the volatilome, i.e., volatile fecal metabolites (chemical substance categories: short-chain fatty acids (SCFAs), 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 the 28th, 56th, and 84th days, ·Evaluation of the presence of anal pain based on the "Rome IV" diagnostic criteria, ·Evaluation of adverse events.

[0105] 7. Statistical analysis Statistical methods Categorical data are presented as counts and percentages, while continuous data are presented as mean and standard deviation or median and range.

[0106] The comparison of categorical data is performed, as appropriate, by chi-square test or Fisher's exact test. Continuous data are compared by Student's t-test (for independent or dependent data), analysis of variance (ANOVA), and analysis of variance for repeated measures, and the normality of the data is determined by appropriate statistical tests. If the data are not normally distributed, nonparametric tests such as the sum of ranks test (Mann-Whitney sum of ranks test) or the signed rank sum test (Wilcoxon signed rank), the Kruskal-Wallis test, or the Friedman test are used. If necessary, multivariate regression techniques are used to evaluate the simultaneous effects of several independent variables on the response variable or to identify possible confounding factors and / or analyses of temporal data. Results are considered statistically significant at P < 0.05.

[0107] All analyses are performed using Stata 15 software (StatCorp).

[0108] 8. Microbiological Results of the Test 8.1 Primary Evaluation Item: Number of Bacterial Genomes of L. casei DG (registered trademark) on the 28th Day by Real-Time PCR

[0109] The results of fecal samples collected during the scheduled hospital visits of V1 to V5 are schematically illustrated in Figures 2A to 2E.

[0110] Such figures (box-and-whisker plots) show the concentration of L. casei DG (registered trademark) in the placebo group and the "Tx" group administered the composition to be analyzed at each follow-up observation time point. For each group, the median, 25th percentile, 75th percentile, minimum value, and maximum value of the L. casei DG (registered trademark) concentration (CFU / ml) are shown.

[0111] The probiotic L. casei DG (registered trademark) began to be detected in the intestines of the subjects in the treatment group 10 days after the first administration and persisted until the 84th day after ingestion, but began to decrease slightly from the V3 time point (day of treatment) of the follow-up observation. 28 day) onwards.

[0112] 8.2 Metagenomic analysis Figures 3A to 3D show histograms of the relative abundances of Lactobacillus species at the time points of V2 to V5 of the scheduled hospital visits.

[0113] From the ecological analysis of the microbiota, it can be inferred that the relative abundance of Lactobacillus species is statistically significantly increased (pFDR ≤ 0.05) by the administration of the composition to be analyzed (Tx group) until 10 days after the start of administration (V2 time point), and this increase continues with a tendency of p < 0.05 at the V4 and V5 time points.

[0114] 8.3 Metabolome analysis Figure 4 illustrates the results of the PLS-DA metabolome analysis. The vertical axis shows the identified metabolites, and the horizontal axis shows the variable importance in projection (VIP).

[0115] By PLS-DA analysis, it was identified that butanoic acid and propanoic acid metabolites (SCFAs) play a major role in the clustering between the placebo group and the Tx group as the concentrations of the metabolites in the Tx samples increase.

[0116] Considering each time point individually by PLS-DA, it can be seen that as the concentration of SCFAs in the Tx samples increases, the maximum separation between the samples occurs between the V2 time point and the V4 time point.

[0117] Such a tendency is consistent with the higher relative abundance of lactobacilli in the subjects of the Tx group administered the composition to be analyzed (section 8.2).

[0118] 8.4 Correlation of all microbiological data obtained The results obtained from the three different approaches (real-time PCR, metagenomic analysis, metabolome analysis) discussed in the previous paragraph were correlated with each other by Spearman correlation.

[0119] Regarding the correlation analysis, metagenomic analysis, metabolomic analysis, and real-time PCR analysis showed the most statistically significant differences between the placebo group and the Tx group treated with the composition to be analyzed. Therefore, the time points V2 and V3 corresponding to the administration of the composition to be analyzed (V2: 10 days; V3: 28 days) were selected.

[0120] By integrating the data obtained from the three approaches, it is shown that the administration of the probiotic L. casei DG (registered trademark) affects both the ecology and function of the microbiota, modifying the ecological status and corresponding functional profiles of the bacteria present in the intestine.

[0121] In particular, certain short-chain fatty acids (SCFAs) such as butyric acid and propanoic acid showed a positive correlation with certain beneficial bacteria of the genera Faecalibactarium, Oscillospira, Eggerthella, Bacteroides, and Bifidobacterium.

[0122] 9. Clinical results of the test: frequency, hardness, amount, and color of feces The frequency of excretion observed at each test visit was substantially higher in the subjects treated with the composition to be analyzed than in the subjects treated with the placebo.

[0123] Regarding the excretion amount, there were no statistically significant differences between the two test groups 10 days, 56 days, and 84 days after the start of treatment (P = 0.12, 0.61, and 0.25, respectively).

[0124] Only at the end of the treatment period (day 28, V3), a statistically significant difference in favor of the subjects treated with the composition to be analyzed was observed for the excretion amount (P = 0.03).

[0125] Regarding the color and hardness of feces (parameters evaluated according to the "Amsterdam Infant stool scale"), for the color of feces, statistically significant differences were observed in favor of the subjects treated with the composition under analysis at 10 days (P<0.0001) and 56 days (P=0.007) after the start of treatment. For the hardness of feces, statistically significant differences were observed at 10 days (P=0.0016), 56 days (P<0.0001) and 84 days (P=0.05) after the start of treatment.

[0126] 10. Results of the safety of the test The results of the safety analysis are summarized in Table 1 below.

[0127]

Table 1

[0128] A total of 37 adverse events (AEs) were recorded, of which 21 were in the placebo treatment group and 16 were in the group of subjects treated with the composition under analysis (Tx).

[0129] Of the 59 subjects who were registered and randomized, 27 showed one or more adverse events (15 subjects in the placebo group and 12 subjects in the Tx group).

[0130] Only two serious adverse events (suspected necrotizing enterocolitis and sepsis) were reported in one subject treated with the composition under analysis. However, neither of these serious adverse events appeared to be related to the administration of the composition under analysis.

[0131] A similar judgment can be made for the serious adverse event (mild gastroesophageal reflux disease requiring hospitalization) observed in the placebo group.

[0132] The deaths recorded in the patients treated with the composition under analysis were caused by disseminated intravascular coagulation syndrome and septic shock, and were classified by the principal investigator of the trial as "probably not related" to the test treatment.

[0133] All other severe and non-severe adverse events were considered either not related (N = 29) or possibly not related (N = 3) to the administered test treatment (placebo or composition under analysis).

[0134] During the study period, eczema or skin diseases were not recorded in either group.

[0135] 11. Conclusions Based on the above, the following conclusions can be drawn: · With respect to the primary evaluation item, in the Tx group treated with the composition under analysis, the probiotic L. casei DG (registered trademark) was detected in the intestine from 10 days after the start of treatment (V2; Figure 2B) and persisted until the end of the observation period V5 (Figure 2E). · With respect to the relative abundance of Lactobacillus species, the composition under analysis was shown to bring about a statistically significant increase in the relative abundance of Lactobacillus until the end of the observation period V5 (Figure 3D). · With respect to the PLS-DA metabolome analysis, butyric acid and propanoic acid metabolites were identified and shown to have higher concentrations in the Tx samples (Figure 4). · From the correlation of previous microbiological analyses, it can be inferred that the presence of butyric acid and propanoic acid showed a positive correlation with specific bacterial species beneficial to the microbiota of the treated subjects. · The subjects treated with the composition under analysis had a statistically significantly higher excretion volume and better stool color and hardness evaluated according to the Amsterdam Stool Scale for Neonates than the placebo treatment group. · Finally, similar to the evaluation of severe adverse events recorded in both test groups, based on the safety and tolerance profiles analyzed above, the composition under analysis was proven to be safe and well-tolerated and to have a safety profile equivalent to that of the placebo.

[0136] The results obtained are far from obvious. This is because, as described above, mammals, especially humans, do not have fully mature internal organs in the first few weeks after birth and cannot even be treated as equals to children. Therefore, the fact that the strains described in this specification were able to provide the pharmacological reactions (responses) obtained and that they also had good tolerance in newborns could never have been foreseen, and thus it was necessary to conduct the above-mentioned clinical trials to confirm the efficacy and safety of these strains.

[0137] Experiment To date, no studies have been conducted to evaluate the effects of LCDG on vulnerable populations such as healthy newborns (in preterm and term newborns).

[0138] The objective of this double-blind placebo-controlled trial was to confirm, by evaluating the ability of the pediatric formulation (or preparation) of LCDG to pass through the gastrointestinal tract of newborns (stratified into three groups according to birth weight: normal birth weight, low birth weight, very low birth weight) alive during and after the administration period, and its ability to have a positive effect on the composition of the microbiota. The safety of the product (monitoring weight, length, and head circumference parameters), the number of bowel movements, the hardness of the stools, and the occurrence of flatulence were also evaluated.

[0139] Materials and Methods Experimental product Lactobacillus paracasei (L. casei DG (registered trademark)-CNCM I-1572; LCDG) was supplied by Sofar Ess P.A.

[0140] Nine drops of Lactobacillus paracasei corresponding to 1 billion colony-forming units (CFU) of LCDG were administered twice a day for 28 days. These droplets were administered directly to the tongue or mixed with cold or lukewarm liquids.

[0141] Subjects of the test This trial was conducted from September 2018 to March 2021. Patients participating in the trial were selected from a group of preterm and term newborns at the Hospital Neonatology Unit, Policlinico Casilino, Rome, Italy. The eligibility criteria for the trial were determined as follows: Neonates of both sexes, born within 0 - 48 hours after birth, with a birth weight of ≥ 1000 g, born by cesarean section, and for whom written informed consent was obtained from the parent(s) / guardian(s). The exclusion criteria were as follows: Vaginal delivery, extremely low birth weight (ELBW) (< 1000 g), established or suspected systemic infection, known severe neurological diseases, known severe metabolic diseases, known genetic diseases and chromosomal disorders, severe malformations (e.g., short bowel syndrome; intestinal obstruction; patent ductus arteriosus may be included if asymptomatic and not requiring treatment), known severe primary or secondary maternal immunodeficiency, known maternal food allergy, maternal diabetes (including gestational diabetes), mothers with a recent history of alcohol or drug abuse or suspicion thereof, severe conditions considered by the principal investigator to potentially interfere with treatment, insufficient reliability, or the presence of conditions that may cause non - compliance or non - observance of the protocol by the patient. Furthermore, for neonates, administration of probiotics other than the investigational product was prohibited throughout the trial period, and for lactating mothers, administration of any probiotics was prohibited throughout the trial period. Systemic treatment or prophylaxis with antibiotics from birth to the end of the trial period was not permitted, except in the case of neonates with normal birth weight (NBW), and for lactating mothers of (NBW infants), systemic treatment or prophylaxis with antibiotics was not permitted for 30 days before the trial and throughout the trial period.

[0142] Each parent / guardian of the neonates signed the informed consent to participate in the trial and received an information sheet describing the conditions of use of the investigational product. Based on the eligibility / exclusion criteria, 60 preterm and term newborns were selected.

[0143] Test design The objective of this 12-week randomized, double-blind, single-center, placebo-controlled trial was to evaluate whether administration of LCDG to neonates within the first 48 hours after birth would change the composition of the fecal gut microbiota after 28 days of product supplementation. The 12-week period was divided into a 4-week treatment period and an 8-week follow-up period.

[0144] The children participated in 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 intake), Visit 4 (V4; 56 days ± 3 days), and Visit 5 (V5; 84 days ± 3 days; end of trial).

[0145] At V1, the neonates were stratified into 3 groups according to birth weight: normal birth weight (>2500 g, NBW), low birth weight (1500 - 2500 g, LBW), and very low birth weight (1000 - 1500 g, VLBW).

[0146] The eligible patients stratified in this way were randomized at a 1:1 ratio into one of the following groups: Group 1: Treatment group: Starting from 48 hours after birth for 4 weeks (28 days), 9 drops of LCDG corresponding to 2 billion CFU (colony-forming units) twice a day; Group 2: Placebo group: Starting from 48 hours after birth for 4 weeks (28 days), placebo (a product indistinguishable from the test product) twice a day, 9 drops each time.

[0147] During the same hospital stay, demographic data of the infants and mothers, as well as stool samples from the children, were collected. The primary outcome measure was the composition of the fecal gut microbiota in terms of the number of bacterial genomes of LCDG on day 28 of treatment (V3) by real-time PCR. Secondary outcome measures evaluated during the trial were as follows: modification of the functional activity of the fecal gut microbiota (metabolomics analysis - alpha diversity and beta diversity) by prediction of the microbiota ecology on days 28, 56, and 84 after the start of administration (V3, V4, and V5, respectively); change in the composition of the fecal gut microbiota (real-time PCR) by prediction of the microbiota ecology on days 56 and 84 from the start of administration (V4 and V5, respectively); changes in the quality and quantity of stool (frequency and hardness); occurrence of neonatal colic defined according to the Rome IV criteria [Castelluzzo]; product safety and tolerability; patient prosperity.

[0148] Ethics This trial was conducted in accordance with ethical principles derived from or based on the Helsinki Declaration and in accordance with the Good Clinical Practice Guidelines for the conduct of clinical trials of medicinal products. The principal investigator submitted all trial documents to the relevant ethics committee and forwarded a copy of the approval / favourable opinion received from the ethics committee to the sponsor of the trial (ASL Roma 2: Prot. n°01220672 / 2018). All parents / guardians of the neonates provided signed informed consent.

[0149] Evaluation of results Stool samples were collected within 24 hours before each hospital visit. For each neonate, the number of bowel movements was recorded, and the color and hardness of the stool samples were evaluated using the Amsterdam Stool Scale (described below).

[0150] Furthermore, physicochemical analysis of stool samples, including stercobilinogen, neutral fat, fatty acids, soaps, amides, meat and vegetable fibers, mucosa, and pH, was performed on each visit day by the Clinical Pathology Laboratory of the Casilino Hospital. To ensure optimal storage conditions, stool samples were stored at -20 °C in the hospital freezer and sent monthly, by courier at the expense of the trial sponsor, together with dry ice, to the Laboratory of Parasitology / Human Microbiome of the Bambino Gesu Paediatric Hospital (San Paolo site) for molecular analysis.

[0151] To evaluate the bacterial load of L. casei DG® strain, real-time PCR analysis was performed (according to the already validated and published method (Ferrario)), and metagenome and metabolome analyses were carried out to characterize the gut microbiota before and after probiotic administration. In particular, the microbiota was evaluated by nucleotide sequence analysis of a portion of the gene encoding the bacterial ribosomal subunit 16S rRNA. Metagenomic DNA was extracted from stool 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. Bioinformatics sequence analysis was performed for characterization of the microbial community, hierarchical clustering, taxonomic analysis, and construction of phylogenetic trees by heatmap.

[0152] For metabolome analysis, microbial-derived 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 using a GC-MS / SPME (gas chromatography - mass spectrometry by solid-phase microextraction) system. For VOC extraction, a carboxyl-polydimethylsiloxane (CAR-PDMS) coated fiber (85 μm) was used in the SPME process. Each sample was analyzed three times, with an average of 100 - 500 mg placed in a 10 ml glass vial, and 4-methyl-2-pentanol was added as an internal standard (IS). Subsequently, the fecal samples were equilibrated at 45 °C for 10 minutes. The fiber was exposed to each sample for 45 minutes before injection into a GC-MS (Hewlett Packard 6890 GC) connected to a 5973C mass selective detector and equipped with a Supelcowax 10 capillary column. The metabolites were detected according to their characteristics. The metabolites were detected according to their retention times (Rt) relative to pure compounds. The chromatograms were integrated and identified by comparing the fragment patterns with those in the NIST library and then visually inspected manually. Quantitative metabolite data were obtained by interpolating the relative area with respect to the IS area.

[0153] Safety was evaluated by monitoring for adverse events during the study and measuring the body weight, length, and head circumference of the neonates on day 1 (V1), day 10 (V2), day 28 (V3), day 56 (V4), and day 84 (V5).

[0154] Finally, the evaluation of the occurrence of colic was performed according to the "Rome IV" diagnostic criteria (described below), and the daily stool frequency and hardness were monitored using a specific diary handed to the mother at V1.

[0155] Amsterdam Stool Scale This scale classifies the amount of feces into four classes: 1: smear, 2: up to 25%, 3: 25 - 50%, and 4: >50%.

[0156] Classify the hardness of feces as follows: A: watery, B: soft, C: formed, and D: hard.

[0157] Finally, classify the color of feces into six classes (I - VI).

[0158] ‘Rome IV’ diagnostic criteria for infantile colic For clinical purposes, the diagnosis of colic in infants must be based on the presence of all of the following criteria: the child is less than 5 months old at the time of onset and resolution of symptoms; prolonged crying (crying), agitation, or irritability occurs repeatedly without an obvious cause and cannot be prevented or resolved by the parent; unsubstantiated failure to thrive, fever, or illness. The term "agitation" refers to intermittent vocalizations and is defined as "behavior that is not exactly crying but is not the behavior of a happy person who is awake." Children often alternate between crying and agitation, and it is difficult to distinguish between the two symptoms.

[0159] For research purposes, the diagnosis of colic in infants needs to include both of the following, along with the previous criteria: during a phone call or one-on-one interview with a researcher or physician, the parent (both parents) reports that crying or agitation has persisted for at least 3 hours per day for at least 3 days per week; the duration of crying and agitation, reported to be continuous over 24 hours in the selected group of children and confirmed to have persisted for at least 3 hours, is prospectively quantified by keeping a daily log.

[0160] Statistical analysis In this working hypothesis, it is assumed that Lactobacillus paracasei DG (Lactobacillus paracasei CNCM I-1572; LCDG) exhibits the same behavior as Lactobacillus salivaris, which showed a difference in stool copies of 2.36 Log10 / 200 mg after 28 days in the same test [Putignani et al., in submission] (from 0.04 at SD 0.4 to 2.5 at SD 2.5). Considering that the treatment group ingests 2 billion CFU / day in each group (NBW, LBW, VLBW), samples of 9 patients in each treatment group provide 80% power for this test with a two-sided t-test and a 95% significance level for each stratum.

[0161] It should be noted that, following 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 strain Lactobacillus casei DG (CNCM I-1572) or Lactobacillus paracasei DG (CNCM I-1572) was re-deposited on February 2, 2022 as Lactiplantibacillus paracasei DG I-1572 DSM 34154. Since the above two names always refer to the same strain (bacterial strain), they are interchangeable with each other.

[0162] Anticipating potential losses (10% dropout) during follow-up, 20 newborns were enrolled in each weight stratum (10 of them received active treatment and 10 were administered placebo), for a total of 60 newborns.

[0163] Categorical data were presented as counts and percentages, while continuous data were presented as mean and standard deviation or median and range.

[0164] The comparison of categorical data was performed as appropriate using the chi-square test or Fisher's exact test. Continuous data were compared using Student's t-test (for independent or dependent data), analysis of variance test (Anova’s test), and repeated measures analysis of variance, and the normality of the data was determined by appropriate statistical tests. When not normally distributed, non-parametric tests such as the Mann-Whitney rank sum test or Wilcoxon signed-rank test, Kruskal-Wallis test, or Friedman test were applied. If necessary, multivariate regression techniques were used to evaluate the simultaneous effects of a series of independent variables on the response variable or to identify possible confounding factors and / or analyses for time data. The results obtained were considered statistically significant with a P-value < 0.05. All analyses were performed using Stata 15 software (StatCorp).

[0165] Results Patient disposition and basic characteristics Sixty subjects were selected from preterm and term neonates at the Neonatology Unit of the Policlinico Casilino Hospital and randomized into two treatment groups at a 1:1 ratio: 30 were treated with LCDG and 30 were administered placebo (Figure 5).

[0166] In the placebo group, 20 subjects (66.7%) completed the trial and 10 (33.3%) did not. Two subjects (6.7%) withdrew their consent, one patient (3.3%) discontinued the trial due to an adverse event (AE), six subjects (20.0%) were lost to follow-up during the follow-up (FUP) period, and one subject (3.3%) discontinued due to the need for surgery for a pharyngeal error swelling.

[0167] In the LCDG group, 26 subjects (86.7%) completed the trial, and 4 (13.3%) did not. Among these, 3 (10.0%) were lost to follow-up during the FUP period, and 1 (3.3%) died due to "disseminated intravascular coagulation syndrome" and "septic shock".

[0168] Regarding demographic and basic characteristics, the mean age of the mothers was 35 years ± 6 years in the placebo group and 35 years ± 5 years in the LCDG (treatment) group (P = 0.94). There were no statistically significant differences between the two treatment groups with respect to the mother's ethnicity, type of breastfeeding, mother's weight and height, type of diet, smoking status, physical activity, presence of pets, and educational level.

[0169] Regarding neonatal characteristics, in the trial, neonates were classified into three groups according to birth weight: normal birth weight (NBW), low birth weight (LBW), and very low birth weight (VLBW). During the trial, 12 pairs of twins (a total of 24 neonates) were born. Specifically, 14 (46.7%) subjects were included in the LCDG group and 10 (34.5%) subjects were included in the placebo group (P = 0.34). 18 (62.1%) neonates in the placebo group and 17 (56.7%) neonates in the LCDG group were female (P = 0.67). The mean birth weight of the neonates was 2050 g ± 784 g in the placebo group and 2005 g ± 768 g in the LCDG group (P = 0.82). There were also no statistically significant differences between the two treatment groups with respect to the APGAR score, gestational (in utero) period, and PN / EG ratio (AGA and SGA).

[0170] L. casei DG (LCDG) was detected in the intestine 10 days after the first administration and persisted until the final visit (84 days).

[0171] The inventors analyzed 251 fecal samples collected at birth from 59 subjects stratified into VLW (n = 20), LW (n = 20) and NW (n = 19). The LCDG strain was detected in fecal samples starting 10 days after the first administration (V2) and persisted until the final visit (84 days; visit at the end of the trial), with a slight decrease starting from the first follow-up time point (day 56 of the trial; 28 days after the final administration of the product) (Figure 6A). The presence of LCDG was significantly higher in the treatment group than in the control group in samples collected at both the treatment periods (10 days and 28 days) and the follow-up visits (56 days and 84 days). This finding was also confirmed by stratifying the samples from each group (NW, LW and VLW) according to birth weight. In particular, a comparison between the three weight classes in the placebo group (within-group analysis) showed a statistical difference in the LCDG concentration at V2 (10 days after the first administration) (p-value = 0.01), while in the treatment group, a statistical difference in the LCDG concentration between the three weight classes (p-value = 0.01) was V4 observed 28 days after the final administration of the product. No statistically significant differences between weight classes were observed at the other time points evaluated (Figure 6B). Furthermore, at day 56, the difference in LCDG concentration between the treatment and placebo groups was statistically significant for the NW and VLW infants (Figure 6B).

[0172] The Wilcoxon signed-rank test was performed to compare the LCDG concentrations at different time points for each group. In the placebo group, the comparisons of V1 vs V2 (p-value = 0.012), V1 vs V3 (p-value = 0.016) and V1 vs V4 (p-value = 0.012) were statistically significant. For the treatment group, the comparisons of V1 vs V2 (p-value = 1.5×10−12), V1 vs V3 (p-value = 5.8×10−9), V1 vs V4 (p-value = 1.8×10−8), V1 vs V5 (p-value = 2.0×10−6), V2 vs V4 (p-value = 1.6×10−3) and V2 vs V5 (p-value = 9.7×10−3) were statistically significant (Figure 7).

[0173] Treatment with LCDG was able to maintain the heterogeneity of the gut microbiota.

[0174] To verify whether LCDG treatment can maintain the heterogeneity of the gut microbiota, 16S-r R N Let A be Using the targeted metagenomic approach, alpha and beta diversity analyses were performed on fecal samples.

[0175] Analysis of alpha diversity at each time point of the subjects divided into the placebo group and the treatment group showed that at time points V2 (10 days) and V3 (28 days), the microbiota of the placebo group seemed to be superior in terms of the richness of microbial species to that of the treatment group (p≥0.05). At the V4 time point, the alpha diversity index seemed to be equivalent (p≥0.05), while at the V5 time point, the treatment group was superior in terms of richness (p≥0.05) (Figure 8A). However, comparisons between the placebo group and the treatment group at various time points were not statistically significant (p≥0.05).

[0176] Regarding beta diversity analysis, the diversity matrix obtained using the Unweighted UniFrac algorithm was represented as PCoA at various time points between the placebo group and the treatment group, indicating that comparisons between these two groups at various time points were not statistically significant (p≥0.05). This result was confirmed by PERMANOVA statistical analysis and applied to the distance matrix calculated using the unweighted Unifrac algorithm (Figure 8B).

[0177] Analysis of the distribution of operational taxonomic units (OTUs) at the phylum level applied to comparisons at various time points and the Kruskal-Wallis test showed no statistically significant differences in any of the placebo / treatment comparisons (p≥0.05) (Figure 8C).

[0178] At the genus level, the comparison between the placebo group and the treatment group was only statistically significant at the V2 time point (p<0.05). In particular, the genus Lactobacillus was higher in the treatment group (Figure 8D).

[0179] Administration of LCDG increases the concentration of short-chain fatty acids (SCFAs).

[0180] Metabolome analysis was performed on fecal samples (n = 85) collected from subjects in whom collection was completed at all time points of follow-up observation (V1 to V5). The fecal samples were analyzed by gas chromatography–mass spectrometry with solid-phase microextraction system (GC-MS-SPME).

[0181] 492 metabolites were identified, quantified, and classified into 15 chemical classes: acids, alcohols, aldehydes, alkanes, alkenes, amines, aromatic hydrocarbons, esters, furans, furanones, indoles, ketones, phenols, pyrazines, and terpenes. As expected, the metabolic profiles of each sample showed considerable variation among subjects. To make the analysis as robust as possible, the raw data matrix was condensed into a matrix of 98 metabolites that included metabolites present in at least 10% of the entire sample set. The entire data analysis was carried out based on this condensed matrix. Multivariate analysis was performed for each time point using a supervised approach, namely, partial least squares discriminant analysis (PLS-DA). The greatest clustering of samples according to treatment (placebo / treatment) occurred between time point V2 (10 days) and time point V4 (56 days), and the SCFA concentration increased in the treatment samples (Figure 9).

[0182] Furthermore, at each test visit, physicochemical evaluation of feces was performed to assess the presence of neutral fat, acidic fat, soap, starch, meat and vegetable fibers, mucus, and pH value. The chi-square test was applied to compare the differences between the two treatment groups, but no significant differences (P>0.05) were found in any of the parameters.

[0183] Administration of LCDG affected both the ecology and function of the microbiota, modifying the ecological status and corresponding functional profile of the bacteria present in the intestine.

[0184] The results obtained by three different approaches, namely real-time PCR, metagenomics, and metabolomics, were correlated with each other by Spearman correlation. The V2 and V3 time points corresponded to the administration of probiotics at 10 days and 28 days, respectively, and these time points were selected for correlation analysis because at these time points, metagenomic analysis, metabolomic analysis, and real-time analysis showed the most statistically significant differences between the placebo group and the treatment group.

[0185] Specifically, at the V2 time point, Lactobacillus paracasei (CFU / mL) showed positive correlations with the metabolites cyclohexanone, n-decanoic acid, and 3,4-dimethylheptane, and with the OTUs Lactobacillus and Granulicatella (p≤0.05). Furthermore, several statistically significant positive correlations were observed between OTUs and metabolites. The most interesting ones after probiotic intake were SCFAs containing butyric acid, which showed significant correlations with Ruminococcus, Prevotella, Collinsella, Faecalibacterium, and Oscillospira (p≤0.05). Pentanoic acid showed significant correlations with Streptococcus, Granulicatella, and Enterococcus (p≤0.05), while propionic acid had significant correlations with Eggerthella, Bacteroides, and Bifidobacterium (p≤0.05).

[0186] At time point V3, Lactobacillus paracasei (CFU / mL) showed a positive correlation with 2,3-dimethylpentanal, 1-hexanol, decanal, and dodecanoic acid (p≤0.05) (Figure 10A), but no significant correlation was found between Lactobacillus paracasei (CFU / mL) and various OTUs (Figure 10B). Finally, several statistically significant positive correlations (p≤0.05) were observed between OTUs and metabolites. In particular, butanoic acid and propanoic acid showed a significant correlation with Collinsella (p≤0.05), while pentanoic acid was positively correlated with Propionibacterium, Faecalibacterium, Pre votella, and Coprococcus (Figure 10C).

[0187] The number of colic episodes did not decrease with the administration of LCDG.

[0188] According to the "Rome IV" diagnostic criteria, the principal investigator recorded colic episodes (occurrences) when crying lasted for at least 3 hours per day and at least 3 days per week for more than 3 hours. During the trial, the mother was required to keep a diary recording the crying of the infant when it lasted for more than 3 hours. At all study visits involved (10 days, 28 days, 56 days, and 84 days after randomization), no statistically significant difference was found between the two study groups (placebo and Enterolactis Baby). In fact, the number of patients crying for more than 3 hours per day and the average crying time in each study group were approximately the same at each study visit, and a total of 8 colic episodes occurred during the trial (4 in the placebo group and 4 in the LCDG group).

[0189] Stool hardness, volume, and color During the trial, stool hardness, volume, and color were evaluated using the Amsterdam Stool Scale. Color and hardness were significantly different between the placebo group and the treatment group on day 10 (V2) and day 56 (V4).

[0190] In V2, the p-value was P<0.0001 for the color of the excrement and P = 0.0016 for the hardness of the excrement. Specifically, among a total of 143 and 221 excrements recorded for PTs belonging to the placebo group and the treatment group, respectively, the excrements classified as color II were 37 (25.9%) in the placebo group and 18 (8.1%) in the treatment group, and the excrements classified as color IV were 7 (4.9%) in the placebo group and 0 in the treatment group. Regarding hardness, the excrements classified as hardness B were 80 (55.9%) in the placebo group and 152 (68.8%) in the treatment group, and the excrements classified as hardness C / D and D were 0 in the placebo group and 6 (2.7%) and 2 (0.9%) in the treatment group, respectively.

[0191] In Visit 4, 119 excrements and 183 excrements occurred for PTs in the placebo group and the treatment group, respectively, and there were statistically significant differences in color and hardness between the treatment groups (using the Amsterdam scale, P = 0.007 for the color of the excrement and P<0.0001 for the hardness of the excrement).

[0192] Regarding the quantity, it was only significant in the samples collected on the 28th day (V3), and the total number of excretions recorded for the placebo group and the treatment group was 122 times and 219 times, respectively.

[0193] Safety evaluation To monitor the growth of the registered neonates, the weight, height, and head circumference parameters were evaluated at each test visit. When comparing the treatment group and the placebo group, no statistically significant differences were found during the test. Finally, no differences were found in the vital signs (except for a higher RR in the placebo group than in the treatment group).

[0194] Regarding adverse events (AEs), a total of 21 AEs in the placebo group and 16 AEs in the LCDG group were recorded during the test. Among these adverse events, 10 SAEs in the placebo group and 6 SAEs in the treatment group were recorded, and 2 severe AEs in the placebo group and 4 severe AEs in the treatment group were recorded.

[0195] In the placebo group, 10 AEs (47.6%) were considered mild, 9 AEs (42.9%) were of moderate severity, and 2 AEs (9.5%) were of severe severity, while in the LCDG group, 8 AEs (50.0%) were considered mild, 4 AEs (25.0%) were of moderate severity, and 4 AEs (25.0%) were of severe severity.

[0196] Among the AEs that occurred in the PTs belonging to the placebo group, 3 AEs (14.3%) were considered probably not related to the treatment (1 was "neonatal respiratory distress syndrome" and 2 were "abdominal pain"), and 1 AE (4.8%) was considered probably related to the treatment ("gastroesophageal reflux disease"). In the PTs belonging to the LCDG group, 2 AEs (12.5%) were considered probably not related to the treatment ("disseminated intravascular coagulation syndrome" and "septic shock" that caused the death of the PT 9 days after the second visit to the hospital), and 2 AEs (12.5%) were considered probably related to the treatment ("necrotizing enterocolitis" and "sepsis").

[0197] Discussion In a specific neonatal trial, the effect of probiotics in the treatment of colic has been studied, and it has been shown that they are effective in shortening the duration of crying episodes and colic symptoms compared to placebo [Lundelin].

[0198] In a long-term follow-up study of probiotic treatment during the neonatal period, it has been shown that the subjects in the probiotic treatment group have a lower incidence of allergies than those assigned to the placebo group [Denkel].

[0199] Furthermore, the administration of probiotics was correlated with a decrease in the incidence of necrotizing enterocolitis and a decrease in mortality in preterm neonates [Lundelin, Strunk, Shane, Costeloe].

[0200] According to these findings, probiotics are characterized by an excellent safety profile. Although cases of bacteremia caused by strains included in probiotics have been recorded, those cases are extremely rare and are determined by the presence of severe immunodeficiency [Lundelin, Salminen, Thomas].

[0201] However, further evidence is needed regarding the safety and potential effects of probiotic supplementation in neonates.

[0202] Considering the increasing use of Lactobacillus in probiotic mixtures, the accurate identification and counting of LCDG cells in feces would improve the study of the actual stability and persistence of these probiotics in the human gut microbiota [Arioli].

[0203] In the present study, for the first time, the safety of LCDG and the ability of LCDG to have a positive impact on the composition of the microbiota were tested in a special population including not only NMW infants but also LW and VLW subjects.

[0204] LCDG was detected in the intestine from 10 days after the first administration and persisted until the final visit (84 days; end of the study), although it decreased slightly from the first follow-up time point (day 56 of the study; 28 days after the final administration of the product). The presence of LCDG was significantly higher in the treatment group than in the control group for NW and VLW infants at V3 (day 28; last day of treatment).

[0205] In the analysis of probiotic strains in stool samples, considering α and β diversity analysis, the changes in the overall profile of the microbiota were time-dependent and did not seem to be affected by treatment (it was demonstrated that treatment could sustain the heterogeneity of the gut microbiota), but at the genus level, according to a temporal analysis, administration of the probiotic led to a statistically significant increase in Lactobacillus species 10 days after the start of administration. This increase was detected at later time points but was no longer statistically significant.

[0206] Regarding metabolome analysis, as expected, the metabolic profiles of each sample showed considerable variation among subjects. PLS-DA analysis revealed that butyric acid (butyrate) and propanoic acid (short-chain fatty acid (SCFA)) metabolites were the main factors for clustering between the placebo group and the treatment group, and their concentrations were increased in samples collected from the treatment group.

[0207] All these results emphasized and confirmed the ability of LCDG to survive gastrointestinal transit while maintaining the heterogeneity of the gut microbiota. These results are consistent with the results of tests conducted in healthy children (3 - 12 years old) [Radicioni] or adult populations [Drago].

[0208] Furthermore, the results obtained by three different approaches (real-time PCR, metagenomics, and metabolomics) were correlated with each other by Spearman correlation.

[0209] Integrating the data obtained from these approaches showed that the administration of probiotic LCDG affected both the ecology and function of the microbiota, altering the ecological status of the bacteria present in the intestine and the corresponding functional profiles. In particular, several SCFAs such as butyric acid and propionic acid showed a positive correlation with certain beneficial bacteria such as Faecalibacterium, Oscillospira, Eggerthella, Bacteroides, and Bifidobacterium. These molecules demonstrated the ability to influence nutrient adsorption [Heimann]. Furthermore, on the 28th day of the test, a positive correlation (p≤0.05) was observed between Lactobacillus paracasei (CFU / mL) and cyclohexanone, n-decanoic acid, and 3,4-dimethylheptane.

[0210] During the clinical trial, the hardness, amount, and color of the feces were also recorded. The amount was significant only in the samples collected on the 28th day, while the color and hardness were significantly different on the 10th and 56th days (4 weeks after the end of administration).

[0211] On the other hand, no difference was observed in the occurrence of abdominal pain based on the "Rome IV" diagnostic criteria, but these results need to be analyzed considering the possibility that both the treatment and the total test period may be insufficient to confirm the improvement of this parameter. For example, Partty et al. [Partty] demonstrated that by providing a follow-up period of up to one year and extending the probiotic supplementation for up to two months (twice the period of this trial), symptoms associated with crying and agitation in premature neonates can be alleviated.

[0212] Regarding safety, as mentioned above, no difference was observed in the vital signs (except for the RR, which was higher in the placebo group than in the treatment group).

[0213] During the trial, to monitor the growth of the enrolled neonates, weight, length, and head circumference parameters were also evaluated at each trial visit. When comparing the treatment group with the placebo group, no statistically significant differences were seen during the trial. Previous literature has shown that a commercially available symbiotic solution containing a combination of probiotics and prebiotics can improve the weight and head circumference of preterm neonates [Guney], while other studies have shown data [Vlieger] that are consistent with the results of the present inventors (i.e., no difference between the treatment group and the placebo group), particularly data [Indrio, Underwood] regarding the weight parameter. Neonatal weight gain is affected by many co-existing medical conditions, as well as the availability of total parental supplementation and / or breast milk fortification [Moni], which may have influenced the weight trends in both groups of that study.

[0214] In recent years, Matin et al. [Matin] published the results of a study in which probiotics (1.5×10 9 CFU / g of Lacticaseibacillus paracasei subsp. paracasei) were orally administered to lactating mothers or their very low birth weight (VLBW) neonates to examine the changes in neonatal serum total bilirubin (TSB) levels and weight gain. Administration of the selected probiotics decreased the TSB levels in neonates but had no significant effect on weight gain after the first week of the intervention.

[0215] Furthermore, based on the evaluation of severe AEs recorded in both study groups and the analyzed safety and tolerability profiles, the test product was shown to be as safe and well-tolerated as placebo in this trial.

[0216] Limitations and future plans Overall, the results of this study confirmed the high safety profile of this product even for vulnerable populations such as NBW, LBW, and VLBW neonates. Although LDCG was confirmed to have the ability to survive in the gastrointestinal tract and affect the gut microbiota and SCFA composition, considering the role of the gut microbiota in nutrient absorption and thus neonatal growth as a result, it would be useful in the future to analyze the long-term effects of this product by considering at least one year of follow-up observation.

[0217] Another interesting aspect not sufficiently emphasized in this trial is the potential effect of this product on the microbiota and serum metabolome under different nutritional conditions. In fact, nutrition in infancy determines the development of the gut microbiota. Unlike breastfeeding, formula feeding has been shown to influence the gut microbiota and serum metabolome towards a less favorable state. In recent years, another Lactobacillus paracasei strain has been shown to affect the microbiota and serum metabolome, although not canceling out the effects of formula feeding, and may reduce some of the unfavorable effects on metabolism caused by formula feeding [Lee].

Claims

Claim 1 A strain for use in a method for treating, preventing and / or curing a subject having an inflammatory and / or functional gastrointestinal disorder, wherein the disorder is selected from the group consisting of or comprising neonatal colic, intestinal colic, chronic enteritis or chronic enteritis in premature neonates, necrotizing enterocolitis, sepsis, irritable bowel syndrome (IBS), IBS with predominantly constipation (prevalent constipation) or constipated bowel, IBS with predominantly diarrhoea (prevalent diarrhoea) or diarrhoeic bowel, IBS with alternating bowel, unclassified IBS, inflammatory bowel disease (IBD), Crohn's disease and ulcerative proctocolitis, wherein the strain belongs to the species Lactobacillus paracasei and is selected from the group consisting of or comprising Lactobacillus paracasei DG (registered trademark) CNCM I-1572, Lactobacillus paracasei LPC-S01 DSM 26760 and mixtures thereof, and wherein the subject is a neonatal subject within 4 weeks of birth and / or is of an age of 1 month to 12 months or less, the strain. Claim 2 The strain is a viable strain of bacteria or a derivative of the strain, and the derivative of the strain is selected from the group consisting of or comprising a tyndallized strain, an ultrasonically treated strain, a radiation-inactivated strain, a lysed strain or a bacterial homogenate, the strain extract or the cell wall fraction, the strain for use according to claim 1. Claim 3 The strain is a viable strain of bacteria or a derivative of the strain, and the derivative of the strain is selected from the group consisting of or comprising a tyndallized strain, an ultrasonically treated strain, a gamma-radiation-inactivated strain, a lysed strain or a bacterial homogenate, the strain extract or the cell wall fraction, the strain for use according to claim 1 or 2. Claim 4 The strain is a viable strain of bacteria or a derivative of the strain, and the derivative of the strain is a tyndallized strain, the strain for use according to claim 1 or 2. Claim 5 The strain for use according to claim 1 or 2, wherein the disorder is selected from the group consisting of or comprising neonatal colic, intestinal colic, chronic enteritis or chronic enteritis in premature neonates, necrotizing enterocolitis, sepsis, irritable bowel syndrome (IBS), IBS mainly accompanied by constipation or with a constipated intestine, IBS mainly accompanied by diarrhea or with a diarrheal intestine, alternating bowel IBS, unclassified IBS, inflammatory bowel disease (IBD), Crohn's disease, ulcerative proctocolitis.

6. A mixture M for use according to claim 1 or 2, a strain belonging to the species Lactobacillus paracasei selected from the group consisting of or comprising Lactobacillus paracasei DG (registered trademark) CNCM I-1572, Lactobacillus paracasei LPC-S01 DSM 26760 and mixtures thereof, and at least one food-grade or pharmaceutical-grade additive and / or excipient Mixture M, which contains or consists of these.

7. A mixture M for use according to claim 6, a strain belonging to the species Lactobacillus paracasei selected from the group consisting of or comprising Lactobacillus paracasei DG (registered trademark) CNCM I-1572, Lactobacillus paracasei LPC-S01 DSM 26760 and mixtures thereof, and (a) Bifidobacterium breve BbIBS01 DSM 33231, (b) Bifidobacterium breve BbIBS02 DSM 33232, (c) Bifidobacterium animalis subsp. lactis BlIBS01 DSM 33233, (d) Lactobacillus plantarum LpIBS01 DSM 33234, (e) Bifidobacterium bifidum BbfIBS01 DSM 32708, and mixtures thereof Mixture M, which contains or consists of at least one additional strain selected from the group consisting of or comprising these.

8. A mixture M for use according to claim 6, (1) at least one vitamin from vitamin groups A, B, C, D, E and / or K One or more polyphenols selected from the group consisting of glutathione, resveratrol, and trans-resveratrol, coenzyme Q10, astaxanthin, lycopene, or one or more antioxidants selected from the group consisting thereof, One or more plant substances having an intestinal relaxation effect, including or consisting of botanicals (crude plant products, botanicals) or extracts thereof, One or more minerals or salts thereof selected from the group consisting of zinc, selenium, magnesium, and potassium, One or more monounsaturated fatty acids including or selected from the group consisting of omega-9, and / or one or more polyunsaturated fatty acids including or selected from the group consisting of omega-3 and / or omega-6, One or more immunostimulants, antidiarrheal substances, and / or nutrients, At least one prebiotic, And mixtures thereof Mixture M further comprising at least one additional active ingredient selected from the group consisting of or including these.

9. (1-1) The vitamin is vitamin B group and / or vitamin D group, and / or (3-1) The plant substance is selected from the group consisting of valerian, passionflower, lemon balm, Japanese quince, and chamomile, and / or (7-1) The prebiotic is selected from the group (II) consisting of inulin, fructooligosaccharide (FOS), galactooligosaccharide (GOS), xylitol-oligosaccharide (XOS), guar gum, lactoferrin, and mixtures thereof. Mixture M for use according to Claim 8.

10. The prebiotic is selected from the group (II) consisting of inulin and mixtures thereof. Mixture M for use according to Claim 9.

11. The disorder includes or is selected from the group consisting of neonatal colic, intestinal colic, chronic enteritis, or chronic enteritis in premature neonates, necrotizing enterocolitis. The strain for use according to Claim 1 or 2.

12. The disorder includes or is selected from the group consisting of neonatal colic, intestinal colic, chronic enteritis, or chronic enteritis in premature neonates, necrotizing enterocolitis. Mixture M for use according to Claim 6.

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