Probiotic Compositions for the Treatment of Intestinal Hyperpermeability - Patent application

Abstract

Probiotic compositions are provided that include Bifidobacterium longum subsp. longum CECT7894. The probiotic compositions are useful for treating, preventing, or ameliorating intestinal barrier dysfunction (e.g., increased intestinal permeability) or related conditions, or symptoms, complications, and / or sequelae thereof, in a subject in need thereof by producing polyphosphate. Combinations of the probiotic compositions and at least one human milk oligosaccharide are also provided.

Description

[Technical field]

[0001] The present invention relates to the fields of medicine and microbiology, in particular to probiotic compositions that benefit human and animal health, in particular useful for the treatment of intestinal barrier dysfunction or related conditions. [Background technology]

[0002] Bifidobacteria are members of the human intestinal microflora that play an important role in human health. In infants, the intestinal microflora is dominated by bifidobacteria, but in adulthood the levels become lower. The presence of different types of bifidobacteria changes with age, from childhood to old age. Bifidobacteria play an important role in the normal development of the intestinal microflora and its barrier effect, which has beneficial effects on the absorption of food compounds and on the maturation of the immune system during critical periods in the early stages of life.

[0003] A reduction in Bifidobacteria is associated with an increased risk of long-term disorders such as allergies, obesity or inflammatory bowel disease, which may be caused by factors such as Caesarean section, premature birth, artificial feeding, or prenatal and postnatal antibiotic treatment. As a result, Bifidobacteria strains are being investigated for use as probiotics in the prevention and treatment of disease.

[0004] WO2015018883A2 discloses a probiotic composition containing Pediococcus pentosaceus CECT8330 and optionally Bifidobacterium longum CECT7894, useful for improving excessive crying in infants. Clinical trials testing both probiotic bacterial compositions showed that ingestion of the probiotics significantly reduced the average daily crying time and the duration of each episode. It also explains that "from the relevant properties of the bacterial composition described above, it follows that administration of the bacterial composition is also useful for treating other conditions characterized by gastrointestinal disorders associated with inflammation as a result of an immature immune system; for treating intestinal hypersensitivity and for balancing the excess of undesirable bacteria in the intestine." Regarding the properties of each probiotic strain included in the composition, WO2015018883A2 discloses that P. pentosaceus CECT 8330 exhibited a higher ability to induce IL-10, which may result in improved inflammation in the intestinal tract, whereas B. longum CECT7894 exhibited a higher ability to inhibit the growth of undesirable bacteria commonly abundant in excessively crying infants.

[0005] JP2006176450A describes a probiotic composition containing lactic acid bacteria such as Bifidobacterium adolescentis JCM1251 or Bifidobacterium breve JCM1273, which can accumulate polyphosphate by absorbing phosphorus. This composition may have the ability to inhibit excessive absorption of phosphorus in the small intestine, and therefore may have a positive effect on the prevention of various diseases, including nephrolithiasis.

[0006] Some strains of Lactobacillus and Bifidobacterium have shown the ability to produce polyphosphate (polyP), which has been found to have a postbiotic effect due to its role in enhancing intestinal barrier function and maintaining host intestinal homeostasis. Host-probiotic interactions are promoted through epithelial endocytosis of probiotic-derived polyP. In intestinal cells, polyP induces cytoprotective factors such as the heat shock protein HSP27 through the integrin β1-p38 MAPK pathway.

[0007] The polyP formation ability of bifidobacteria was suggested by Qian et al. (2011). The authors show that the bifidobacterial strains B. adolescentis ATCC15703 (JCM1275), B. longum ATCC15707, B. longum ATCC55816, Bifidobacterium sp. BAA-718 and B. skaldvii BAA-773 produce observable granules that may correspond to polyP granules, but this has not been proven. Furthermore, no quantification or characterization of the granules has been performed. These granules may also correspond to granules containing, for example, metal or protein granules. In addition, the expression of the ppk gene encoding the polyP biosynthetic enzyme PPK has been studied in the non-probiotic strain B. skaldvii BAA-773 in response to oxidative stress.

[0008] Another study also assessed the ability of Lactobacillus, Bifidobacterium, Lactococcus and Streptococcus to form polyP through an indirect assay measuring the amount of phosphate remaining in the medium after an incubation period (Anand et al., 2019). B. adolescentis JCM1275 shows the highest phosphorus accumulation capacity, although no quantification of polyP was performed in this experiment.

[0009] Furthermore, Saiki et al., 2016 indirectly quantified polyP production by Lactobacillus and Bifidobacterium through direct quantification of ATP after addition of polyphosphate kinase (PPK). PPK is an enzyme that catalyzes a reversible reaction that generates polyP and ADP from ATP and phosphate. The results show a wide diversity of polyP production capabilities between species and strains. Lacticasei Bacillus paracasei subsp. paracasei JCM 1163 shows the highest polyP concentration.

[0010] These studies represent an initial step towards the use of probiotic bacteria capable of producing polyP. Nevertheless, probiotic characteristics are strain dependent, even among bacteria of the same species. It is therefore important to discover strains capable of producing significant amounts of polyP in order to have a beneficial effect on the host. Furthermore, they should perform well in all probiotic requirements, such as resistance to gastrointestinal conditions, proper growth, and be suitable for large-scale production.

[0011] The abstract by Xiao et al., accepted May 30, 2022, concludes that B. longum CECT7894 improved the efficacy of infliximab against dextran sulfate sodium (DSS)-induced colitis in mice via regulating gut microbiota and bile acid metabolism. Summary of the Invention [Problem to be solved by the invention]

[0012] The problem to be solved by the present invention is to provide new compositions capable of positively affecting intestinal barrier dysfunction in subjects in need thereof. [Means for solving the problem]

[0013] The present inventors have found a new probiotic composition capable of producing large amounts of polyphosphate (polyP), which has a positive effect on the intestinal barrier. The probiotic composition comprises a Bifidobacterium longum subsp. longum strain, which is a strain of human intestinal origin adapted to the conditions of the human intestine. In particular, the bifidobacterium strain of the present invention is a Bifidobacterium longum subsp. longum strain deposited under the Budapest Treaty at the Spanish Collection of Type Cultures (CECT) under the accession number CECT7894 (also referred to herein as KABP-042). Surprisingly, in addition to being capable of producing large amounts of polyP, the strain of the present invention is also capable of growing while producing polyP. Moreover, it belongs to B. longum subsp. longum, which is present in the human microbiota at all stages of life, and therefore has the potential to have a positive effect on people from newborns to the elderly. In addition, the inventors of the present invention have demonstrated that the strain is well adapted to infant and adult gastrointestinal conditions, e.g. resistance to gastric and bile salt stress, good adhesion to the intestinal epithelium, utilisation of complex sugars from human milk and, surprisingly, also has a good stability with only a one-third decrease over 12 months, unlike other bifidobacteria known in the art. Effect of the Invention

[0014] The examples herein provide detailed experimental data that demonstrate the ability of the probiotic composition of the present invention to produce significant amounts of polyP without compromising its growth rate.Continuous growth of this strain allows the production of increasing levels of polyP, a postbiotic molecule that has a protective effect on the intestinal barrier.Furthermore, as will be understood by those skilled in the art in this context, the natural habitat of this bifidobacterial strain is the human intestine.Therefore, this strain clearly shows the potential to produce polyP while growing under these optimal environmental conditions.

[0015] Example 1 shows that B. longum subsp. longum CECT7894 has the highest ability to produce polyP compared to some test strains (e.g., B. animalis BB-12, B. adolescentis JCM1275, L. plantarum WCFS1 and B. scardovi BAA-773). Furthermore, B. longum subsp. longum CECT7894 shows a high potential to grow while producing polyP, which is essential for intestinal engraftment and may enable the strain to grow from early postnatal life. Therefore, early administration of this health-promoting strain in infants can benefit the intestine and maintain its positive effect in further stages of life.

[0016] From a long-term perspective, the fact that the growth rate of the strains of the present invention is not compromised by high polyP production is advantageous for obtaining higher amounts of polyP in the human intestine thereafter. Figure 2 and Table 2 show the excellent ability of B. longum subsp. longum CECT7894 to synthesize large amounts of polyP while growing at all time points considered in this study. Similarly, B. longum subsp. longum 36524™, B. longum subsp. longum ATCC15707 and B. animalis BB-12 also produce large amounts of polyP and have high growth rates. However, the two B. longum strains cannot maintain high polyP production at 16 hours, and only B. animalis BB-12 can produce detectable amounts of polyP at 16 hours.

[0017] Furthermore, B. longum subsp. longum CECT7894 is a human resident bifidobacterium (HRB) strain, whereas B. animalis BB-12 is classified as a non-HRB strain. HRB strains are frequently isolated from healthy human feces and oral cavity, exert better health-promoting effects, and are therefore characterized as better probiotic candidates for human use because their metabolism is adapted to the human gastrointestinal tract. In contrast, B. animalis BB-12 does not adapt and engraft properly in the human intestine, cannot tolerate the human intestinal conditions, and cannot maintain proliferation ability while producing large amounts of polyP.

[0018] B. breve JCM1273 shows a similar growth rate at 6 h and a faster growth rate at 16 h compared to B. longum subsp. longum CECT7894. Nevertheless, the ability to generate polyP is rather low at both time points.

[0019] B. adolescentis JCM1275 is also capable of producing some amount of polyP, but at the same time does not have the ability to grow. As a result, the production may be impaired overall, since the aim is to achieve a sustained existence of the strain, i.e., sustained production of polyP. It should be noted that this strain is considered an adult-type HRB, since it is abundant in adults and elderly people, but almost absent in infants.

[0020] In particular, genomic analysis and in vitro experiments have shown in Example 3 that B. longum subsp. longum CECT7894 has the potential to adapt well to the gastrointestinal tract of infants and adults. In addition, since subsp. longum subsp. longum is a long-term colonizer with a higher distribution and abundance in infants than other strains and species, the strain of the present invention has a high potential to colonize the intestines of babies. Furthermore, B. longum subsp. longum is also abundant in the intestines of adults and elderly humans, thus providing beneficial effects to the host.

[0021] In addition, B. skaldovii is known to carry an active ppk gene and has exceptional growth potential (as shown in Example 1 with B. skaldovii strain BAA-773), but has minimal ability to produce polyP. Furthermore, B. skaldovii is known to be a pathogenic strain, making it unsuitable for probiotic compositions.

[0022] Finally, L. plantarum WCFS1 is known to protect the intestinal barrier through polyP production, whereas B. longum subsp. longum CECT7894 produces even larger amounts of polyP. In addition, L. plantarum is not the predominant species in the infant gut.

[0023] Overall, the strains of the present invention will be able to produce the greatest amount of polyP when administered to subjects with the same initial dose of the probiotic composition. For example, when comparing tablets containing the same cfu of the different studied strains, the strains of the present invention have the highest potential to produce the greatest amount of polyP.

[0024] Furthermore, B. longum subsp. longum CECT7894 in the pharmaceutical composition demonstrated stable viable cell counts over time, as shown in Example 2 and Figure 4. These results indicate that a 3-fold overdose at the time of manufacture resulted in a 10 9 It has been shown that this is sufficient to ensure viable cfu, thereby allowing large-scale production and long-term storage of the probiotic composition.

[0025] This long-term stability of the probiotic strain B. longum subsp. longum CECT7894 is unexpected, because it is well known in the prior art that many probiotic bifidobacteria strains have low tolerance to oxygen and therefore do not show suitable stability. Some bifidobacteria strains, such as B. pyschroaerophilum, B. indicum and B. asteroides, have higher stability, but they are not suitable HRB strains for probiotic compositions. In contrast, B. longum subsp. longum CECT7894 is not only HRB, but also shows high stability and therefore shows tolerance to oxygen. Therefore, this strain is suitable for the manufacture of probiotic compositions that may require long-term storage.

[0026] Furthermore, as shown in Example 4, the effect of polyP produced by B. longum CECT7894 has been shown to have a positive effect on barrier integrity, intestinal permeability and intestinal barrier homeostasis. In addition, such effects have been shown to involve the induction of other markers of barrier integrity, including the production of heat shock proteins (HSP27) and tight junction proteins, all of which are induced by the presence of polyP from B. longum CECT7894.

[0027] In addition, the inventors have demonstrated in Example 5 the ability of B. longum CECT7894 to produce polyP in the presence of breast milk, which indicates the beneficial effect of B. longum CECT7894 in lactating infants. Breast milk contains carbohydrate HMOs. The HMO lacto-N-tetraose (LNT) is used by B. longum CECT7894, as confirmed in Example 3. Furthermore, LNT has been demonstrated to have a positive effect on polyP biosynthesis in the B. longum CECT7894 strain. Surprisingly, Example 6 shows that B. longum CECT7894 can grow in the presence of the supernatant of other bifidobacteria that can utilize the HMO 2'-fucosyllactose (2'-FL). Overall, these results demonstrate that B. longum CECT7894 is able to grow and increase polyP production in the presence of the two most abundant HMOs in breast milk (LNT and 2'-FL), thus highlighting the beneficial role of B. longum CECT7894 supplementation in infants, for example.

[0028] Overall, it has been elegantly demonstrated that B. longum CECT7894 produces large amounts of polyP during growth and has a positive effect on intestinal permeability. Furthermore, it has been shown that the addition of HMOs positively affects polyP biosynthesis in B. longum CECT7894.

[0029] The abstract by Xiao et al., accepted May 30, 2022, concludes that B. longum CECT7894 improved the efficacy of infliximab against dextran sulfate sodium (DSS)-induced colitis via regulating gut microbiota and bile acid metabolism. The experimental model used is DSS-induced acute colitis in mice. Ulcerative colitis is considered an inflammatory bowel disease characterized by overt intestinal inflammation and alterations of normal gut bacteria. The treatments described in the abstract are infliximab (a monoclonal antibody with immunosuppressive effects used to treat inflammatory conditions such as colitis), and infliximab + B. longum CECT7894. No animal group received B. longum CECT7894 alone. Infliximab is highly effective in treating colitis in both humans and animal models, but its use has been associated with an increased risk of infection in some clinical trials (Shah et al., 2017).

[0030] The authors explain that the addition of B. longum CECT7894 to infliximab alters the microbiota and bile acid metabolism. The authors acknowledge that the change in bile acids may explain the effect. B. longum CECT7894 increased the relative abundance of Bifidobacteria, Blautia, Butyricicoccus, Clostridium, Coprococcus, Gemmiger, and Parabacterioides, and decreased the relative abundance of Enterococcus and Pseudomonas bacteria. Considering that enterococci, especially Pseudomonas, can be pathogenic, and that the use of infliximab is known to reduce inflammation but increase the risk of infection, the mere fact of adding B. longum CECT7894 could compensate for the shortcomings of infliximab therapy and thus facilitate faster healing of the intestine by reducing the level of pathogenic bacteria already present in the intestine. Of note, the observed effects were dependent on the pre-existing gut flora and on the combination with infliximab.

[0031] Moreover, the authors report that improvement in the DSS colitis model is associated with changes in some bile acids, but the generalizability of this finding to humans is limited because the bile acid compositions of mice and humans are very different, with the former containing reasonable amounts of alpha- and beta-murocholic acid and the latter practically absent.

[0032] On the other hand, they disclose that some parameters, such as tight junctions (ZO-1, occludin), improve in the infliximab + B. longum CECT7894 group, but there is no data to fully prove this effect.In summary, this abstract presents some results on the effect of B. longum CECT7894 on the efficacy of infliximab in a specific experimental (DSS)-induced colitis model in mice by modulating the gut microbiota and bile acid metabolism.

[0033] In particular, in tight junction experiments where the results were inconclusive in combination with infliximab, the effect of B. longum CECT7894 alone cannot be derived without treatment with infliximab. Furthermore, as discussed, the effect observed in combination with infliximab depends on the pre-existing gut microbiota and therefore cannot be derived from the effect of B. longum CECT7894 in diseases other than the experimental colitis model used in this study.

[0034] Surprisingly, the effect of B. longum CECT7894 on disease improvement (through improving the efficacy of infliximab by regulating microbiota and bile acid metabolism as mentioned above) can be considered as an indirect effect. In contrast, the present invention shows a direct effect of B. longum CECT7894, namely, protection of intestinal permeability through direct delivery of polyphosphate to the intestinal epithelium. Moreover, the effect is independent of the disease model and the surrounding microbiota.

[0035] Overall, the inventors have found that the Bifidobacterium longum subsp. longum CECT7894 strain encompasses all the key properties desired for a probiotic composition to exert beneficial effects in the intestine, especially in humans suffering from intestinal barrier dysfunction. These include resistance to gastrointestinal conditions (such as resistance to gastric stress and bile salts), long-term stability, belonging to a species present in all stages of life, and a remarkable ability to generate polyP during growth. Thus, a probiotic formulation containing B. longum subsp. longum CECT7894 according to the present invention is useful for improving any clinical condition in which intestinal permeability is impaired.

[0036] Accordingly, the present invention relates to a method for treating, preventing or ameliorating intestinal barrier dysfunction or a related condition, or symptoms, complications and / or sequelae thereof, in a subject in need thereof, by producing polyphosphate, comprising the steps of: (a) has a genome that is at least 99% identical to the genome of the corresponding deposited strain; and (b) the corresponding deposited strain retains the ability to produce polyphosphate and probiotic compositions comprising the derived bacterial strains.

[0037] "The Bifidobacterium longum subsp. longum strain deposited under the Budapest Treaty in the Spanish Type Culture Collection (CECT) under accession number CECT7894, or a derived bacterial strain thereof which (a) has a genome that is at least 99% identical to the genome of the corresponding deposited strain; and (b) retains the ability of the corresponding deposited strain to produce polyphosphate" is hereinafter abbreviated as B. longum CECT7894 or a derived bacterial strain thereof.

[0038] In another aspect, the present invention provides a probiotic composition comprising B. longum CECT7894 or a derived bacterial strain thereof for use in the treatment of intestinal hyperpermeability and related conditions in a subject, wherein the treatment of intestinal hyperpermeability is by producing polyphosphate and the related condition is an extra-intestinal condition.

[0039] It is understood herein that the probiotic composition is useful for treating intestinal barrier dysfunction, particularly intestinal hyperpermeability, and also for treating related conditions per se, i.e. conditions associated with intestinal barrier dysfunction, particularly intestinal hyperpermeability, which may alternatively be expressed as a probiotic composition for use in treating the conditions described herein by treating intestinal hyperpermeability through the production of polyphosphates.

[0040] Another aspect of the present invention is a method for producing a (i) B. longum CECT7894 or a derived bacterial strain thereof, and (ii) at least one human milk oligosaccharide; which is configured for simultaneous, separate or sequential administration.

[0041] This aspect can alternatively be described in relation to a probiotic composition comprising B. longum CECT7894 or a derived bacterial strain thereof as described herein for use in combination with at least one human milk oligosaccharide, the combination being configured for simultaneous, separate or sequential administration.

[0042] In another aspect, the invention relates to a combination as provided herein for use in the treatment of intestinal hyperpermeability and related conditions in a subject, wherein the treatment of intestinal hyperpermeability is by generating polyphosphate, and the related condition is selected from the group consisting of an immune disorder or disease, a metabolic or cardiovascular disorder or disease, a neurological or psychiatric disorder or disease, and a gastrointestinal disorder or disease.

[0043] In another aspect, the present invention provides a method for producing a composition comprising: (i) B. longum CECT7894 or a derived bacterial strain thereof; and (ii) at least one human milk oligosaccharide; A composition comprising:

[0044] The probiotic compositions, combinations, and compositions according to aspects of the present invention can be used for different medical applications / uses as detailed herein. All uses described herein can be alternatively described as the use of any of the compositions described herein for the manufacture of a pharmaceutical, nutraceutical, veterinary, or food / nutritional composition for the treatment, prevention, or amelioration of intestinal barrier dysfunction or related conditions, or symptoms, complications, and / or sequelae thereof, as disclosed herein. This can also be alternatively described as a method for treating, preventing, or ameliorating intestinal barrier dysfunction or related conditions, or symptoms, complications, and / or sequelae thereof, in a subject in need thereof, comprising administering to the subject a composition described herein according to aspects of the present invention.

[0045] The terms used in the claims and aspects of the present invention are understood in their broad and general sense in this description. Nevertheless, they are defined hereafter in the detailed description of the present invention. Throughout the specification and claims, the word "comprises" and its variations are not intended to exclude other technical features, additives, ingredients, or steps. Additional objects, advantages, and features of the present invention will become apparent to those skilled in the art upon studying this description or can be learned by practicing the present invention. Moreover, the present invention encompasses all possible combinations of the specific and preferred embodiments described herein. The following examples and figures are provided herein for illustrative purposes and are not intended to limit the present invention. [Brief description of the drawings]

[0046] [Figure 1]Figure 1 shows the growth curves of the test strains. PolyP was extracted and quantified at 6 and 16 hours. OD means optical density (measured at 595 nm) and t (hr) means time in hours. [Diagram 2] FIG. 2 shows polyP biosynthesis (nmol) of the test strains after 6 and 16 hours of growth. [Diagram 3] FIG. 3 shows a neighbor-joining tree showing the relationships between the PPK proteins in the bifidobacterial strains examined. [Figure 4] Figure 4 shows the stability of B. longum subsp. longum KABP-042 (CECT7894) over time in the final product. Viable bacteria are expressed as log cfu over time in months (t(months)). [Diagram 5] Figure 5 shows the growth of B. longum subsp. longum KABP-042 (CECT7894) in the presence of the HMO lacto-N-tetraose (LNT), glucose (Gluc) and in the absence of carbon source (C-). OD means optical density (measured at 595 nm) and t (hr) means time in hours. [Figure 6] Figure 6 shows the apparent permeability coefficient (Papp) (left) and transepithelial electrical resistance (TEER) (right) of Caco-2 barrier exposed to supernatants of B. longum CECT7894 with high (sb_MEI) and low (sb_LP) amounts of polyP. Cells were exposed to MEM, non-fermented MEI and LP media as controls. [Figure 7] Figure 7 shows the relative expression of HSP27 protein in Caco-2 cells exposed to the supernatant of B. longum CECT7894 with high (MEI) and low (LP) amounts of polyP. The amount of HSP27 was normalized to the amount of β-actin (left). Correlation (Pearson r=0.87, p=0.01) between the relative expression of HSP27 and the amount of polyP expressed as nmol P in the supernatant (right). [Figure 8]Figure 8 shows the relative expression (RE) of tight junction proteins Zonula ocludens-1 (ZO1), junction adhesion protein-1 (JAM1) and occluding in Caco-2 cells exposed to supernatants of B. longum CECT7894 with mei and lp polyP. Expression was normalized to 18S rRNA and GADPH gene expression. [Figure 9] FIG. 9 shows polyP biosynthesis (nmol) of B. longum CECT7894 cultures incubated for 6 and 16 h under different conditions: control (C), breast milk (BM), LNT, polyamines (Polya). [Figure 10] Figure 10 shows the growth of B. longum subsp. longum KABP-042 (CECT7894) in the presence of supernatant of B. bifidum Bb01 cultured with HMO 2'-fucosyllactose (SN B. bifidum 2'-FL), glucose (Gluc), and in the absence of carbon source (C-). OD means optical density (measured at 595 nm) and t (hr) means time in hours. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0047] Detailed Description of the Invention definition Probiotics : As used herein, this term refers to live non-pathogenic microorganisms, e.g., bacteria, that can confer health benefits to a host organism that carries an adequate amount of the microorganism. In some embodiments, the host organism is a mammal. In some embodiments, the host organism is a human. Some species, strains, and / or subtypes of non-pathogenic bacteria are now recognized as probiotics. Probiotics can be variants or mutant strains of bacteria. Probiotic bacteria can be naturally mutated or genetically engineered to retain, enhance, or improve desired biological properties, e.g., viability, to provide probiotic properties or to retain, enhance, or improve probiotic properties.

[0048] Derived As used herein, the terms "derived", "derivative", "variant", "mutant" (e.g., "mutant strain"), or any grammatical variation thereof, refer to a component isolated from or made using a particular molecule / substance (e.g., a strain of the present disclosure). For example, a bacterial strain derived from a first bacterial strain (e.g., a deposited strain) can be a strain that is identical or substantially similar to the first strain. In the case of bacterial strains, the derivative strain can be obtained, for example, by naturally occurring mutagenesis, artificially directed mutagenesis, artificially random mutagenesis, or other genetic engineering techniques that retain, enhance, or improve at least one capability of the deposited strain.

[0049] Excipients / Carriers : These terms are used interchangeably and refer to inert substances added, for example, to pharmaceutical compositions to further facilitate administration of a compound, for example, a bacterial strain of the present disclosure. Examples include, but are not limited to, calcium bicarbonate, calcium phosphate, various sugars and various types of starch, cellulose derivatives, gelatin, vegetable oils, polyethylene glycols, and surfactants, including, for example, polysorbates. The terms "physiologically acceptable excipient / carrier" and "pharmaceutical acceptable excipient / carrier" may be used interchangeably and refer to substances or diluents that do not cause significant irritation to the organism and do not impair the biological activity and properties of the administered bacterial compound. Adjuvants are included in these terms.

[0050] composition : As used herein, this term refers to different compositions and combinations according to the aspects of the present invention. In addition, it refers to product forms such as a mixture of at least one compound useful within the present invention and excipients / carriers. For example, a "pharmaceutical composition" refers to a preparation of the bacteria of the present invention and other components such as pharmaceutical acceptable carriers and / or excipients. A pharmaceutical composition facilitates administration of a compound to a patient or subject.

[0051] identity: As used herein, this term refers to the overall conservation of monomeric sequences between polymer molecules, e.g., between DNA molecules and / or RNA molecules. The term "identical" without any additional qualifier means that the sequences are 100% identical (100% sequence identity). Describing two sequences as, for example, "70% identical" is equivalent to describing them as having, for example, "70% sequence identity."

[0052] The calculation of the percent identity of two polymer molecules, e.g., polynucleotide sequences, can be performed, for example, by aligning the two sequences for optimal comparison purposes (e.g., for optimal alignment, gaps can be introduced into one or both of the first and second polynucleotide sequences). In certain embodiments, the length of the aligned sequence for comparison purposes is at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90% or about 100% of the length of the reference sequence. In the case of polynucleotides, the bases at corresponding base positions are then compared.

[0053] The percent identity between two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps and the length of each gap, and can be determined using a mathematical algorithm. Suitable software programs are available for alignment of both protein and nucleotide sequences. One suitable program for determining percent sequence identity is bl2seq, which uses either the BLASTN (used to compare nucleic acid sequences) or BLASTP (used to compare amino acid sequences) algorithms to perform a comparison between two sequences. Other suitable programs are, for example, Needle, Stretcher, Water, or Matcher, which are part of the EMBOSS suite of bioinformatics programs. Sequence alignment can be performed using methods known in the art, such as MAFFT, Clustal (ClustalW, Clustal X, or Clustal Omega), MUSCLE, MAUVE, MUMMER, RAST, etc.

[0054] In certain embodiments, the percentage identity (%ID) of a first sequence to a second sequence is calculated as %ID=100×(Y / Z), where Y is the number of amino acid residues or nucleobases scored as identical matches in the alignment of the first and second sequences (e.g., aligned by visual inspection or by a specific sequence alignment program), and Z is the total number of residues in the second sequence. When comparing complete or near-complete genomic nucleobase sequences, %ID may be referred to as ANI (Average Nucleotide Identity). Calculating ANI usually involves fragmenting the genomic sequence, followed by nucleotide sequence search, alignment, and identity calculation.

[0055] Prevent As used herein, the terms "prevent", "preventing", "prevention" and variations thereof refer to, e.g., (i) partially or completely delaying the onset of the diseases, disorders and / or conditions disclosed herein; (ii) partially or completely delaying the onset of one or more symptoms, characteristics, or clinical signs, complications, or sequelae of the specific diseases, disorders, and / or conditions disclosed herein; (iii) partially or completely delaying the onset of one or more symptoms, characteristics, or signs, complications, or sequelae of the specific diseases, disorders, and / or conditions disclosed herein; (iv) partially or completely slowing the progression of certain diseases, disorders and / or conditions disclosed herein; and / or (v) reducing the risk of developing symptoms associated with the diseases, disorders, and / or conditions disclosed herein. Refers to...

[0056] subject The terms "subject," "patient," "individual," and "host," and variations thereof, are used interchangeably herein and refer to any mammalian subject, particularly humans, for whom diagnosis, treatment, or therapy is desired, including, but not limited to, humans, domestic animals (e.g., dogs, cats, etc.), livestock (e.g., cows, sheep, pigs, horses, etc.), and laboratory animals (e.g., monkeys, rats, mice, rabbits, guinea pigs, etc.). The compositions described herein are applicable to both human therapy and veterinary applications.

[0057] infant The term "infant" is to be understood in this description as a very young offspring of a human or animal, e.g. a child under the age of 1 year. When applied to humans, this term is considered synonymous with the term "baby". The term "child" refers to a human between the birth stage and the adolescent stage. "Toddler" refers to a child between 1 and 7 years of age, and "toddler" refers to a child between 1 and 3 years of age. However, in this description, the terms "infant", "baby", "toddler" and "toddler" are considered synonymous and used interchangeably.

[0058] Humans other than infants or non-infant: As used herein, these terms refer to humans over the age of 7. Non-infant humans can be teenagers, adults, or seniors (65 years and older). This category also includes athletes and infirm non-infant individuals.

[0059] Who needs it? As used herein, a "subject in need thereof" includes a subject, such as a mammalian subject, that would benefit from the administration of a composition of the present disclosure.

[0060] Therapeutically Effective Dose The terms "therapeutically effective dose" and "therapeutically effective amount" are used to refer to an amount of a composition of the present disclosure that is sufficient to produce a desired therapeutic, pharmacological and / or physiological effect on a subject in need thereof. In particular, the term refers to an amount of a compound that results in the prevention of a condition, e.g., diarrhea, delaying the onset of symptoms, or ameliorating symptoms. A therapeutically effective amount may be sufficient, for example, to treat, prevent, reduce the severity of, delay the onset of, and / or reduce the risk of developing one or more symptoms of a disease or condition associated with impaired intestinal barrier function. Therapeutically effective amounts, as well as therapeutically effective administration frequencies, are known in the art and can be determined by methods discussed below.

[0061] treatment As used herein, the terms "treat", "treatment", "therapy" refer to, for example, reducing the severity of a disease or condition disclosed herein; alleviating / ameliorating or eliminating one or more symptoms, complications, or sequelae associated with a disease disclosed herein (e.g., intestinal barrier dysfunction or related conditions); providing a beneficial effect to a subject having a condition / disease disclosed herein, without necessarily curing the disease or condition. The term also includes prophylaxis or prevention of a disease or condition, or symptoms, complications, or sequelae thereof. Thus, as used herein, the expression "treating" encompasses treating, preventing, or ameliorating a disease, or its symptoms, complications, and / or sequelae.

[0062] The term may refer to, for example, with respect to what would be expected in the absence of treatment with a composition of the disclosure, preventing a disease or condition; curing a disease or condition; delaying the onset of a disease or condition; delaying the onset of a symptom, complication, or sequelae; reducing the severity of a disease or condition; reducing the severity of a symptom, complication, or sequelae; ameliorating one or more symptoms; ameliorating one or more complications; ameliorating one or more sequelae; preventing one or more symptoms; preventing one or more complications; preventing one or more sequelae; delaying one or more symptoms; delaying one or more symptoms; delaying one or more complications; delaying one or more sequelae; "relieve / ameliorate one or more symptoms; reduce / ameliorate one or more complications; reduce / ameliorate one or more sequelae; shorten the duration of one or more symptoms; shorten the duration of one or more complications; shorten the duration of one or more sequelae; reduce the frequency of one or more symptoms; reduce the frequency of one or more complications; shorten the frequency of one or more sequelae; reduce the severity of one or more symptoms; reduce the severity of one or more complications; reduce the severity of one or more sequelae; improve quality of life; increase survival; prevent recurrence of a disease or condition; delay the recurrence of a disease or condition; or any combination thereof."

[0063] Dietary management and / or secondary dietary prevention : These terms refer to specialized or partial diets of patients who have a limited, impaired or hindered ability to ingest, digest, absorb, metabolize or excrete normal foods or certain nutrients or metabolites contained therein due to a disease, disorder or condition from which the patient suffers, or have other medically determined nutritional requirements. As used herein, "treating" or "treatment" includes dietary management and / or secondary prevention through diet.

[0064] Symptoms: As used herein, this term refers to subjective or physical signs, symptoms, or evidence of a disease or physical disorder observed by a subject. In general, this term refers to any pathological phenomenon or deviation from normal in structure, function, or sensation experienced by a patient that indicates a disease. A symptom may be felt or noticed by the individual experiencing the symptom, but may not be easily noticed by others. In some embodiments, a symptom may be a mild symptom, a moderate symptom, or a severe symptom. As used herein, the term "mild symptom" refers to a symptom that is not life-threatening, e.g., does not require intensive care. As used herein, the term "moderate symptom" refers to a symptom that may become life-threatening and require monitoring, e.g., hospitalization. As used herein, the term "severe symptom" refers to a symptom that is life-threatening, e.g., requires intensive care.

[0065] complications : As used herein, this term refers to a pathological process or event that occurs during a disease or condition that is not an essential part of the disease or condition; it may result from the disease / condition or an independent cause. For example, treating a condition with antibiotics or nonsteroidal anti-inflammatory drugs can cause epithelial damage in the intestine as a side effect, resulting in increased permeability. This increased permeability can lead to an increased risk of allergic, inflammatory or metabolic diseases as long-term complications. In some aspects, the complications can be temporary. In some aspects, the complications can be chronic or permanent. As used herein, the term "sequelae" refers to long-term, chronic or permanent complications.

[0066] Intestinal Barrier : As used herein, this term refers to the functional entity that separates the intestinal lumen from the internal host and consists of mechanical elements (mucus, epithelial layer), humoral elements (defensins, IgA), immunological elements (lymphocytes, innate immune cells), muscles, neurological elements and the microbiota.

[0067] intestinal permeability: As used herein, this term refers to the functional characteristics of the intestinal barrier at a given site, which can be measured, inter alia, by analyzing the flux rate across the entire intestinal wall or wall components. Intestinal permeability refers to the control of the passage of materials from within the digestive tract through the cells lining the intestinal wall to the rest of the body. A healthy intestine exhibits selective permeability, which allows nutrients to pass through the intestine while maintaining a barrier function that prevents potentially harmful substances (such as antigens) from leaving the intestine and moving more widely into the body.

[0068] Normal intestinal permeability : As used herein, this term refers to the stable permeability seen in healthy individuals with no signs of intoxication, inflammation or intestinal dysfunction.

[0069] Intestinal barrier dysfunction The terms "intestinal barrier dysfunction", "intestinal permeability disorder", "intestinal permeability imbalance" and "abnormal intestinal permeability" are used interchangeably and refer to permeability disturbances that are not transiently altered compared to normal permeability and that lead to loss of intestinal homeostasis, dysfunction and disease.

[0070] Increased intestinal permeability As used herein, the term "intestinal hyperpermeability" refers to a condition in which junctions in the intestinal epithelial wall lose their integrity, allowing materials from the lumen to pass into the bloodstream, other organs, or adipose tissue. When tight junctions in the intestinal wall loosen, the intestine becomes more permeable, allowing bacteria and toxins to pass from the intestine into the bloodstream. This phenomenon, for example, is commonly referred to as "leaky gut."

[0071] Intestinal hyperpermeability is a factor in several diseases, such as Crohn's disease, celiac disease, type 1 diabetes, type 2 diabetes, rheumatoid arthritis, spondyloarthropathy, inflammatory bowel disease, irritable bowel syndrome, schizophrenia, certain types of cancer, obesity, fatty liver, atopic and allergic diseases. In most cases, hyperpermeability precedes the disease, but the causal relationship of hyperpermeability to most of these diseases is unclear. For this reason, the term "intestinal barrier dysfunction (e.g., hyperpermeability and related conditions)" is used herein.

[0072] "Intestinal barrier", "intestinal permeability", "normal intestinal permeability", "intestinal barrier dysfunction", and "reduced / enhanced intestinal permeability" are terms also defined in Bischoff et al., 2014.

[0073] Human Milk Oligosaccharides :The term HMO, abbreviated as HMO and also known as "human milk glycans", collectively refers to the oligosaccharides present in human milk, which constitute the third largest solid component in human milk after lactose and fat. HMOs are short polymers of monosaccharides, usually consisting of a carbohydrate core that often contains lactose at the reducing end and fucose or sialic acid at the non-reducing end. HMOs are present in human milk at concentrations between 11.3 and 17.7 g / L, depending on the stage of lactation. About 200 HMOs with different structures are known and can be classified according to different classifications, for example, fucosylated, sialylated and neutral core HMOs. The composition of human milk oligosaccharides in breast milk varies for each mother and changes over the lactation period. The predominant oligosaccharide in 80% of all women is 2'-fucosyllactose, which is present in human breast milk at a concentration of about 2.5 g / L. Other abundant oligosaccharides include lacto-N-tetraose, lacto-N-neotetraose, and lacto-N-fucopentaose.

[0074] synthetic mixture : means a mixture obtained by chemical and / or biological means and may be chemically identical to a mixture naturally occurring in, for example, mammalian milk. All compositions described herein are synthetic mixtures.

[0075] Nutritional composition : This term refers to a composition that provides nutrition to a subject. The nutritional composition should usually be administered orally or intravenously and usually contains a lipid or fat source and a protein source. In particular, the nutritional composition is a complete nutritional mixture (e.g., infant formula) that meets all or most of the nutritional needs of a subject. The nutritional composition includes food.

[0076] Infant formula: This term, as used herein, refers to food intended for specific nutritional use by infants during the first few months of life and which alone meets the nutritional requirements of this category of person (Article 2(c) of Commission Directive 91 / 321 / EEC 2006 / 141 / EC of 22 December 2006 on infant formulae and follow-on formulae). It also refers to nutritional compositions intended for infants and defined in Codex Alimentarius (Codex STAN 72-1981) and special products for infants (including foods for special medical purposes). The term "infant formula" encompasses, but is not limited to, the following forms:

[0077] Starter formula : means a food intended for a specific nutritional use by infants aged 6 months.

[0078] Follow-up or follow-on formula It may be given from the sixth month onwards. It constitutes the main liquid component of the increasingly diversified diet of this category of individuals.

[0079] Infant formulas, follow-on formulas and starter infant formulas can be either in liquid ready-to-consume or concentrated form, or in dry powder form that can be reconstituted to form formula upon addition of water. Such formulas are well known in the art.

[0080] baby food : means a food intended for a specific nutritional use by an infant or young child during the first few years of life.

[0081] Infant cereal composition : means a food intended for a specific nutritional use by an infant or young child during the first few years of life.

[0082] Reinforcement :Refers to liquid or solid nutritional compositions suitable for mixing with breast milk or infant formula.

[0083] Growing Up Milk : means a milk-based beverage adapted to the specific nutritional needs of young children.

[0084] Weaning : The period during which breast milk is replaced by other foods in an infant's diet.

[0085] Enteral Administration By "administration" is meant any conventional form for delivering a composition to a non-infant that deposits the composition in the digestive tract (including the stomach).

[0086] Oral route : means any conventional form for delivering a composition to a non-infant via the mouth. Thus, oral administration is a form of enteral administration.

[0087] Probiotic Composition In one embodiment, the probiotic composition comprises Bifidobacterium longum subsp. longum, deposited under accession number CECT7894.

[0088] Bifidobacterium longum subsp. longum CECT7894 strain has been described in WO2015018883A2, the contents of which are incorporated herein by reference in their entirety. This strain was deposited at the Spanish Type Culture Collection (CECT, Parc Cientific de la Universitat de Valencia, Carrer del Catedratic Agustin Escardino Benlloch, 9, 46980 Paterna, Valencia, Spain) on March 30, 2011 (30.03.2011) under the accession number CECT7894. The deposit was made under the conditions of the Budapest Treaty and is viable and maintains all its functions related to the deposit. It was deposited by the same applicant.

[0089] Bifidobacterium longum subsp. longum CECT7894 (also referred to herein as KABP-042) was isolated from the feces of a healthy breast-fed infant. In silico and in vitro analyses of CECT7894 have been performed to study the probiotic properties of this strain, confirming that it can withstand human gastrointestinal challenges (gastric conditions and bile salts) and adhere to the intestinal epithelium. Genotypic analysis confirmed these characteristics.

[0090] Human milk oligosaccharides (HMOs) are complex sugars found in human milk, and their utilization is strain-specific in bifidobacteria. It has been found herein that B. longum subsp. longum CECT7894 is able to utilize the HMO lacto-N-tetraose in vitro, one of the most common HMOs found in breast milk. At the same time, its genome carries most of the typical HMO degradation genes, including lacto-N-biosidase, beta-galactosidase, alpha-galactosidase, hexosaminidase and beta-glucuronidase. This analysis confirms that this strain is able to utilize HMOs and thus is compatible with the infant gut.

[0091] Furthermore, B. longum subsp. longum CECT7894 has a diverse carbohydrate metabolism, as other genes in its genome code for carbohydrate-active enzymes (CAZy), suggesting that it can degrade a wide range of complex substrates. In addition, genes encoding lantipeptide B, serpins and adhesins are also present in the genome of B. longum subsp. longum CECT7894. Lantipeptide B (a lantibiotic) is a class I bacteriocin that exhibits potent antibacterial activity against a range of Gram-negative and Gram-positive pathogens. Serpins selectively inactivate human neutrophil and pancreatic elastase (a protease), providing anti-inflammatory effects and contributing to the maintenance of intestinal homeostasis.

[0092] Overall, the phenotypic and genotypic analysis of B. longum subsp. longum CECT7894 herein confirms that the strain has the ability to degrade HMOs and is therefore well adapted to the human gastrointestinal tract, including the infant gut.

[0093] As will be understood by those skilled in the art in this context, a bacterial strain is isolated from its natural environment, i.e. it is not present in its natural environment and therefore does not contain other organisms and substances present in its natural environment.

[0094] The emergence and spread of resistance to antimicrobial drugs in bacteria poses a threat to human and animal health and results in large financial and societal costs. Whole genome sequencing revealed that the novel strain B. longum subsp. longum CECT7894 does not harbor transferable antibiotic resistance genes to commonly used antibiotics. Overall, these results rule out the risk of potential transfer of antibiotic resistance to pathogenic species.

[0095] It is clear that by using the deposited strains as starting material, the skilled artisan can routinely obtain further variants or mutants thereof by conventional mutagenesis or reisolation techniques, which retain, enhance or improve the relevant characteristics and advantages described herein of the strains forming the compositions of the present invention. Therefore, the present invention also relates to variants / mutants of the strains disclosed herein. In an embodiment, the probiotic composition comprises a bacterial strain derived from Bifidobacterium longum subsp. longum CECT7894 strain, the derived bacterial strain being (a) has a genome that has at least 99% average nucleotide identity (ANI) with the genome of the corresponding deposited strain CECT7894; and (b) maintain, enhance or improve the polyphosphate producing ability of the corresponding deposited strain.

[0096] In certain embodiments, bacterial strains derived from the deposited strains have genomes that have an average nucleotide identity (ANI) of at least 99% with the genome of the corresponding deposited strain, more particularly the percentage (%) of identity is 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8% or 99.9%. In particular, the % ANI is at least 99.5%. More specifically, the % of ANI is 99.50%, 99.51%, 99.52%, 99.53%, 99.54%, 99.55%, 99.56%, 99.57%, 99.58%, 99.59%, 99.60%, 99.61%, 99.62%, 99.63%, 99.64%, 99.65%, 99.66%, 99.67%, 99.68%, 99.69%, 99.70%, 99.71%, 99.72%, 99.73%, 99. .74%, 99.75%, 99.76%, 99.77%, 99.78%, 99.79%, 99.80%, 99.81%, 99.82%, 99.83%, 99.84%, 99.85%, 99.86%, 99.87%, 99.88%, 99.89%, 99.90%, 99.91%, 99.92%, 99.93%, 99.94%, 99.95%, 99.96%, 99.97%, 99.98% or 99.99%. In another embodiment, the % of ANI is at least 99.9%; in particular, the % of ANI is 99.91%, 99.92%, 99.93%, 99.94%, 99.95%, 99.96%, 99.97%, 99.98% or 99.99%.

[0097] In some embodiments, the mutants are obtained by naturally occurring mutagenesis, artificial directed mutagenesis, or artificial random mutagenesis. In one particular embodiment, the bacterial strain derived from the deposited strain is obtained by using recombinant DNA technology. Therefore, another aspect of the present invention relates to a method for obtaining a strain derived from the deposited strain, comprising using the deposited strain as starting material and applying mutagenesis, wherein the obtained variants or mutants further retain, enhance, or improve at least one capability of the deposited strain disclosed herein.

[0098] The strain-forming portion of the composition of the present invention may be in the form of viable cells. Alternatively, the strain may be in the form of non-viable cells. This may include microorganisms killed by heat, or by exposure to pH changes, sonication, radiation, or high pressure. With non-viable cells, product preparation is simpler, as the cells can be easily incorporated into dietary, pharmaceutical, or edible products, and storage requirements are much less restrictive than with viable cells. Compositions containing the strains of the present invention as non-viable cells may include products derived from the strains in culture media.

[0099] The strains disclosed herein are produced by culturing (or fermenting) bacteria in a suitable artificial medium and under suitable conditions. The expression "artificial medium" is understood to be a medium that contains natural substances and optionally synthetic chemicals such as polyvinyl alcohol, a polymer that can reproduce some of the functions of serum. A generally suitable artificial medium is a nutrient broth that contains elements necessary for bacterial growth, including a carbon source (e.g., glucose), a nitrogen source (e.g., amino acids and proteins), water and salts. The growth medium can be in liquid form or can often be mixed with agar or another gelling agent to obtain a solid medium. The strains can be cultivated alone to form a pure culture, or as a mixed culture with other microorganisms, or by culturing different types of bacteria separately and then combining them in the desired ratio. After cultivation and depending on the final preparation, the strains may be used as purified bacteria, or alternatively the bacterial culture or cell suspension may be used as such or after suitable post-treatment. In this specification, the term "biomass" is understood to be the bacterial strain culture obtained after cultivation (or fermentation, a term synonymous with cultivation).

[0100] In a particular embodiment, the strain is fermented in an artificial medium and subjected to a post-treatment after fermentation to obtain bacterial cells, the bacterial cells being in a liquid medium or in a solid form, in particular the post-treatment is selected from the group consisting of drying, freezing, lyophilization, fluidized bed drying, spray drying and refrigeration in a liquid medium, more particularly lyophilization.

[0101] The term "post-treatment" should be understood in the present context as any treatment carried out on a biomass with the aim of obtaining bacterial cells that can be preserved. The purpose of the post-treatment is to reduce the metabolic activity of the cells in the biomass and thus slow down the rate of harmful reactions of the cells. As a result of the post-treatment, the bacterial cells can be in solid or liquid form. In solid form, the preserved bacterial cells can be powder or granules. In any case, both solid and liquid forms containing bacterial cells do not exist in nature and therefore do not exist in nature, since they are the result of an artificial post-treatment process(es). The post-treatment process may require the use of one or more so-called post-treatment agents in certain embodiments. In the context of the present invention, the expression "post-treatment agent" refers to the compounds used to carry out the post-treatment process described herein. Post-treatment agents include, but are not limited to, dehydrating agents, bacteriostatic agents, cryoprotective agents (cryoprotectants), inert fillers (also known as lyoprotectants), carrier materials (also known as core materials), etc., used alone or in combination.

[0102] There are two basic approaches to reduce the metabolic activity of bacterial cells, and therefore two approaches to perform post-treatment. The first is to reduce the rate of all chemical reactions, which can be done by reducing the temperature by refrigeration or freezing using refrigerators, mechanical freezers, and liquid nitrogen freezers. Alternatively, reducing the rate of all chemical reactions can be achieved by adding substances that inhibit the growth of bacterial cells, i.e. bacteriostatic agents, abbreviated as Bstatic.

[0103] The second approach to post-treatment is to remove water from the biomass, a process that may involve sublimation of water using a freeze dryer. Suitable techniques for removing water from the biomass are drying, freeze drying, spray drying or fluidized bed drying. Post-treatments that result in a solid form may be drying, freezing, freeze drying, fluidized bed drying, or spray drying.

[0104] A post-treatment is in particular freeze-drying, which involves the removal of water from the frozen bacterial suspension by sublimation under reduced pressure. This process consists of three steps: pre-freezing the product to form a frozen structure, primary drying to remove most of the water, and secondary drying to remove the bound water. Due to the objective and expected variability of the industrial processes for the production and isolation of freeze-dried bacterial cultures, the latter generally contain a certain amount of inert filler, also known as cryoprotectant. Its role is to standardize the content of live probiotic bacteria in the product. In commercially available freeze-dried cultures, the following inert fillers are used: sucrose, saccharose, lactose, trehalose, glucose, maltose, maltodextrin, corn starch, inulin, and other pharma- ceutically acceptable non-hygroscopic fillers. If necessary, other stabilizers or cryoprotectants, such as ascorbic acid, are also used to form a viscous paste, which is then subjected to freeze-drying. In any case, the material so obtained can be ground to a suitable size, including a powder.

[0105] As an alternative to storing the biomass in solid form, it may be stored in liquid form, which can be done by adding a bacteriostatic agent to the medium as described above to stop bacterial growth, or by an intermediate step of harvesting the cells and resuspending the pellet in saline with a bacteriostatic agent, optionally refrigerated.

[0106] Sometimes, the probiotic composition is subjected to an immobilization and / or coating or encapsulation process to improve shelf life and / or functionality, for example as described above in the fluidized bed drying process. Several techniques for immobilization, coating or encapsulation of bacteria are known in the art.

[0107] In other embodiments, the probiotic composition is formulated for sustained release administration, for example, by encapsulation in liposomes, microbubbles, microparticles, or microcapsules. Suitable sustained release forms and materials and methods for their preparation are well known in the art. Thus, any orally administrable form of the probiotic composition of the present invention is a sustained release form further comprising at least one coating or matrix. The sustained release coating or matrix includes, but is not limited to, natural semi-synthetic or synthetic polymers, water-insoluble or processed waxes, fats, fatty alcohols, fatty acids, natural, semi-synthetic or synthetic plasticizers, or combinations of two or more thereof. The enteric coating can be applied using conventional processes known to those skilled in the art.

[0108] The effective amount of colony forming units (cfu) for a bacterial strain in a composition is determined by one of skill in the art and depends on the final formulation. The term "colony forming unit" ("cfu") is defined as the number of bacterial cells revealed by the microbial count on an agar plate.

[0109] As known to those skilled in the art, the effective amount of colony units can also be measured by the effective amount of active fluorescent units. The term "active fluorescent units" ("afu") is defined as the number of bacterial cells revealed by flow cytometry counting within a gate specific to the fluorescent properties of presumably viable cells. Thus, those skilled in the art consider the above-mentioned specific amount of cfu to be approximately the same amount of afu.

[0110] In one embodiment, the probiotic composition is a solid composition, hi another embodiment, the probiotic composition is a liquid composition.

[0111] In another embodiment, the probiotic composition comprises about 10 5 cfu ~ approx. 10 12 cfu of the strain, more particularly about 10 8 cfu ~ approx. 10 11The freeze-dried bacterial biomass containing strains of cfu.

[0112] In an embodiment, the probiotic composition comprises a cryoprotectant. In particular, the probiotic composition comprises at least one cryoprotectant that is an allergen-free cryoprotectant. In some embodiments, the probiotic composition comprises at least one cryoprotectant, such as maltose, trehalose, mannitol (particularly d-mannitol), sucrose, lactose, dextrose, sodium ascorbate, sodium citrate, L-cysteine, maltodextrin, anhydrous dextrose, starch, cellulose, and inulin. In a particular embodiment, the cryoprotectant and / or the pharma- ceutically acceptable carrier is selected from the group consisting of trehalose, D-mannitol, dextrose, sodium ascorbate, sodium citrate, L-cysteine, maltodextrin, starch, and cellulose. In particular, the starch is corn, corn starch, and / or potato starch.

[0113] More particularly, the composition further comprises a pharma- ceutically acceptable carrier selected from an emulsion, a suspension, a gel, a paste, a granule, a powder, and a gum. In particular, the carrier is an allergen-free carrier.

[0114] In some embodiments, the probiotic composition comprises one or more carriers selected from the group consisting of maltodextrin, cellulose, various starches, inulin, lactose, or reduced water activity carriers.

[0115] In certain embodiments, the probiotic composition comprises: -about 10 5 cfu ~ approx. 10 12 freeze-dried bacterial biomass containing strains of cfu; - a cryoprotectant and / or a pharma- ceutically acceptable carrier selected from emulsions, suspensions, gels, pastes, granules, powders, and gums; A composition comprising:

[0116] Polyphosphate Production In one embodiment, the production of polyphosphate by Bifidobacterium longum subsp. longum CECT7894 or a derived bacterial strain thereof comprises the steps of: (a) culturing the strain inoculated at OD 0.1 in malic enzyme induction medium (MEI) containing 0.5% yeast extract, 0.5% tryptone, 0.4% K2HPO4, 0.5% KH2PO4, 0.02% MgSO4·7H2O, 0.005% MnSO4, 1 ml Tween 80, 0.05% cysteine, and 0.5% glucose at 37°C under anaerobic conditions; (b) harvesting the cells by centrifugation and lysing them in 1 ml of 5% sodium hypochlorite with gentle agitation at room temperature for 45 minutes; (c) centrifuging the insoluble material at 16,000 g for 5 minutes at 4° C. to obtain a pellet, which is then washed twice with 1 ml of 1.5 M NaCl plus 1 mM EDTA, with centrifugation at 16,000 g for 5 minutes at 4° C. in between; (d) extraction of polyP from the pellet by two successive washes with 1 ml of water, with centrifugation at 16,000 g for 5 min at 4° C. in between; (e) precipitating polyP in the pooled aqueous extracts by adding 0.1 M NaCl and 1 volume of ethanol, followed by incubation on ice for 1 hour; (f) centrifugation at 16,000 g for 10 minutes and resuspending the polyP pellet in 50 μL of water; (g) Process: i. Hydrolysis of serial dilutions of samples of polyP isolated from a polyP-producing control strain, such as Lactobacillus plantarum WCFS1 (Alcantara et al. 2014), with one volume of 2 M HCl and incubation at 95 °C for 15 min; ii. neutralizing the dilution by adding half the volume of 2M NaOH; iii. measuring the released phosphate with BIOMOL Green kit to obtain the amount of phosphate in each dilution; iv. measuring the released phosphate by fluorescence using 4',6-diamidino-2-phenylindole, DAPI, at a final concentration of 10 μM in 50 mM Tris-HCl pH 7.5, 50 mM NaCl buffer, at an excitation wavelength of 415 nm and an emission wavelength of 550 nm in a fluorometer, obtaining a fluorescence value for each dilution; and v. generating a calibration curve using the phosphate values ​​obtained in (iii) and the corresponding fluorescence values ​​obtained in (iv); preparing a calibration curve relating the amount of phosphate derived from polyP to fluorescence intensity according to the method of the present invention; and (h) Quantifying polyP from the resuspended fraction of step (f): 1) measuring polyP by fluorescence using DAPI at a final concentration of 10 μM in 50 mM Tris-HCl pH 7.5, 50 mM NaCl buffer at an excitation wavelength of 415 nm and an emission wavelength of 550 nm in a fluorometer; 2) calculating the amount of polyP using a calibration curve; and 3) Expressing polyP values ​​in nmol phosphate This is greater than the polyphosphate production of the control strain as determined by HPLC at 6 and / or 16 h incubation.

[0117] Example 1 herein (Materials and Methods section 1.1.2) provides a detailed description of a suitable assay to quantify polyP and thus assess the ability of a bacterial strain to produce polyP, with reference to steps (a)-(h) of an embodiment of the invention.

[0118] It is pertinent to note that the description and conditions of the polyP quantification assay disclosed in steps (a)-(h) of the embodiment of the present invention are not intended to limit the scope of the present invention. The assay is one suitable method to test the ability of a bacterial strain (e.g., B. longum subsp. longum CECT7894) to produce polyP. The detailed conditions of this Example 1 form herein a specific assay for determining whether a (derived) bacterial strain of interest meets the criteria of the embodiment of the present invention.

[0119] Thus, based on the detailed assay described herein, one of skill in the art can routinely repeat this assay to objectively determine whether a specific bacterial strain of interest has the ability to produce the polyP of the present embodiments.

[0120] As mentioned before, polyP production can be quantified by the above method. Such a method consists of three main steps, starting with polyP extraction from cells with sodium hypochlorite, staining the extracted polyP with DAPI, and quantifying the fluorescence of the samples. The amount of PolyP is inferred from a standard curve correlating the amount of phosphate derived from polyP with fluorescence units. This method is an indirect polyP quantification method through the measurement of phosphate by fluorescence.

[0121] In some embodiments, quantification of polyP can be performed by alternative indirect polyP quantification methods. In a particular embodiment, the amount of phosphate released from polyP hydrolysis is measured with the BIOMOL Green kit for all samples, i.e., both the control strain and the strain of the invention. In another particular embodiment, quantification of polyP is performed by adding PPK enzyme to obtain phosphate from polyP catabolism.

[0122] In some embodiments, polyP production of the bacterial strains of the invention or derived bacterial strains thereof is greater than the control strain when determined at 6 and / or 16 hours of culture, considering the same initial inoculum size for all strains. In particular, polyP production is greater when determined at the 6 and 16 hour time points. In other embodiments, polyP production is greater when determined at one or more time points, e.g., at 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 and / or 20 hours of culture.

[0123] As understood herein and in accordance with the present invention, a control strain is, for example, at least one of the following control strains: L. plantarum WCFS1 and L. paracasei JCM1163, which are known to produce polyP; B. breve JCM1273, B. adolescentis JCM1275 and B. longum subsp. longum ATCC15707, which are known to be able to remove phosphate; and B. scardovi DSMZ13734 (BAA-773), which is known to carry the ppk gene.

[0124] In certain embodiments, the control strain is, for example, L. plantarum WCFS1, L. paracasei JCM1163, B. breve JCM1273, B. adolescentis JCM1275, B. longum subsp. longum ATCC15707, or B. scardovi DSMZ13734 (BAA-773).

[0125] When using the described assay, in some embodiments, the level of polyP produced by B. longum subsp. longum CECT7894 or its derived bacterial strain at 6 and 16 hours is higher than the polyP production of the control strain L. plantarum WCFS1 at the same time points.

[0126] In some embodiments, the level of polyP produced by B. longum subsp. longum CECT7894 or a derived bacterial strain thereof at 6 hours is at least, e.g., 1.2-fold, 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 15-fold, 20-fold, 30-fold, 50-fold or 100-fold higher than polyP production of the control strain.

[0127] In certain embodiments, the level of polyP produced by B. longum subsp. longum CECT7894 or a derived bacterial strain thereof is at least 10-fold higher than the polyP production of the control strain L. plantarum WCFS1. In particular, the level of polyP produced by B. longum subsp. longum CECT7894 or a derived bacterial strain thereof at 6 hours is at least 15-fold or 18-fold higher than the polyP production of the control strain L. plantarum WCFS1.

[0128] In certain embodiments, the level of polyP produced by B. longum subsp. longum CECT7894 or a derived bacterial strain thereof at 6 hours is at least 3-fold higher than the polyP production of the control strain B. breve JCM1273.

[0129] In certain embodiments, the level of polyP produced by B. longum subsp. longum CECT7894 or its derived bacterial strain at 6 hours is at least 4-fold higher than the polyP production of the control strain B. adolescentis JCM1275. In particular, the level of polyP produced by B. longum subsp. longum CECT7894 or its derived bacterial strain at 6 hours is at least 4.5-fold higher than the polyP production of the control strain B. adolescentis JCM1275.

[0130] In certain embodiments, the level of polyP produced by B. longum subsp. longum CECT7894 or a derived bacterial strain thereof at 6 hours is at least 100-fold higher than the polyP production of the control strain B. scardovii DSMZ13734 (BAA-773). In particular, the level of polyP produced by B. longum subsp. longum CECT7894 or a derived bacterial strain thereof at 6 hours is at least 120-fold, 130-fold, or 140-fold higher than the polyP production of the control strain B. scardovii DSMZ13734 (BAA-773).

[0131] In some embodiments, the level of polyP produced by B. longum subsp. longum CECT7894 or a derived bacterial strain thereof at 16 hours is at least, e.g., 1.2-fold, 1.5-fold, 2-fold, 2.5-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 15-fold, 20-fold, 30-fold, 50-fold, or 100-fold, 200-fold, 300-fold, 400-fold, 500-fold, or 600-fold higher than polyP production of the control strain.

[0132] In certain embodiments, the level of polyP produced by B. longum subsp. longum CECT7894 or a derived bacterial strain thereof at 16 hours is higher than the polyP production of the control strain, and the level of polyP by the control strain at 16 hours is absent, particularly when the control strain is L. plantarum WCFS1.

[0133] In certain embodiments, the level of polyP produced by B. longum subsp. longum CECT7894 or a derived bacterial strain thereof at 16 hours is at least 2-fold higher than the polyP production of the control strain B. breve JCM1273.

[0134] In certain embodiments, the level of polyP produced by B. longum subsp. longum CECT7894 or a derived bacterial strain thereof at 16 hours is at least 2.5-fold higher than the polyP production of the control strain B. adolescentis JCM1275.

[0135] In certain embodiments, the level of polyP produced by B. longum subsp. longum CECT7894 or a derived bacterial strain thereof at 16 hours is at least 500-fold higher than the polyP production of the control strain B. scardovii DSMZ13734 (BAA-773).

[0136] In certain embodiments, the level of polyP produced by B. longum subsp. longum CECT7894 or a derived bacterial strain thereof is at least 10-fold higher than the polyP production of the control strain L. plantarum WCFS1 at 6 hours, and higher at 16 hours, and the polyP production of the control strain L. plantarum WCFS1 and the level of polyP by the control strain are absent at 16 hours. In particular, the level of polyP produced by B. longum subsp. longum CECT7894 or a derived bacterial strain thereof is at least 15-fold or 18-fold higher than the polyP production of the control strain L. plantarum WCFS1 at 6 hours, and higher at 16 hours, and the polyP production of the control strain L. plantarum WCFS1 and the level of polyP by the control strain are absent at 16 hours.

[0137] In certain embodiments, the level of polyP produced by B. longum subsp. longum CECT7894 or a derived bacterial strain thereof is at least 100-fold higher at 6 hours and at least 500-fold higher at 16 hours than the polyP production of the control strain B. scardovii DSMZ13734 (BAA-773). In particular, the level of polyP produced by B. longum subsp. longum CECT7894 or a derived bacterial strain thereof is at least 120-fold, 130-fold or 140-fold higher at 6 hours and at least 500-fold higher at 16 hours than the polyP production of the control strain B. scardovii DSMZ13734 (BAA-773).

[0138] Combination of probiotic composition and HMO The term probiotic composition as used herein refers to a composition comprising Bifidobacterium longum subsp. longum strain CECT7894 or a derived bacterial strain thereof in the above terms.

[0139] As mentioned above, according to aspects of the present invention, the probiotic compositions described herein may further comprise at least one human milk oligosaccharide in combination.

[0140] Because the probiotic composition and the HMO can be formulated together in a single composition or in separate compositions, when the embodiments described below refer to a "composition of the invention" or simply a "composition," they refer to a probiotic composition, a composition comprising an HMO, and a composition comprising both.

[0141] In some embodiments, the compositions of the invention comprise an additional probiotic different from B. longum CECT7894 or a derived bacterial strain thereof capable of degrading HMOs, i.e., lacto-N-tetraose (LNT). In certain embodiments, the additional probiotic strain is a bifidobacterium, more particularly Bifidobacterium bifidum or Bifidobacterium longum subsp. infantis.

[0142] In a particular embodiment, the Bifidobacterium bifidum is B. bifidum deposited under CECT30646. The strain was deposited at the Spanish Type Culture Collection (CECT, Parc Cientific de la Universitat de Valencia, Carrer del Catedratic Agustin Escardino Benlloch, 9, 46980 Paterna, Valencia, Spain) on May 17, 2022 (17.05.2022) under the accession number CECT30646. The deposit was made under the conditions of the Budapest Treaty and is viable and maintains all its characteristics pertaining to the deposit. It was deposited by the same applicant. B. bifidum CECT30646 (also referred to herein as Bb01) was isolated from human breast milk.

[0143] In one embodiment, the composition of the invention comprises an HMO selected from the group consisting of fucosylated oligosaccharides, sialylated oligosaccharides, N-acetyllactosamines, and combinations thereof.

[0144] In certain embodiments, the composition comprises fucosylated oligosaccharides (particularly 2'-fucosyllactose (2-FL) and / or difucosyllactose (DFL)) and N-acetyllactosamine (particularly lacto-N-tetraose (LNT)).

[0145] HMOs can be isolated or concentrated from milk(s) secreted by mammals, including but not limited to human, bovine, ovine, porcine, or caprine species, particularly humans, by well-known processes. HMOs can also be produced by well-known processes using microbial fermentation, enzymatic processes, chemical synthesis, or a combination of these techniques.

[0146] The HMO may, for example, be dissolved, emulsified, or suspended in water in the compositions of the present invention.

[0147] In one embodiment, the HMOs are present in the composition in a total amount of 0.1-50 g / L, or 0.3-5 g / L, or 0.5-1 g / L, or 0.25 or 0.5 or 1 or 1.5 or 2 g / L.

[0148] Fucosylated oligosaccharides The composition according to the invention may comprise one or more fucosylated oligosaccharides. In particular, the fucosylated oligosaccharides comprise 2'-fucosyllactose (2'-FL) and / or difucosyllactose (DFL).

[0149] In some embodiments, the fucosylated oligosaccharides are selected from the group including 2'-fucosyllactose (2'-FL), 3-fucosyllactose (3-FL), difucosyllactose (DFL), lacto-N-fucopentaose (i.e., LNFP I, II, III and V), lacto-N-difucohexaose (LNDFH I and II), lacto-N-difucohexaose III (LNDFH-III), fucosyl-lacto-N-hexaose (FLNH I and II), fucosyl-lacto-N-neohexaose (FLNnH), difucosyllacto-N-hexaose I, difucosyllacto-N-neohexaose (I and II) and fucosyl-paralacto-N-hexaose (FpLNH I and II). Particular fucosylated oligosaccharides are 2-FL or DFL or a mixture thereof.

[0150] Fucosylated oligosaccharides can be isolated from natural sources, such as animal milk, by chromatography or filtration techniques. Alternatively, they can be produced by biotechnological means using specific fucosyltransferases and / or fucosidases, either through the use of enzyme-based fermentation techniques (recombinant or natural enzymes) or microbial fermentation techniques. In the latter case, microorganisms can express the native enzymes and substrates or can be engineered to produce the respective substrates and enzymes. Monomicrobial cultures and / or mixed cultures can be used. Alternatively, fucosylated oligosaccharides are produced by chemical synthesis from lactose and free fucose. Fucosylated oligosaccharides are also available, for example, from Kyowa Hakko Kogyo Co., Ltd., Japan.

[0151] In particular, the composition according to the invention comprises from 0.02 to 10 g of fucosylated oligosaccharide(s) per 100 g of composition on a dry weight basis, most particularly 2FL, for example from 0.2 to 0.5 g or from 0.3 to 5 g of 2FL per 100 g of composition on a dry weight basis, and in particular from 0.1 to 3 g of 2FL per 100 g of composition on a dry weight basis.

[0152] In some embodiments, the composition comprises the following ranges or amounts of 2FL: 0.05-20g or 0.1-5g or 0.2-3g or 0.1-2g or 0.25g-1g or 0.25g or 1g per liter of composition if in ready-to-use liquid form, or 0.05-20g or 0.1-5g or 0.2-3g or 0.1-2g or 0.25g-1g or 0.25g or 1g per liter of composition (in liquid diluted form) if the composition is in powder form and intended to be reconstituted into a diluted liquid form, or 0.05-20g or 0.1-5g or 0.2-3g or 0.1-2g or 0.25g-1g or 0.25g or 1g per liter of composition ... (in liquid diluted form), or 0.05-20g or 0.1-5g or 0.2-3g or 0.1-2g or 0.25g-1g or 0.25g or 1g per liter of composition if the composition is in powder form and intended to be reconstituted into a diluted liquid form, or 0.05-20g or 0.1-5g or 0.2 If the nutritional composition is in the form of a concentrated composition intended to be diluted (0, 50, or 100 times) or intended to be used directly as a concentrated form, it contains the above multiplied by 2, 5, 10, 20, 50 or 100, or if the nutritional composition is in the form of a dry powder, it contains 0.04g to 1.5g per 100g of nutritional composition powder, or 0.08 to 1.2g per 100g of nutritional composition powder, or 0.1 to 1g per 100g, or 0.2 to 0.8g per 100g, or 0.2g per 100g, or 0.4g per 100g, or 0.8g per 100g, or 1g per 100g, or 1g per 100g.

[0153] N-acetyllactosamine In some embodiments, the compositions of the present invention comprise at least one N-acetyllactosamine, i.e., the compositions comprise N-acetyllactosamine and / or oligosaccharides containing N-acetyllactosamine. Suitable oligosaccharides containing N-acetyllactosamine include lacto-N-tetraose (LNT), lacto-N-neotetraose (LNnT), lacto-N-neohexaose (LNnH), paralacto-N-neohexaose (pLNnH), paralacto-N-hexaose (pLNH) and lacto-N-hexaose (LNH).

[0154] In one embodiment, the composition according to the invention comprises an N-acetyllactosamine, in particular selected from the group comprising lacto-N-tetraose (LNT) and lacto-N-neotetraose (LNnT).

[0155] LNT and LNnT can be chemically synthesized by enzymatic transfer of a glycan unit from a donor moiety to an acceptor moiety using glycosyltransferases. Alternatively, LNT and LNnT can be prepared by chemical conversion of a ketohexose (e.g., fructose) either free or bound to an oligosaccharide (e.g., lactulose) to an N-acetylhexosamine or an N-acetylhexosamine-containing oligosaccharide. The N-acetyllactosamine thus produced can be transferred to lactose as an acceptor moiety. LNT can also be produced by microbial fermentation, for example, using a genetically engineered strain of Escherichia coli K-12 recently approved by EFSA.

[0156] In particular, the composition according to the invention comprises 0.01-3 g N-acetyllactosamine per 100 g composition on a dry weight basis, in particular 0.1-3 g LNnT per 100 g composition on a dry weight basis, for example 0.1-0.25 g or 0.15-0.5 g LNnT per 100 g composition on a dry weight basis.

[0157] In some embodiments, the composition comprises the following ranges or amounts of LNnT: 0.02-10 g, or 0.05-2.5 g, or 0.1-1.5 g, or 0.05-1 g, or 0.12 g-0.5 g, or 0.12 g, or 0.5 g, or 1 g per liter of composition if in ready-to-use liquid form, or 0.02-10 g, or 0.05-2.5 g, or 0.1-1.5 g, or 0.05-1 g, or 0.12 g-0.5 g, or 0.12 g, or 0.5 g, or 1 g per liter of composition if the composition is in powder form and intended to be reconstituted into a diluted liquid form (in liquid diluted form), or when the composition is diluted with water or human breast milk (2, or 0.05-0.5g per 100g, or 0.1-0.4g per 100g, or 0.1g per 100g, or 0.2g per 100g, or 0.25g per 100g, or 0.5g per 100g, or 1g per 100g, or 3g per 100g, if the nutritional composition is in the form of a concentrated composition intended to be diluted (5, 10, 20, 50 or 100 times) or intended to be used directly as a concentrated form, the nutritional composition comprises an amount equal to the above multiplied by 2, 5, 10, 20, 50 or 100, or if the nutritional composition is in the form of a dry powder, the nutritional composition comprises 0.02g to 0.75g per 100g of nutritional composition powder, or 0.04-0.6g per 100g of nutritional composition powder, or 0.05-0.5g per 100g, or 0.1-0.4g per 100g, or 0.1g per 100g, or 0.2g per 100g, or 0.25g per 100g, or 0.5g per 100g, or 1g per 100g, or 3g per 100g.

[0158] Sialylated Oligosaccharides Compositions according to the present invention may, in some embodiments, include one or more sialylated oligosaccharides.

[0159] Examples of acidic HMOs include 3'-sialyllactose (3'-SL), 6'-sialyllactose (6'-SL), 3-fucosyl-3'-sialyllactose (FSL), LST a, fucosyl-LST a (FLST a), LST b, fucosyl-LST b (FLST b), LST c, fucosyl-LST c (FLST c), sialyl-LNH (SLNH), sialyl-lacto-N-hexaose (SLNH), sialyl-lacto-N-neohexaose I (SLNH-I), sialyl-lacto-N-neohexaose II (SLNH-II) and disialyl-lacto-N-tetraose (DS-LNT).

[0160] In one embodiment, the composition according to the invention comprises sialylated oligosaccharides selected from the group comprising in particular 3'-sialyllactose and 6'-sialyllactose. More particularly, the composition comprises both 3'-sialyllactose and 6'-sialyllactose, the ratio of 3'-sialyllactose to 6'-sialyllactose being in the range of in particular between 100:1 and 1:100, more particularly between 10:1 and 1:10, even more particularly between 5:1 and 1:2.

[0161] 3' and 6' sialyllactose can be isolated from natural sources such as animal milk by chromatography or filtration techniques. Alternatively, they can be produced by biotechnological means using specific sialyltransferases or sialidases, neuraminidases, either by enzyme-based fermentation techniques (recombinant or natural enzymes), chemical synthesis or microbial fermentation techniques. In the latter case, the microorganism can express either its natural enzymes and substrates, or can be engineered to produce the respective substrates and enzymes. Single microbial cultures or mixed cultures can be used. Alternatively, sialyllactose can be produced by chemical synthesis from lactose and free N'-acetylneuraminic acid (sialic acid). Sialyllactose is also commercially available, for example, from Kyowa Hakko Kogyo Co., Ltd., Japan.

[0162] In particular, the composition according to the invention comprises, on a dry weight basis, from 0.05 to 10 g, more particularly from 0.1 to 5 g, even more particularly from 0.1 to 2 g of sialylated oligosaccharide(s) per 100 g of composition.

[0163] Specific product form As will be understood by those skilled in the art in this context, in the context of the combinations provided herein, it is not important whether the two "compounds" referred to herein (i.e., B. longum CECT7894 or its derived bacterial strain, and HMO) are administered simultaneously, e.g., as a single intake or in a single composition, or administered sequentially, e.g., as two separate compositions. What is important is that the two compounds can exert their effects together in the patient's body. In particular, the two compounds are administered within a time frame, e.g., within a digestive period that may take up to 18 hours in an adult.

[0164] Thus, the term "combination" as used herein refers to various combinations of two compounds, for example, two compounds in a single composition, two compounds in a combination mixture consisting of separate compositions of a single compound, such as a "tank mix," and two compounds in combination in a single compound when applied sequentially, i.e., in a reasonably short period of time, such as a few hours, or simultaneously. The order in which the B. longum CECT7894 or its derived bacterial strain and the HMO are administered is not important.

[0165] Thus, the combination of the probiotic composition and the HMO can be formulated for simultaneous, separate, or sequential administration. In particular, if administration is not simultaneous, the compounds are administered relatively close in time to each other. Furthermore, the compounds are administered in the same or different dosage forms, or by the same or different routes of administration, in particular orally. In some embodiments, the combination of two compounds can be, for example, - The two compounds are always administered simultaneously, as a combination that is part of the same composition; - As a combination of two units / compositions, each of which allows for the possibility of simultaneous, sequential or separate administration of one of the substances is administered.

[0166] For example, B. longum CECT7894 or a derived bacterial strain thereof is administered independently (ie, in 2 units) but simultaneously with the HMO.

[0167] The B. longum CECT7894 or a derived bacterial strain thereof and the HMO may be formulated in any form as described herein. Examples of different combinations are provided herein.

[0168] In an embodiment, the combination comprises a probiotic composition comprising B. longum CECT7894 or a derived bacterial strain thereof administered to a breast-fed infant, wherein the HMO is present in breast milk.

[0169] In another embodiment, the combination comprises a probiotic composition comprising B. longum CECT7894 or a derived bacterial strain thereof and an infant formula comprising HMO. Thus, the composition comprising B. longum CECT7894 or a derived bacterial strain thereof is administered to infants fed with infant formula. In particular, the composition comprising B. longum CECT7894 or a derived bacterial strain thereof is in the form of oil drops.

[0170] In an embodiment, the combination comprises a single composition comprising B. longum CECT7894 or a derived bacterial strain thereof, and an HMO, in any of the product forms described herein.

[0171] In an embodiment, the combination is for non-infant use and comprises a single composition comprising B. longum CECT7894 or a derived bacterial strain thereof and an HMO. In another embodiment, the combination comprises a composition comprising an HMO and a composition comprising B. longum CECT7894 or a derived bacterial strain thereof, for example in the form of an effervescent tablet or energy bar.

[0172] As mentioned above, the combination may also include additional strains of Bifidobacterium, which may be formulated for simultaneous, separate or sequential administration with the other two compounds described herein.

[0173] Use of the composition The embodiments in this section refer to any "composition" according to the invention, i.e., a probiotic composition comprising B. longum CECT7894 or a derived bacterial strain thereof, as well as combinations and compositions comprising a probiotic composition and an HMO.

[0174] As discussed herein, the probiotic composition shows high efficacy in terms of polyphosphate production during growth. The mechanism of action of polyP is known to be related to its protective effect on epithelial cells by preventing intestinal permeability. Thus, probiotic-derived polyP enhances intestinal barrier function and maintains intestinal homeostasis. The relationship between polyP production and protective effect in preventing / treating intestinal permeability is demonstrated by the examples provided herein (e.g., Example 4). Furthermore, it is plausible for those skilled in the art that B. longum CECT7894, through the production of polyP, may have a positive effect on intestinal barrier function and related conditions described herein.

[0175] For example, Saiki et al., 2016 show that polyP extracted from L. paracasei JCM1163 suppresses oxidant-induced intestinal permeability in mouse small intestine. Segawa et al., 2011 show that polyP inhibits mucosal permeability in in vitro experiments with small intestinal tissue. They first expose the tissue to a permeability-enhancing oxidant and then add polyP to confirm the protective effect. Permeability is measured by quantifying the flux of mannitol. PolyP reduces the flux of mannitol and therefore reduces permeability. Similarly, Tanaka et al., 2015 demonstrate in vitro that polyP reduces the flux of mannitol through Caco-2 intestinal epithelial cells and therefore reduces permeability. Finally, Fujiya et al., 2020 test permeability by measuring the resistance of the barrier with TEER (as assessed in Example 4, Figure 6 herein). They demonstrate that Caco-2 intestinal epithelial cells are treated with TNF-alpha to increase permeability, and then polyP improves resistance (improves TER).

[0176] Thus, the experimental data herein provided plausible evidence that the probiotic composition, by producing polyP, has a significant positive effect in treating intestinal barrier dysfunction and related conditions in subjects in need thereof.

[0177] In certain embodiments, the probiotic composition is for use in a method of treating intestinal barrier dysfunction. In embodiments, the intestinal barrier gate dysfunction is associated with increased intestinal permeability. In embodiments, the probiotic composition is for use in a method of treating increased intestinal permeability. In another embodiment, the probiotic composition is for use in a method of treating increased intestinal permeability and related conditions.

[0178] In a particular embodiment, the subject is a mammal. In a more particular embodiment, the mammal is a human. In particular, the human is an infant. In another embodiment, the human is not an infant. In another embodiment, the human is selected from the group consisting of the elderly, premature infants, infants, athletes, and frail individuals.

[0179] In some embodiments, the intestinal barrier dysfunction (e.g., increased intestinal permeability) and related conditions are associated with premature birth, aging, intense physical activity, an unbalanced diet, infection, drug treatment, or stress. In certain embodiments, the (e.g., increased intestinal permeability) and related conditions are associated with aging.

[0180] Related States A healthy intestinal barrier is believed to protect against conditions such as bacterial translocation, bacteremia, autoimmunity, brain damage, heart disease and liver disease, and obesity. Intestinal barrier dysfunction is strongly associated with several other diseases and conditions, including immune diseases such as autoimmune diseases (Crohn's disease, celiac disease, multiple sclerosis, rheumatoid arthritis, ulcerative colitis), other immune diseases (allergies / hypersensitivities such as asthma, allergic rhinoconjunctivitis, atopic dermatitis, food allergies / hypersensitivities), metabolic diseases such as non-alcoholic fatty liver disease, liver cirrhosis, type II diabetes and obesity, gastrointestinal diseases such as irritable bowel syndrome (IBS) or celiac disease, and pancreatitis, polycystic ovarian syndrome and autism. In particular, barrier dysfunction due to mucosal damage is also known to result from several drug treatments, such as oral antibiotics or nonsteroidal anti-inflammatory drugs.

[0181] In some embodiments, the associated condition is an immune disorder or disease, a metabolic or cardiovascular disorder or disease, a neurological or psychiatric disorder or disease, or a gastrointestinal disorder or disease. In particular, the immune disorder or disease is a non-intestinal immune disorder or disease. In another embodiment, the associated condition is a non-intestinal immune disorder or disease, a metabolic or cardiovascular disorder or disease, or a neurological or psychiatric disorder or disease.

[0182] In some embodiments, intestinal barrier dysfunction (e.g., intestinal hyperpermeability) is associated with conditions that occur primarily in organs other than the intestine, and are referred to herein as "non-intestinal conditions" or "indirectly intestinal-related conditions." Of note, minimal hyperactivation or infiltration of immune cells may sometimes occur in some areas of the intestine in such conditions due to hyperpermeability. However, such localized events may be asymptomatic, if at all, and are not a major cause of health concern for those skilled in the art in patients with such conditions. Clear examples of such extraintestinal conditions for those skilled in the art are neurological or psychiatric conditions (such as Alzheimer's disease, autism spectrum disorder, schizophrenia or depression), metabolic or cardiovascular conditions (such as prediabetes, diabetes, obesity, fatty liver disease, cirrhosis, atherosclerosis, hypertension, stroke or chronic heart failure), or immune disorders occurring at the systemic level or in body sites distal to the intestine (lupus erythematosus, multiple sclerosis, immunosenescence, rheumatoid arthritis, asthma, allergic rhinoconjunctivitis, atopic dermatitis or other non-food allergies / hypersensitivities). In such conditions, bacterial toxins (such as, but not limited to, lipopolysaccharide (LPS) or trimethylamine N-oxide (TMAO)) can enter the systemic blood circulation due to increased permeability in the intestine and cause inflammation and other adverse health effects in organs far from the intestine, such as the heart, brain, lungs or skin, as well as in the walls of blood vessels or immune cells in various locations in the body.

[0183] The skilled artisan will recognize that increased intestinal permeability is associated with non-intestinal diseases as described herein, such as allergies, arthritis and metabolic diseases (Bischoff et al., 2014), psychiatric disorders (Kelly et al., 2015), hypertension and atherosclerosis (Verharr et al., 2020), cardiovascular disorders (Rogler et al., 2014), Alzheimer's disease (Jiang et al., 2017), obesity (Cox et al., 2015), atopic dermatitis (Pike et al., 1986), arthritis (Tajik et al., 2020) or metabolic diseases (Massier et al., 2021).

[0184] In certain embodiments, the relevant condition is an immune disorder or disease selected from the group consisting of autoimmune diseases, such as, but not limited to, Crohn's disease, multiple sclerosis, rheumatoid arthritis, ulcerative colitis, and allergic reactions / hypersensitivities (e.g., food allergies / hypersensitivities, asthma, atopic dermatitis, or allergic rhinoconjunctivitis). In particular, the immune disorder or disease is a non-intestinal immune disorder or disease, such as an autoimmune disease other than the intestine (especially multiple sclerosis, lupus erythematosus, or rheumatoid arthritis); immunosenescence, non-food allergies / hypersensitivities, asthma, atopic dermatitis, or allergic rhinoconjunctivitis.

[0185] In certain embodiments, the relevant condition is a metabolic or cardiovascular disorder or disease, particularly selected from the group including, but not limited to, stroke, chronic heart failure, atherosclerosis, hypertension, insulin resistance (pre-diabetes), diabetes, obesity, non-alcoholic fatty liver disease, and cirrhosis.

[0186] In certain embodiments, the relevant condition is a neurological or psychiatric disorder or disease, particularly selected from the group including, but not limited to, Alzheimer's disease, autism spectrum disorder, schizophrenia and depression.

[0187] In some embodiments, the extra-intestinal condition is selected from the group consisting of obesity, diabetes, insulin resistance, non-alcoholic fatty liver disease, cirrhosis, non-food allergies / hypersensitivities, immunosenescence, multiple sclerosis, rheumatoid arthritis, lupus erythematosus, sarcopenia, asthma, allergic rhinoconjunctivitis, atopic dermatitis, Alzheimer's disease, atherosclerosis, hypertension, chronic heart failure, stroke, autism spectrum disorder, schizophrenia, and depression.

[0188] In certain embodiments, the relevant condition is a gastrointestinal disorder or disease, particularly selected from the group including, but not limited to, early onset inflammatory bowel disease (such as Crohn's disease, ulcerative colitis, pouchitis or lymphocytic colitis), irritable bowel syndrome (IBS), leaky gut syndrome, villous atrophy, necrotizing enterocolitis, intestinal ischemic injury, epithelial injury induced by nonsteroidal anti-inflammatory drugs and celiac disease.

[0189] IBS is one of the most prevalent gastrointestinal disorders in high-income countries and is commonly associated with the presence of altered intestinal barrier. Altered intestinal barrier has been reported to be associated with GI symptoms in IBS patients, such as diarrhea and abdominal pain. It appears that barrier dysfunction is an early event in IBS and may contribute to low-grade intestinal inflammation and increased visceral sensation. Furthermore, intestinal permeability in IBS subtypes, such as diarrhea-predominant IBS (IBS-D) and post-infectious IBS, is often related to altered intestinal barrier function.

[0190] In addition, ulcerative colitis (UC) and Crohn's disease (CD), classified as chronic inflammatory bowel disease (IBD), have similar symptoms and result in digestive disorders including diarrhea, abdominal pain, rectal bleeding and weight loss. Epithelial integrity is disturbed in IBD patients who also exhibit intestinal hyperpermeability. Loss of the intestinal barrier is a potentially contributing factor to the multi-hit mechanism of IBD pathogenesis. Moreover, many IBS patients with healed mucosa still have ongoing intestinal symptoms, which are associated with impaired intestinal permeability.

[0191] In another embodiment, the associated condition is characterized by microinflammation of the gastrointestinal tract, vascular damage and / or swallowing disorders.

[0192] In particular, the associated condition is directly related to the intestinal tract. In a more particular embodiment, the intestinal barrier dysfunction or associated condition is selected from the group consisting of irritable bowel syndrome (IBS), inflammatory bowel disease (IBD), intestinal infection, gastric ulcer, diarrhea (e.g. gastric or infectious, such as recurrent Clostridium difficile diarrhea), celiac disease, cancer associated with the digestive tract, colitis, ulcerative colitis, Crohn's disease, mitochondrial neurogastrointestinal encephalopathy (MNGIE), leaky gut syndrome, villous atrophy, necrotizing enterocolitis (NEC), intestinal ischemic injury, chronic enteropathy, chronic constipation, and intestinal mucosal injury. In particular, it is known that intestinal barrier dysfunction due to mucosal injury also results from some drug treatments, such as oral antibiotics or nonsteroidal anti-inflammatory drugs. In particular, the associated condition is irritable bowel syndrome (IBS). In particular, the associated condition is inflammatory bowel disease (IBD). In particular, the associated condition is cancer. More particularly, the cancer of the digestive tract is selected from the group consisting of esophageal cancer, gastric cancer and colon cancer.

[0193] In some embodiments, the probiotic composition is for use in the treatment of at least one symptom, complication and / or sequelae selected from the group consisting of abdominal pain, constipation, weight loss, rectal bleeding, sarcopenia, frailty, cachexia, gastrointestinal disorders, cramps, swelling, flatulence, vomiting, nausea, stomach pain, fatigue, fever, altered absorption of certain nutrients, loss of appetite, systemic inflammation, and heat stroke. In particular, the symptom, complication and / or sequelae is selected from the group consisting of weight loss, sarcopenia, frailty, cachexia, fatigue, fever, systemic inflammation, and heat stroke.

[0194] In an embodiment, administration of the probiotic composition comprises: - Reduction of intestinal permeability, improvement of gastrointestinal barrier function, improvement of intestinal epithelial integrity or protection of the intestinal mucosa; -Reduced intestinal sensitivity or improved intestinal tolerance; -Improved intestinal motility; and -Maintaining intestinal balance The method produces at least one result selected from the group consisting of:

[0195] The terms "reducing intestinal permeability", "improving gastrointestinal barrier function", "improving intestinal epithelial integrity" and "protecting the intestinal mucosa" are understood as the proper containment of unwanted luminal contents in the intestine.

[0196] The terms "reduced intestinal sensitivity" and "improved intestinal tolerance" are understood as the normal visceral response to painful stimuli.

[0197] The term "improvement of intestinal motility" is understood as the regular movement of the digestive tract and the transfer of contents therein.

[0198] The term "maintenance of intestinal balance" is understood as the equilibrium of the intestinal ecosystem.

[0199] In another embodiment, administering the composition comprises: - Decreased levels of intestinal permeability-related biomarkers; - Mitigation or reduction of increases in intestinal permeability-related biomarkers due to intestinal mucosal injury; and - Reduction of serum tight junction protein levels caused by intestinal mucosal injury The method produces at least one result selected from the group consisting of:

[0200] Biomarkers may include circulating indicators such as intestinal fatty acid binding protein (I-FABP, also known as FABP-2), zonulin, claudin 3 (or other tight junction proteins), citrulline, lipopolysaccharide (LPS) or bacterial DNA; urinary indicators such as oligosaccharides (e.g., lactulose, mannitol, sucralose, cellobiose, and ratios such as lactulose / mannitol distribution), polyethylene glycol (PEG), chromium-ethylenediaminetetraacetic acid (Cr-EDTA); or fecal markers including calprotectin, zonulin, alpha (α)-1-antitrypsin (AAT), diamine oxidase (DAO), or lipocalin-2 (LCN-2).

[0201] Use in infants Increasing evidence suggests that multiple disorders, including inflammatory diseases such as allergies, contribute to the disruption of the microbiota early in life. Children exposed to antibiotics early (first 2 years of life) are at increased risk for allergic rhinitis, atopic dermatitis, childhood-onset asthma, celiac disease, and obesity. In addition, babies born by Caesarean section are more susceptible to allergic rhinoconjunctivitis and asthma than vaginally delivered babies, and a reduction in Bifidobacteria is directly linked to atopic dermatitis and allergic asthma. Finally, infants fed formula have a higher incidence of atopic dermatitis compared to breastfed infants. These results are consistent with the fact that the gut microbiota plays an important role in the formation and permeability of the intestinal barrier structure, and changes in the gut microbiota are associated with increased intestinal permeability in several disorders.

[0202] Indeed, abnormal intestinal permeability is implicated in allergies. For example, intestinal permeability is abnormally increased in 80% of children with food allergies and digestive symptoms. Furthermore, impaired intestinal barrier is implicated in the development of atopic dermatitis. Similarly, babies with early allergic symptoms have increased intestinal permeability to proteins compared to non-allergic infants.

[0203] Therefore, the probiotic composition described herein produces molecules (polyP) capable of restoring the intestinal barrier and is a therapeutic option for treating allergies. Babies born by Caesarean section, fed formula or given antibiotics and premature infants could also benefit from this probiotic treatment as a preventative that could reduce the onset of allergies.

[0204] Thus, in an embodiment, the subject is an infant. In particular, the infant is a premature infant, a weak infant, an infant born under birth weight, an intrauterine growth restricted infant subject, an infant born by Caesarean section, an infant administered antibiotics, an infant fed with formula or an infant fed with breast milk. More particularly, the infant is a premature infant.

[0205] More particularly, the intestinal barrier dysfunction (e.g., increased intestinal permeability) and associated conditions are associated with preterm birth, Caesarean section delivery, formula feeding, under birth weight, and / or antibiotic administration. In certain embodiments, the intestinal barrier dysfunction (e.g., increased intestinal permeability) and associated conditions are associated with preterm birth. In certain embodiments, the intestinal barrier dysfunction (e.g., increased intestinal permeability) and associated conditions are associated with Caesarean section delivery. In certain embodiments, the intestinal barrier dysfunction (e.g., increased intestinal permeability) and associated conditions are associated with formula feeding. In certain embodiments, the intestinal barrier dysfunction (e.g., increased intestinal permeability) and associated conditions are associated with antibiotic administration.

[0206] In addition, the probiotic compositions of the present invention are not only useful for treating these conditions and restoring abnormal infant microbiota, but also for preventing these conditions in the future by enhancing a healthy infant microbiota. Thus, in certain embodiments, the probiotic compositions are for use in preventing infant-related conditions.

[0207] In some embodiments, the relevant condition relating to the infant is selected from the group consisting of Crohn's disease, multiple sclerosis, lupus erythematosus, rheumatoid arthritis, ulcerative colitis, obesity, insulin resistance (pre-diabetes), diabetes, irritable bowel syndrome, celiac disease, early inflammatory bowel disease, allergic reactions / sensitivities such as, but not limited to, food allergies / sensitivities, asthma, atopic dermatitis or allergic rhinoconjunctivitis, non-alcoholic fatty liver disease, autism spectrum disorder, schizophrenia, and depression.

[0208] In some embodiments, the relevant condition relating to infants is selected from the group consisting of lupus erythematosus, multiple sclerosis, rheumatoid arthritis, non-food allergies / hypersensitivities, asthma, atopic dermatitis, allergic rhinoconjunctivitis, insulin resistance (pre-diabetes), diabetes, obesity, non-alcoholic fatty liver disease, autism spectrum disorder, schizophrenia, and depression.

[0209] In particular, relevant conditions relating to infants are selected from the group consisting of autism spectrum disorder, non-food allergies / hypersensitivities, asthma, atopic dermatitis, allergic rhinoconjunctivitis, insulin resistance (pre-diabetes), diabetes, fatty liver disease and obesity.

[0210] In a more particular embodiment, the associated condition is associated with premature birth and is allergy. In another embodiment, the associated condition is associated with infants receiving antibiotics and is selected from the group including, but not limited to, allergic rhinoconjunctivitis, atopic dermatitis, childhood onset asthma, and obesity. In another embodiment, the associated condition is associated with infants born by Caesarean section and is selected from the group consisting of allergic rhinoconjunctivitis, atopic dermatitis, and asthma. In another embodiment, the associated condition is associated with infants fed formula and is atopic dermatitis.

[0211] Use in athletes Symptoms of gastrointestinal disorders, such as diarrhea, cramps, vomiting, nausea and stomach pain, are common in athletes during high-intensity training and competition. Heat stress and oxidative damage during exercise disrupt tight junction proteins in intestinal epithelial cells, leading to increased permeability to luminal toxins. Prolonged intense exercise is associated with increased core body temperature and increased intestinal permeability. Therefore, the degree of exercise-induced hyperthermia is directly related to increased intestinal permeability and can cause systemic inflammation that can affect physical performance and induce heatstroke in severe cases.

[0212] Administration of the probiotic compositions described herein may combat exercise-induced leaky gut, improve intestinal barrier integrity, reduce gastrointestinal disorders, and improve performance during exercise at high temperatures in athletes.

[0213] Thus, in certain embodiments, the subject is an athlete. In certain embodiments, intestinal barrier dysfunction (e.g., increased intestinal permeability) and related conditions are associated with high intensity physical activity.

[0214] In some embodiments, the probiotic compositions of the present invention are for use in a method for treating intestinal barrier dysfunction (e.g., increased intestinal permeability) and associated conditions, or symptoms, complications and / or sequelae selected from the group consisting of diarrhea, cramps, vomiting, nausea, stomach pain, altered absorption of certain nutrients, systemic inflammation (which may affect physical performance), and, in severe cases, heat stroke.

[0215] Use in older adults The aging process is associated with natural changes in the composition of the gut microbiota, low-grade chronic inflammation, and increased intestinal permeability, all of which are linked. Changes in the gut microbiota include increased intestinal epithelial permeability, subsequent leakage of gut bacteria and their metabolic products, and resulting inflammation. Furthermore, local inflammation can also be directly regulated by changes in the microbiota.

[0216] Thus, in certain embodiments, the subject is elderly or infirm. In certain embodiments, the intestinal barrier dysfunction (e.g., increased intestinal permeability) and related conditions are associated with aging.

[0217] In particular, the intestinal barrier dysfunction (e.g., increased intestinal permeability) and associated conditions associated with aging are selected from the group consisting of constipation, diarrhea, sarcopenia, frailty, recurrent Clostridium difficile diarrhea, Alzheimer's disease, atherosclerosis, stroke, cancer and cachexia, and more particularly sarcopenia, frailty, Alzheimer's disease, atherosclerosis, chronic heart failure, immunosenescence, and stroke.

[0218] Product forms containing the composition The embodiments in this section also refer to all compositions according to the invention, i.e. probiotic compositions comprising B. longum CECT7894 or a derived bacterial strain thereof, compositions comprising HMOs and compositions comprising both.

[0219] Pharmaceutical Form In some embodiments, the compositions described herein are in a pharmaceutical form, such as a capsule, powder, suspension, tablet, topical cream or ointment.

[0220] The term "pharmaceutical form" is understood in its broadest sense to include any composition containing an active ingredient, in this case a strain or composition as described herein, together with at least a pharma- ceutical (also referred to as nutraceutical or veterinary) acceptable excipient. The term "pharmaceutical form" is not limited to drugs, but includes, for example, pharmaceutical, nutraceutical or veterinary compositions. Pharmaceutical forms may adopt different names depending on the regulatory approval route and country of the product.

[0221] Nutraceutical compositions can also be named, for example, as dietary supplements or dietary supplements. Nutraceutical compositions are understood as preparations or products made from compounds normally used in foods that are intended to supplement the diet and provide nutrients or beneficial components that may not normally be consumed in the normal diet or in sufficient amounts. Nutraceutical compositions are usually sold "over the counter", i.e. without a prescription.

[0222] In some embodiments, the composition is formulated as a pharmaceutical form in which the strain is the only active agent or is mixed with one or more other active agents and / or mixed with pharma- ceutical / nutraceutical / veterinary acceptable excipients. In particular, the additional active agent or agents are other probiotic bacteria that are not antagonistic to the strain forming the composition of the present invention. Depending on the formulation, the strain may be added as purified bacteria, as a bacterial culture, as part of a bacterial culture, as a post-treated bacterial culture, and alone or with suitable carriers or ingredients. Examples of other active ingredients added to the composition are prebiotics such as fructooligosaccharides (e.g., inulin), galactooligosaccharides, xylooligosaccharides, arabinoxylan oligosaccharides, pectin, beta-glucan, human milk oligosaccharides (e.g., lacto-N-tetraose) or partially hydrolyzed guar gum.

[0223] The term "pharmaceutical / nutraceutical / veterinary acceptable" is recognized in the art and includes excipients, compounds, materials, compositions, carriers, vehicles and / or dosage forms that are, within the scope of sound medical judgment, suitable for use in contact with the tissues of a subject (e.g., a human or animal) without undue toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit / risk ratio. Each carrier, excipient, etc. must be "acceptable" in the sense of being compatible with the other ingredients of the formulation. Suitable carriers, excipients, etc. can be found in standard pharmaceutical, nutritional, and veterinary texts.

[0224] As such, some embodiments of the present invention relate to pharmaceutical, nutraceutical, and veterinary compositions comprising the compositions described herein together with at least one pharma- ceutical / nutritional / veterinarily acceptable excipient as described above.

[0225] Some non-limiting examples of materials that may function as pharma- ceutically / nutraceutical / veterinarily acceptable excipients or carriers include sugars such as lactose, glucose and sucrose; starches such as corn starch and potato starch; cellulose and its derivatives such as sodium carboxymethylcellulose, ethylcellulose and cellulose acetate; powdered tragacanth; malt; gelatin; talc; cocoa butter and suppository wax; oils such as peanut oil, cottonseed oil, sunflower oil, sesame oil, olive oil, corn oil and soybean oil; glycols such as propylene glycol; polyols such as glycerin, sorbitol, mannitol and polyethylene glycol; esters such as ethyl oleate and ethyl laurate; agar; buffers such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline; Ringer's solution; ethyl alcohol; or phosphate buffer solutions.

[0226] The excipients are selected from the group including, but not limited to, fillers / diluents / bulking agents, binders, anti-adherents, disintegrants, coating agents, anti-caking agents, antioxidants, lubricants, sweeteners, flavors, colorants, or surfactants.

[0227] The filler is selected from the group including, but not limited to, inulin, oligofructose, pectin, modified pectin, crystalline cellulose, lactose, starch, maltodextrin, saccharose, glucose, fructose, mannitol, xylitol, amorphous sorbitol, calcium carbonate, dicalcium phosphate, other inert inorganic and organic pharmacologically acceptable fillers, and mixtures of these substances. In oral suspension dosage forms, the filler or diluent is selected from the group including vegetable oils, oleic acid, oleyl alcohol, liquid polyethylene glycols, other pharmacologically acceptable inert liquids, or mixtures of these substances.

[0228] Binders are used in solid dosage forms, for example, to hold the ingredients in a tablet together, to ensure that tablets and granules can be formed with the necessary mechanical strength, and to provide volume to tablets with low active doses. Binders in solid dosage forms, such as tablets, are lactose, sucrose, corn (maize) starch, modified starch, microcrystalline cellulose, modified cellulose (e.g., hydroxypropyl methylcellulose (HPMC) and hydroxyethyl cellulose), other water-soluble cellulose ethers, polyvinylpyrrolidone (PVP), also known as povidone, polyethylene glycol, sorbitol, maltitol, xylitol, and dibasic calcium phosphate; other suitable pharmacologically acceptable binders, or mixtures of these substances.

[0229] Anti-adherents are used to reduce adhesion between the powder (granules) and the punch faces, to prevent adhesion to the tablet punches, and to prevent inter-tablet adhesion. The most commonly used is magnesium stearate.

[0230] As disintegrants and superdisintegrants in solid dosage forms such as tablets and capsules, the following substances are used, but are not limited to: cross-linked polyvinylpyrrolidone, sodium starch glycolate, sodium carboxymethylcellulose, calcium carboxymethylcellulose, and formaldehyde-casein, other suitable pharmacologically acceptable disintegrants and superdisintegrants, or mixtures thereof.

[0231] Coatings for solid dosage forms such as tablets and granules for capsule filling protect the ingredients from degradation due to moisture in the air, make large, unpleasant-tasting tablets easier to swallow, and / or, in the case of enteric coatings, ensure intact passage through the strongly acidic medium of gastric juice (pH of about 1) and release in the duodenum or ileum (small intestine). For most coated tablets, cellulose ether hydroxypropylmethylcellulose (HPMC) film coatings are used. Occasionally, other coating materials are used, such as synthetic polymers and copolymers such as polyvinyl acetate phthalate (PVAP); copolymers of methyl acrylate-methacrylic acid; copolymers of methyl methacrylate-methacrylic acid; shellac, corn protein zein or other polysaccharides; waxes or wax-like substances such as beeswax, stearic acid; higher fatty alcohols such as cetyl alcohol or stearyl alcohol; solid paraffin; glycerol monostearate; glycerol distearate, or combinations thereof. Capsules are coated with gelatin or hydroxypropylmethylcellulose.

[0232] Enteric coatings control the rate of drug release and determine where in the digestive tract the drug is released. Materials used in enteric coatings include fatty acids, waxes, shellac, plastics, and vegetable fibers, as well as combinations with the other coatings mentioned above.

[0233] Anticaking agents are additives added to powdered or granular materials to prevent the formation of lumps (caking) and to facilitate packaging, shipping, and consumption. The following are used as anticaking agents in solid dosage forms such as tablets, capsules, or powders: magnesium stearate, colloidal silicon dioxide, talc, other pharmacologically acceptable anticaking agents, or mixtures thereof.

[0234] Lubricants are used in solid dosage forms, especially tablets and capsules, to prevent ingredients from clumping and sticking to tablet punches or capsule filling machines, and also in hard capsules. As lubricants, talc or silica, and fats such as vegetable stearin, magnesium stearate or stearic acid, and mixtures thereof, are the most frequently used lubricants in tablets or hard gelatin capsules.

[0235] Sweeteners are added to make ingredients more palatable, especially in solid dosage forms, such as chewable tablets, as well as liquid dosage forms, such as cough syrups. The sweetener can be selected from artificial, natural or synthetic sweeteners or semi-synthetic sweeteners; non-limiting examples of sweeteners are aspartame, acesulfame potassium, cyclamate, sucralose, saccharin, sugars or any mixture thereof.

[0236] Flavorings can be used to mask unpleasant tasting active ingredients in any dosage form. Flavorings can be natural (e.g., fruit extracts) or artificial. For example, (1) mint, cherry or anise can be used to improve a bitter product; (2) peach or apricot or licorice can be used to improve a salty product; (3) raspberry can be used to improve a sour product; and (4) vanilla can be used to improve a product that is too sweet.

[0237] Except for auxiliary substances from the class of excipients, formulations from the present invention may contain other pharmacologically active substances or nutritional substances, including, but not limited to, vitamins, such as vitamin D (calciferol), in a pharma- ceutically acceptable chemical form, salts or derivatives; minerals, in a pharma- ceutically and nutritionally acceptable chemical form; and L-amino acids.

[0238] In each case, the presentation of the composition is adapted to the type of administration used by means known to those skilled in the art. Thus, the composition can be presented in a therapeutically effective amount in the form of a solution or any other form of clinically acceptable administration. The composition can therefore be formulated into solid, semi-solid or liquid preparations such as tablets, capsules, powders (such as those derived from lyophilization (freeze-drying) or air-drying), granules, solutions, suppositories, gels or microspheres. In certain embodiments, the composition is formulated for administration in liquid or solid form.

[0239] In certain embodiments, the composition is in solid form, such as tablets, lozenges, confectionery, chewable tablets, chewing gum, capsules, sachets, powders, granules, coated particles or coated tablets, tablets, pills, lozenges, gastroresistant tablets and capsules, dispersible strips and films. More particularly, the composition is in the form of capsules, powders, tablets, pills, lozenges, sachets, or granules. In embodiments, the composition is in the form of a powder that is contacted with an aqueous phase to form a solution. The aqueous phase can include fibers, such as inulin. The two components (powder and aqueous phase) can be placed in separate compartments / containers, and the two components are mixed to reconstitute on the spot.

[0240] In an embodiment, the composition is in the form of a gelatin capsule. In a particular embodiment, the composition is in the form of a vegetable capsule and comprises hydroxypropyl methylcellulose (HPMC).

[0241] In another embodiment, the composition is in a liquid form, such as an oral solution, drops, suspension (e.g., oil), emulsion, and syrup. In particular, the composition is in the form of drops. More particularly, the composition is in the form of oily drops.

[0242] In some embodiments, the composition is in the form of an oil suspension to be administered alone or mixed with a liquid. The oil suspension comprises at least one edible oil, such as olive oil, corn oil, soybean oil, linseed oil, sunflower oil or rice oil. The oil is present in an amount of at least 70% w / w. In certain embodiments, the oil suspension also comprises at least one excipient that is an emulsifier, stabilizer or anti-caking agent in an amount of 0.1-15% w / w. Suitable agents are silicon dioxide, silica gel, colloidal silica, precipitated silica, talc, magnesium silicate, lecithin, pectin, starch, modified starch, konjac gum, xanthan gum, gellan gum, carrageenan, sodium alginate, mono- or diglycerides of fatty acids such as glycerol monostearate or glycerol monooleate, as well as citric acid esters of mono- or diglycerides.

[0243] In particular, the composition is in the form of an oily suspension, in particular in the form of an infant nutritional supplement in the form of oily drops.In a particular embodiment, the oily suspension comprises sunflower oil and in particular 1% by weight colloidal silica, and bacterial cells.In another embodiment, the oily suspension comprises sunflower oil and an agent selected from lecithin, mono- or diglycerides of fatty acids, carrageenan, and sodium alginate, and bacterial cells.

[0244] In particular, for example, the capsule, sachet or stick, tablet or pill has a weight of about 150 mg to about 8000 mg. More particularly, the capsule has a weight of about 200 mg to about 600 mg. More particularly, the sachet or stick has a weight of about 1.5 g to about 6 g. More particularly, the tablet or pill has a weight of about 400 mg to about 1200 mg.

[0245] In particular, for example, the spray, oil drops (e.g., sunflower oil drops) have a volume of about 3 ml to about 50 ml. More particularly, the spray has a volume of about 5 ml to about 50 ml. More particularly, the oil drops have a volume of about 3 ml to about 30 ml.

[0246] Regarding the preparation of the formulation of the present invention, this is within the scope of those skilled in the art and depends on the final dosage formulation. For example, but not limited to, when the final dosage form is an oral solid dosage form such as a tablet, capsule, powder, granule, oral suspension, the preparation process of the solid dosage form of the formulation includes (1) homogenizing an effective amount of the active ingredient(s), including the post-treated probiotic bacteria of the present invention, with (2) one or more excipients to form a homogenous mixture, which is subjected to lubrication, for example, with magnesium stearate or other lubricants according to requirements, to produce the final dosage form of powder. Such homogenous powder is filled into a normal gelatin capsule or filled into an enteric capsule. In the case of tablets, they are manufactured by direct compression or granulation. In the first example, a homogenous mixture of the active ingredient and suitable excipients such as anhydrous lactose, amorphous sorbitol, and others is prepared. In the second example, the tablet is processed into a mixture in granule form. The granules are prepared by a granulation process of the active ingredients of the formulation with suitable fillers, binders, disintegrants, and a small amount of purified water. The granules thus prepared are sieved and dried until the moisture content is less than 1% w / w.

[0247] With regard to the preparation process of liquid dosage forms (e.g., oral suspensions), this involves homogenizing the active ingredient(s) of the formulation, including an effective amount of the post-treated probiotic bacteria of the present invention, in an inert liquid diluent (filler), such as various vegetable oils, such as sunflower oil, soybean oil, or olive oil; oleic acid; oleyl alcohol; liquid polyethylene glycols, such as PEG200, PEG400, or PEG600; or other inert pharmacologically acceptable liquids. This process further involves treating the homogenous mixture with one or more processes selected from the group including: (1) stabilizing the formulation by adding a suspension stabilizer, such as beeswax, colloidal silicon dioxide, and the like, and homogenizing; (2) sweetening the formulation by adding a sweetener and homogenizing; and (3) flavoring the formulation by adding a flavoring and homogenizing.

[0248] Food / nutritional composition In some embodiments, the composition is in the form of a food or edible composition, such as an infant formula or food, a milk-based fermented product (e.g., yogurt, cheese, curd), a plant-based fermented product, bread, a bar (e.g., energy bar), a spread, a biscuit, a syrup, a beverage, a dressing, a sauce, a filling, a soup, an ice cream, an oil, a dressing or a confectionery.

[0249] The term "food or edible composition" is used herein in its broadest sense to include any type of product in any presentation form that can be ingested by an animal, particularly a human, but to exclude pharmaceuticals, dietary supplements and veterinary products.

[0250] In particular, the composition is included in an infant formula or food product, in particular, the composition is included in a beverage.

[0251] Other examples of food products include meat products, chocolate spreads, fillings and frostings, chocolates, confectionery, baked goods, sauces and soups, fruit juices, and whiteners for coffee. Food products include carrier materials such as oatmeal porridge, lacto-fermented foods, resistant starches, dietary fibers, carbohydrates, proteins, and glycosylated proteins, among others. In certain embodiments, the strains of the present invention are encapsulated or coated. In particular, the milk can be of either animal or vegetable origin.

[0252] In an embodiment the food or edible composition is a nutritional composition commonly used in the field of infant nutrition, but also for the elderly and frail population.

[0253] In certain embodiments, the composition of the present invention is an infant formula. In some embodiments, the composition is, for example, a starter infant formula, an infant food, an infant cereal composition, a follow-up infant formula or a growing-up milk, or a nutritional supplement. The composition can also be used before and / or during the weaning period.

[0254] In one embodiment, the nutritional composition can be a complete nutritional composition or a supplement for the aging, elderly or infirm. In some embodiments, the compositions of the invention are hydration solutions or dietary maintenance or supplements, for example, for the elderly, athletes or immunocompromised individuals.

[0255] The composition according to the invention may be a complete composition that provides 100% or a majority of the nutritional requirements of the target population (e.g., in terms of caloric requirements, or in terms of vitamin or mineral requirements, in terms of protein, lipid or carbohydrate requirements). Alternatively, the composition of the invention may be a supplement that is consumed in addition to the normal diet. However, in that case, the dose and overall consumption of the composition is adapted to provide the claimed benefits for emotional processing (e.g., in proportion to the caloric load and caloric requirements of the subject).

[0256] The use of the composition of the present invention can include the example where the composition is a supplement, preferably provided in unit dose form (e.g., tablet, capsule, powder sachet, etc.). In one embodiment, the composition is a supplement for human breastfeeding. The unit dose form can contain acceptable carriers, such as phosphate buffered saline, mixtures of ethanol in water, water and emulsions such as oil / water or water / oil emulsions, as well as various wetting agents or excipients. Examples of carriers and excipients are described herein above.

[0257] The composition may be in the form of, for example, a powder composition intended to be diluted with water or mixed with milk (e.g., human breast milk) or ingested as a powder. In one embodiment, the composition of the present invention is in a ready-to-drink or liquid form to be diluted with water or mixed with milk (e.g., human breast milk).

[0258] The composition may be in the form of a ready-to-use liquid, or may be a liquid concentrate or powdered formula that can be reconstituted into a ready-to-use liquid by adding the required amount of water.

[0259] Administration In some embodiments, the composition is administered in a single dose or multiple doses at specific time intervals, for example, daily for a specific number of days or according to a specific administration schedule. In particular, the composition is administered for 10 to 90 days. More particularly, the composition is administered for 10 to 60 days or 15 to 45 days, more particularly, for 30 days.

[0260] In some embodiments, the composition is administered once every three days to three times a day, particularly once a day.

[0261] In some embodiments, the composition can be administered orally, enterally, parenterally, topically, ophthalmically, auricularly, nasally, vaginally or bucally to provide a local and / or systemic effect. In particular, the composition is administered orally. In embodiments, a unit dose of the composition of the present invention is administered orally as a tablet, capsule, or pellet, or as a powder or granule, or in any of the above forms, such as a gel, paste, solution, suspension, emulsion, syrup, bolus, electuary, or slurry in aqueous or non-aqueous liquid.

[0262] In one embodiment, the composition is administered enterally. Methods of enteral administration include feeding via nasogastric or jejunal tube, oral, sublingual, and rectal. Thus, the unit dosage form of the composition can also be administered in elderly or infirm individuals via rectal suppository, aerosol tube, nasogastric tube, or direct injection into the gastrointestinal tract or stomach.

[0263] In other embodiments, the compositions may be administered by nasal inhalation, oral spray, or via the nasal route, hi other embodiments, the compositions may be administered in the form of oral drops.

[0264] Working Example Example 1: Polyphosphate biosynthesis ability of Bifidobacterium longum subsp. longum KABP-042 (CECT7894) 1.1 Materials and Methods 1.1.1 Strains and culture conditions Nineteen strains were evaluated for their ability to biosynthesize polyP (Table 1). Strains included B. longum subsp. longum KABP-042 (CECT7894), other bifidobacterial strains, and other strains belonging to the Lactobacillus group and the Saccharomyces genus. Strains included infant and adult human resident bacterial (HRB) and non-HRB strains from the AB-Biotics SL collection or commercial products.

[0265] The analysis included the following control strains: L. plantarum WCFS1, known to produce polyP (Alcantara et al. 2014) and L. paracasei JCM1163 (Saiki et al. 2016), B. breve JCM1273, B. adolescentis JCM1275 and B. longum subsp. longum ATCC15707, known to be able to remove phosphate (Anand et al. 2019), and B. scardovi DSMZ13734 (BAA-773), known to harbor the ppk gene (Qian et al. 2011).

[0266] Strains were isolated from commercial sources as indicated by inoculating appropriate agar plates. After cultivation, single colonies were grown and stored in glycerol stocks, and species identity (ID) was confirmed by PCR amplification of the 16S rRNA gene and Sanger sequencing. Control strains were purchased from culture collections and species identity was confirmed.

[0267] Bifidobacterial strains were precultured in Man, Rogosa and Sharpe agar (MRS) containing 0.05% cysteine ​​(MRScys) under anaerobic conditions at 37°C. Lactobacillus strains were precultured in MRS under aerobic conditions at 30°C. Saccharomyces boulardii CNCM I-754 was precultured in YPD medium at 37°C under aerobic conditions with shaking.

[0268] For polyP production assays, malic enzyme induction (MEI) medium was used (Alcantara et al. 2014), containing (per L, w / v) 0.5% yeast extract, 0.5% tryptone, 0.4% K2HPO4, 0.5% KH2PO4, 0.02% MgSO4·7H2O, 0.005% MnSO4, 1 ml Tween 80, 0.05% cysteine, and 0.5% glucose. Strains unable to grow in MEI were grown in MRScys. Cultures were inoculated at an OD (595 nm) of 0.1, and each strain was grown under the conditions indicated above. Growth was monitored by measuring the OD for 16 h.

[0269] Table 1. Characterization of strains. HRB, human resident bifidobacteria; nHRB, non-HRB; CECT, Spanish Type Culture Collection; DSMZ, German Collection of Microorganisms and Cell Cultures; ATCC, American Type Culture Collection. Strains classified as controls have some published evidence for polyP metabolism. [Table 1]

[0270] 1.1.2 Quantification of polyphosphate (polyP) PolyP was isolated from cells by its resistance to hydrolysis by sodium hypochlorite as previously described (Alcantara et al. 2014). Cells were harvested by centrifugation and dissolved in 1 ml of 5% sodium hypochlorite with gentle agitation at room temperature for 45 min. Insoluble material was pelleted by centrifugation at 16,000 g at 4 °C for 5 min and washed twice with 1 ml of 1.5 M NaCl plus 1 mM EDTA, with an intervening centrifugation step at 16,000 g at 4 °C for 5 min. PolyP was extracted from the pellet and washed twice with 1 ml of water, with an intervening centrifugation step at 16,000 g at 4 °C for 5 min. PolyP in the pooled water extracts was precipitated by the addition of 0.1 M NaCl and 1 volume of ethanol, followed by incubation on ice for 1 h. After centrifugation at 16,000 g for 10 min, the polyP pellet was resuspended in 50 μL of water.

[0271] A calibration curve relating the amount of phosphate to the fluorescence intensity was constructed to quantify the polyP extracted from the strains. In the first step, serial dilutions of polyP samples isolated from the polyphosphate-producing control strain Lactiplantibacillus plantarum strain WCFS1 (Alcantara et al. 2014) were prepared. Secondly, the dilutions were hydrolyzed with one volume of 2 M HCl and incubated at 95 °C for 15 min to release phosphate, then neutralized by adding half a volume of 2 M NaOH. Thirdly, the released phosphate from each dilution was quantified with the BIOMOL Green kit (Enzo Life Sciences) according to the manufacturer's recommendations. In parallel, the released phosphate from each dilution was stained using 4',6-diamidino-2-phenylindole (DAPI) at a final concentration of 10 μM in 50 mM Tris-HCl pH 7.5, 50 mM NaCl buffer and the fluorescence was measured in a fluorometer at an excitation wavelength of 415 nm and an emission wavelength of 550 nm. Finally, a calibration curve was constructed with the obtained phosphate values ​​and the corresponding fluorescence values.

[0272] After obtaining the standard curve, the amount of polyP from the samples can be quantified according to the fluorescence value without using the BIOMOL Green kit. Therefore, the quantification of polyP from the strain samples was indirectly measured by DAPI fluorescence using the standard curve. First, the extracted polyP was measured by fluorescence at an excitation wavelength of 415 nm and an emission wavelength of 550 nm in a fluorometer using DAPI at a final concentration of 10 μM in 50 mM Tris-HCl pH 7.5, 50 mM NaCl buffer. Then, the amount of polyP was calculated as nmol phosphate by the standard curve. Biological replicates were performed at least three times.

[0273] 1.1.3 Detection of ppk gene by in silico analysis The nucleotide sequences of the ppk genes of Bifidobacterium and Lactobacillus species were retrieved from NCBI with accession numbers AE014295.3 (version 3, updated on January 31, 2014, genome of B. longum NCC2705) and AL935263.2 (version 2, updated on February 28, 2015, genome of L. plantarum WCFS1), respectively, and subjected to BLAST analysis against the test genomes. The amino acid sequences of the PPK proteins detected in Bifidobacterium species were aligned, and a tree was constructed using ClustalW.

[0274] 1.2. Results The polyP production ability and associated growth of B. longum subsp. longum KABP-042 (CECT7894) was compared with 12 bifidobacterial strains belonging to six different species, six lactobacillus strains belonging to five species, and one yeast strain (Table 1 ).

[0275] Strains were inoculated into MEI or MRScys at the same OD (0.1) and grown for 16 h. OD was monitored and polyP formation was examined at 6 and 16 h, when significant growth was observed for most strains (Fig. 1). PolyP synthesis and OD values ​​varied widely among strains (Fig. 1, Fig. 2, and Table 2).

[0276] In general, bifidobacteria showed a higher polyP formation capacity than Lactobacillus strains. The levels of PolyP produced by L. plantarum 299v, L. brevis KABP-052 (CECT7840), L. rhamnosus GG, L. reuteri DSM17938 and S. boulardii CNCM I-754 cells were very low (<2 nmol at 16 h).

[0277] In bifidobacteria, all strains were able to produce some amount of polyP, but B. bifidum ABP671, B. breve ABP734, B. breve M16-V and B. skaldovii BAA-773 produced the lowest amounts (less than 25 nmol at 16 h, Figure 2 and Table 2). This result indicated that polyP synthesis in bifidobacteria varied greatly between different strains, as previously observed in lactobacilli.

[0278] Comparing polyP production at 6 and 16 h time points, all B. longum strains, except B. scardovi and B. longum subsp. longum KABP-042 (CECT7894), showed higher polyP values ​​at 6 h than at 16 h (Figure 2 and Table 2). The remaining strains produced more polyP at 16 h, whereas B. longum subsp. longum KABP-042 (CECT7894) produced similar amounts at both time points. Thus, it can be concluded that polyP production in bifidobacteria varies along the growth curve and that this growth-related variation is also strain-dependent, highlighting the importance of analyzing more than one time point along the growth curve.

[0279] In particular, B. longum subsp. longum KABP-042 (CECT7894) showed high polyP production at 6 h (Table 2). Surprisingly, at 16 h, B. longum subsp. longum KABP-042 (CECT7894) also showed the highest polyP formation capacity. Interestingly, B. longum subsp. longum KABP-042 was the only B. longum strain to show this behavior, and high polyP production was observed regardless of the culture period. In contrast, other polyP-producing strains showed production capacity only at short culture periods (e.g., B. longum subsp. longum ATCC15707) or at long culture periods (e.g., B. animalis BB12). Therefore, the ability to constantly produce polyP represents an additional advantage of the B. longum subsp. longum KABP-042 (CECT7894) strain.

[0280] Notably, unlike B. adolescentis JCM1275, B. longum subsp. longum KABP-042 (CECT7894) was able to grow while producing polyP. In addition, B. longum subsp. longum KABP-042 (CECT7894) produced more polyP than the other strains that were able to grow further. This demonstrates that B. longum subsp. longum KABP-042 (CECT7894) has the highest ability to grow and colonize in the intestine while eliciting beneficial effects through efficient production of the postbiotic molecule polyP.

[0281] Furthermore, B. longum subsp. longum KABP-042 (CECT7894) was able to produce 140-fold more polyP (1.6 vs. 230.9 nmol, Table 2) in 6 h than B. skaldovii BAA-773, which is known to express ppk (Qian et al., 2011). B. longum subsp. longum KABP-042 (CECT7894) was able to produce 18-fold more PolyP (12.7 vs. 230.9 nmol, Table 2) than L. plantarum WCFS1, which is known to produce PolyP.

[0282] Table 2. PolyP quantification (nmol) and growth (OD ) of the strains analyzed in this study at 6 and 16 h. 550 ppk, polyphosphate kinase gene; NA, not applicable. [Table 2]

[0283] In addition, the presence of the ppk gene was assessed by BLAST in the available genomes of the strains under study (Tables 2 and 3). In agreement with the phenotypic results, ppk sequences were found in all Bifidobacterial and some Lactobacillus genomes tested. However, considering the differences in polyP production between the strains, the data support that the regulatory mechanisms differ between the strains. Indeed, polyP biosynthesis in bacteria seems to be regulated at the post-transcriptional and / or post-translational level.

[0284] Table 3. Identification of ppk genes in available genomes by BLAST. ND: not detected. [Table 3]

[0285] Considering the differences observed among the ppk sequences of bifidobacterial strains, their amino acid sequences were aligned and a tree was constructed. The results showed that bifidobacterial PPKs could be classified into two clades (Fig. 3), one including B. animalis and B. adolescentis strains and the other including B. skaldovii, B. longum and B. breve strains.

[0286] Example 2: Stability of Bifidobacterium longum subsp. longum KABP-042 (CECT7894) in the final product The stability of a probiotic product depends on several factors, including the industrial processes of manufacture and storage, and the intrinsic properties of the probiotic strain.

[0287] Industrial processes are optimized to reduce the loss of viability of the strains during production and storage. In addition, manufacturers tend to start with a high dose of probiotic bacteria to combat losses during the shelf life of the product. However, bifidobacteria have a naturally reduced aerotolerance, making them more difficult to maintain stability over extended product shelf life compared to other probiotic species.

[0288] This study examined the stability of B. longum subsp. longum KABP-042 (CECT7894) in the final product.

[0289] 2.1 Materials and Methods The final product of B. longum subsp. longum KABP-042 (CECT7894) was treated with the active ingredient (min. 10 9The product was formulated in a matrix containing colony forming units (cfu), sunflower oil (up to 10 mL) and DL-alpha tocopherol (4 mg). The product was packaged in amber glass bottles and stored at Zone II conditions (25° C., 60% relative humidity (RH)).

[0290] The amount of active ingredient (probiotic strain) was selected to meet the recommended cfu / dose according to available guidelines.

[0291] The stability of the strains was examined by measuring cfu by plate count according to ISO 29981 at 0, 1, 3 and 6 months after production. Results were expressed as log(cfu). Trend lines were obtained to extrapolate expected cfu at 12 months. Fold reduction and log reduction comparing cfu between 0 and 12 months were calculated.

[0292] 2.2 Results Figure 4 shows the viable B. longum subsp. longum KABP-042 (CECT7894) found in the final product over time (0-6 months) and the predicted trend line. At 12 months, the log(cfu) was estimated to be 9.01. The results revealed a 3-fold decline (i.e., approximately 0.5 log loss) over 12 months, indicating good stability of the product. Thus, the 3-fold excess at manufacture resulted in 10% of viable bacteria at the 12-month time point. 9 is sufficient to ensure cfu.

[0293] Example 3: Further probiotic properties of Bifidobacterium longum subsp. longum KABP-042 (CECT7894) 3.1 Materials and Methods The ability of B. longum subsp. longum KABP-042 (CECT7894) to resist gastrointestinal conditions, adhere to the intestinal epithelium, and utilize human milk oligosaccharides (HMOs) was characterized. L. rhamnosus GG (ATCC53103) and B. longum subsp. longum ATCC15707 were used as controls as indicated. Lactobacillus strains were routinely grown in anaerobic conditions at 37°C in MRS. Bifidobacterial strains were grown under the same conditions except that the MRS was supplemented with 0.1% (w / v) cysteine-HCl (MRScys).

[0294] Gastric stress resistance and bile salt survival were investigated by exposing the strains to simulated gastric fluid (per liter: 7.3 g NaCl, 0.52 g KCl, 3.78 g NaHCO3 and 3 g pepsin) and to medium containing 0.3% (w / v) bile salts for 180 min, at pH 2.3 for 30 min and at pH 3 for 90 min. Vegetative bacteria were counted by serial dilution and counting method before and after the incubation period. The commercial probiotic strain L. rhamnosus GG was used as reference.

[0295] Adhesion to the intestinal epithelium was studied in vitro using Caco-2 intestinal epithelial cells. Bacterial suspensions were added to Caco-2 monolayers (at a multiplicity of infection (MOI) of cells to probiotics of 1:5) and to wells without Caco-2 cells as controls. After 1 h of incubation at 37 °C, the medium was removed, cells were detached, and the suspension was collected. Bacteria in the resulting suspension were counted by serial dilution and plate counting. Bacteria in the medium of control wells were also quantified. B. longum subsp. longum ATCC15707, known for its adhesion ratio of 47-55, was used for quality control.

[0296] The ability to degrade HMO was tested by growing the strains on MRS with the HMO lacto-N-tetraose (1%) as the sole carbon source. MRS with 1% glucose was used as a positive control. MRS without carbon source was used as a negative control. Growth was monitored for 24 h.

[0297] The genome sequence of B. longum subsp. longum KABP-042 (CECT7894) was obtained from Illumina Hiseq, and the reads were assembled and annotated. The genome was searched for genes of interest, such as adhesins, bacteriocins, HMO-degrading enzymes, and bile salt hydrolases, by BLAST.

[0298] 3.2 Results Resistance to gastric conditions was evaluated by simulating fast gastric transit without pH buffer (pH 2.3 for 30 min) and slow postprandial digestion with pH buffer (pH 3 for 90 min). B. longum subsp. longum KABP-042 (CECT7894) and the well-known probiotic strain L. rhamnosus GG showed less than 1 log cfu / mL loss upon gastric challenge at pH 2.3 and pH 3 (Table 4). In addition, B. longum subsp. longum KABP-042 (CECT7894) showed high resistance to bile salts, with losses less than 0.5 log cfu / mL, the same level as L. rhamnosus GG. Furthermore, one copy of the bsh gene encoding bile salt hydrolase was found in the genome of B. longum subsp. longum KABP-042 (CECT7894), confirming that this strain is well adapted to the gastrointestinal tract.

[0299] Table 4. Resistance to gastric stress and bile salts and adhesion to the intestinal epithelium. Values ​​presented are the mean and standard deviation of log cfu / mL or % of log cfu / mL. L. rhamnosus GG and B. longum subsp. longum ATCC15707 were used as controls. NA, not applicable. [Table 4]

[0300] B. longum subsp. longum KABP-042 (CECT7894) was confirmed to adhere to the intestinal epithelium with an adhesion capacity of 70.8% (Table 4). This strain adhered more than the moderately adherent control strain B. longum subsp. longum ATCC15707 (51.2%). Genomic analysis confirmed that this strain is well endowed with several adhesion proteins and domains. Bacterial adhesion to human tissues is a prerequisite for effective bacterial engraftment, which is a desirable trait to achieve sustained health benefit effects.

[0301] B. longum subsp. longum KABP-042 (CECT7894) was able to grow in the presence of the HMO lacto-N-tetraose (LNT) as the sole carbon source (Figure 5). The genome was confirmed to harbor HMO degradation genes including lacto-N-biosidase, beta-galactosidase, alpha-galactosidase, hexosaminidase, and beta-glucuronidase. Thus, HMO utilization by B. longum subsp. longum KABP-042 (CECT7894) was phenotypically and genotypically confirmed, proving that it is well adapted to the infant gut.

[0302] In addition, the B. longum subsp. longum KABP-042(CECT7894) genome harbors other genes encoding carbohydrate-active enzymes (CAZy), suggesting that it has the ability to degrade a wide range of complex substrates, including those derived from various human foods. B. longum subsp. longum KABP-042(CECT7894) appears to have a versatile carbohydrate metabolism.

[0303] Further analysis showed the presence of genes encoding lantipeptide B, a serpin, and an adhesin. Lantipeptide B (lantibiotic) is a class I bacteriocin produced by B. longum strains that exhibits potent antibacterial activity against a range of Gram-negative and Gram-positive pathogens. Serpins (derived from serine protease inhibitors) selectively inactivate human neutrophils and pancreatic elastase (a protease), providing anti-inflammatory effects and contributing to the maintenance of intestinal homeostasis.

[0304] Overall, the in vitro and in silico analyses of B. longum subsp. longum KABP-042 (CECT7894) confirmed the probiotic properties of this strain and demonstrated that it is well adapted to the human gastrointestinal tract, including the infant gut, due to its ability to degrade HMOs.

[0305] Example 4: Effect of polyP from B. longum CECT7894 in protecting the intestinal barrier The postbiotic effect of polyP is related to its role in maintaining intestinal homeostasis and protecting the intestinal barrier function. One of the mechanisms of action is the induction of the heat shock protein HSP27, a cytoprotective factor in enterocytes (Alcantara et al., 2018).

[0306] We investigated whether polyP produced by B. longum CECT7894 affects barrier integrity and intestinal permeability, and whether its effects are related to HSP27 production or the induction of other barrier integrity markers, including tight junction proteins.

[0307] 4.1. Materials and Methods 4.1.1 Preparation of B. longum CECT7894 samples and quantification of polyP production B. longum CECT7894 was grown in MEI and low phosphate (LP) medium. The latter medium has the same composition as MEI but is supplemented with no polyP precursors (K2HPO4 and KH2PO4) so ​​that this strain cannot produce significant amounts of polyP. After 16 h of growth, the cultures were centrifuged and the supernatants were collected, filtered and adjusted to neutral pH. The amount of PolyP was measured as described in Example 1.

[0308] 4.1.2. Assessment of Barrier Integrity and Permeability The integrity of the Caco-2 cell monolayer was assessed by measuring the transepithelial electrical resistance (TEER) and permeability was assessed by the apparent permeability coefficient (Papp) of the paracellular transport marker Lucifer Yellow.

[0309] Caco-2 cells were seeded on porous membrane inserts with apical (top) and basolateral (bottom) compartments. Medium Essential Medium Eagle (MEM) was added to both compartments. Cells were treated with supernatants of B. longum CECT7894 grown in MEI and LP media. Additional cells were treated with MEM, non-fermented MEI and LP media and used as controls.

[0310] After 72 hours of treatment, TEER and permeability were determined. TEER was measured with a Millicell® ERS voltammetry. For the permeability assay, Lucifer Yellow was added to the apical compartment. Aliquots were taken from the basolateral compartment at 15, 30, 45, 60, 90 and 120 minutes and the fluorescence of transported Lucifer Yellow was measured with a fluorescence microplate reader at excitation / emission wavelengths of 485 / 520 nm.

[0311] Quantification of HSP27 production The production of HSP27 was examined in confluent Caco-2 intestinal epithelial cells by Western blot assay as described by Alcantara et al., 2018, with some modifications. Bacterial supernatant was added to the cell culture and incubation was allowed to proceed for 16 h. MEI medium and LP medium were used as controls. To recover HSP27, cells were lysed by SDS-PAGE and boiled for 5 min. Proteins were separated on SDS-PAGE gels and then transferred to nylon membranes (blots). Blots were incubated with rabbit polyclonal anti-HSP27 serum or mouse monoclonal anti-β-actin antibody (protein used for normalization). After washing, the respective secondary antibodies peroxidase-conjugated anti-rabbit IgG and anti-mouse IgG were used. Blot images were captured and proteins were quantified with an Imagin680 system.

[0312] 4.1.4. Expression of genes encoding tight junction proteins Caco-2 cells were exposed to the supernatant of B. longum CECT7894 grown in MEI and LP media for 16 h. Cells were then harvested and RNA was extracted with TRIZOL reagent. cDNA was obtained from the RNA using the SuperScript VILO cDNA synthesis kit. Quantitative PCR (qPCR) reactions were performed using SYBR Green under conditions specified by the manufacturer. Expression of the tight junction proteins zonula occludens-1 (ZO1), junction adhesion protein-1 (JAM1) and occludin was quantified. Expression of 18S rRNA and GADPH genes was used for normalization.

[0313] 4.2 Results First, the amount of polyP in the supernatants grown in MEI medium was higher than that in the supernatants grown in LP medium (Table 5). Notably, the amount in MEI was lower than that quantified in Example 1 in the same medium. However, in Example 1, polyP is measured intracellularly, whereas in Example 4, polyP is measured extracellularly. Here, we investigated extracellular production, i.e., extracellular polyP in contact with the intestinal barrier, to mimic the conditions in the intestine.

[0314] Table 5. PolyP amount (nmol) in the supernatant of B. longum CECT7894 grown for 16 h under high phosphate (MEI medium) or low phosphate (LP medium) conditions. Growth (OD 550 ) is shown. [Table 5]

[0315] Experiments with Caco-2 monolayers in a two-compartment system showed that apical exposure to B. longum CECT7894 supernatants (i.e., from cultures in MEI medium) with high concentrations of polyP showed higher TEER (an indicator of greater resistance of the cell barrier) compared to supernatants with lower amounts of polyP and to control supernatants. Experiments measuring the flux of Lucifer Yellow from the apical to the basolateral compartment also showed that high polyP concentrations derived from B. longum CECT7894 significantly reduced the permeability of compounds compared to low polyP supernatants and controls (shown in Figure 6). These results indicate that polyP produced by B. longum CECT7894 promotes a stronger functional barrier that impedes intestinal permeability. Importantly, even though the amount of polyP in the supernatant was lower than in the cells, the effect was significant, suggesting that even the small amount of polyP produced by B. longum CECT7894 is sufficient to have a beneficial effect on barrier integrity.

[0316] Western blot analysis of HSP27 production in intestinal epithelial cells showed that the supernatants with high concentrations of polyP produced by B. longum CECT7894 (i.e., from cultures in MEI medium) induced significantly higher HSP27 production compared to the supernatants from cultures with low concentrations of polyP (i.e., from cultures in LP medium). In addition, a correlation was also observed between HSP27 expression and polyP concentration in the supernatants of B. longum CECT7894 using different samples with different polyP amounts (shown in Figure 7). These results indicate that B. longum CECT7894 can affect the production of HSP27 through the synthesis of polyP and thus this strain has a protective effect on the intestinal epithelium.

[0317] Furthermore, the expression of tight junction proteins ZO1, JAM1 and occludin, which are important for maintaining barrier integrity, was induced by the presence of high polyP content in the supernatant of B. longum CECT7894 compared to the supernatant with low polyP content (shown in Figure 8 ).

[0318] Overall, these results confirm that B. longum CECT7894 is able to increase barrier integrity and reduce intestinal permeability by inducing the production of the cytoprotective protein HSP27 and tight junction proteins via the production of polyP, thus exerting a positive effect on intestinal barrier homeostasis.

[0319] Example 5: Effect of breast milk HMO lacto-N-tetraose and polyamines on PolyP production capacity of B. longum CECT7894 B. longum is naturally found in human breast milk and in the intestine of infants. Human milk contains a certain amount of phosphate (the substrate for polyP). We investigated whether B. longum CECT7894 can produce polyP in the presence of breast milk. In addition, some evidence in other bacteria suggests that polyamines and carbon sources can affect polyP metabolism (Anand et al., 2019). Because breast milk contains polyamines and carbohydrate HMOs, we tested whether the polyamines and HMO lacto-N-tetraose (LNT) utilized by B. longum CECT7894 (as confirmed in Example 3) can affect polyP biosynthesis in the test strain.

[0320] 5.1. Materials and Methods B. longum CECT7894 was grown in glucose-free MEI medium supplemented with i) breast milk (1% v / v); ii) LNT (1% w / v); iii) glucose (0.5% w / v) and the amounts of polyamines found in breast milk: putrescine, spermidine, and spermine at 70.0, 424.2, and 610.0 nmol / dl, respectively; and iv) glucose (0.5% w / v) as a positive control. Growth (OD 550 ) and polyP production was determined after 6 and 16 h of incubation.

[0321] 5.2 Results Analysis of the growth of B. longum CECT7894 in the presence of breast milk (with sugars present in breast milk as the sole carbon source) showed that, even though the strain only reached a low OD, it was still able to produce some amount of PolyP at 6 h. Growth in LNT as the sole carbon source was lower than growth in control conditions at 6 h (OD 1.8 vs. 2.9). However, the strain produced a higher amount of polyP (117.0 vs. 110.2). In addition, polyP persisted for a longer time in the LNT compared to the control (145.0 vs. 70.2 at 16 h). The presence of polyamines in MEI medium with glucose had no effect on growth or polyP production (see Table 6 and Figure 9).

[0322] Table 6. PolyP quantification (nmol) and growth (OD ) at 6 and 16 h of B. longum CECT7894 cultures incubated under different conditions. 550 ) [Table 6]

[0323] In conclusion, these results show that B. longum CECT7894 can produce polyP in the presence of breast milk and that HMO LNTs enhance polyP biosynthesis, suggesting an LNT-dependent regulation of polyP metabolism in the tested strain. Importantly, this is the first time that an interaction between HMO and polyP has been shown, highlighting the beneficial role that B. longum CECT7894 supplementation may have, for example, in infants.

[0324] Example 6: Symbiosis between Bifidobacteria utilizing 2FL and B. longum CECT7894 We investigated whether B. longum CECT7894 could grow on HMO 2'-FL, for example due to syntrophy with other bifidobacteria present in human milk or the human intestine.

[0325] 6.1. Materials and Methods B. bifidum Bb01 (CECT30646) was grown for 48 h in MRS medium with 2'-fucosyllactose (2'-FL) (4% wt / vol) as the sole carbon source. The supernatant was collected and filtered to remove cells. The supernatant was mixed (1:1) with fresh MRS medium without a carbon source. B. longum CECT7894 was grown in this mixture for 24 h and the OD was monitored.

[0326] 6.2. Results B. longum CECT7894 was able to grow in the presence of the supernatant of B. bifidum Bb01 (CECT30646) cultured with 2'-FL until it reached an OD of 0.5 (Figure 10). This result demonstrates that B. longum CECT7894 can be a food source for other bifidobacteria that utilize 2'-FL. Therefore, together with the results of Example 3 (Figure 5), B. longum CECT7894 is able to grow in the presence of the two most abundant HMOs in breast milk (LNT and 2'-FL).

Claims

1. A probiotic composition for use in the treatment of extra-intestinal conditions associated with enhanced intestinal permeability in a subject, comprising a strain of Bifidobacterium longum subsp. longum deposited under accession number CECT7894 at the Spanish Type Culture Collection (CECT) under the Budapest Treaty, or a derivative bacterial strain thereof, wherein the treatment is by generating polyphosphates, and the derivative bacterial strain has (a) a genome having an average nucleotide identity (ANI) of at least 99% with the genome of the corresponding deposited strain; and (b) retains the polyphosphate-generating ability of the corresponding deposited strain, the probiotic composition.

2. The probiotic composition for use according to claim 1, wherein the extra-intestinal condition is an immune disorder or disease, a metabolic or cardiovascular disorder or disease, or a neurological or mental disorder or disease.

3. The probiotic composition for use according to claim 2, wherein the metabolic or cardiovascular disorder or disease is selected from the group consisting of obesity, diabetes, insulin resistance, non-alcoholic fatty liver disease, cirrhosis, atherosclerosis, hypertension, chronic heart failure, and stroke; the immune disorder or disease is selected from the group consisting of non-food allergies / hypersensitivities, immune senescence, multiple sclerosis, rheumatoid arthritis, lupus erythematosus, sarcopenia, asthma, allergic rhinitis, and atopic dermatitis; and the neurological or mental disorder or disease is selected from the group consisting of Alzheimer's disease, autism spectrum disorder, schizophrenia, and depression.

4. The extra-intestinal condition is an immune disorder or disease, or a metabolic or cardiovascular disorder or disease, the immune disorder or disease is selected from the group consisting of non-food allergies / hypersensitivities, immune senescence, multiple sclerosis, rheumatoid arthritis, lupus erythematosus, sarcopenia, asthma, allergic rhinitis, and atopic dermatitis, and the metabolic or cardiovascular disorder or disease is selected from the group consisting of obesity, diabetes, insulin resistance, non-alcoholic fatty liver disease, cirrhosis, atherosclerosis, hypertension, chronic heart failure, and stroke, the probiotic composition for use according to claim 2.

5. The probiotic composition for use according to claim 1, further comprising at least one human milk oligosaccharide.

6. The probiotic composition for use according to claim 1, wherein the subject is a human, and the human is selected from the group consisting of an elderly person, a premature infant, an infant, an athlete, and a debilitated person.

7. The probiotic composition for use according to claim 6, wherein the infant is selected from the group consisting of a premature infant, a weak infant, an infant born with a birth weight below the normal range, an infant subject with intrauterine growth retardation, an infant born by cesarean section, an infant administered with an antibiotic, an infant raised on formula milk, or an infant breastfed.

8. The probiotic composition for use according to claim 1, wherein the derived bacterial strain has a genome having an average nucleotide identity of at least 99.5% with the genome of the corresponding deposited strain.

9. The production of polyphosphate of the Bifidobacterium longum subsp. longum CECT7894 strain or its derived bacterial strain comprises the following steps: (a) The strain inoculated with OD0.1 was cultured at 37°C under anaerobic conditions in a malic enzyme induction medium containing 0.5% yeast extract, 0.5% tryptone, 0.4% K ２ HPO ４ , 0.5% KH ２ PO ４ , 0.02% MgSO ４ ·7H ２ O, 0.005% MnSO ４ , 1 ml of Tween 80, 0.05% cysteine, and 0.5% glucose; (b) Recovering the cells by centrifugation and dissolving them in 1 ml of 5% sodium hypochlorite while gently stirring at room temperature for 45 minutes; (c) Centrifuging the insoluble material at 16,000 g for 5 minutes at 4°C to obtain a pellet, washing it twice with 1 ml of 1.5 M NaCl plus 1 mM EDTA, and centrifuging at 16,000 g for 5 minutes at 4°C in between; (d) Washing twice successively with 1 ml of water and centrifuging at 16,000 g for 5 minutes at 4°C in between to extract polyphosphate from the pellet; (e) Precipitating the polyphosphate in the pooled water extract by adding 0.1 M NaCl and one volume of ethanol, and then incubating on ice for 1 hour; (f) Centrifuging at 16,000 g for 10 minutes and resuspending the polyphosphate pellet in 50 μL of water; (g) The step: i. Hydrolyzing the serial dilutions of the polyphosphate sample isolated from the control strain Lactobacillus plantarum WCFS1 with one volume of 2 M HCl and incubating at 95°C for 15 minutes; ii. Adding half a volume of 2 M NaOH to neutralize the dilution; iii. Measuring the released phosphate with a BIOMOL Green kit to obtain the amount of phosphate in each dilution; iv. Measuring the fluorescence of the released phosphate using 4',6-diamidino-2-phenylindole, DAPI, at a final concentration of 10 μM in a buffer of 50 mM Tris-HCl pH 7.5 and 50 mM NaCl with a fluorescence spectrometer at an excitation wavelength of 415 nm and an emission wavelength of 550 nm to obtain the fluorescence value of each dilution; and v. Creating a calibration curve using the phosphate value obtained in (iii) and the corresponding fluorescence value obtained in (iv) to create a calibration curve relating the amount of phosphate from polyphosphate to fluorescence intensity; and (h) A step of quantifying polyphosphate from the resuspended fraction of step (f): 1) Measuring the fluorescence of phosphate using DAPI at a final concentration of 10 μM in a buffer of 50 mM Tris-HCl pH 7.5 and 50 mM NaCl with a fluorescence spectrometer at an excitation wavelength of 415 nm and an emission wavelength of 550 nm; 2) Calculating the amount of polyphosphate using the calibration curve; and 3) Expressing the polyphosphate value as nmol phosphate By which, when the polyphosphate production is determined at 6 hours and / or 16 hours of culture, a probiotic composition for use according to any one of claims 1 to 8, which has more polyphosphate production than that of the control strain.

10. B. The polyphosphate production of Bifidobacterium longum subsp. longum CECT7894 or its derived bacterial strain is at least 10 times higher at 6 hours than the polyphosphate production of the control strain L. plantarum WCFS1, and is higher at 16 hours, and the polyphosphate production of the control strain L. plantarum WCFS1 and the level of polyphosphate by the control strain do not exist at 16 hours. A probiotic composition for use according to claim 9.

11. A probiotic composition for use according to claim 1, comprising a Bifidobacterium longum subsp. longum strain deposited under the accession number CECT7894.

12. (i) A Bifidobacterium longum subsp. longum strain deposited with the Spanish Type Culture Collection CECT under the accession number CECT7894 based on the Budapest Treaty, or a derived bacterial strain thereof, (a) having a genome with an average nucleotide identity ANI of at least 99% with the genome of the corresponding deposited strain; and (b) retaining the polyphosphate-producing ability of the corresponding deposited strain A probiotic composition comprising a derived bacterial strain, and (ii) at least one human milk oligosaccharide A combination comprising the following, configured for simultaneous, separate or sequential administration. **Claim 13** The combination according to claim 12, wherein the human milk oligosaccharide is selected from the group consisting of fucosylated oligosaccharides, sialylated oligosaccharides, N-acetyl lactosamine and combinations thereof. **Claim 14** The combination according to claim 13, comprising 2'-fucosyllactose and / or lacto-N-tetraose. **Claim 15** The combination according to claim 12, further comprising a Bifidobacterium strain, particularly B. bifidum CECT30646. **Claim 16** A combination according to any one of claims 12 to 15 for use in the treatment of a condition associated with enhanced intestinal permeability in a subject, wherein the treatment is by producing polyphosphate, and the condition is selected from the group consisting of immune disorders or diseases, metabolic or cardiovascular disorders or diseases, neurological or psychiatric disorders or diseases, and gastrointestinal disorders or diseases.

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