Lactic acid bacteria species, strains and compositions that allow for regulation of in vivo oxygenation by increasing HIF-1α levels

Abstract

The present invention relates to specific genera, species, strains and compositions of lactic acid bacteria capable of increasing cellular levels of the hypoxia-inducible factor HIF-1α, for example for maintaining or enhancing normoxia in hypoxic-inducible conditions such as physical exertion, fatigue, chronic fatigue, oxygen deficiency, travel beyond the limits of the Earth's atmosphere, ocular oxidative stress and scuba diving, or for the treatment of hypoxia in hypoxic-inducible conditions such as neurodegenerative diseases, pulmonary effects associated with respiratory failure, neonatal hypoxia-ischemia, myocardial ischemia, metabolic disorders, chronic cardiac and renal diseases, reproductive disorders such as pre-eclampsia and endometriosis, exacerbation of postural and kinetic tremors, cerebral hypoxia.

Description

[Technical field]

[0001] The present invention relates to specific genera, species, strains and compositions of lactic acid bacteria capable of increasing cellular levels of the hypoxia-inducible factor HIF-1α for maintaining or enhancing normoxia in hypoxic-induced conditions such as, for example, physical exertion, lethargy, chronic fatigue, oxygen deficiency, travel beyond the limits of the Earth's atmosphere, ocular oxidative stress, scuba diving, or for treating hypoxia in hypoxic-induced conditions such as neurodegenerative diseases, pulmonary effects associated with respiratory failure, neonatal hypoxia-ischemia, myocardial ischemia, metabolic disorders, chronic cardiac and renal diseases, reproductive disorders such as pre-eclampsia and endometriosis, exacerbation of postural and kinetic tremors, cerebral hypoxia. [Background technology]

[0002] Oxygen (O2) is an essential nutrient that serves as a key substrate in cellular metabolism and energy production by aerobic organisms. In various physiological and pathological conditions, organisms can experience limited / insufficient oxygen availability, a condition called hypoxia.

[0003] Oxygen deficiency poses a great stress to living cells. This condition is associated with the inappropriate accumulation of free radicals, which cause further stress on the protein components and genetic material of the cells. To cope with the hypoxic stress condition, cells activate a series of adaptive responses that match the O2 supply with their metabolic, bioenergetic, and redox requirements. In particular, they temporarily arrest the cell cycle, reduce energy expenditure, and secrete survival and pro-angiogenic factors. The intestinal mucosa receives 10% to 35% of the total cardiac output, and the estimated surface area of ​​the digestive tract is approximately 250-300 m under normal conditions. 2(Lundquist et al., 2016). The intestine is characterized by a unique oxygenation profile that results from a combination of factors, including large fluctuations in blood perfusion due to food intake (Matheson et al., 2000). Changes in the amount of blood reaching the intestine greatly affect the amount of oxygen available to the remaining body compartments. Therefore, the intestine plays a key role in determining the distribution of total O2 available to the organism.

[0004] In the small intestine, increased oxygen availability sustains the intense energy expenditure of highly proliferative stem cells and differentiated postmitotic cells with high energy demands from digestive, secretory and absorptive processes (Rangel-Huertara et al., 2017; Van Der Schoorra, 2002). The characteristics of oxygenation and oxygen consumption in the small intestine allow the hypothesis that their regulation may have a major impact on the redistribution of globally available oxygen in the body. Under basal physiological conditions, intestinal mucosal epithelial cells are exposed to relatively low oxygen levels, previously described as “physiological hypoxia” (Karhausen et al., 2005). To this condition, intestinal epithelial cells are persistently adapted (Shepherd, 1982; Albenberg et al., 2014).

[0005] Hypoxia-inducible factors (HIFs) constitute key mediators of intestinal epithelial adaptation to oxygen-poor microenvironments (Ramakrishnanra, 2016). These mediators are involved in the reduction of oxygen consumption in mitochondria through the inhibition of pyruvate to acetyl-CoA conversion, suppression of mitochondrial biogenesis, and activation of mitochondrial autophagy (Goda and Kanai, 2012). The HIF-associated reduction in cellular oxygen consumption and the subsequent redistribution of oxygen in the pericellular microenvironment are supported by evidence obtained using PHD inhibitors (Susser et al., 2020). HIFs are heterodimers composed of two subunits, called α and β, respectively, with the second subunit constitutively expressed in eukaryotic cells. The HIF-α subunit belongs to the helix-loop-helix Per-Arnt-Sim (bHLH-PAS) family of basic transcription factors (Schito et al., 2016). Vertebrates have three subunit α, HIF-1α, HIF-2α, and HIF-3α. The N-terminal regions of these subunits contain domains required for DNA binding and heterodimerization (Wu et al., 2015). HIF-α subunits have a highly conserved oxygen-dependent degradation (ODD) domain. The ODD domain contains two hydroxylated prolines in both HIF-1α and HIF-2α (Chan et al., 2005). Hydroxylation of HIF-α leads to proteasomal degradation. HIF-α subunits are hydroxylated by specific enzymes PHD1 (EGLN2), PHD2 (EGLN1), and PHD3 (EGLN3), which belong to the prolyl hydroxylase domain (PHD) enzyme family, which are the main oxygen sensors in cells. Under normoxic conditions, PHDs use oxygen to hydroxylate HIF-α subunits at the proline level present in the ODDs. Hydroxylation allows the binding and degradation of HIF-α by the Von Hippel-Lindau tumor suppressor protein (VHL), which acts as an E3 ubiquitin ligase (Ivan et al., 2001).Under conditions of low oxygen availability, PHD enzymes are unable to hydroxylate HIF-α, which is stabilized by heterodimerization with HIF-β subunits (Wang et al., 1995). The resulting heterodimers can bind to genetic elements called HIF response elements (HREs) present in the promoters of target genes. Such binding results in the expression of target genes and allows cells to mount an adaptive response to hypoxia (Toescu et al., 2004; Wiener et al., 1996). Although HIF-1α and HIF-2α are closely related and can activate HRE-dependent expression, both subunits differ in their transcriptional domains, suggesting that they have distinct gene targets. In particular, scientific evidence has shown that HIF-1α preferentially induces the glycolytic pathway, adapting to energy production during oxygen deficiency (Hu et al., 2003). Adaptation to hypoxia associated with HIF activity involves many changes in cellular metabolism. Chief among these is the reduction of oxygen consumption by shifting energy production from mitochondrial oxidative phosphorylation to anaerobic glycolysis.In the intestinal environment, HIF regulation is influenced by multiple factors related to both cellular metabolism and microbial action (Singhal et al., 2020).

[0006] Hypoxia is frequently observed in the presence of various acute and chronic pathologies. Pathologies associated with hypoxia include neonatal hypoxia-ischemia, myocardial ischemia, metabolic disorders, chronic cardiac and renal diseases, reproductive disorders such as preeclampsia and endometriosis, exacerbation of postural and kinetic tremors, cerebral hypoxia, and neurodegenerative diseases (Chen et al., 2020; Legros et al., 2010; Nalivaeva et al., 2019; Merelli et al., 2020). Hypoxia is considered important in pathological conditions resulting from the loss of respiratory function of the lung surface. In this context, attention should be paid to the acute respiratory distress caused by infection with the novel pandemic coronavirus Sars-CoV-2 (Gibson et al., 2020; Ramirez et al., 2020). In addition, hypoxia is closely related to the altered physiological state and the manifestation of pathological symptoms associated with prolonged stays with low oxygen availability. In this regard, emphasis should be placed on the pulmonary, neurological and muscular disorders associated with activities at high altitude.In contrast to reports in the scientific literature on probiotic microorganisms (Esfandiary et al., 2016; Deepak et al., 2015; Chen et al., 2020; Han et al., 2020), the Applicant has surprisingly found that Lactobacillus acidophilus, or Lactobacillus acidophilus and Streptococcus thermophilus and / or Bifidobacterium animalis subsp. lactis, preferably Levilactobacillus brevis (formerly known as Lactobacillus brevis), Lactiplantibacillus plantarum subsp. plantarum (formerly known as Lactobacillus plantarum ... (formerly known as Lactobacillus plantarum), Lactobacillus helveticus, Lacticaseibacillus paracasei subsp. paracasei (formerly known as Lactobacillus paracasei subsp. It has been discovered that oral administration of Lactobacillus casei (known as Lactobacillus subtilis paracasei) is able to increase the expression / stability of HIF-1α and therefore that certain genera, species, strains and compositions of Lactobacillus casei are able to maintain or enhance normoxia under hypoxic-inducing conditions such as, for example, for the treatment of hypoxia in conditions involving hypoxia such as physical exertion, fatigue, chronic fatigue, oxygen deficiency, travel beyond the limits of the Earth's atmosphere, ocular oxidative stress and scuba diving, or neurodegenerative diseases, pulmonary effects associated with respiratory failure, neonatal hypoxia-ischemia, myocardial ischemia, metabolic disorders, chronic cardiac and renal diseases, reproductive disorders such as pre-eclampsia and endometriosis, exacerbation of postural and kinetic tremors, cerebral hypoxia, etc. Summary of the Invention

[0007] One object of the present invention is Lactobacillus acidophilus capable of positively regulating cellular levels of the hypoxia inducible factor HIF-1α associated with a decrease in cellular oxygen consumption for use in the treatment of hypoxia induced conditions such as physical exertion, fatigue, chronic fatigue, oxygen deficiency, travel beyond the limits of the Earth's atmosphere, ocular oxidative stress and scuba diving, or in the treatment of hypoxia in conditions related to hypoxia such as neurodegenerative diseases, pulmonary effects associated with respiratory failure, neonatal hypoxia-ischemia, myocardial ischemia, metabolic disorders, chronic cardiac and renal diseases, reproductive disorders such as pre-eclampsia and endometriosis, exacerbation of postural and kinetic tremors, cerebral hypoxia.

[0008] According to one aspect of the present invention, the Lactobacillus acidophilus shown above is a Lactobacillus acidophilus strain deposited by the applicant under the Budapest Treaty on September 1, 2020 at the Collection National des Cultures de Microorganisms (hereinafter CNCM), Institut Pasteur, 75724 Paris Cedex 15, Rue du Doctor Rue 25, and bearing the accession number CNCM I-5567.

[0009] A further object of the present invention is a composition comprising the above-mentioned Lactobacillus acidophilus and, optionally, one or more pharma- ceutically acceptable excipients.

[0010] According to one embodiment of the invention, the composition further comprises Streptococcus thermophilus and / or Bifidobacterium animalis subsp. lactis.

[0011] According to a further aspect of the invention, the Streptococcus thermophilus is a Streptococcus thermophilus strain deposited by the applicant at the CNCM under the Budapest Treaty on September 1, 2020 and having the accession number CNCM I-5570, and the Bifidobacterium animalis subsp. lactis is a Bifidobacterium animalis subsp. lactis strain deposited by the applicant at the CNCM under the Budapest Treaty on September 1, 2020 and having the accession number CNCM I-5571, and / or a Bifidobacterium animalis subsp. lactis strain deposited by the applicant at the CNCM under the Budapest Treaty on September 1, 2020 and having the accession number CNCM I-5572.

[0012] According to a further aspect of the invention, the composition comprises, by weight of the composition, 30% to 50% Lactobacillus acidophilus, 25% to 35% Streptococcus thermophilus, and 25% to 35% Bifidobacterium animalis subsp. lactis.

[0013] According to further aspects of the invention, the composition may further comprise Leviractobacillus brevis (formerly known as Lactobacillus brevis), Lactiplantibacillus plantarum subsp. plantarum (formerly known as Lactobacillus plantarum), Lacticaseibacillus paracasei subsp. paracasei (formerly known as Lactobacillus paracasei subsp. paracasei), and Lactobacillus helveticus.

[0014] According to a further aspect of the present invention, the Leviractobacillus brevis strain is the Leviractobacillus brevis strain deposited by the applicant at the CNCM under the Budapest Treaty on September 1, 2020 and having the accession number CNCM I-5566, the Lactiplantibacillus plantarum subsp. plantarum is the Lactiplantibacillus plantarum subsp. plantarum strain deposited by the applicant at the CNCM under the Budapest Treaty on September 1, 2020 and having the accession number CNCM I-5569, and the Lacticaseibacillus paracasei subsp. paracasei is the Lacticaseibacillus paracasei strain deposited by the applicant at the CNCM under the Budapest Treaty on September 1, 2020 and having the accession number CNCM I-5569. paracasei strain, which is Lacticaceae, Bacillus paracasei subsp. paracasei strain I-5568, and Lactobacillus helveticus strain deposited by the applicant at the CNCM under the Budapest Treaty on September 1, 2020 and bearing the accession number CNCM I-5573.

[0015] According to a further aspect of the invention, the composition comprises, by weight of the composition, 30% to 50% Lactobacillus acidophilus, 1% to 10% Streptococcus thermophilus, 1% to 20% Bifidobacterium animalis subsp. lactis, 1% to 10% Leviractobacillus brevis (formerly known as Lactobacillus brevis), 1% to 10% Lactiplantibacillus plantarum subsp. plantarum (formerly known as Lactobacillus plantarum), 1% to 10% Lacticaseibacillus paracasei subsp. paracasei (formerly known as Lactobacillus paracasei subsp. paracasei), and 1% to 10% Lactobacillus helveticus.

[0016] According to a further aspect, the composition according to the invention is suitable for oral administration, for example in the form of a powder, capsule or granules. The composition preferably has a high concentration of bacteria, at least 10 billion for adults and at least 100 million for infants.

[0017] According to a further aspect, the composition according to the invention is suitable for oral administration to animals kept and / or maintained under oxygen-deficient conditions, e.g. animals kept and / or maintained on farms located very high above sea level, e.g. in the form of a powder, capsule, granules or spray.

[0018] The probiotic bacteria used may be viable or non-viable, sonicated, intermittently sterilized or freeze-dried.

[0019] Finally, the present invention relates to a method for identifying Lactobacillus acidophilus capable of positively regulating the cellular levels of the hypoxia-inducible factor HIF-1α associated with a decrease in cellular oxygen consumption, comprising the steps of evaluation of the cellular levels of HIF-1α, by Western Blot technique, in an in vitro cell model suitable for a suitable physiological representation of the target tissue, in the presence and absence of a pretreatment for at least 24 hours with a bacterial lysate specific for the Lactobacillus acidophilus strain to be evaluated, and evaluation of the extracellular acidification rate (ECAR), the oxygen consumption rate (OCR) and the relative glycolytic rate (ECAR / OCR ratio) in the same cell model, in the presence and absence of a 24 hour treatment with a bacterial lysate specific for the Lactobacillus acidophilus strain to be evaluated, using a Seahorse XFe96 analyzer (Agilent) according to the manufacturer's instructions or an equivalent technique. [Brief description of the drawings]

[0020] [Figure 1]FIG. 1 shows the effect of treatment with probiotic strains on HIF-1α levels under normoxic and hypoxic conditions. Western blotting of HIF-1α in Caco-2 cells differentiated for 6 days after 24 h incubation under normoxic and hypoxic conditions: (a), (b) in the presence (100 μg / ml) and absence of soluble fractions of bacterial lysates of Lactobacillus brevis strain CNCM I-5566, Lactobacillus acidophilus strain CNCM I-5567, Lactiplantibacillus plantarum subsp. plantarum CNCM I-5569, Lactobacillus helveticus CNCM I-5573, Lacticaseibacillus paracasei subsp. paracasei CNCM I-5568, Bifidobacterium animalis subsp. lactis CNCM I-5571, and Streptococcus thermophilus strain CNCM I-5570; (c), (d) In addition to those listed above, in the presence or absence of specific combinations of probiotic strains, including the bacterial strain Bifidobacterium animalis subsp. lactis CNCM I-5572. After densitometric analysis, the values ​​obtained were normalized to β-actin. Data are presented as mean ± SD of duplicate experiments. Data were compared by one-way analysis of variance (ANOVA) and Dunnett's test. * p < 0.05, ** p < 0.01, *** p < 0.001. Representative immunoblots showing quantification of HIF-1α, β-actin are also shown in the figures.

[0021] [Diagram 2]Figure 2 shows the effect of treating Caco-2 cells with bacterial lysate of the probiotic strain mixture on (a) L-lactate levels, (b) extracellular acidification rate (ECAR), (c) oxygen consumption rate (OCR), and (d) ECAR / OCR ratio. Caco-2 cells were treated with or without bacterial lysate (100 μg / ml) for 24 h. Lactate levels were analyzed by colorimetric assay. ECAR and OCR values ​​were obtained with a Seahorse XF extracellular flow analyzer. Values ​​are expressed as mean ± SEM of triplicates from three independent experiments. Student's t-test for unpaired data was used for comparison between two means. (*p<0.05; **p<0.01).

[0022] [Diagram 3] Figure 3 shows the effect of treatment with the mixture of probiotic strains described above on HIF-1α levels in brain homogenates of wild-type (wt) mice and 3xTg-AD mice used as Alzheimer's disease model mice. The analysis was performed on brain extracts of guinea pigs killed at 8 weeks of age and of guinea pigs at 16 and 48 weeks of age after the start of treatment. After densitometric analysis, the obtained values ​​were normalized to glyceraldehyde 3-phosphate dehydrogenase (GAPD). Data are presented as mean ± SD of triplicates of three experiments. Data were compared by one-way analysis of variance (ANOVA) with Bonferroni test. * p < 0,05. Representative immunoblots showing quantification of HIF-1α and GAPD are also shown in the figure.

[0023] [Figure 4] Figure 4 shows the effect of the intake of a specific probiotic formulation on (a) oxygen consumption, (b) mean heart rate during the last 5 minutes of exercise, and (c) blood lactate levels in subjects performing endurance sports. Data are presented as mean ± SD of triplicate experiments. Statistical significance between experimental groups performed in the absence of probiotic intake and those performed after receiving a specific oral bacterial therapy was determined by Student's t-test for paired data. *: p ≤ 0.05.

[0024] [Diagram 5] Figure 5 shows the distribution of values ​​of the considered blood parameters obtained from the general laboratory analysis. When present, statistically significant differences between the MTD and MTD+BO groups at each time point and over time for each group were indicated. *: p ≤ 0.05, **: p ≤ 0.001, ***: p ≤ 0.0001. Detailed Description of the Invention

[0025] In vitro experiments HIF-1α is a key mediator in the regulation of oxygen homeostasis and preferential induction of glycolysis, thereby adapting to energy production during oxygen deficiency and reducing oxygen consumption by shifting energy metabolism towards that pathway ( Hu et al., 2003 ).

[0026] The inventors performed in vitro assays using intestinal-derived cell models to evaluate the ability of specific bacterial strains, alone or in combination, to modulate the accumulation of HIF-1α associated with reduced cellular oxygen consumption. The results obtained show that exposure of Caco-2 cells to bacterial lysates of L. brevis CNCM I-5566, L. acidophilus CNCM I-5567, L. plantarum CNCM I-5569, L. helveticus CNCM I-5573, L. paracasei CNCM I-5568, B. lactis CNCM I-5571, and S. thermophilus CNCM I-5570 under normoxic conditions was associated with a significant increase in intracellular HIF-1α levels compared to untreated controls (Figure 1a). S. thermophilus CNCM I-5570, B. lactis CNCM I-5571, and L. acidophilus CNCM I-5567 were the most effective (L. acidophilus CNCM I-5567 increased approximately 2.2-fold, S. thermophilus CNCM I-5570 and B. lactis CNCM I-5571 increased approximately 2-fold). Under hypoxic conditions, each strain did not induce significant changes compared to untreated cells, except that the addition of L. acidophilus CNCM I-5567 significantly increased HIF-1α levels compared to the control (Figure 1b). Exposure of Caco-2 cells to the combined bacterial extract at concentrations of 50 and 100 μg / ml was associated with a significant increase in the intracellular accumulation of HIF-1α, both under normoxic and hypoxic conditions (Figures 2a and b). It is noteworthy that under normoxic conditions, the HIF-1α accumulation level recorded for the combined bacterial lysate at a concentration of 100 μg / ml was comparable to that measured at the same concentration for the lysate of L. acidophilus CNCM I-5567 alone. Under hypoxic conditions, already at a concentration of 50 μg / ml, the bacterial lysate of the probiotic strain combination induced a cellular accumulation of HIF-1α similar to that recorded for L. acidophilus CNCM I-5567 alone, but at higher concentrations.Considering that L. acidophilus strain CNCM I-5567 is a quantitative minority among the bacteria included in the combination of probiotic strains, the observed results suggest that the combined use of the specific probiotic strains tested is characterized by a synergistic effect with regard to the induction of cellular accumulation of HIF-1α.

[0027] We investigated the effect of lysed bacterial strains on cellular energy metabolism and cellular oxygen consumption. For this purpose, the levels of L-lactate, the main metabolic product of glycolysis, the extracellular acidification rate (ECAR), a parameter reflecting glycolysis, and the oxygen consumption rate (OCR), used to determine oxidative phosphorylation, were evaluated in the medium. Exposure of the Caco-2 cell line to total bacterial lysate for 24 h resulted in a significant reduction in OCR values, evidencing a reduction in cellular oxygen consumption, compared to untreated controls (Figure 2c). In contrast, exposure of the cell line to bacterial lysate was associated with a significant increase in lactate levels, ECAR values, and ECAR / OCR ratio, evidencing an increase in glycolysis (Figures 2a, 2b, 2d).

[0028] conclusion The amount of oxygenated blood collected at the intestinal level regulates the availability of oxygen to extraintestinal body compartments, including vital organs such as the brain, heart, kidneys, and liver.

[0029] In the intestinal tract, oxygen homeostasis is highly dependent on HIF. Probiotic microbes may modulate HIF and affect processes controlled by HIF. Strains belonging to the bacterial genera L. paracasei, L. acidophilus, L. crispatus, L. rhamnosus, and B. longum are able to inhibit HIF-1α expression in vitro and in various cell models (Han et al., 2020; Esfandiary et al., 2016; Deepak et al., 2015; Chen et al., 2020). Contrary to reports in the literature, our results surprisingly showed that the tested probiotic microbes were able to positively modulate HIF-1α accumulation. This contrasting effect can be explained by the fact that the regulation of HIF-1α reflects the involvement of different molecular mechanisms. Moreover, the beneficial effects exerted by probiotics depend on the specific physiological state of the host cells (McFarland et al., 2018). The increased accumulation of HIF-1α induced by the tested bacterial species was associated with a significant decrease in oxygen consumption and induction of anaerobic metabolism in intestinal cells, which allows survival under oxygen-poor conditions. The oxygen conservation induced by the tested bacteria may regulate oxygen consumption in the intestine. The amount of oxygen not consumed in that body compartment may become available for other vital organs and tissues.

[0030] Materials and Methods Cell culture and treatment Human colon adenocarcinoma cell line (Caco-2) was cultured in DMEM (Dulbecco's Modified Eagle Medium) medium containing 10% (v / v) fetal bovine serum, 1% non-essential amino acids, 1 mM sodium pyruvate, 2 mM glutamine, 100 U / ml penicillin, and 100 μg / ml streptomycin at 37°C in a humidified atmosphere of 5% CO2. After reaching 80% confluence, cells were detached and plated at 6x10 in 6-well multiwell plates. 4 cells / cm 2Cell proliferation was monitored by light microscopy. To assess cellular HIF-1α levels, extracellular acidification rates and oxygen consumption rates, cells differentiated at day 14 post-confluence were pretreated for 30 min with the indicated concentrations of probiotic or incubated for 24 h without added probiotic at normoxia (~21% O2) under standard culture conditions or at hypoxia using a "hypoxic incubation chamber" (1% O2).

[0031] Preparation of soluble fractions from bacterial lysates The soluble fraction of bacterial lysates was prepared as follows: each sample was washed three times (8,600 × g, 20 min, 4°C) and suspended in phosphate-buffered saline (PBS). The bacterial suspension was sonicated for 30 min with alternating 10 s sonication and 10 s rest periods and centrifuged at 17.949 × g, 4°C for 20 min. The supernatant was filtered through a 0.22 μm filter to remove remaining intact bacteria, and the protein concentration was determined. For testing of individual bacterial strains, the concentration of the soluble fraction of bacterial lysates was 100 μg / ml. Assays for combinations of probiotic strains were performed using increasing concentrations of the total bacterial lysate soluble fraction of 10, 50, and 100 μg / ml. The assays carried out to evaluate the effect of the combination of strains were carried out on a probiotic preparation in which the proportion of bacterial cells of each individual strain relative to the total bacterial cells in the composite was as follows: 35.46% L. brevis CNCM I-5566, 1.42% L. acidophilus CNCM I-5567, 5.32% L. plantarum CNCM I-5569, 0.71% L. helveticus CNCM I-5573, 2.13% L. paracasei CNCM I-5568, 17.73% B. lactis CNCM I-5571, 1.77% B. lactis CNCM I-5572, 35.46% S. thermophilus CNCM I-5570. The samples thus prepared were frozen at -80°C until use. Untreated cells served as control.

[0032] Western blot HIF-1α expression was evaluated by Western blotting. Cells were lysed for 30 min on ice using RIPA buffer containing protease inhibitors. After cell lysis, samples were centrifuged at 17,949 × g for 20 min at 4°C. The supernatant was collected and a total protein assay was performed. Sample buffer and mercaptoethanol were added to the supernatant volume equivalent to 25 g of protein, samples were boiled for 5 min, and separated by sodium dodecyl sulfate (SDS)-polyacrylamide 10% gel electrophoresis (SDS-PAGE). Transfer of samples to nitrocellulose membranes (0.45 μm) was performed at 70 volts constant for 1 h at 4°C, and the nitrocellulose filters were incubated with nonspecific site blocking solution for 1 h at room temperature, followed by incubation with monoclonal anti-HIF-1α or anti-β-actin antibodies overnight at 4°C. Immunoreactive bands were visualized by chemiluminescence after incubation with horseradish peroxidase (HRP)-conjugated secondary antibodies. Densitometric analysis of the band corresponding to HIF-1α was then performed, and the values ​​obtained were normalized to those of β-actin.

[0033] L-lactate production assay L-lactate concentrations in cell culture supernatants were measured using an L-lactate assay kit (Abcam, Cambridge, UK) according to the manufacturer's instructions. Supernatants were deproteinized with 10-kDa NMWCO centrifugal filter units (Amicon, Millipore), and the filtrates were added to reaction wells. Absorbance was measured at 570 nm using a spectrophotometer.

[0034] Metabolic testing The extracellular acidification rate (ECAR) and oxygen consumption rate (OCR) were assessed for the cells treated as above using a Seahorse XFe96 analyzer (Agilent) according to the manufacturer's instructions, and the glycolytic rate (ECAR / OCR) was calculated. Briefly, on the day of the test, the medium was replaced with Seahorse XF DMEM medium pH 7.4 (Agilent) supplemented with glucose (10 mmol / L), pyruvate (1 mmol / L), and glutamine (2 mmol / L), and the cells were allowed to equilibrate in a non-CO2 incubator for 1 h, after which the OCR and ECAR were measured. Mitochondrial function was examined using the XFp Mito Stress Test Kit. Injections of oligomycin (1 μM), carbonyl cyanide-4(trifluoromethoxy)phenylhydrazone (FCCP, 1 μM), and a mixture of rotenone and antimycin A (1 μM) allowed the determination of the main bioenergetic parameters: basal respiration, ATP production-linked respiration (ATP production), maximal respiration, spare respiratory capacity, non-mitochondrial respiration, proton leak, and coupling efficiency.

[0035] statistical analysis ANOVA followed by Dunnett's test or Tukey's post-hoc test was used to check for statistically significant differences between the different conditions tested, and in case of two groups, comparisons between the means were performed with unpaired Student's t-test. A p-value ≤ 0.05 was considered statistically significant. Analyses were performed using R 4.0.3 statistical software.

[0036] In vivo experiments A reduction in oxygen supply to the brain plays a key role in neurodegeneration in the aging process (Ogunshola and Antoniou, 2009). Pathological processes such as oxidative stress, impaired oxygen or glucose supply, and disruption of iron homeostasis are common in neurodegenerative diseases (Correia and Moreira, 2010; Gironi et al., 2011; Benarroch, 2009). A reduction in brain levels of HIF-1α is associated with a reduced expression of GLUT1 and GLUT2 receptors responsible for glucose uptake, which has been previously demonstrated in Alzheimer's disease (AD) models. We performed an in vivo assay to evaluate the ability of specific bacterial combinations to modulate HIF-1α accumulation in brain tissue of 3xTg-AD mice. This reliable model of human AD shows both plaque and tangle pathology, intracellular Aβ immunoreactivity is detectable at 3 months of age, and tau protein hyperphosphorylation occurs between 12 and 15 months of age (Oddo et al., 2003). The experimental design features, as well as the preparation of brain extracts, were consistent with those previously reported by Bonfili et al. in 2018 (Bonfili et al., 2018). Forty-eight 8-week-old 3xTg-AD guinea pigs were divided into two groups: The first group (n=24) was treated with a specific probiotic preparation including Streptococcus thermophilus CNCM I-5570, Bifidobacterium animalis subsp. lactis CNCM I-5571, Bifidobacterium animalis subsp. lactis CNCM I-5572, Lactobacillus acidophilus CNCM I-5567, Lactobacillus helveticus CNCM I-5573, Lacticaseibacillus paracasei subsp. paracasei CNCM I-5568, Lactiplantibacillus plantarum subsp. plantarum CNCM I-5569 and Lactobacillus brevis CNCM I-5566, whereas the other group (n=24) was untreated and served as control. Concurrently, 48 age-matched wild-type mice were divided into two equal groups, one of which was treated with the same probiotic formulation, and the dosage (200 billion / kg / day) was determined using body surface area normalization as previously reported (Crawford et al., 1950).Mice were killed for biochemical analysis at 24 and 56 weeks of age (16 and 48 weeks from the start of treatment) and brains were stored at -80 °C until preparation of brain homogenates. ANOVA followed by Bonferroni's test was used to test for statistically significant differences in HIF-1α expression levels. A p-value ≤ 0.05 was considered statistically significant. HIF-1α subunit levels in brain homogenates were analyzed by Western blotting assay as previously described (Bonfili et al., 2018). The results obtained showed that untreated 3xTg-AD mice had significantly lower brain levels of HIF-1α than age-matched wt mice. Surprisingly, administration of the probiotic formulation to 3xTg-AD mice significantly increased brain levels of HIF-1α. Unexpectedly, treatment with the probiotic restored HIF-1α expression to the levels recorded in age-matched wt mice (Figure 3). The observed increase in brain levels of HIF-1α suggests that administration of the specific probiotic preparation tested could improve oxygen homeostasis and glucose metabolism in the brain, making it a viable therapeutic approach for neurodegenerative diseases.

[0037] Studies conducted on humans First human trials This study evaluated the effect of the intake of a specific probiotic preparation on respiratory, cardiac, and metabolic parameters in subjects performing endurance sports.For this purpose, four male triathlete subjects (age: mean ±DS, 37 ± 5 years; weight: mean ±DS, 70 ± 3 kg) were recruited.Two sets of studies were performed, the first set, called "pre", performed the protocol without the intake of the specific probiotic preparation. In a second set of studies, called "post", the same subjects were tested after ingestion of a bacterial combination consisting of Streptococcus thermophilus CNCM I-5570, Bifidobacterium animalis subsp. lactis CNCM I-5571, Bifidobacterium animalis subsp. lactis CNCM I-5572, Lactobacillus acidophilus CNCM I-5567, Lactobacillus helveticus CNCM I-5573, Lacticaseibacillus paracasei subsp. paracasei CNCM I-5568, Lactiplantibacillus plantarum subsp. plantarum CNCM I-5569 and Lactobacillus brevis CNCM I-5566. The probiotic intake amounted to approximately 400 billion bacterial cells per dose. Subjects were asked to consume their last meal at dinner on the day before the test, to fast at the designated time, and to consume only water. In the "post" test set, the probiotic preparation was required to be consumed within 5 hours of the last meal and at least 5 hours before the test time. To minimize the influence of weekend physical activity, the tests were performed mid-week. Each subject repeated two sets of tests on the same day and at the same time. The tests were performed on a Panatta Treadmill model T-190 (Panatta, Italy) with a fixed belt inclination of 1%. After the subjects were fitted with a metabolometer mask, they performed a 5-minute warm-up at a free intensity lower than the test intensity. The intensity was then increased without interruption to the threshold and continued for another 10 minutes. This value was selected using data on the subjects' individual anaerobic threshold intensity and corresponds to an exercise intensity appropriate for running a total distance of 20 km.Measurements of the average heart rate during the last 5 min of exercise, and the amount of oxygen the body can extract and use per unit time of muscular contraction (VO2) were obtained using a Fitmate PRO device (COSMED, Italy) coupled to a heart rate monitor band (POLAR, Italy). Blood lactate concentrations were determined by capillary sampling from the earlobe performed at the end of the threshold intensity step. The presence of significant differences between the pre- and post-test groups, with regard to the parameters studied, was assessed by paired Student's t-test. A p-value ≤ 0.05 was considered statistically significant. Since lactate is the end product when glycolysis is carried out in oxygen deprivation, its concentration reflects the level of anaerobic metabolism. Heart rate and VO2 are additional parameters indicating the level of aerobic metabolism, and a decrease in these parameters indicates an improved aerobic metabolism and a higher oxygen utilization rate. In general, the decrease in heart rate, VO2, and blood lactate concentrations suggests an improved efficiency of aerobic metabolism associated with the intake of the probiotic preparation (Figure 4).

[0038] These results are consistent with the hypothesis that the positive regulation of HIF-1α induced by the action of specific probiotic strains in the intestine allows a reduction in O2 consumption at that site. The oxygen sparing induced by probiotic intake makes this gas more available in the blood circulation, resulting in its redistribution to other body sites.

[0039] Secondary experiment on humans Hypoxia is a common condition in many disease states, and is particularly relevant in those associated with acute lung injury (Lee et al., 2019). The aim of this study was to investigate the baseline effect of the bacterial strain in alleviating the respiratory condition affecting the lungs in subjects associated with Sars-CoV-2 infection. In addition, we also evaluated how quickly this beneficial effect manifests. For this purpose, the response of two patient groups was tested: one group was treated with the best currently available treatment (BAT) and the other group additionally received oral bacteriotherapy (BAT+OB). The effect of probiotic intake was evaluated by comparing the blood oxygenation parameters partial pressure of oxygen (pO2), fraction of inspired oxygen (FiO2), oxygenated hemoglobin (O2Hb), pO2 / FiO2 ratio, and hemoglobin saturated with oxygen (SaO2) between the two groups at the start of treatment and after 24 hours. Oxygen delivery (l / min) was additionally measured. Oxygen administration (l / min) was also measured. The main characteristics of patients in both groups are summarized in Table 1.

[0040] [Table 1]

[0041] Except for gender, the two groups determined by the administration of the probiotic preparation were homogenous in all clinically considered variables, including medication for the treatment of SARS-CoV-2 infection, blood oxygenation parameters, and oxygen dosage (median; IQR BAT 4; 1-6 l / min; BAT+OB 1.5; 1-6 l / min, p = 0.31). 24 hours after the first probiotic administration, the BAT+OB group showed significantly higher values ​​of pO2 / FiO2 ratio and pO2 than the BAT group, whereas the opposite situation was observed for FiO2 values ​​(Figure 5a-c). Analysis of O2Hb and SaO2 levels gave results consistent with those previously described for pO2 and pO2 / FiO2 ratio (Figure 5e and 5f). The overall results showed that 24 hours after the start of treatment, the group that received the probiotic formulation had better blood oxygenation levels than the group that received only standard treatment, but the BAT group saw a notable increase in the amount of oxygen delivered over time (Figure 5d).

[0042] The improvement in blood oxygenation parameters observed in the BAT+OB group is consistent with the hypothesis that, at the intestinal level, the positive regulation of HIF-1α induced by the action of specific probiotic strains allows a reduction in oxygen consumption that is redistributed by making it more available at the bloodstream level.

[0043] Patients and methods Experimental design, population, data collection, and procedures This study was performed in adult patients (>18 years) infected with SARS-CoV-2, undergoing spontaneous ventilation with oxygen therapy delivered by a Venturi mask. A diagnosis of SARS-CoV-2 infection was defined by dual positive oropharyngeal and nasopharyngeal swabs for SARS-CoV-2 E and S genes by reverse transcription-polymerase chain reaction (RT-PCR). The patients included in the experiment were housed in two different wards dedicated to COVID-19 response: in the first ward, as proposed in the interim guidelines of the Societe Italiana di Malattie Infettive e Tropicali (SIMIT) (Italian Society of Infectious Diseases and Tropical Diseases) and the Italian Medicines Agency (AIFA), they received only BAT, dexamethasone (6 mg per day for 10 days) + low molecular weight heparin (prophylactic administration) + / - azithromycin (500 mg per day); (including remdesivir according to AIFA guidelines). In the second ward, they received BAT and the following antibiotics: S. thermophilus CNCM I-5570, B. lactis CNCM I-5571, B. lactis CNCM I-5572, L. acidophilus CNCM I-5567, L. helveticus CNCM I-5573, L. paracasei CNCM I-5568, L. The study combined oral bacteriotherapy with a total of 400 billion bacteria per day, including L. plantarum CNCM I-5569, and L. brevis CNCM I-5566 strains. Variables examined included: 1) medical history data, 2) past medical history (complications), 3) current medical history, treatment history, and laboratory data. Arterial blood gas analysis (ABG test) was performed using blood drawn from the radial artery 24 hours after the start of treatment.

[0044] statistical analysis Categorical variables, including gender, antiviral treatment, and antibiotic administration, were compared using chi-squared tests with Yates' continuity correction, given the limited sample size, and presented as absolute frequencies and percentages. Two-tailed Mann-Whitney U tests were used for all continuous variables, including respiratory variables (pO2, FiO2, pO2 / FiO2 change in oxygen delivery compared to start of treatment, O2Hb, SaO2), biochemical variables (blood glucose, lactate, hematocrit), demographics, and clinical variables (age, BMI, ALT, AST, Charlson index) to determine statistically significant differences between groups at each time point. Meanwhile, Wilcoxon tests were used to assess significant differences between consecutive time points for each group. In all cases, a p-value ≤ 0.05 was considered statistically significant.

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Claims

1. A composition for use in the treatment of hypoxia in a hypoxia-induced state selected from the group consisting of chronic fatigue, oxygen deficiency, neurodegenerative diseases, neonatal hypoxic-ischemia, myocardial ischemia, metabolic disorders, chronic heart diseases and kidney diseases, reproductive disorders such as preeclampsia and endometriosis, exacerbation of postural tremor and kinetic tremor, and cerebral hypoxia, comprising Lactobacillus acidophilus deposited by the applicant with the CNCM on September 1, 2020 under the Budapest Treaty, with the accession number CNCM I-5567, which is capable of increasing the cellular level of the hypoxia-inducible factor HIF-1α associated with a decrease in oxygen consumption of cells.

2. The composition according to claim 1, further comprising one or more pharmaceutically acceptable excipients.

3. The Streptococcus thermophilus strain deposited by the applicant with the CNCM on September 1, 2020 under the Budapest Treaty, with the accession number CNCM I-5570, The Bifidobacterium animalis subsp. lactis strain deposited by the applicant with the CNCM on September 1, 2020 under the Budapest Treaty, with the accession number CNCM I-5571, and / or The Bifidobacterium animalis subsp. lactis strain deposited by the applicant with the CNCM on September 1, 2020 under the Budapest Treaty, with the accession number CNCM I-5572, The composition according to claim 2, further comprising the above.

4. Based on the weight of the composition, 30% to 50% Lactobacillus acidophilus, 25% to 35% Streptococcus thermophilus, and 25% to 35% Bifidobacterium animalis subsp. lactis, The composition according to claim 1, comprising.

5. Based on the weight of the composition, 30% to 50% Lactobacillus acidophilus, 25% to 35% Streptococcus thermophilus, and 25% to 35% Bifidobacterium animalis subsp. lactis, The composition according to claim 2, comprising.

6. Based on the weight of the composition, 30% to 50% Lactobacillus acidophilus, 25% to 35% Streptococcus thermophilus, and 25% to 35% Bifidobacterium animalis subsp. lactis, The composition according to claim 3, comprising.

7. The Levilactobacillus brevis strain deposited by the applicant with the CNCM on September 1, 2020 under the Budapest Treaty, with the accession number CNCM I-5566, The Lactiplantibacillus plantarum subsp. plantarum strain deposited by the applicant with the CNCM on September 1, 2020 under the Budapest Treaty, with the accession number CNCM I-5569, Under the Budapest Treaty, on September 1, 2020, by the applicant, Lactiplantibacillus plantarum subsp. plantarum strain deposited with the CNCM under the Budapest Treaty, with the accession number CNCM I-5568, and Lactobacillus helveticus, under the Budapest Treaty, on September 1, 2020, by the applicant, Lactobacillus helveticus strain deposited with the CNCM, with the accession number CNCM I-5573, The composition according to claim 1, further comprising.

8. Under the Budapest Treaty, on September 1, 2020, by the applicant, Levilactobacillus brevis strain deposited with the CNCM, with the accession number CNCM I-5566, Under the Budapest Treaty, on September 1, 2020, by the applicant, Lactiplantibacillus plantarum subsp. plantarum strain deposited with the CNCM, with the accession number CNCM I-5569, Under the Budapest Treaty, on September 1, 2020, by the applicant, Lactiplantibacillus plantarum subsp. plantarum strain deposited with the CNCM, with the accession number CNCM I-5568, and Lactobacillus helveticus, under the Budapest Treaty, on September 1, 2020, by the applicant, Lactobacillus helveticus strain deposited with the CNCM, with the accession number CNCM I-5573, The composition according to claim 2, further comprising.

9. Under the Budapest Treaty, the strain of Levilactobacillus brevis deposited with the CNCM by the applicant on September 1, 2020, with the accession number CNCM I-5566, Under the Budapest Treaty, the strain of Lactiplantibacillus plantarum subsp. plantarum deposited with the CNCM by the applicant on September 1, 2020, with the accession number CNCM I-5569, Under the Budapest Treaty, the strain of Lacticaseibacillus paracasei subsp. paracasei deposited with the CNCM by the applicant on September 1, 2020, with the accession number CNCM I-5568, and the strain of Lactobacillus helveticus, Lactobacillus helveticus, deposited with the CNCM by the applicant on September 1, 2020, with the accession number CNCM I-5573 under the Budapest Treaty, The composition according to claim 3, further comprising.

10. Under the Budapest Treaty, the strain of Levilactobacillus brevis deposited with the CNCM by the applicant on September 1, 2020, with the accession number CNCM I-5566, Under the Budapest Treaty, the strain of Lactiplantibacillus plantarum subsp. plantarum deposited with the CNCM by the applicant on September 1, 2020, with the accession number CNCM I-5569, Under the Budapest Treaty, on September 1, 2020, the applicant deposited with the CNCM a strain of Lacticaseibacillus paracasei subsp. paracasei with the deposit number CNCM I-5568, and Lactobacillus helveticus, which, under the Budapest Treaty, on September 1, 2020, was deposited by the applicant with the CNCM as a strain of Lactobacillus helveticus with the deposit number CNCM I-5573, The composition according to claim 4, further comprising

11. Under the Budapest Treaty, on September 1, 2020, the applicant deposited with the CNCM a strain of Levilactobacillus brevis with the deposit number CNCM I-5566, Under the Budapest Treaty, on September 1, 2020, the applicant deposited with the CNCM a strain of Lactiplantibacillus plantarum subsp. plantarum with the deposit number CNCM I-5569, Under the Budapest Treaty, on September 1, 2020, the applicant deposited with the CNCM a strain of Lacticaseibacillus paracasei subsp. paracasei with the deposit number CNCM I-5568, and Lactobacillus helveticus, which, under the Budapest Treaty, on September 1, 2020, was deposited by the applicant with the CNCM as a strain of Lactobacillus helveticus with the deposit number CNCM I-5573, The composition according to claim 5, further comprising

12. Under the Budapest Treaty, the strain of Levilactobacillus brevis deposited with the CNCM by the applicant on September 1, 2020, with the deposit number CNCM I-5566, Under the Budapest Treaty, the strain of Lactiplantibacillus plantarum subsp. plantarum deposited with the CNCM by the applicant on September 1, 2020, with the deposit number CNCM I-5569, Under the Budapest Treaty, the strain of Lacticaseibacillus paracasei subsp. paracasei deposited with the CNCM by the applicant on September 1, 2020, with the deposit number CNCM I-5568, and the strain of Lactobacillus helveticus, Lactobacillus helveticus, deposited with the CNCM by the applicant on September 1, 2020, under the Budapest Treaty, with the deposit number CNCM I-5573, The composition according to claim 6, further comprising

13. Based on the weight of the composition, 30% to 50% Lactobacillus acidophilus, 1% to 10% Streptococcus thermophilus, 1% to 20% Bifidobacterium animalis subsp. lactis, 1% to 10% Levilactobacillus brevis (formerly known as Lactobacillus brevis), 1% to 10% Lactiplantibacillus plantarum subsp. plantarum (formerly known as Lactobacillus plantarum), 1% to 10% Lacticaseibacillus paracasei subsp. paracasei (formerly known as Lactobacillus paracasei subsp. paracasei), and 1% to 10% Lactobacillus helveticus, The composition according to claim 7, comprising.

14. Based on the weight of the composition, 30% to 50% Lactobacillus acidophilus, 1% to 10% Streptococcus thermophilus, 1% to 20% Bifidobacterium animalis subsp. lactis, 1% to 10% Levilactobacillus brevis (formerly known as Lactobacillus brevis), 1% to 10% Lactiplantibacillus plantarum subsp. plantarum (formerly known as Lactobacillus plantarum), 1% to 10% Lacticaseibacillus paracasei subsp. paracasei (formerly known as Lactobacillus paracasei subsp. paracasei), and 1% to 10% Lactobacillus helveticus, The composition according to claim 8, comprising.

15. Based on the weight of the composition, 30% to 50% Lactobacillus acidophilus, 1% to 10% Streptococcus thermophilus, 1% to 20% Bifidobacterium animalis subsp. lactis, 1% to 10% Levilactobacillus brevis (formerly known as Lactobacillus brevis), 1% to 10% Lactiplantibacillus plantarum subsp. plantarum (formerly known as Lactobacillus plantarum), 1% to 10% Lacticaseibacillus paracasei subsp. paracasei (formerly known as Lactobacillus paracasei subsp. paracasei), and 1% to 10% Lactobacillus helveticus, the composition according to claim 9, comprising

16. Based on the weight of the composition, 30% to 50% Lactobacillus acidophilus, 1% to 10% Streptococcus thermophilus, 1% to 20% Bifidobacterium animalis subsp. lactis, 1% to 10% Levilactobacillus brevis (formerly known as Lactobacillus brevis), 1% to 10% Lactiplantibacillus plantarum subsp. plantarum (formerly known as Lactobacillus plantarum), 1% to 10% Lacticaseibacillus paracasei subsp. paracasei (formerly known as Lactobacillus paracasei subsp. paracasei), and 1% to 10% Lactobacillus helveticus, the composition according to claim 10, comprising

17. Based on the weight of the composition, 30% to 50% Lactobacillus acidophilus, 1% to 10% Streptococcus thermophilus, 1% to 20% Bifidobacterium animalis subsp. lactis, 1% to 10% Levilactobacillus brevis (formerly known as Lactobacillus brevis), 1% to 10% Lactiplantibacillus plantarum subsp. plantarum (formerly known as Lactobacillus plantarum), 1% to 10% Lacticaseibacillus paracasei subsp. paracasei (formerly known as Lactobacillus paracasei subsp. paracasei), and 1% to 10% Lactobacillus helveticus, The composition according to claim 11, comprising.

18. Based on the weight of the composition, 30% to 50% Lactobacillus acidophilus, 1% to 10% Streptococcus thermophilus, 1% to 20% Bifidobacterium animalis subsp. lactis, 1% to 10% of Levilactobacillus brevis (formerly known as Lactobacillus brevis), 1% to 10% of Lactiplantibacillus plantarum subsp. plantarum (formerly known as Lactobacillus plantarum), 1% to 10% of Lacticaseibacillus paracasei subsp. paracasei (formerly known as Lactobacillus paracasei subsp. paracasei), and 1% to 10% of Lactobacillus helveticus, The composition according to claim 12, comprising

19. The composition according to any one of claims 1 to 18, which is suitable for oral administration and is in the form of, for example, powder, capsule, or granule, or spray.

20. The composition according to claim 19, having a high concentration of bacteria, at least 10 billion in adults and at least 100 million in infants.

21. The composition according to claim 19, suitable for administration to animals reared and / or maintained under hypoxic conditions for use in the treatment of hypoxic states or for use in the treatment of hypoxia in conditions associated with hypoxia.

22. The composition according to any one of claims 1 to 18, wherein the bacteria used are alive, dead, ultrasonicated, intermittently sterilized, or lyophilized.

23. The composition according to claim 19, wherein the bacteria used are living, non-living, sonicated, intermittently sterilized, or lyophilized.

24. The composition according to claim 20, wherein the bacteria used are living, non-living, sonicated, intermittently sterilized, or lyophilized.

25. The composition according to claim 21, wherein the bacteria used are living, non-living, sonicated, intermittently sterilized, or lyophilized.

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