WO2025181405A1 - Use of a bacterial composition - Google Patents

Abstract

The invention relates to use of a bacterial composition in a healthy subject for increasing or maintaining athletic endurance and/or athletic performance during a physical activity and/or for enhancing recovery from a physical activity. Further the invention relates to a bacterial composition for use in treating or preventing disorders occurring during or after physical activity, to a non-therapeutic method for supplementing the microbiome of a healthy subject, and to a non-therapeutic method for reducing lactate levels in a healthy subject, during or after physical activity.

Description

Use of a bacterial composition

FIELD OF THE INVENTION

The present invention relates to use of a bacterial composition for improving or maintaining athletic endurance and/or performance, for enhancing recovery from physical activity, and for treating or preventing disorders occurring during or after physical activity.

BACKGROUND OF THE INVENTION

Athletes require high-quality nutrition to fuel their intense training and support recovery. Lactic acid is produced in abundance during intense bouts of exercise and is a primary aspect of fatigue in endurance sports (Rauch et al., 1998).

Blood lactic acid concentrations increase during progressive exercise. While the muscles, the heart, the liver, and the kidney cortex can remove lactic acid from the circulation, increased production leads to a rise in blood levels which can lead to acidosis. Low muscle pH, which causes fatigue at high exercise intensities is a consequence of the accumulation of lactic acid (Moxnes and Sandbakk, 2012). Furthermore, increased concentrations of lactate in plasma have been positively associated with nausea-level post exercise (Merrells et al., 2019).

In athletes, circulating lactic acid is able to cross the epithelial barrier into the intestinal lumen, becoming available as a substrate for the gut microbiota (Scheiman et al., 2019). The intestinal lactate could be metabolized by members of the gut microbiota such as lactate-utilizing bacteria (LUB) or accumulate with possible detrimental effects. Lactate utilization by some taxa within the LUB is accompanied by formation of H2 gas. Veillonella, Anaerostipes and Anaerobutyricum hallii, utilize different pathways for H2 gas production (Hoshino and Sato 1986; Marquet et al. 2009).

WO 2017/180501 A1 discloses a probiotic formulation including one or more bacteria, bacterial strains, or bacterial species of the genus Veillonella, genus Faecalibacterium, genus Phascolarctobacterium, genus Oscillospira, genus Ruminococcus, genus Bacteroides, genus Blautia, family Christensenellaceae, genus Dialister, or phylum Cyanobacteria.

Although Veillonella spp. can efficiently utilize lactate and metabolize it into propionate, its high production of H2 gas represents a disadvantage as it can produce bloating and intestinal discomfort. Stomach cramping has been reported as an adverse effect in humans (n=7) in a study looking at the effects of 14 days oral supplementation with Veillonella atypica FB0054 (Gross et al., 2024).

Apart from producing extremely high amounts of H2 gas, Veillonella have been described as causing agents in diverse infections (Hirai et al., 2016; Kolenbrander, 2006). Moreover, recent studies have identified positive correlations of Veillonella abundance with blood systolic pressure (Hogue et al., 2024; Sohail et al., 2021 ). Sohail et al. (2021 ) reported higher abundance of Veillonella in the oral microbiome of hypertensive (n=56) vs. normotensive donors (n=40). Hogue, et al. (2024) investigated the gut microbiome of normotensive (n=15) vs. hypertensive (n=15) African American collegiate athletes, and Veillonella was one of the main microbes discriminating between normal versus high blood pressure donors. In a recent study looking at pediatric patients suffering from Ulcerative Colitis, an expansion of Veillonella in fecal abundance was detected in moderate/severe disease (Schirmer et al. , 2024). Although these studies cannot infer causality, the pathogenic and hypertensive potential of Veillonella are negative aspects for the regulatory recognition of Veillonella as a safe food for human consumption.

Veillonella also represents a technological disadvantage due to its strict anaerobiosis which requires maintaining an oxygen-free atmosphere in order to keep it viable during production, storage, and handling (Kolenbrander 2006). In a study looking at the safety and effect in humans (n=7) of 14 days oral supplementation with Veillonella atypica FB0054, no increased performance was observed and metagenomics analyses of collected stool samples from the participants could not detect changes in the bacterial communities (Gross et al., 2024). The previous indicates limited intestinal viability or effect of Veillonella when supplemented orally.

For the purpose of reducing intestinal lactic acid, supplementation with lactic acid bacteria that could increase intestinal lactic acid concentration is not desirable. Moreover, once orally ingested, the live bacteria have to be active in the gastrointestinal tract. In currently available bacterial compositions, only a small portion of the compositions can meet industrial and commercial standards.

Therefore, there is a need for bacterial compositions that are highly capable of reducing intestinal lactic acid and improving athletic endurance, performance, and recovery.

SUMMARY OF THE INVENTION

The inventors identified P. freudenreichii JS27 having a high capacity of survival, growth, and metabolism in the intestinal milieu over other intestinal bacteria and also other P. freudenreichii strains, such as P. freudenreichii JS, DSM20271T and DSM4902T. P. freudenreichii JS27 shows exceptional capacity of reducing intestinal lactic acid accumulation and transforming lactic acid into acetate and propionate which is a sport enhancing molecule.

The metabolic capacity of lactate metabolism of P. freudenreichii JS27 can thus prevent lactate accumulation and divert lactate from the production of detrimental metabolites. By reducing excess lactate, P. freudenreichii helps to sustain a higher lactate threshold, allowing athletes to perform at greater intensities for longer durations. No H2 or other detrimental gas is produced by P. freudenreichii during metabolism of lactic acid. Thereby, physical performance is optimized, and recovery is promoted.

Moreover, the inventors found that P. freudenreichii JS27 has a unique capacity to alter the gut microbiome and colonize the microbiota composition as compared to other intestinal bacteria.

For the purpose of reducing intestinal lactate in athletes, supplementation with common probiotics such as Lactobacillus or Bifidobacterium could worsen the acidosis status, since these are lactic acid producing bacteria. This is confirmed by a negative correlation that was observed between the relative abundance of lactobacilli in the native gut microbiota of trained mice and its endurance performance. P. freudenreichii strains are instead specialized in metabolizing lactate into propionate and thus can prevent lactate accumulation, without the production of H2 gas. Additional benefits provided by P. freudenreichii strains are their capability of producing cobalamin (Vitamin B12). Recognized beneficial health effects for food containing Vitamin B12 include reduction of tiredness and fatigue, and contribution to normal neurological and psychological functions.

Thus, when administered before, during or after physical activity, the composition of the invention provides targeted support for high performance, muscle recovery and energy production.

Accordingly, the invention relates to, inter alia, the following embodiments:

1 . Use of a bacterial composition in a healthy subject for increasing or maintaining athletic endurance and/or athletic performance during a physical activity and/or for enhancing recovery from a physical activity, said composition comprises bacteria comprising or preferably consisting of bacteria of the order Propionibacteriales, preferably bacteria of the genus Propionibacterium, more preferably bacteria of the species P. freudenreichii, again more preferably bacteria of strain P. freudenreichii JS27.

1.1 A non-therapeutic use of a bacterial composition in a healthy subject for increasing or maintaining athletic endurance and/or athletic performance during a physical activity and/or for enhancing recovery from a physical activity, said composition comprises bacteria comprising or consisting of bacteria of the order Propionibacteriales, preferably bacteria of the genus Propionibacterium, more preferably bacteria of the species P. freudenreichii, again more preferably bacteria selected from the group consisting of P. freudenreichii JS, JS27, and DSM20271 T again more preferably bacteria of P. freudenreichii JS27.

2. A bacterial composition comprising bacteria comprising or preferably consisting of bacteria of the order Propionibacteriales, preferably bacteria of the genus Propionibacterium, more preferably bacteria of the species P. freudenreichii, again more preferably bacteria of strain P. freudenreichii JS27 for use in treating or preventing a disorder in a subject, preferably an athlete, occurring during or after physical activity, wherein preferably the disorder is selected from the group consisting of acidosis, preferably lactic acidosis, weakness attack, muscle weakness, muscles cramps, muscle pain, inflammation, infection, immune deficiency, vitamin B12 deficiency, metabolic endotoxemia, hemolysis, nausea, vomiting, gastritis, gastrointestinal bleeding, stomach pain, exercise-related transient abdominal pain (ETAP), colic, intestinal discomfort, intestinal pain, visceral sensitivity, and intestinal cramp more preferably the disorder is selected from the group consisting of acidosis, preferably lactic acidosis, weakness attack, muscle weakness, muscles cramps, muscle pain, inflammation, infection, immune deficiency, vitamin B12 deficiency, metabolic endotoxemia, hemolysis, and exercise-related transient abdominal pain (ETAP), again more preferably the disorder is selected from the group consisting of acidosis, preferably lactic acidosis, weakness attack, muscle weakness, muscles cramps, and muscle pain. A bacterial composition comprising bacteria comprising or preferably consisting of bacteria of the order Propionibacteriales, preferably bacteria of the genus Propionibacterium, more preferably bacteria of the species P. freudenreichii, again more preferably bacteria of strain P. freudenreichii JS27 for use in treating or preventing a disorder in a subject, preferably an athlete, occurring during or after physical activity, wherein preferably the disorder is selected from the group consisting of acidosis, preferably lactic acidosis, myasthenia, muscle fatigue, preferably metabolic muscle fatigue, muscles cramps, muscle pain, inflammation, infection, immune deficiency, vitamin B12 deficiency, metabolic endotoxemia, hemolysis, nausea, vomiting, gastritis, gastrointestinal bleeding, stomach pain, exercise-related transient abdominal pain (ETAP), colic, and intestinal pain or intestinal spasm. more preferably the disorder is selected from the group consisting of acidosis, preferably lactic acidosis, myasthenia, muscle fatigue, preferably metabolic muscle fatigue, muscles cramps, muscle pain, inflammation, infection, immune deficiency, vitamin B12 deficiency, metabolic endotoxemia, hemolysis, and exercise-related transient abdominal pain (ETAP), again more preferably the disorder is selected from the group consisting of acidosis, preferably lactic acidosis, myasthenia, muscle fatigue, preferably metabolic muscle fatigue, muscles cramps, and muscle pain.

2.2 A bacterial composition comprising bacteria comprising or preferably consisting of bacteria of the order Propionibacteriales, preferably bacteria of the genus Propionibacterium, more preferably bacteria of the species P. freudenreichii, again more preferably bacteria of strain P. freudenreichii JS27 for use in treating or preventing a disorder in a subject, preferably an athlete, occurring during or after physical activity, wherein preferably the disorder is selected from the group consisting of acidosis, preferably lactic acidosis, muscles cramps, muscle pain, inflammation, infection, immune deficiency, vitamin B12 deficiency, metabolic endotoxemia, hemolysis, nausea, vomiting, gastritis, gastrointestinal bleeding, stomach pain, exercise- related transient abdominal pain (ETAP), and intestinal pain. more preferably the disorder is selected from the group consisting of acidosis, preferably lactic acidosis, muscles cramps, muscle pain, inflammation, infection, immune deficiency, vitamin B12 deficiency, metabolic endotoxemia, hemolysis, and exercise-related transient abdominal pain (ETAP), again more preferably the disorder is selected from the group consisting of acidosis, preferably lactic acidosis, muscles cramps, and muscle pain.3. A non- therapeutic method for supplementing the microbiome of a healthy subject, the method comprises the step of administering an effective dose of bacteria comprising or preferably consisting of bacteria of the order Propionibacteriales, preferably bacteria of the genus Propionibacterium, more preferably bacteria of the species P. freudenreichii, again more preferably bacteria of strain P. freudenreichii JS27, to the healthy subject before, during or after physical activity, wherein a population of bacteria of the order Propionibacteriales, preferably of the genus Propionibacterium, more preferably of species P. freudenreichii, more preferably of strain P. freudenreichii JS27 in the gut of the subject is increased. A non-therapeutic method for reducing level of lactic acid and/or lactates, for increasing level of propionic acid and/or propionates and/or increasing the level of acetic acid and/or acetates, in a healthy subject, preferably in the blood of a healthy subject, during or after physical activity, the method comprises the step of administering an effective dose of bacteria comprising or preferably consisting of bacteria of the order Propionibacteriales, preferably bacteria of the genus Propionibacterium, more preferably bacteria of the species P. freudenreichii, again more preferably bacteria of the strain P. freudenreichii JS27, to the healthy subject before, during or after physical activity, wherein preferably the physical activity generates increased lactate levels reduced by the administered bacteria. The method or use of any one of the preceding embodiments, wherein the bacteria of the order Propionibacteriales comprise a 16S rDNA sequence as defined in SEQ ID NO: 1 , or a 16S rDNA sequence having at least 95% sequence identity to a 16S rDNA sequence as defined in SEQ ID NO: 1 , preferably the bacteria of the order Propionibacteriales comprise a 16S rDNA sequence as defined in SEQ ID NO: 1. The method or use of any one of the preceding embodiments, wherein the bacteria of the order Propionibacteriales comprise a DNA sequence comprising or consisting of SEQ ID NO: 1 or a DNA sequence comprising or consisting of a sequence having at least 95% sequence identity to SEQ ID NO: 1 , preferably the bacteria of the order Propionibacteriales comprise or consist of bacteria which comprise a DNA sequence comprising or consisting of SEQ ID NO: 1 . The method or use of any one of the preceding embodiments, wherein the physical activity generates increased lactate levels, preferably the lactate levels are increased as compared levels without physical activity. The method or use of any one of the preceding embodiments, wherein the bacteria are capable of growing in a colon and/or surviving gastro-intestinal conditions. The method or use of any one of the preceding embodiments, wherein the composition further comprises one or more compounds selected from the group consisting of an amino acid, polypeptide, protein, preferably whey protein, vitamin, preferably vitamin B12, mineral, such as a salt of sodium, potassium, magnesium, calcium, carbohydrate, lipid, and levocarnitine (L-carnitine). The method or use of any one of the preceding embodiments, wherein the composition is administered before, during or after physical activity. The method or use of any one of the preceding embodiments, wherein the composition is administered enterally, preferably the composition is administered orally. The method or use of any one of the preceding embodiments, wherein the composition is formulated for delivering the bacteria of the order Propionibacteriales, preferably the bacteria of the genus Propionibacterium, more preferably the of species P. freudenreichii, again more preferably the bacteria of strain P. freudenreichii JS27 to the gut, preferably the proximal gut. The method or use of any one of the preceding embodiments, wherein the composition is formulated for delivering the bacteria of the order Propionibacteriales, preferably the bacteria of the genus Propionibacterium, more preferably the of species P. freudenreichii, again more preferably the bacteria of strain P. freudenreichii JS27 to the gut, preferably the colon, more preferably the proximal colon. The method or use of any one of the preceding embodiments, wherein the composition is formulated as a product selected from the group consisting of a food product, food supplementary product, dietary supplement, and beverage. The method or use of any one of the preceding embodiments, wherein the composition is formulated as solid or liquid formulation selected from the group consisting of powder, pill, capsule, gastro-resistant capsules, slow-release formulation, tablet, gelatin, chewable formulation, dissolvable formulation, time release formulation, microencapsulated formulation, and solution. The method or use of any one of the preceding embodiments, wherein the composition increases the population of bacteria of the order Propionibacteriales, preferably of the genus Propionibacterium, more preferably of the species P. freudenreichii, again more preferably of strain P. freudenreichii JS27. The method or use of any one of the preceding embodiments, wherein the level of lactic acid and/or lactates in the subject is decreased. The method or use of any one of the preceding embodiments, wherein the level of propionic acid and/or propionates in the subject is increased. The method or use of any one of the preceding embodiments, wherein the level of acetic acid and/or acetates in the subject is increased. The method or use of any one of the preceding embodiments, wherein the bacteria of the composition, preferably the bacteria of the order Propionibacteriales, more preferably the bacteria of the genus Propionibacterium, again more preferably the bacteria of the species P. freudenreichii, again more preferably the bacteria of the strain P. freudenreichii JS27, are administered in a dose from about 10⁴ CFU to about 10¹⁶ CFU, about 10⁹ CFU to about 10¹¹, or about 10⁹ CFU to about 1 O¹⁰ CFU. The method or use of any one of the preceding embodiments, wherein the bacteria of the composition, preferably the bacteria of the order Propionibacteriales, more preferably the bacteria of the genus Propionibacterium, again more preferably the bacteria of the species P. freudenreichii, again more preferably the bacteria of the strain P. freudenreichii JS27, are administered in a single daily dose of 10⁹ CFU or at least 10⁹ CFU, preferably for at least 14 days. The method or use of any one of the preceding embodiments, wherein the bacteria of the composition, preferably the bacteria of the order Propionibacteriales, more preferably the bacteria of the genus Propionibacterium, again more preferably the bacteria of the species P. freudenreichii, again more preferably the bacteria of the strain P. freudenreichii JS27, are administered in a single daily dose, preferably for at least 14 days; or in two daily doses before and during or during and after physical activity; or in three, ten or more daily doses before, during and after physical activity.

22. The method or use of any one of the preceding embodiments, wherein the bacteria of the composition, preferably the bacteria of the order Propionibacteriales, more preferably the bacteria of the genus Propionibacterium, again more preferably the bacteria of the species P. freudenreichii, again more preferably the bacteria of the strain P. freudenreichii JS27, are administered in a single daily dose of 10⁹ cfu, preferably for at least 14 days; or in two daily doses of 10⁹ cfu each, before and during or during and after physical activity; or in three, ten or more daily doses of 10⁹cfu each, before, during and after physical activity.

23. The method or use of any one of the preceding embodiments, wherein the subject is a mammalian, preferably a horse, dog, human, more preferably a human.

DESCRIPTION OF THE INVENTION

In a first aspect, the present invention relates to use of a bacterial composition in a healthy subject, i.e., a non-therapeutic use, for increasing or maintaining athletic endurance and/or athletic performance during a physical activity and/or for enhancing recovery from a physical activity, wherein the composition comprises bacteria comprising or consisting of bacteria of the order Propionibacteriales, preferably the bacteria of the order Propionibacteriales comprise or consist of propionibacteria.

Further, the invention relates to a method for increasing or maintaining athletic endurance and/or athletic performance during a physical activity and/or for enhancing recovery from a physical activity, wherein an effective dose of a bacterial composition comprising or consisting of bacteria of the order Propionibacteriales is administered to a subject, preferably the bacteria of the order Propionibacteriales comprise or consist of propionibacteria.

In the invention, the taxonomy is based on the List of Prokaryotic names with Standing in Nomenclature (LPSN) and National Center for Biotechnology Information (NCBI). In certain embodiments, the bacteria of the order Propionibacteriales comprise propionibacteria, more preferably the Propionibacteriales comprise bacteria of the species P. freudenreichii., again more preferably Propionibacteriales comprise bacteria of strain P. freudenreichii JS27. In a preferred embodiment, the bacteria of the order Propionibacteriales are propionibacteria, more preferably the bacteria of the order Propionibacteriales are bacteria of the species P. freudenreichii. Even more preferably, the bacteria of the order Propionibacteriales are bacteria of the strain P. freudenreichii JS27. Preferably, the bacteria of the composition of the invention are isolated and purified bacteria. Preferably, the bacteria of the composition of the invention are lyophilized bacteria.

In a preferred embodiment, the Propionibacteriales, propionibacteria or P. freudenreichii are capable of producing vitamin B12. More preferably the strain of P. freudenreichii is capable of producing vitamin B12.

The inventors found that Propionibacteriales, preferably propionibacteria, more preferably P. freudenreichii, again more preferably P. freudenreichii JS27 have a high capacity to reduce the level of lactic acid and/or lactates, to increase the level of propionic acid and/or propionates and the level of acetic acid and/or acetates in the gut of a subject.

In preferred embodiments, the bacterial composition is a non-pharmaceutical composition, such as a nutritional composition that may be consumed in any form.

In preferred embodiments, the composition is formulated for non-therapeutic uses as a product selected from the group consisting of a food product, food supplementary product, dietary supplement, and beverage, such as protein bars or sport gels.

In a further aspect, the present invention relates to a bacterial composition comprising bacteria comprising or preferably consisting of bacteria of one or more species of the order Propionibacteriales, preferably of the genus Propionibacterium, more preferably of species P. freudenreichii, more preferably of strain P. freudenreichii JS27, for use in treating or preventing a disorder occurring during or after physical activity.

In a further aspect, the present invention relates to a method for treating or preventing a disorder occurring during or after physical activity, wherein an effective dose of a bacterial composition comprising bacteria, wherein the bacteria comprise or consist of bacteria of the order Propionibacteriales, preferably of the genus Propionibacterium, more preferably of the species P. freudenreichii, again more preferably of strain P. freudenreichii JS27, is administered to a subject. Preferably, the bacteria of the composition of the invention are isolated and purified bacteria.

Preferably, the disorder occurring during or after physical activity is induced or caused at least partly by the physical acidity Preferably, the disorder is an exercise-induced disease or disorder.

Preferably the disorder is selected from the group consisting of acidosis, preferably lactic acidosis, weakness attack, muscle weakness, muscles cramps, muscle pain, inflammation, infection, immune system deficiency, vitamin B12 deficiency, metabolic endotoxemia, hemolysis, nausea, vomiting, gastritis, gastrointestinal bleeding, stomach pain, exercise-related transient abdominal pain (ETAP), colic, intestinal discomfort, intestinal pain, visceral sensitivity or intestinal cramp, and combinations thereof.

More preferably, the disorder is selected from the group consisting of acidosis, preferably lactic acidosis, weakness attack, muscle weakness, muscles cramps, muscle pain, inflammation, infection, immune system deficiency, vitamin B12 deficiency, metabolic endotoxemia, hemolysis, and exercise-related transient abdominal pain (ETAP).

More preferably the disorder is selected from the group consisting of acidosis, preferably lactic acidosis, weakness attack, muscle weakness, muscles cramps, muscle pain, inflammation, infection, immune system deficiency, vitamin B12 deficiency, hemolysis, and metabolic endotoxemia. Again more preferably, the disorder is selected from the group consisting of acidosis, preferably lactic acidosis, weakness attack, muscle weakness, muscles cramps, muscle pain, inflammation, metabolic endotoxemia, hemolysis, and vitamin B12 deficiency.

Again more preferably, the disorder is selected from the group consisting of acidosis, preferably lactic acidosis, weakness attack, muscle weakness, muscles cramps, and muscle pain.

In a preferred embodiment, the immune system deficiency occurring during or after physical activity refers to a subject’s immune system impaired by high-volume or high- intensity training loads. The immune deficiency can be determined by a significantly increased expression of proinflammatory cytokines as compared to an immune system of a subject without training loads. Preferably, the immune system deficiency is defined by acute neutrophilia and lymphopenia.

In a preferred embodiment, the inflammation occurring during or after physical activity is an exercise-related inflammation. Preferable, the exercise-related inflammation is a systemic inflammation, such as a metabolic endotoxemia and/or systemic low-grade inflammation. The systemic low-grade inflammation is preferably through the LPS-Toll- like receptor 4 (LPS/TLR4) signal transduction pathways. Strenuous exercise can directly affect the immune system; thus, microorganisms and microbial metabolites translocate to the systemic circulation and generate paracrine and endocrine effects, leading to a systemic inflammatory response. Heavy training can decrease splanchnic blood flow and, consequently, gastrointestinal hypoperfusion, which aggravates the damage of intestinal layer integrity. Intestinal permeability allows lipopolysaccharides (LPS) to enter the portal system, promoting a metabolic endotoxemia and systemic low-grade inflammation through the LPS-Toll-like receptor 4 (LPS/TLR4) signal transduction pathways (Mancin et al., 2020).

In preferred embodiments of the invention, the infection is an upper respiratory tract infection.

In preferred embodiments of the invention, the subject to which the bacterial composition is administered is an athlete. In preferred embodiments, the bacterial composition is a pharmaceutical composition. A pharmaceutical composition may include the bacteria in combination with a pharmaceutically acceptable carrier which may include one or more excipients. Pharmaceutically acceptable carriers or excipients are well known in the pharmaceutical art, and are described, for example, in Remington's Pharmaceutical Sciences, Mack Publishing Co. (A. R. Gennaro edit. 1985). For example, saline and phosphate-buffered saline at physiological pH may be used. Stabilizers, dyes, and even flavoring agents may be provided in the nutritional or pharmaceutical compositions describe herein.

In a further aspect, the present invention relates to a non-therapeutic method for supplementing the microbiome of a healthy subject. The method comprises the step of administering an effective dose of bacteria, wherein the bacteria comprise or consist of bacteria of the order Propionibacteriales, preferably of the genus Propionibacterium, preferably of species P. freudenreichii, more preferably of strain P. freudenreichii JS27, to the healthy subject before, during or after physical activity, wherein a population of Propionibacteriales, preferably Propionibacteriaceae, more preferably bacteria of genus Propionibacteria, again more preferably P. freudenreichii, again more preferably P. freudenreichii JS27 in the gut of the subject is increased.

The inventors found that the composition of the invention increases the availability of viable Propionibacteriaceae in the human gut and allows for enhanced survival of these bacteria in conditions resembling the gut. Especially, P. freudenreichii JS27 has an exceptional capacity to alter the gut microbiome and colonize the microbiota when administered to a subject.

In a further aspect, the present invention relates to a non-therapeutic method for reducing level of lactic acid and/or lactates, for increasing level of propionic acid and/or propionates, and/or for increasing the level of acetic acid and/or acetates, in a healthy subject, preferably in the blood of a healthy subject, during or after physical activity, the method comprises the step of administering an effective dose of bacteria comprising or preferably consisting of bacteria of the order Propionibacteriales, preferably of the genus Propionibacterium, more preferably of the species P. freudenreichii, again more preferably strain P. freudenreichii JS27, to the healthy subject before, during or after physical activity.

Tests for measuring the level of lactic acid, lactates, propionic acid, propionates, acetic acid and acetates are described herein in the example section and are known in the art, such as the blood lactate tests (doi: 10.1177/193229680700100414) for measuring lactate levels and chromatographic methods for measuring propionate levels, such as high-pressure liquid chromatography analysis with refractive index detection (HPLC- doi: 10.1126/scitranslmed.aav012; cf.

Examples 2-4 as described herein).

In a further aspect, the present invention relates to a non-therapeutic method for reducing the level of lactic acid and/or lactates in a healthy subject, preferably in the blood of a healthy subject, the method comprises the step of administering an effective dose of bacteria to the healthy subject, wherein said bacteria comprise or consist of the order Propionibacteriales, preferably of genus Propionibacterium, more preferably species P. freudenreichii, again more preferably strain P. freudenreichii JS27. In a preferred embodiment, the increased lactate levels are reduced by the administered bacteria of the order Propionibacteriales, preferably of genus Propionibacterium, more preferably species P. freudenreichii, again more preferably strain P. freudenreichii JS27.

In a preferred embodiment , the present invention relates to a non-therapeutic method for reducing the level of lactic acid and/or lactates in a healthy subject, preferably in the blood of a healthy subject, during or after physical activity, during or after physical activity, the method comprises the step of administering an effective dose of bacteria to the healthy subject before, wherein said bacteria comprise or consist of Propionibacteriales, preferably of Propionibacteria, more preferably P. freudenreichii, again more preferably strain P. freudenreichii JS27, wherein the physical activity generates increased lactate levels reduced by the administered bacteria.

Propionibacterium spp. are Gram-positive, nonmotile, pleomorphic rod-shaped bacteria, non-spore-forming, aerotolerant/anaerobic or microaerophilic bacteria of the order Propionibacteriales and the family Propionibacteriaceae (Patrick and McDowell 2012). The different species of the genus Propionibacterium are P. freudenreichii (type species), Propionibacterium cyclohexanicum, Propionibacterium acidifaciens and Propionibacterium australiense. P. freudenreichii species have been used over long time for food fermentation due to their capacity of converting lactate into propionate, acetate, and CO2 (Piveteau 1999). Propionate is a gluconeogenic short chain fatty acid (SCFA) with performance-enhancing properties which can improve the intestinal barrier and decrease fat formation and serum cholesterol levels. P. freudenreichii is used in the cheese industry and is responsible for the development of the characteristic eyes and flavor of Swiss-type cheeses. The metabolic capacity of lactate metabolism of Propionibacterium is particularly interesting as could prevent lactate accumulation and divert lactate from the production of detrimental metabolites. P. freudenreichii taxonomical unit has received qualified presumption of safety (QPS) status by the European Food Safety Authority (EFSA) (EFSA BIOHAZ Panel 2018). The QPS classification provides a generic pre-assessment of the safety of microorganisms intended for use in food. Some species of propionibacteria are proposed probiotics due to their effects on the modulation of the gut microbiota and the immune response (reviewed by Cousin et al. (2011 ) and Rabah et al. (2017)).

The bacteria of the strain P. freudenreichii JS27 (Propionibacterium freudenreichii JS27, as characterized in MIESCHER, Susanne. Antimicrobial and autolytic systems of dairy propionibacteria. 1999. Diss. ETH No. 13486; cheese LFB strain ID: BT-120). is preferably a bacterium comprising a 16S rDNA sequence as defined in SEQ ID NO: 1 . P. freudenreichii JS27 is preferably a bacterium comprising a genome of a sequence as defined by SEQ ID NO: 1

The bacteria of the genus Propionibacterium, more preferably of the species P. freudenreichii, again more preferably of the strain P. freudenreichii JS27 preferably comprise a 16S rDNA sequence as defined in SEQ ID NO: 1 or a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1 %, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9% sequence identity to the 16S rDNA sequence as defined in SEQ ID NO: 1 , wherein P. freudenreichii JS27 maintains being capable of growing in a colon and metabolizing lactic acid. The bacteria of the genus Propionibacterium, more preferably of the species P. freudenreichii, again more preferably of the strain P. freudenreichii JS27 preferably comprise a sequence as defined by SEQ ID NO: 1 or comprising a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1 %, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9% sequence identity to SEQ ID NO: 1 , wherein the P. freudenreichii JS27 maintains being capable of growing in a colon and metabolizing lactic acid.

Preferably, the 16S rDNA sequence as defined in SEQ ID NO: 1 or the sequence of SEQ ID NO: 1 is comprised in the genome of the bacteria.

The capability of growing in a colon is preferably examined in an assay for colon growth at 37°C, more preferably examined in an assay for infant colon growth at 37°C, more preferably in an assay known in the art. The capability of metabolizing lactic acid is preferably examined as described in Example 3.

"Percent (%) sequence identity" with respect to a reference sequence is defined as the percentage of residues in a candidate sequence that are identical with the residues in the reference sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity. Alignment for purposes of determining percent sequence identity can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software. Those skilled in the art can determine appropriate parameters for aligning sequences, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared.

The differences compared to the sequence of SEQ ID NO: 1 may be the result of natural or engineered mutations that do not or not substantially limit the technical effect(s) described herein. As such, the mutations can comprise insertions, deletions and/or replacements of nucleotides in the reference genome sequences as defined by SEQ ID NO: 1. Preferably, the physical activity generates increases lactate levels in the subject. Preferably, the physical activity generates increased lactate levels in the blood and colon of the subject. Preferably, the physical activity generates increased lactate levels in the colon, preferably the proximal colon of the subject.

The bacteria included in the composition can be dead or alive. Preferably the bacteria of the composition are viable. In preferred embodiments, the bacteria described herein may be in a dried form. In more preferred embodiments, the composition is formulated as freeze-dried bacteria. The drying or freeze drying of bacteria after production by fermentation is known to those of skill in the art. Freeze-drying technology is widely considered as a suitable technique to preserve microorganisms. In preferred embodiments, protective agents are added prior to freeze drying to improve the survival and storage stability of the bacteria.

In preferred embodiments, the composition of the invention further comprises one or more additional compounds, such as nutritional supplements. Preferably, these additional compounds are selected from the group consisting of an amino acid, polypeptide, vitamin, preferably vitamin B12, whey protein, mineral, such as a salt of sodium, potassium, magnesium und calcium, carbohydrate, lipid, levocarnitine (L- carnitine), and combinations thereof.

In preferred embodiments, the carbohydrate included in the composition is or comprises a saccharide, preferably a nutritive saccharide, for example maltodextrin. Said saccharides, especially maltodextrin is produced by controlled enzymatic hydrolysis, purification, and spray-drying of food maize starch. In preferred embodiments, the polypeptide included in the composition is or comprises proteins, such as a whey protein. In preferred embodiments, the carbohydrate included in the composition is or comprises microcrystalline cellulose.

In preferred embodiments, the composition of the invention further comprises vitamin B12 which is produced by the propionibacteria, preferably by P. freudenreichii JS27. Vitamin B12, also known as cobalamin, is produced by P. freudenreichii JS27. In a freeze-dried bacterial culture of Propionibacterium freudenreichii JS27 vitamin B12 was identified, for example at concentrations between 2800-3200 pg/100 g, without carrier. The European Food Safety Authority (EFSA) has recognized that there are beneficial health effects for food formulations containing vitamin B12 (EFSA NDA Panel 2010). Thus, supplementation with vitamin B12 can improve work endurance in situations of inadequate micronutrient status.

In preferred embodiments, the composition of the invention further comprises maltodextrin, whey protein and vitamin B12.

The composition can be administered before, during or after physical activity. Preferably, it is administered during physical activity.

The composition can be administered by any route that allows the bacteria to colonize the gut of the subject. Preferably, the composition is administered enterally. The term “enteral administration” includes administration via the gastrointestinal tract including oral administration, sublingual administration, rectal administration, administration via the esophagus, stomach, small and large intestine tract. Preferably, the composition is administered orally.

In preferred embodiments, the composition is formulated and administered for delivering bacteria of the order Propionibacteriales, genus Propionibacterium, preferably species P. freudenreichii, more preferably strain P. freudenreichii JS27 to the gut, preferably the colon, more preferably the proximal colon. The proximal colon preferably includes the transverse colon, ascending colon, and the cecum. More preferably, the proximal colon is the transverse colon.

In preferred embodiments, the composition is formulated as solid or liquid. In preferred embodiments, the composition is a formulation selected from the group consisting of powder, pill, capsule, preferably a gastro-resistant or slow-release capsule, tablet, gelatin, gel, chewable formulation, bars, such as protein bars, dissolvable formulation, time release formulation, microencapsulated formulation, and solution.

In preferred embodiments, the composition of the invention is/are administered after a meal, preferably the composition is formulated in gastro-resistant capsules and orally administered.

The composition can be formulated in that the bacteria are capable of growing in a colon and/or surviving gastro-intestinal conditions. Microencapsulation formulations and gastro-resistant capsules may protect the live bacteria from the digestive actions of the stomach, duodenum, and jejunum of the intestine and allow administration, delivery, or release to the gut, preferably the colon, more preferably the proximal colon of an individual.

Preferably, the proximal colon includes the cecum (a pouch that connects the small intestine to the colon), the ascending colon (the right side of the colon), and the transverse colon (the part of the colon that goes across the body between the right and left sides of the colon).

Two or more coatings can be used to cover a tablet or capsule like form comprising the bacteria, wherein the outside coating is degraded in a pH environment of 5 to 6 and the inside coating is degraded in a pH environment of about 7.

In preferred embodiments, the bacteria of the composition increase the population of bacteria of the order Propionibacteriales, preferably the genus Propionibacterium, more preferably species P. freudenreichii, again more preferably strain P. freudenreichii JS27. Preferably, the population of bacteria is increased in the subject. Preferably, the population of bacteria is increased in the colon of the subject, preferably the proximal colon of the subject.

In preferred embodiments, the composition of the invention decreases the level of lactic acid and/or lactates in the subject, preferably in the colon of the subject. In preferred embodiments, the composition of the invention increases the level of propionic acid and/or propionates in the subject, preferably in the colon of the subject. In preferred embodiments, the composition of the invention increases the level of acetic acid and/or acetates in the subject, preferably in the colon of the subject. In preferred embodiments, the composition of the invention decreases the level of lactic acid and/or lactates in the subject’s blood.

As demonstrated by the inventors, the composition of the invention comprising bacteria of the order Propionibacteriales, preferably of the genus Propionibacterium, more preferably species P. freudenreichii, more preferably strain P. freudenreichii JS27 metabolizes and reduces lactic acid and/or lactates and transforms it into propionic acid and/or propionates and acetic acid and/or acetates in a highly efficient manner in a model mimicking the colon of a subject after physical activity.

Levels of lactic acid, lactates, propionic acid, propionates, acetic acid and/or acetates were and can be measured in a continuous in vitro intestinal fermentation model (PolyfermS fermentation model corresponding to Fermentation 1 described in Pham et al. (2019)), preferably in presence of sterile fermentation effluent. The fermentation model mimics the colon, especially the proximal colon, in a healthy donor and allows observing effects of bacterial supplementation. Levels of lactic acid, lactates, propionic acid, propionates, acetic acid and/or acetates can be measured in effluent, YEL media and supplemented YEL media with effluent via high-pressure liquid chromatography analysis with refractive index detection (HPLC-RI). Increased lactate production as it occurs during physical exercises is mimicked in the model by spiking the medium with sterile skim milk. Further conditions are described in Examples 2-4.

In preferred embodiments, the bacteria in the composition of the invention are administered in a dose from of about 10⁴ CFU to about 10¹⁶ CFU, about 10⁹ CFU to about 10¹¹ , or about 10⁹ CFU to about 10¹° CFU.

The bacteria in the composition of the invention can be administered in a single or multiple daily doses, such as three daily doses. Preferably the bacteria are administered in a single daily dose of 10⁹ cfu, preferably for at least 10 days, more preferably for at least 14 days. This allows the bacteria to establish in the gut microbiome. Preferably, one to ten daily doses are taken during or before physical activity. In another preferred embodiment, the bacteria in the composition of the invention requires 2 weeks of a daily dose of 10⁹ cfu per day to establish in the gut.

In preferred embodiments, the bacteria in the composition of the invention are administered in two, three, five, ten or more daily doses of 10⁹ cfu each, before, during and after physical activity. Multiple doses before and during or during and after physical activity are preferred in case the physical activity is high performance sport.

Viability through gastrointestinal passage can be increased by a) ingestion after a meal by avoiding ingestion with an empty stomach; b) incorporating the lyophilized powder in gastro-resistant capsules or with microencapsulation; and c) adding it to compounds such as whey powder that could protect from gastric pH during digestion. Thus, in preferred embodiments, the bacteria are administered after a meal, preferably via oral administration. In preferred embodiments, the bacteria are administered after a meal, preferably the bacteria are formulated in gastro-resistant capsules or using microencapsulation and orally administered. In preferred embodiments, the composition of the invention comprises one or more compounds, such as whey powder that could protect from gastric pH during digestion.

The “subject” can be defined as mammalian. In preferred embodiments, the subject is a mammalian, such as a dog, horse, or human. More preferably, the subject is a human. More preferably, the subject is an athlete. Again more preferably, the subject is a high-performance athlete.

The term "athlete", as used herein, refers preferably to a person who is proficient in sports and other forms of physical exercise or a person who is trained or skilled in exercises, sports, or games requiring physical strength, agility, endurance, speed or stamina or a person who possesses physical skills such as strength, agility, endurance, speed, or stamina, on average and is suited for physical competition. An athlete is preferably physically active for at least 1 , 2, 3 or 4 times per week for at least 20 minutes.

In some embodiments, the subject or athlete follows a regular exercise regimen. The term “regular exercise regimen” is not limited and may be determined by a person of ordinary skill in the art, a physician or an athletic trainer. For instance, a regular exercise regimen may comprise performing a “physical exercise” at least once a week, for example twice a week, thrice or four times a week, e.g., for 20 minutes or more. In some embodiments, the subject or athlete possesses enhanced physical strength, agility, endurance, speed and/or stamina as compared to the non-athlete.

The term "physical exercise" or “physical activity”, used interchangeably herein, refers preferably to any bodily activity that enhances or maintains physical fitness and overall health and wellness. It is performed for various reasons, including strengthening muscles and the cardiovascular system, honing athletic ability or skills, weight loss or maintenance, and merely enjoyment. Physical activity comprises but is not limited to an activity selected from the group consisting of running, jogging, weight training, strength training, high-intensity interval training (HIIT), CrossFit, yoga, Pilates, gymnastics, swimming, cycling, hiking, climbing, bouldering, and team sports (e.g., football, basketball). During "physical exercise" or “physical activity”, heart rate is preferably about 50% or more of maximum heart rate.

The term “physical activity” refers preferably to physical activity for a period of at least about 20 minutes. The physical activity can be a frequent physical activity practiced at least 3 times/week. Preferably, the physical activity is a frequent physical activity of at least about 20 minutes practiced at least 3 times/week. The increased or maintained athletic endurance and/or athletic performance during a physical activity and/or enhanced recovery from a physical activity is preferably an improved physical performance during a physical activity, reduced post-workout fatigue, shorter recovery time and/or less muscle soreness. More preferably, the increased or maintained athletic endurance and/or athletic performance during a physical activity and/or enhanced recovery from a physical activity is an improved physical performance and/or shorter recovery time.

The term "increasing or maintaining athletic endurance and/or athletic performance" refers preferably to a comparison of athletic endurance or performance using objective factors for a particular individual. Such objective factors may include ability to continue a particular exercise or training event over a given period of time. Such exercises or training events are known to those of skill in the art and can be determined based on the particular athletic event or sport engaged in by the individual.

The increased or maintained athletic endurance and/or athletic performance during a physical activity is preferably an improved physical performance.

The term “enhancing recovery" refers preferably to a comparison of recovery using objective factors for a particular individual. Such objective recovery factors include the length of time it takes for an individual to perform at the same or similar level between an athletic event, training, performance or physical activity.

The enhanced recovery from a physical activity is preferably an improved post-workout fatigue and/or shorter recovery time.

The term “improved” relates to an improvement as compared to a group or subject not consuming the bacterial composition of the invention before, during or after physical activity. Preferably, the increased athletic endurance, increased athletic performance, enhanced recovery, improved physical performance, reduced post-workout fatigue, shorter recovery time and less muscle soreness relates preferably to an improvement of 5% or more. The improvement can also be detected in a consumer perception study as described in Example 5.

The term “treating” (and its grammatical variations thereof such as “treat” or “treating”), as used herein, refers to a clinical intervention in an attempt to alter the natural course of the individual being treated, and can be performed either for prophylaxis or during the course of clinical pathology. The term “preventing” refers to, but is not limited to, inhibition or the averting of symptoms associated with a particular disease or disorder. Desirable effects of treatment include, but are not limited to, alleviation of symptoms, diminishing of any direct or indirect pathological consequences of the disorder, or preventing occurrence or recurrence of at least one of the disorders.

Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. The publications and applications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein is to be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention. In addition, the materials, methods, and examples are illustrative only and are not intended to be limiting.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described below. In case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.

While aspects of the invention are illustrated and described in detail in the drawings and foregoing description, such illustration and description are to be considered illustrative or exemplary and not restrictive. It will be understood that changes and modifications may be made by those of ordinary skill within the scope and spirit of the following claims. In particular, the present invention covers further embodiments with any combination of features from different embodiments described above and below. The invention also covers all further features shown in the figures individually, although they may not have been described in the previous or following description. Also, single alternatives of the embodiments described in the figures and the description and single alternatives of features thereof can be disclaimed from the subject matter of the other aspect of the invention.

The general methods and techniques described herein may be performed according to conventional methods well known in the art and as described in various general and more specific references that are cited and discussed throughout the present specification unless otherwise indicated.

Throughout this specification and the claims, unless the context requires otherwise, the word "comprise" or “include”, and variations such as "comprises/includes" and "comprising/including" are to be understood to imply the inclusion of an element, stated integer, step or a group thereof but not the exclusion of any other element, stated integer, step or a group thereof.

As used in this specification and the appended claims, the singular forms "a", "an", and "the" include plural referents, unless the content clearly dictates otherwise.

A single unit may fulfill the functions of several features recited in the claims.

The term “essentially”, “about” or “approximately” and the like when used in connection with an attribute or numerical value particularly defines exactly the attribute or value and additionally encompass, unless the context indicates otherwise, numerical values within a range having a lower limit that is 0-10% smaller than the indicated numerical value and having an upper limit that is 0-10% larger than the indicated numerical value. The term “about” or “approximately” means preferably ±10%, more preferably ±5%, again more preferably ±3% or most preferably ±0% (referring to the given numeric value, respectively). In each of the invention embodiments, „about” can be deleted. All ranges of values disclosed herein, should refer to and include any and all values falling within said range including the values defining the range.

BRIEF DESCRIPTION OF DRAWINGS

Figure 1 : Metabolic activity of P. freudenreichii strains in culture medium (YEL), in culture medium with 20% (YEL 20%), or 40% (YEL 40%) v/v fermentation effluent (mimicking colon milieu) after 48h incubation. Reported values are single HPLC-RI measurements on combined samples from 3 biological replicates.

Figure 2: Bacterial growth (optical density) of P. freudenreichii strains in culture medium (YEL), in culture medium with 20% (YEL 20%), or 40% (YEL 40%) v/v fermentation effluent (mimicking colon milieu) after 24h and 48h incubation. Reported values are means and standard deviations for samples from 3 biological replicates.

Figure 3: (A) Set up of the PolyfermS fermentation model. The fermentation model was inoculated with immobilized fecal microbiota from a 2 months old infant and consisted of a first-stage inoculation reactor (IR) containing fecal beads and three second-stage reactors, one control reactor (CR) and two treatment reactors (TR1 , and TR3) continuously inoculated with 5% IR effluent. All reactors operated under the same conditions, modelling the infant proximal colon.

Figure 4: Lactate accumulation in the fermentation effluent of an in vitro continuous fermentation model mimicking the proximal colon of a healthy donor and effects of supplementation with P. freudenreichii JS27 or L reuteri during period 1 (first intervention period).

Figure 5: Lactate accumulation in the fermentation effluent of an in vitro continuous fermentation model mimicking the proximal colon of a healthy donor and effects of supplementation with P. freudenreichii JS27 or L reuteri during period 2 (second intervention period).

Figure 6: Differentially abundant families visualization of DESeq2 outcome. Donormicrobiota taxonomic response in TR1. Only the families with both significant and consistent log2Fold Changes between treatment and stabilization in period 1 (A) and period 2 (B) are presented. Differential abundance analysis was performed on non rarefied relative abundance datasets from samples of the last 3 days of stabilization and intervention phases.

Figure 7: Abundance of P. freudenreichii JS27 in TR1 after 7 days (grey) and 14 days of supplementation (black) by qPCR utilizing strain specific primers.

Figure 8: Results of the Consumer Perception Study

EXAMPLES

Aspects of the present invention are additionally described by way of the following illustrative non-limiting examples that provide a better understanding of embodiments of the present invention and of its many advantages. The following examples are included to demonstrate preferred embodiments of the invention. It should be appreciated by those skilled in the art that the techniques disclosed in the examples which follow represent techniques used in the present invention to function well in the practice of the invention, and thus can be considered to constitute preferred modes for its practice. However, those skilled in the art should appreciate, in light of the present disclosure that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention. Several documents including patent applications, manufacturer’s manuals and scientific publications are cited herein. The disclosure of these documents, while not considered relevant for the patentability of this invention, is herewith incorporated by reference in its entirety. More specifically, all referenced documents are incorporated by reference to the same extent as if each individual document was specifically and individually indicated to be incorporated by reference.

Example 1

The bacteria were grown in medium. The bacterial strains were obtained from the German Collection of Microorganisms and Cell Cultures (DSMZ, Braunschweig, Germany), the strain collection of SURI BioTech GmbH or isolated from commercial products. Table 1. List of strains used in this study.

Strain name Isolation Source

Propionibacterium freudenreichii JS27 (cf. Proprietary strain SURI BioTech,

MIESCHER, Susanne. Antimicrobial and comprising SEQ ID NO: 1 autolytic systems of dairy propionibacteria. LFB strain ID' BT 1205

1999. Diss. ETH No. 13486)

Propionibacterium freudenreichii JS HOLDBAC ^TM YM-B by Danisco

Propionibacterium freudenreichii DSM4902^T DSMZ

Propionibacterium freudenreichii DSM20271 ^T DSMZ

Limosilactobacillus reuteri DSM17938 Biogaia drops by Biogaia AB

^T Type strain; DSMZ - Leibniz Institute DSMZ: Deposit of a Proposed Type Strain

Glycerol stocks stored at -80°C were re-activated on agar plates and incubated in anaerobic jars (Mitsubishi AnaeroPack, Thermo Fisher Diagnostics AG, Pratteln, Switzerland) containing the AnaeroGen system (Oxoid, Thermo Fisher Diagnostics AG). Limosilactobacillus reuteri DSM 17938 were incubated at 37°C for two days and Propionibacterium for five days. Subsequently, a single colony was picked, transferred into conical polypropylene tubes containing 10 mL of sterile broth and Limosilactobacillus were incubated for 48 h and Propionibacterium for 72 h at 37°C. Strains were sub-cultured twice in liquid media before being used as working cultures.

P. freudenreichii were grown in yeast extract sodium lactate medium (YEL) consisting of:

- 1 % (w/v) trypticase soy broth without dextrose (Becton Dickinson AG, Allschwil, Switzerland);

- 1 % (w/v) yeast extract (Merck, Darmstadt, Germany);

- sodium DL- lactate 50% (Sigma-Aldrich, Buchs, Switzerland).

Limosilactobacillus reuteri DSM17938 (L. reuteri) were grown in Man-Rogosa-Sharp (MRS) commercial media.

Example 2 - Growth and metabolism of Propionibacterium freudenreichii strains in media mimicking proximal colon conditions

To investigate growth and metabolic ability in presence of sterile fermentation effluent produced in an intestinal fermentation model, 20 pL of each working culture was used to inoculate in triplicate wells in 96-well microtiter plates, each well containing 180 pL of YEL broth containing 55 mM sodium-DL-lactate, and different proportions of fermentation effluent. Water was replaced in YEL media by fermenter effluent in the following proportions at 20% or 40% v/v. Fermentation effluent was collected from the inoculum and/or control reactor of an in vitro continuous fermentation model PolyFermS mimicking the proximal colon of healthy donor corresponding to Fermentation 1 described in Pham et al. (2019). Concentrations of carbohydrates, short chain fatty acids (SCFA) and fermentation metabolites in effluent, YEL media and supplemented YEL media with effluent are shown in Table 2.

Table 2. Concentration of carbohydrates, SCFA and fermentation metabolites in effluent, YEL media and supplemented YEL media with effluent.

Cultures in YEL and YEL with 20% or 40% v/v effluent media were incubated for 48 h at 37°C in anaerobic jars (Mitsubishi AnaeroPack, Thermo Fisher Diagnostics AG) containing the AnaeroGen system (Oxoid, Thermo Fisher Diagnostics AG). Cell growth was assessed in triplicates for each strain and condition by measuring culture optical density at 600 nm (ODeoo) at 24 h and 48 h. Concentrations of lactate and SCFA in pooled supernatant from cultures from same strains and in same media were determined by high-pressure liquid chromatography analysis with refractive index detection (HPLC-RI) after 48 h.

High-Pressure Liquid Chromatography analysis with refractive index detection (HPLC-RI):

Concentrations of glucose, galactose, acetate, propionate, butyrate, formate, lactate, succinate, isobutyrate, isovalerate, and valerate were determined in the supernatant. For the analysis, 400 pL of the supernatants were filtered through a 0.45 pm membrane (Millipore AG, Zug, Switzerland), transferred into glass HPLC vials (Infochroma, Hitachi LaChrome, Merck, Dietikon, Switzerland), and sealed with crimpcaps.

The HPLC (Hitachi LaChrome) system was equipped with a refractive index (Rl) detector and the used column consisted of two parts (stationary phase):

- Security Guard Cartridges Carbo-H column (4 x 3 mm; Phenomenex Inc., Torrance, CA, USA);

- Rezex ROA-Organic Acid H⁺ column (8%, 300 x 7.8 mm; Phenomenex).

The mobile phase consisted of 10 mM H2SO4 (Fluka, Buchs, Switzerland) solvent. The elution was performed at a flow rate of 0.4 mL/min at 25°C. Detection limit was 5 mM.

Results

Propionibacteria strains (JS, JS27, DSM20271T and DSM4902T) were analyzed upon their growth and metabolic ability in order to identify their capacity of survival and activity in intestinal milieu. It was observed that JS27 had the highest capacity of metabolism and cell growth over other P. freudenreichii strains in culture media YEL with and without the addition of fermentation effluent (Figures 1 and 2).

Example 3 - Effect of Propionibacterium freudenreichii JS27 and commercial Limosilactobacillus reuteri DSM 17938 as representative of lactobacilli bacteria, in gut proximal colonic conditions

An in vitro gut modeling system was used to mimic human intestinal microbiota and proximal colon conditions and compare the impact of a daily supplementation of bacterial strains Limosilactobacillus reuteri DSM17938 (L reuteri) and P. freudenreichii JS27 on the gut microbiota composition and metabolism.

The utilization of in vitro intestinal fermentation model inoculated with immobilized fecal microbiota obtained from a fecal sample of a donor has been previously described (Pham et al., 2019). According to Scheiman et al., (2019), circulating lactic acid in athletes is able to cross the epithelial barrier into the intestinal lumen, becoming available as a substrate for the gut microbiota (Scheiman et al., 2019). In order to mimic human intestinal microbiota with an active lactate metabolism, the intestinal model chosen was that of a 2-month-old infant which has been previously shown to harbor a high-lactate gut environment and an abundant lactate-utilizing bacteria (Pham et al., 2019; Rocha Martin et al., 2019). Increased lactate production as it occurs during physical exercises was mimicked in the model by spiking the medium with sterile skim milk. For this experiment, the fermentation system consisted of an inoculum reactor (IR) containing immobilized fecal microbiota and 3 second-stage reactors: 1 control reactor (CR) and 2 treatment reactors (TRs) (Figure 3). The second stage reactors were continuously inoculated with 95% fresh medium and 5% IR effluent. The fermentation medium composition, fecal immobilization procedure and reactors conditions is shown in Table 3 and described in Rocha Martin et al. (“Colonization of Cutibacterium avidum during infant gut microbiota establishment,” FEMS Microbiol Ecol, vol. 95, no. fiy215, 2019). Conditions set for all the reactors were: temperature 37°C, pH 6.0, retention time of 5 h, stirring at 100 rpm and a working volume of 200 mL. Reactors were kept anaerobic by constantly flushing CO2 in the headspace. Fermentation stability was monitored daily by HPLC-RI analysis of concentration of SCFA and intermediate metabolites in the effluent of reactors.

After fecal immobilization in gellan-xanthan gel beads and IR inoculation, an initial batch fermentation was carried out for 48 hours to allow colonization of the microbial population immobilized on the beads. Afterwards, the fermentation media inflow was switched to continuous mode and the microbiota in IR was allowed to stabilize for a phase of 10 days before connecting the second-stage reactors. The second-stage reactors were tested in two consecutive periods: period 1 and period 2. Each period was divided into a stabilization of 7 days followed by an intervention phase of 14 days. Samples for metabolic analysis were taken during the last 3 days of each phase. During the intervention period, bacterial strains were daily spiked in the TR1 and TR3. In-between periods, the second-stage reactors were disconnected, washed, and reconnected to repeat the described phases in fermentation period 2.

Bacterial suspension preparation and supplementation of second-stage reactors

Bacterial suspensions of Limosilactobacillus reuteri DSM 17938 (/_. reuteri) for supplementing treatment reactor 3 (TR3) in the intestinal fermentation model were prepared by centrifuging cells from a 24-hours working cultures (128 pL) at 7000 rpm for 10 min and washing with a phosphate buffered saline (PBS) solution supplemented with L-cysteine hydrochloride (L-cysteine HCI). PBS was composed of 8 g liter-1 NaCI, 0.2 g liter-1 KCI, 1.42 g liter-1 Na2HPO4, 0.24 g liter-1 KH2PO4 (VWR International, Dietikon, Switzerland). L-cysteine HCI was added to have a final concentration of

O.05% (v/v). If not differently specified, all components were from Sigma-Aldrich, Buchs, Switzerland.

P. freudenreichii JS27 used for the spiking of the treatment reactor 1 (TR1 ) were produced in a batch fermentation and stored at -80°C. Bacterial concentration was calculated in working cultures or frozen cryostock by plating serial dilutions in duplicates on agar plates.

During intervention period, TR1 was spiked daily with 820 pL of P. freudenreichii JS27 corresponding to 5x10⁹ CFU from frozen stocks, resuspended in sterile skim milk to a total volume of 2.72 mL. TR3 was used to test the effects of daily supplementation of 5x10⁸ CFU L reuteri. Considering the bacterial enumeration performed on working cultures, 128 pL of a 24h culture in MRS was resuspended in sterile skim milk to a total volume of 2.72 mL. The Control Reactor (CR) was spiked daily with 2.72 mL of sterile skim milk.

Statistical analysis

Statistical comparison using Repeated measures ANOVA and pairwise t-tests followed by Bonferroni correction were conducted using Python with the pingouin library.

Results

Increased intestinal lactate concentrations, as it occurs during physical exercises, was successfully induced in the fermentation model by the supplementation of sterile milk in second-stage reactors. Supplementation of sterile milk in second-stage reactors induced an increased lactate production in both intervention periods compared to respective stabilization phases (Figures 4 and 5). During period 1 , lactate accumulation was significantly higher in TR3 supplemented with L reuteri compared to TR1 under supplementation with P. freudenreichii JS27 (p 0.03). During period 2, TR1 supplemented with P. freudenreichii JS27 showed a significantly lower lactate accumulation when compared to supplementation with sterile skim milk (CR, control reactor; p 0.02) and supplementation with L reuteri (TR3; p 0.005). Furthermore, in the second intervention period, supplementing L reuteri in TR3 lead to significantly increased lactate accumulation compared to the CR (p 0.02).

Daily supplementation for 14 days of P. freudenreichii JS27 shows exceptional capacity of reducing intestinal lactic acid accumulation compared to lactobacilli. The metabolic capacity of lactate metabolism of P. freudenreichii JS27 can thus prevent lactate accumulation and divert lactate from the production of detrimental metabolites. Lactobacilli supplementation increases intestinal lactic acid concentration which could lead to lactic acidosis when supplemented to athletes, which is not desirable.

Table 3. Composition of fermentation medium (a) and vitamin solution (b). FOS and vitamin solutions were filter-sterilized and added to the medium after it was autoclaved. All components were from Sigma-Aldrich (Buchs, Switzerland) unless differently specified. a) Fermentation medium

D-Lactose monohydrate 3.95

Vivinal GOS (COSUCRA, Warcoing, Belgium) 10.3

Amicase (Sigma Aldrich, Buch, Switzerland) 0.5

Whey protein (Emmi Schweiz AG, Dagmarsellen,

Switzerland) 8.1

Peptone water (Thermo Fisher Diagnostics AG, Pratteln,

Switzerland) 0.5

Bacto tryptone (Becton, Dickinson AG,

Allschwil, Switzerland) 0.5

Mucin from porcine stomach (type II) 4

Yeast extract (ProCel 251 MG, Procelys,Maisons-Alfort,

France) 2.5

L-cysteine HCI monohydrate 0.8

Bile salts (Oxoid, Thermo Fisher Diagnostics AG) 0.05

KH2PO4 (VWR international) 0.5

NaHCO3 (Acres Organics BVBA, Geel, Belgium) 1.5

NaCI 4.5

KCI 4.5

MgSO4 ■ 7H2O 1.25 CaCI2 ■ 2H2O 0.15

FeS04 ■ 7H2O 0.005

Hemin 100 mg/mL (mL) 0.01

Tween 80 (mL) 1

Vitamin solution (mL) 0.5

Fibrulose F97 (FOS; COSUCRA, Warcoing,Belgium)16 g/L (mL) 11 b) Vitamin solution

Pyridoxine-HCI 100 100

4-Aminobenzoic acid (PABA) 50

Nicotinic acid (Vit. B3) 50

Biotine (Vit. H/B7) 20

Folic acid 20

Cyanocobalamin (Vit B12) 5

Thiamine (Vit B1 HCI) 50

(-)-Riboflavin 50

Phylloquinone (Vit. K1 ) 0.075

Menadione 10

D-Pantothenic acid hemicalcium salt (Vit B5) 100

Colonization capacity of Propionibacterium freudenreichii JS27

Quantification of P. freudenreichii JS27 in TR1 effluent revealed high abundance levels after 7 days (half intervention) and 14 days of supplementation in both periods (Figure 7).

Conclusion

The inventors evaluated the effect of daily supplementation of 5x10⁹ CFU P. freudenreichii JS27, after 14 days of intervention on gut microbiota composition and metabolism in an in vitro fermentation model. The inventors showed that accumulation of lactate could be simulated, as it occurs in athletes during intense exercise, by adding skim milk in the control reactor. P. freudenreichii JS27 can prevent intestinal lactate accumulation and showed exceptional capacity of reducing intestinal lactic acid accumulation and transforming lactic acid into acetate and propionate when supplemented in a complex microbiome ecosystem.

The efficacy of probiotics to exert a health benefit highly depends on their capacity to colonize the gut microbiome. Colonization capacity of P. freudenreichii JS27 was demonstrated in this study, as a high abundance of the strain was identified in the reactor effluent.

Example 4 - Effect of Propionibacterium freudenreichii JS27 on the gut microbiota analyzed by 16S RNA gene sequencing

DNA extraction and amplicon Illumina sequencing

Effluent samples were centrifuged for 10 min at 14’000 rpm and 4°C. The DNA from TR1 effluent pellets was extracted using the Fast DNA TM Spin Kit for Soil (MP Biomedicals, lllkirch-Graffenstaden, France) following the supplier’s protocol. The DNA concentration was measured with Nanodrop (ND-1000, Witec AG, Sursee, Switzerland), and samples were diluted to 20 ng DNA/pL using DES water (Fast DNA TM Spin Kit, MP Biomedicals) and stored at -20°C until further processing.

Bacterial communities in TR1 effluent samples were identified by amplifying the V3 region of the 16S rRNA gene followed by sequencing with an Illumina Miseq platform (Genetic Diversity Centre, ETH Zurich, Switzerland) as described in Rocha Martin et al., (2019).

Microbiota composition analysis

Atropos software was used as a trimming tool to remove Illumina adaptors and genespecific PCR primers. The DADA2-pipeline was used to generate amplicon sequencing variants (ASVs). Chimeric sequences were identified and removed, and taxonomy was assigned using the SILVA database (v.132) as previously described (Isenring et al., 2021 ).

Data analysis

Microbiota community analysis was done using R packages phyloseq (version 1 .40.0). Differential abundance analyses were performed using DESeq2 (version 1.36.0). The statistical analysis was based on the comparison between treatment and respective stabilization phases from each individual period of the TRs and CR. Differential abundance analysis was performed on non-rarefied relative abundance datasets using the Wald test with DESeq2 (McMurdie & Holmes, 2013).

The analysis of the microbiota composition in TR1 by 16S rRNA gene sequencing also revealed significant increases of Propionibacteriaceae upon supplementation. Upon supplementation in TR1 , significant increases of Propionibacteriaceae family abundance within TR1 compared to stabilization in period 1 (Iog2 FC +1.6 ± 0.3; padj 2.3E-07) (Figure 6A) and period 2 (Iog2 FC +1 .6 ± 0.2; padj 8.2.E-20) (Figure 6B) were detected by DESeq2 analysis. This indicates the unique capacity of P. freudenreichii JS27 to colonize the microbiota composition.

Example 5 - Consumer Perception Study

In a survey, consumers’ feedback is gathered and analyzed for the impact of Propionibacterium freudenreichii JS27 on digestion, fitness and physical performance, energy levels, feeling during workouts, post-workout fatigue, muscle soreness, recovery, and overall well-being. Participants rated these parameters without and with taking the formulation RecoBiotic (composition comprising Propionibacterium freudenreichii JS27 as described herein in Example 6).

The RecoBiotic was consumed by the participants daily for at least for 2 weeks. The target group was made-up of athletes training >3 days/week, typically at least 20 min, and aged 25-50 years. The survey was completed after >2 weeks of daily use (self- reported outcomes, anonymous questionnaire, comparative assessment).

So far, 15 women and 17 men completed the survey, and about 70% reported improved physical performance, and 75% reported less post-workout fatigue, shorter recovery times and less muscle soreness. No negative impact on overall health and digestion has been observed. Athletes sharing their experience with RecoBiotic report that they felt extremely energetic, and that muscle soreness has been reduced, especially after heavy exercising. RecoBiotic helps to recover exceptionally well. Since lactate clears faster, the athletes report that they have less water retention and no more sore muscles.

Example 6 - Exemplary formulation

The following formulation RecoBiotic was tested in the Consumer Perception Study: 1. Excipients (127 mg): a. Microcrystalline cellulose: a bulking agent, binder, and disintegrant. b. Silicon dioxide functions as a lubricant and anti-caking agent.

2. Lyophilized bacterial culture of Propionibacterium freudenreichii, such as Propionibacterium freudenreichii JS27: at least 10⁹ cfu per capsule

3. Capsules shell Enteric-coated DR hard capsules, 2, natural - weight 59 mg

It will be understood that other formulations can be likewise used, and the skilled person knows how to formulate bacteria of the order Propionibacteriales, genus Propionibacteria and the species P. freudenreichii in an effective formulation (see for example, Aljaberi A et al., Functional performance of silicified microcrystalline cellulose versus microcrystalline cellulose: a case study. Drug Dev Ind Pharm. 2009 Sep; 35(9): 1066-71. doi: 10.1080/03639040902774131 ).

Preferred formulations are oral formulations, such as oral enteric formulations. Preferably, oral enteric formulations or oral enteric dosage forms comprise an enteric coating that prevents its dissolution or disintegration in the gastric environment.

Preferably, the formulation comprises a lyophilized bacterial culture of Propionibacteriales, Propionibacteria, Propionibacterium freudenreichii, or P. freudenreichii JS27 , JS, DSM4902T and DSM20271T.

References

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Claims

1. A non-therapeutic use of a bacterial composition in a healthy subject for increasing or maintaining athletic endurance and/or athletic performance during a physical activity and/or for enhancing recovery from a physical activity, said composition comprises bacteria, wherein said bacteria comprise or consist of bacteria of the order Propionibacteriales, preferably bacteria of the genus Propionibacteria, more preferably bacteria of the species P. freudenreichii, again more preferably bacteria of strain P. freudenreichii JS27.

2. A bacterial composition comprising bacteria comprising or consisting of bacteria of the order Propionibacteriales, preferably of the genus Propionibacteria, more preferably bacteria of the species P. freudenreichii, again more preferably bacteria of strain P. freudenreichii JS27, for use in treating or preventing a disorder in a subject, preferably an athlete, the disorder occurs during or after physical activity, wherein preferably the disorder is selected from the group consisting of acidosis, preferably lactic acidosis, weakness attack, muscle weakness, muscles cramps, muscle pain, inflammation, infection, immune system deficiency, vitamin B12 deficiency, metabolic endotoxemia, hemolysis, nausea, vomiting, gastritis, gastrointestinal bleeding, stomach pain, exercise-related transient abdominal pain (ETAP), colic, intestinal discomfort, intestinal pain, visceral sensitivity, and intestinal cramp, more preferably the disorder is selected from the group consisting of acidosis, preferably lactic acidosis, weakness attack, muscle weakness, and muscle pain.

3. A non-therapeutic method for supplementing the microbiome of a healthy subject, the method comprises the step of administering an effective dose of bacteria, wherein said bacteria comprise or consist of bacteria of the order Propionibacteriales, preferably bacteria of the genus Propionibacteria, more preferably bacteria of the species P. freudenreichii, again more preferably bacteria of strain P. freudenreichii JS27, to the healthy subject before, during or after physical activity, wherein the population of bacteria of the order Propionibacteriales, preferably bacteria of genus Propionibacterium, preferably bacteria of species P. freudenreichii, again more preferably bacteria of strain P. freudenreichii JS27 in the gut of the subject is increased.

4. A non-therapeutic method for reducing level of lactic acid and/or lactates in a healthy subject, during or after physical activity, the method comprises the step of administering an effective dose of bacteria comprising or consisting of bacteria of the order Propionibacteriales, preferably bacteria of the genus Propionibacteria, more preferably bacteria of the species P. freudenreichii, again more preferably bacteria of strain P. freudenreichii JS27, wherein the effective dose of bacteria is administered to the healthy subject before, during or after physical activity, preferably the physical activity generates increased lactate levels reduced by the administered bacteria.

5. The method or use of any one of the preceding claims, wherein the bacteria of the order Propionibacteriales comprise a 16S rDNA sequence as defined in SEQ ID NO: 1 , or the bacteria of the order Propionibacteriales comprise a 16S rDNA sequence having at least 95% sequence identity to a 16S rDNA sequence as defined in SEQ ID NO: 1 , preferably the bacteria of the order Propionibacteriales comprise a 16S rDNA sequence as defined in SEQ ID NO: 1.

6. The method or use of any one of the preceding claims, wherein the physical activity generates increased lactate levels.

7. The method or use of any one of the preceding claims, wherein the composition further comprises one or more compounds selected from the group consisting of an amino acid, polypeptide, protein, preferably whey protein, vitamin, preferably vitamin B12, mineral, such as a salt of sodium, potassium, magnesium und calcium, carbohydrate, lipid, and levocarnitine (L-carnitine).

8. The method or use of any one of the preceding claims, wherein the composition is administered before, during or after physical activity, preferably during or after physical activity.

9. The method or use of any one of the preceding claims, wherein the composition is administered enterally, preferably the composition is administered orally.

10. The method or use of any one of the preceding claims, wherein the composition is formulated for delivering bacteria of the order Propionibacteriales, preferably bacteria of the genus Propionibacterium, more preferably bacteria of species P. freudenreichii, again more preferably bacteria of strain P. freudenreichii JS27 to the gut, preferably the colon, more preferably the proximal colon.

11 . The method or use of any one of the preceding claims, wherein the composition is formulated as a product selected from the group consisting of a food product, food supplementary product, dietary supplement, and beverage.

12. The method or use of any one of the preceding claims, wherein the composition increases the population of bacteria of the order Propionibacteriales, preferably bacteria of the genus Propionibacterium, more preferably bacteria of the species P. freudenreichii, again more preferably bacteria of strain P. freudenreichii JS27.

13. The method or use of any one of the preceding claims, wherein the composition decreases the level of lactic acid and/or lactates in the subject.

14. The method or use of any one of the preceding claims, wherein the bacteria of the composition, preferably the bacteria of the order Propionibacteriales, more preferably the bacteria of the genus Propionibacterium, again more preferably the bacteria of the species P. freudenreichii, again more preferably the bacteria of strain P. freudenreichii JS27, are administered in a dose from about 10⁴ CFU to about 10¹⁶ CFU, about 10⁹ CFU to about 10¹¹ , or about 10⁹ CFU to about 10¹° CFU, preferably about at least 10⁹ CFU per dosage unit.

5. The method or use of any one of the preceding claims, wherein the bacteria of the composition, preferably the bacteria of the order Propionibacteriales, more preferably the bacteria of the genus Propionibacterium, again more preferably the bacteria of the species P. freudenreichii, again more preferably the bacteria of strain P. freudenreichii JS27, are administered in a single daily dose, preferably for at least 14 days; or in two daily doses, before and during or during and after physical activity; or in three, ten or more daily doses, before, during and after physical activity, wherein preferably each dose is of about at least 10⁹ CFU.