Probiotic compositions and methods for preventing muscle loss in individuals

Abstract

The present invention relates to probiotic compositions comprising at least one Lactobacillus helveticus, at least one Bifidobacterium longum, and at least one Bifidobacterium animalis. The present invention further relates to methods of using said compositions for preventing muscle loss, improving or maintaining muscle strength, and maintaining intestinal barrier integrity.

Description

[0001] PROBIOTIC COMPOSITIONS AND METHODS FOR PREVENTING MUSCLE LOSS IN INDIVIDUALS

[0002] FIELD

[0003] The present invention relates to probiotic mixtures and uses thereof for preventing muscle loss and improving or maintaining muscle strength in individuals, particularly in aging or elderly individual, performance athletes and more generally in individuals suffering from muscle loss. BACKGROUND

[0004] Sarcopenia is the age-related loss of muscle mass, strength, and function, predominantly affecting older adults but it is well established that individuals gradually begin losing muscle mass and strength sometime in their 30s or 40s. This condition significantly impacts quality of life by reducing mobility and increasing the risk of falls and fractures. Recent studies have highlighted a potential link between sarcopenia and gut microbiota. For instance, Liu et al. (J Cachexia Sarcopenia Muscle 2021. 12(6): 1393-1407) reported a significant correlation between sarcopenia and the diversity of specific bacterial populations in older individuals. Furthermore, it is hypothesized that inflammation may play a critical role in the muscle wasting characteristic of sarcopenia. Under normal physiological conditions, muscle protein balance is regulated by external stimuli such as exercise and dietary protein intake. Disruptions to this balance, particularly in aging populations, contribute to the progression of sarcopenia.

[0005] Another emerging area of interest is the potential role of probiotics in mitigating muscle loss or maintenance of muscle strength, particularly with performance athletes or individuals that suffered from muscle loss due to immobility. Probiotics may exert their effects by reducing gut permeability and modulating systemic inflammation, thereby addressing key factors linked to muscle deterioration (van Krimpen et al., 2021, Nutrients 29; 13(4): 1115).

[0006] Probiotic supplementation has shown promise as a therapeutic strategy for preventing the muscles loss related to sarcopenia and immobility in the population in general. Studies suggest that probiotics can enhance muscle strength, increase muscle mass, and improve overall functionality. Additionally, probiotics may reduce inflammation and improve gut health - two factors implicated in the progression of muscle decline. These findings underscore the potentialof probiotics as a complementary approach to managing sarcopenia and improving the quality of life in aging populations

[0007] SUMMARY

[0008] The present disclosure relates to a method for preventing muscle loss and improving or maintaining muscle strength in individuals, particularly in aging or elderly individual, performance athletes and individuals that suffered from muscle loss, such as, for example, due to immobility. The present disclosure is based on the discovery that a probiotic composition is unexpectedly effective in preventing muscle loss and improving or maintaining muscle strength in individuals.

[0009] The invention is defined by the claims. Thus, the present description provides a probiotic composition comprising at least one Lactobacillus helvelicus. at least one Bifidobacterium longum, and at least one Bifidobacterium animalis. The present description also provides a probiotic composition consisting essentially of at least one Lactobacillus helvelicus. at least one Bifidobacterium longum. and at least one Bifidobacterium animalis. The present description also provides a probiotic composition consisting of at least one Lactobacillus helvelicus., at least one Bifidobacterium longum. and at least one Bifidobacterium animalis.

[0010] The present description also provides uses of the probiotic composition as defined above for stimulating muscle strength in individuals; preventing or reducing the loss of muscle morphology in individuals; preventing locomotion syndrome; increasing or maintaining muscle strength in individuals; or preventing or reducing symptoms associated with sarcopenia.

[0011] The present description also provides uses of the probiotic composition as defined above for maintaining intestinal barrier integrity in individuals; or reducing the loss of intestinal barrier integrity in individuals.

[0012] The present description further provides a method for preventing or reducing the loss of muscle morphology, for increasing muscle morphology and / or for preventing or treating muscle atrophy, e.g. improving muscle recovery after muscle atrophy, comprising administering the probiotic composition as defined above.The present description further provides a method for maintaining intestinal barrier integrity or reducing the loss of intestinal barrier integrity, comprising administering the probiotic composition as defined above.

[0013] The present description also provides a method for preventing or reducing symptoms associated with sarcopenia comprising administering a probiotic composition as defined above.

[0014] The present description further provides with a method for preventing locomotive syndrome comprising administering a probiotic composition as defined above.

[0015] The present description also provides a probiotic composition as defined above for use in: preventing or reducing the loss of muscle morphology in an individual; increasing or maintaining muscle strength in an individual; stimulating muscle strength in an individual; preventing locomotion syndrome in an individual; preventing or reducing symptoms associated with sarcopenia in an individual; increasing muscle morphology in an individual; treating and or preventing muscle atrophy in an individual, e.g. improving muscle recovery after muscle atrophy; preventing or alleviating symptoms associated with sarcopenia in an individual; and / or preventing locomotive syndrome. Additionally or alternatively, the description also provides a probiotic composition as defined above for use in maintaining intestinal barrier integrity in an individual and / or reducing the loss of intestinal barrier integrity in an individual.

[0016] The present description further provides the use of a probiotic composition as defined above for the manufacture of a medicament for: preventing or reducing the loss of muscle morphology in an individual; increasing or maintaining muscle strength in an individual; stimulating muscle strength in an individual; preventing locomotion syndrome in an individual; preventing or reducing symptoms associated with sarcopenia in an individual; increasing muscle morphology in an individual; treating and or preventing muscle atrophy in an individual, e.g. improving muscle recovery after muscle atrophy; preventing or alleviating symptoms associated with sarcopenia in an individual; and / or preventing locomotive syndrome. Additionally or alternatively, the description also provides a probiotic composition as defined above for the manufacture of a medicament for maintaining intestinal barrier integrity in an individual and / or reducing the loss of intestinal barrier integrity in an individual.In some embodiments, the uses and methods described above may be non-therapeutic, e.g. for the purposes of increasing muscle strength in a performance athlete.

[0017] In other embodiments, the uses and methods described above may be therapeutic, e.g. for the purposes of treating or preventing muscle atrophy resulting from immobility following an injury.

[0018] In the embodiments of the probiotic compositions, uses and methods described herein, the individuals to be administered with the probiotic composition are aging or elderly individuals, performance athletes or individuals suffering from muscle loss due to immobility.

[0019] In an embodiment of the probiotic compositions, uses and methods described herein, the at least one Lactobacillus helveticu . the at least one Bifidobacterium longum, and the at least one Bifidobacterium animalis are each individually present in an amount ranging from 0,5 to 99,5 dry weight percent based on the total weight of the probiotic composition. In a further embodiment of the probiotic compositions, uses and methods described herein, the at least one Lactobacillus helveticus is present in an amount ranging from 40,0 to 50,0 dry weight percent based on the total weight of the probiotic composition, the at least one Bifidobacterium longum is present in an amount ranging from 0,5 to 10,0 dry weight percent based on the total weight of the probiotic composition, and the at least one Bifidobacterium animalis is present in an amount ranging from 45,0 to 55,0 dry weight percent based on the total weight of the probiotic composition. In a further embodiment of the probiotic compositions, uses and methods described herein, the at least one Lactobacillus helveticus is present in an amount of 45,0 dry weight percent based on the total weight of the probiotic composition, the at least one Bifidobacterium longum is present in an amount of 5,0 dry weight percent based on the total weight of the probiotic composition, and the at least one Bifidobacterium animalis is present in an amount of 50,0 dry weight percent based on the total weight of the probiotic composition. In a further embodiment of the probiotic compositions, uses and methods, the probiotic compositions is formulated as a food composition, a dietary supplement, a nutritional composition, a nutraceutical, a powdered nutritional product to be reconstituted in a liquid (such as water or milk) before consumption, a food additive, a medicament or a drink.BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 A illustrates a life span study with wildtype (N2) C. elegans;

[0021] Figure IB illustrates an endurance study with wildtype (N2) C. elegans,'

[0022] Figure 2A illustrates a life span study with transgenic (RW1596) C. elegans,- Figure 2B illustrates an endurance study with transgenic (RW1596) C. elegans,'

[0023] Figure 3 illustrates the protocol of the Burrowing assay;

[0024] Figure 4 illustrates the results of a neuromuscular health assay (Burrowing assay) with wildtype C. elegans worms fed on the probiotic composition in accordance with an embodiment of the present disclosure and subjected to an exercise regimen;

[0025] Figure 5 illustrates the results of a neuromuscular health assay (Burrowing assay) with transgenic C. elegans worms fed on the probiotic composition in accordance with an embodiment of the present disclosure and subjected to an exercise regimen;

[0026] Figure 6 illustrates the results of a gut integrity assay with wildtype (N2) C. elegans,' Figure 7 illustrates the results of a gut integrity assay with transgenic (RW1596) C. elegans,' and

[0027] Figure 8 illustrates the results of a pharyngeal pumping rate study with wildtype (N2) C. elegans, where in panels A-D, from left to right, the boxes correspond to E. coli OP50, B94, R0175, R0052 and Mix, and where in panel E, from left to right, the boxes correspond to E. coli OP 50 and Mix; and

[0028] Figure 9 illustrates the results of a pharyngeal pumping rate study with transgenic (RW1596) C. elegans, where in panels A-E, from left to right, the boxes correspond to E. coli OP50, B94, R0175, R0052 and Mix.

[0029] DESCRIPTION

[0030] It has been discovered that a composition comprising at least one Lactobacillus helveticus, at least one Bifidobacterium longum, and at least one Bifidobacterium animalis is beneficial in assisting to prevent muscle loss and improving or maintaining muscle strength in individuals.As is apparent from the examples below, it was found that a probiotic composition comprising at least one Lactobacillus helvelicus, at least one Bifidobacterium longum, and at least one Bifidobacterium animalis proves to be especially useful for preventing or reducing the loss of muscle morphology, for increasing the muscle morphology and / or for improving the muscle recovery after muscle atrophy, preventing or reducing symptoms associated with sarcopenia, and preventing locomotive syndrome. The probiotic composition comprising at least one Lactobacillus helvelicus. at least one Bifidobacterium longum, and at least one Bifidobacterium animalis has also proven useful for maintaining intestinal barrier integrity and reducing the loss of intestinal barrier integrity.

[0031] The term "probiotic" as used herein refers to a live microorganism which, when administered in adequate amounts to a host, confers a health benefit to the host. A probiotic must fulfil several requirements related to lack of toxicity, viability, adhesion and beneficial effects.

[0032] As used herein, the term "Bifidobacterium" refers to members of the genus Bifidobacterium. These bacteria are Gram-positive anaerobic bacteria that are one of the major strains of bacteria present in the gastrointestinal flora. Specific examples of Bifidobacterium species include B. bifidum, B. animalis subsp. lactis, B. breve, B. longum, or B. longum subsp. infantis. The Bifidobacterium species used in the invention are set out in claim 1. Exemplary species and strains of Bifidobacterium include the following well-known strains: B. bifidum HA-132 (sold by Lallemand Health Solutions ("LHS")), B. bifidum Rosell®-71 (LHS), B. breve HA-129 (LHS), B. breve Rosell®-70 (LHS), B. infantis HA-116 (LHS), B. infantis Rosell®-33 (LHS), B. lactis HA- 194 (LHS), B. longum HA-135 (LHS), B. longum Resell®- 175 (LHS), B. animalis subsp. lactis Rosell®-421 (LHS). One of the Bifidobacterium used in the invention may be B. longum Rosell®-175 (LHS). B. longum Rosell®-175 (LHS) is also referred to as B. longum susp. longum RO 175, B. longum susp. longum Resell®- 175, B. longum RO 175, and Resell®- 175. These terms are used interchangeably herein. Another Bifidobacterium used in the invention is Bifidobacterium animalis ssp. lactis LAFTI® B94. Other bacterial strains having a name including an "R" code, e.g. "R0123", may be commercially known with respect to the name "Resell®", e.g. "Resell® -123". It is intended that the genus include species that have been reclassified (e.g., due to changes in the speciation of organisms as the result of genetic and other investigations) or renamed for marketing and / or other purposes.As used herein, the term "Lactobacillus" refers to members of the genus Lactobacillus, in the family Lactobacillaceae. These bacteria are Gram-positive facultatively anaerobic bacteria that represent a major part of the bacterial group often referred to as "lactic acid bacteria." Lactobacillus species include L. acidophilus, L. brevis, L. bulgaricus, L. casei, L. crispatus, L. delbrueckii, L. fermentum, L. gasseri, L. helveticus, L. lactis, L. plantarum, L. reuteri, L. rhamnosus, L. salivarius or L. paracasei. The Lactobacillus species used in the invention is set out in claim 1. Exemplary species and strains of Lactobacillus include the following well-known strains: L. acidophilus HA- 122 (LHS), L. acidophilus R0418 (LHS), L. brevis HA- 112 (LHS), L. casei HA- 108 (LHS), L. casei R0215 (LHS), L. delbrueckii bulgaricus HA- 137 (LHS), L. fermentum HA-179 (LHS), L. helveticus HA-128 (LHS), L. helveticus HA-501 (LHS), L. helveticus R0052 (LHS), L. helveticus Lafti LIO R0419 (LHS), L. paracasei HA- 196 (LHS), L. paracasei HA-274 (LHS), L. paracasei Lafti L26 R0422 (LHS), L. plantarum R0403 (LHS), L. plantarum R0202 (LHS), L. plantarum RIO 12 (LHS), L. reuteri HA- 188 (LHS), L. rhamnosus HA-114 (LHS), L. rhamnosus HA-500 (LHS), L. rhamnosus R0011 (LHS), L. rhamnosus R0049 (LHS), L. rhamnosus R0343 (LHS), L. rhamnosus R1039 (LHS), L. salivarius HA-118 (LHS), L. salivarius R0078 (LHS), L. bulgaricus R0440 (LHS) or L. lactis R1087 (LHS). The Lactobacillus used in the invention is preferably L. helveticus R0052 (LHS) (interchangeably referred to as L. helveticus Rosell®-52 or Rosell®-52). It is intended that the genus include species that have been reclassified (e.g., due to changes in the speciation of organisms as the result of genetic and other investigations) or renamed for marketing and / or other purposes.

[0033] The effective amount of colony forming units ("cfu") for each strain in the composition will be determined by the skilled in the art and will depend upon the final formulation. The term "colony forming unit" is defined herein as number of bacterial cells as revealed by microbiological counts on agar plates. For instance, the total probiotic may be provided in an amount of from about 105to 1012colony forming units (cfu) per dose, from about 106to 1011cfu per dose, from about 107to IO10cfu per dose or from about 108to IO10cfu per dose. The probiotic may be provided in an amount greater than about 1.0 x 109cfu total probiotic per dose. The individual may be administered greater than 5.0 x io9cfu total probiotic per dose. However, it is not intended that the present disclosure be limited to a specific dosage as it is contemplated that dosages of total probiotic will vary depending upon a number of factors such as the identity and number of individual probiotic strains employed, the subject being treated,the nature of the symptoms experienced by the subject that is to be treated, the general health of the subject, and the form in which the composition is administered.

[0034] Each of individual strains constituting the probiotic composition may be present in an amount ranging from 0,5 to 99,5 dry weight percent based on the total weight of the probiotic composition. The total dry weight of the probiotic composition being always 100%. Each of the strains constituting the probiotic composition may further be present in the following amounts: the at least one Lactobacillus helveticus is present in an amount ranging from 40,0 to 50,0 dry weight percent based on the total weight of the probiotic composition, the at least one Bifidobacterium longum is present in an amount ranging from 0,5 to 10,0 dry weight percent based on the total weight of the probiotic composition, and the at least one Bifidobacterium animalis is present in an amount ranging from 45,0 to 55,0 dry weight percent based on the total weight of the probiotic composition. Each of the strains constituting the probiotic composition may further be present in the following amounts: the at least one Lactobacillus helveticus is present in an amount of at least 40,0 dry weight percent based on the total weight of the probiotic composition, the at least one Bifidobacterium longum is present in an amount of at least 0,5 dry weight percent based on the total weight of the probiotic composition, and the at least one Bifidobacterium animalis is present in an amount of at least 40,0 dry weight percent based on the total weight of the probiotic composition. Each of the strains constituting the probiotic composition may further be present in the following amounts: the at least one Lactobacillus helveticus is present in an amount of at least 40,0 dry weight percent based on the total weight of the probiotic composition, the at least one Bifidobacterium longum is present in an amount of at least 0,5 dry weight percent based on the total weight of the probiotic composition, and the at least one Bifidobacterium animalis is present in an amount of at least 45,0 dry weight percent based on the total weight of the probiotic composition. Each of the strains constituting the probiotic composition may further be present in the following amounts: the at least one Lactobacillus helveticus is present in an amount of 45,0 dry weight percent based on the total weight of the probiotic composition, the at least one Bifidobacterium longum is present in an amount of 5,0 dry weight percent based on the total weight of the probiotic composition, and the at least one Bifidobacterium animalis is present in an amount of 50,0 dry weight percent based on the total weight of the probiotic composition. The at least one Lactobacillus helveticus may be individually present in an amount greater than 10,0, 20,0, 30,0, 40,0 or 50,0 dry weight percent based on the total weight of the probiotic composition. The atleast one Bifidobacterium animalis may be individually present in an amount greater than 10,0, 20,0, 30,0, 40,0 or 50,0 dry weight percent based on the total weight of the probiotic composition. The at least one Bifidobacterium longum may be individually present in an amount greater than 0,5, 1,0, 2,0, 3,04,0, 5,0, 6,0, 7,0, 8,0, 9,0 or 10,0 dry weight percent based on the total weight of the probiotic composition.

[0035] As used herein, the term “locomotive syndrome” refers to the functional impairment of one or more locomotive organs such as bones, joints, cartilage, intervertebral discs, and muscles due to but not limited to aging or because of immobilization, resulting in decreased functions such as “standing” and “walking”.

[0036] As used herein, the term “intestinal barrier integrity” refers to the performance of the intestinal epithelium to maintain selective permeability, thereby allowing efficient absorption of nutrients and water while preventing translocation of harmful substances such as pathogens and toxins. As used herein, the term “muscle morphology” refers to structural characteristics of a muscle, including its cross-sectional area, muscle thickness, muscle fiber organization and / or echo intensity. Thus, a “loss of muscle morphology” refers to a decrease in muscle cross-sectional area, muscle thickness, and / or echo intensity, with respect to a control. The control may be for example: C. elegans fed a control diet such as A. coli OP50, or a cohort of human subjects dosed with a placebo. Muscle morphology can be assessed in humans, for example and without limitation, by using muscle ultrasonography. Muscle morphology can be assessed in Caenorhabditis elegans, for example and without limitation, by visual assessment of a reporter strain expressing fluorescent markers such as GFP-tagged myosin heavy chain (MHC) A (MYO-3) in the body wall muscle cells. As used herein, the phrase ‘with respect to a control’ refers to a comparison made between a test condition and a reference condition (the control). The control may be for example C. elegans fed a control diet such as E. coli OP50, or a cohort of human subjects dosed with a placebo. The test condition refers to the population receiving the probiotic composition in accordance with an embodiment of the present disclosure.

[0037] The term "individual" will in the context of the present invention mean a person above the age of 18. In the context of the present invention, the individual may be any person of any sex, i.e. male and / or female. In an embodiment of the present description, the individuals are aging or elderly humans. An elderly human may be at least about 60 years old. An elderly human may be about 60 years old or greater. In another embodiment, the elderly human is a frail elderlyhuman. In the context of the present description, the term "frail" refers to an individual who is physically weak, i.e. not strong, but fragile. The term “aging” refers to individuals not physically weak but taking preventive actions to prevent for example loss of muscle morphology, to increase the muscle morphology and / or improve the muscle recovery after muscle atrophy.

[0038] The term "prevention" or "preventing" as used herein means preventing the disease state, i.e. causing the clinical symptoms of the disease state not to develop in an individual that may be exposed or predisposed to the disease state but does not yet experience or display symptoms of the disease state.

[0039] The term “alleviate” or “alleviating” as used herein means making the disease state less intense or severe, i.e. causing the clinical symptoms of the disease state to be reduced, relieved, mitigated, or made less intense or severe in an individual that has already been exposed or predisposed to the disease state and is experiencing or displaying symptoms of the disease state. As used herein, "sarcopenia" refers to a syndrome characterized by age-related skeletal muscle atrophy, accompanied by a decrease in skeletal muscle mass and skeletal muscle strength, or a decrease in physical function but is well established that individuals gradually begin losing muscle mass and strength sometime in their 30s or 40s. The degree of sarcopenia is determined by two factors: the initial amount of muscle mass and the rate at which muscle mass declines. Due to variations in these factors across the population, the rate of progression and the threshold at which muscle loss becomes apparent is variable. Immobility dramatically increases the rate of muscle loss, even in younger people.

[0040] The probiotic compositions of the present disclosure are typically administered on or in a support as part of a product, in particular as a component of a food product, a dietary supplement, medicament, a powdered nutritional product to be reconstituted in a liquid such as water or milk before consumption or a pharmaceutical formulation. These products typically contain additional components, acceptable excipients, carriers or adequate additives well known to those skilled in the art. The term "acceptable excipients and carriers" as used herein pertains to those that are compatible with the other ingredients in the formulation and biologically acceptable. The products may additionally contain one or more further active agents. The additional active agent or agents may be other probiotic bacteria or yeasts which are not antagonist to the strains forming the composition of the present disclosure.In another embodiment, the composition may be provided as a powder, a dry composition, a suspension, a liquid concentrate, an emulsion or a gel comprising a mixture of the probiotics and another active ingredients (if applicable). The formulation may be a ready to use formulation, such as a unit dosage form, i.e., a capsule, tablet, gummy, soft chew or sachet / stick pack or a formulation that needs to be dissolved in a liquid prior to use. The composition may also be in the form of a kit of parts with the probiotic in one compartment and another active ingredient in another compartment and an instruction describing the best intake form. Examples of functional preparations are cosmetic or pharmaceutical compositions containing active ingredients such as hormone preparations, vitamin preparations, vegetable extract preparations, anti -aging preparations, and / or antimicrobial (antibacterial or antifungal) preparations without being limited thereto.

[0041] In a further embodiment, the probiotic composition in accordance with the present description can also be in a nutritional composition, such as a nutrition product. It can contain sources of protein, lipids, vitamins, minerals and / or digestible carbohydrates and can be in powdered or liquid forms. The composition can be designed to be a nutritional supplement. In particular, such non-medical use can be in a dietary supplement or a nutricosmetic (non-therapeutic) treatment.

[0042] Depending on the formulation, the strains may be added as purified bacteria, as a bacterial culture, as part of a bacterial culture, as a bacterial culture which has been post-treated. Prebiotics could be also added. The food product, the dietary supplement or the pharmaceutical formulation may be prepared in any suitable form which does not negatively affect the bioavailability of the strains forming the composition and is within the scope of ordinary persons skilled in the art.

[0043] For example, the probiotic compositions of the present disclosure can be formulated to be administered orally in the form of freeze-dried power, tablet, capsules, pills, suspension, lozenge, emulsion, liquid preparations, gel, syrup etc. The probiotic compositions of the present disclosure can be used as an ingredient in food products such as milk products, yogurt, curd, cheese (e.g. quark, cream, processed, soft and hard), fermented milk, milk powder, milk based fermented product, ice-cream, a fermented cereal based product, milk based powder, a beverage, a dressing, meat products (e.g. liver paste, frankfurter and salami sausages or meatspreads), spreads, fillings, frostings, chocolate, confectionery (e.g. caramel, candy, fondants or toffee), baked goods (cakes, pastries), sauces and soups, fruit juices or coffee whiteners.

[0044] The probiotic microorganisms are produced by cultivating the microorganisms in a suitable medium and under suitable conditions as known in the art. The probiotic microorganisms can be cultivated alone to form a pure culture, or as a mixed culture together with other microorganisms, or by cultivating probiotic microorganisms of different types separately and then combining them in the desired proportions. After cultivation until a predetermined CFU / g concentration is reached, the cell suspension is recovered and used as such or treated in the desired manner, for instance, by concentrating, spray drying, lyophilization, flatbed oven drying or freeze-drying, to be further employed in the preparation of composition and can be blend with a carrier medium. Sometimes the probiotic preparation is subjected to an immobilization or encapsulation process in order to improve the shelf life. Several techniques for immobilization or encapsulation of bacteria are known in the art.

[0045] The probiotic strains used in the present disclosure are in the form of viable cells. However, the probiotic strains of the present disclosure can also be in the form of non-viable cells such as killed cultures or compositions containing beneficial factors produced by the probiotics. This could include thermally killed micro-organisms or micro-organisms killed by exposure to altered pH, sonication, radiation or subjection to pressure. With non-viable cells product preparation is simpler, cells may be incorporated easily into commercial products and storage requirements are much less limited than viable cells.

[0046] In one embodiment of the present disclosure, the probiotic composition can be used in conjunction with another active ingredient such as, but not limited to Fructooligosaccharide (FOS), Galactooligosaccharides (GOS), Xylooligosaccharides (XOS), Inulin or mixtures thereof. Fructans are the most used fibers and are able to improve the viability of the probiotics. In a further embodiment of the present disclosure, the probiotic composition can be used in conjunction with other compounds such like, for example, resveratrol.

[0047] In accordance with the present disclosure, probiotic compositions will normally be administered so that a symptom-ameliorating effective daily dose is received by the subject. The daily dose may be given in divided doses as necessary, the precise amount of the compound or agent received and the route of administration depending on the general health of the subjectbeing treated according to principles known in the art. A typical dosage regime is once, twice or three dailies. An appropriate dose may be administered once, twice, or three times per day. The word "comprising" in the claims may be replaced by "consisting essentially of or with "consisting of," according to standard practice in patent law.

[0048] The present invention will be more readily understood by referring to the following examples which are given to illustrate the invention rather than to limit its scope.

[0049] EXAMPLES

[0050] Example 1: Effect of a probiotic composition in accordance with an embodiment of the present disclosure on wildtype Caenorhabditis elegans

[0051] The objective of this study was to evaluate the effect of a probiotic composition in accordance with an embodiment of the present disclosure on the behavior of wildtype C. elegans submitted to a swim exercise regimen.

[0052] Synchronization of C. elegans:

[0053] Experiments were carried out with the wildtype strain N2 of C. elegans. Worms were age synchronized by isolating eggs from gravid adults, collecting the eggs in M9 buffer, hatching the eggs overnight in NGM plates and sterilely isolating LI -stage worms with M9 buffer. The LI -stage worms were transferred to NGM (Nematode Growth Medium) plates inoculated with Escherichia coh strain OP50 and incubated for three days at 20°C to reach adulthood. Similar results are also expected if Ll-stage worms are transferred to NGM (Nematode Growth Medium) plates inoculated with Escherichia coli strain OP50 and incubated for four days at 15 °C to reach adulthood.

[0054] The lifespan of wildtype C. elegans (N2) was assessed from day 1 of adulthood until death, following feeding on different monocultures and the probiotic composition in accordance with an embodiment of the present disclosure (Bifidobacterium animalis ssp. lactis LAFTI® B94 50% weight vol + Lactobacillus helveticus Rosell®-52 45% weight vol + Bifidobacterium longum Rosell®-175 5% weight vol). As shown in Figure 1A, the probiotic composition inaccordance with an embodiment of the present disclosure (referred to as MIX) significantly extended the lifespan of the worms when compared to the E. coli OP50 control.

[0055] Locomotion and endurance

[0056] A swim exercise regimen was implemented for young adult wildtype worms fed on the probiotic composition in accordance with an embodiment of the present disclosure to test their muscle endurance. During this period, the worms swam for at least 40 minutes, typically 40 minutes or 45 minutes, (suspended in liquid buffer) on days 2, 3, 4, 7, and 14, while continuing their probiotic composition intake. Their locomotion was then evaluated using the Micro tracker (assays the speed and distance travelled by the worm). The findings revealed, as shown in Figure IB, that the probiotic composition in accordance with an embodiment of the present disclosure outperformed the control diet.

[0057] As seen above, the wildtype N2 worms, both the probiotic composition in accordance with an embodiment of the present disclosure (Bifidobacterium animalis ssp. lactis LAFTI® B94 50% weight vol + Lactobacillus helveticus Rosell®-52 45% weight vol + Bifidobacterium longum Rosell®-1755% weight vol) and Bifidobacterium animalis ssp. lactis LAFTI® B94 significantly extended lifespan compared to the control (p < 0.05). Locomotion was assessed through a swim exercise regimen and subsequent tracking of worm movement using the Micro Tracker apparatus. Wildtype worms fed on the probiotic composition in accordance with an embodiment of the present disclosure (Bifidobacterium animalis ssp. lactis LAFTI® B94 50% weight vol + Lactobacillus helveticus Rosell®-52 45% weight vol + Bifidobacterium longum Resell®- 175 5% weight vol) and B94 exhibited superior speed and distance traveled compared to the control group, suggesting enhanced muscle endurance.

[0058] Example 2: Effect of a probiotic composition in accordance with an embodiment of the present disclosure on a transgenic Caenorhabditis elegans sarcopenia model

[0059] The RW1596 strain carries a mutation in the myo-3 gene, this gene is involved in muscle function, and the mutation affects muscle integrity and health. Additionally, RW1596 contains an extrachromosomal array stEx30, which includes a myo-3p::GFP::myo-3 construct thatallows for visualization of muscle cells. It carries a genetic modification that causes the worms to move in a distinctive rolling motion. This is due to the presence of the rol-6 (sul006) gene in the strain. The rolling movement is a result of changes in the worm’s muscle structure, which affects how it moves.

[0060] Ageing in C. elegans experience gradual, progressive muscle deterioration, resembling human sarcopenia. In young nematodes, body wall muscle myofilaments are well organized in a tight, parallel manner, whereas in older animals they show progressive disorganization, irregular orientation, breaks and abnormal bends. Nematode lacking muscle stem cells allows to focus on muscle deterioration during ageing without the confounding influence of muscle regeneration.

[0061] Worm myofilament organization is often studied by visual assessment of a reporter strain expressing fluorescent markers such as GFP-tagged myosin heavy chain (MHC) A (MYO-3) in the body wall muscle cells. The defect in the myosin heavy chain, specifically in the myo-3(st386) mutation, affects the structure and function of the myosin protein. Myosin is a crucial component of muscle fibers, responsible for converting chemical energy into mechanical force, which is essential for muscle contraction. In the case of the myo-3(st386) mutation, the defect leads to improper folding or assembly of the myosin protein. This results in muscle fibers that are structurally compromised and unable to function correctly, leading to muscle weakness and other related symptoms. This mutation is particularly useful to study the effects of muscle degeneration and potentially prevent symptoms associated with muscles loss due to age, trauma or immobility

[0062] A lifespan assay with this transgenic strain was conducted, and the results, as shown in Figure 2A, demonstrated that the probiotic composition in accordance with an embodiment of the present disclosure (referred to as MIX) significantly extended lifespan compared to the control bacteria. Similarly, the swim exercise regimen revealed that the probiotic composition in accordance with an embodiment of the present disclosure increased both the speed and distance traveled by these transgenic worms.

[0063] As seen above, in RW1596 worms, the probiotic composition in accordance with an embodiment of the present disclosure (Bifidobacterium animalis ssp. lactis LAFTI® B94 50% weight vol + Lactobacillus helveticus Rosell®-52 45% weight vol + Bifidobacterium longum Resell®- 175 5% weight vol) showed a pronounced lifespan extension compared to the control,indicating the potential of probiotics in mitigating age-related muscle deterioration. In RW1596 worms, the probiotic composition in accordance with an embodiment of the present disclosure (B9450% + R005245% + R01755%) significantly improved locomotion metrics as the worms aged, highlighting its protective effect on muscle function (maintenance of the muscle strength). These results strongly suggested that transgenic worms, despite the induced muscle weakness and other related symptoms, had an extended life and were also able to increase their speed and maintain their locomotion for a longer period during their extended life and moreover capable of travelling longer distances. These results also suggest that the probiotic composition in accordance with an embodiment of the present disclosure could also improve or maintain muscle strength in individuals, particularly in aging or elderly individual, performance athletes and individuals that suffered from muscle loss due to immobility.

[0064] Example 3: Assessment of neuromuscular health of wildtype C. elegans - Burrowing assay The burrowing assay utilizes a Pluronic gel, a transparent, semi-solid medium that solidifies at room temperature but remains liquid at 4°C. This assay measures the ability of C. elegans to travel through the gel toward a chemoattractant over at least a 90-minute period, preferably over a 120-minute period, which reflects their neuromuscular strength. Age-synchronized young adult worms were either fed a probiotic (selected from B94, R0052, R0175 and the probiotic composition in accordance with an embodiment of the present disclosure (Bifidobacterium animalis ssp. lactis LAFTI® B9450% weight vol + Lactobacillus helveticus Rosell-5245% weight vol + Bifidobacterium longum Rosell-175 5% weight vol, referred to as MIX)) or a control diet (E. coli Op50) and were submitted to an exercise regimen for at least 20 minutes, preferably for 40 minutes, in a standard buffer solution M9 at 20°C at days 2, 3, 4, 7, 11 and 14 of their adulthood. Plates were prepared for each time point as the worms cannot be recovered after the burrowing assay. The burrowing assay was performed as previously described and the number of worms that appear on top over a 2-hour period were counted and analyzed.

[0065] As shown in Figure 4, the burrowing assay measured neuromuscular strength in wildtype worms on various diets with an added exercise regimen. Wildtype worms fed the probiotic composition in accordance with an embodiment of the present disclosure (Bifidobacterium animalis ssp. lactis LAFTI® B94 50% weight vol + Lactobacillus helveticus Rosell®-52 45%weight vol + Bifidobacterium longum Rosell®-1755% weight vol) displayed significantly better burrowing efficiency (p < 0.0001) toward the chemoattractant from the young adult stage (Day 2) through Day 14 compared to those on the control diet. Statistical analysis was performed using Two-way ANOVA followed by Dunnett's multiple comparisons test to assess the significance of the diets relative to the control. By Day 21, although worms remained alive in the probiotic diets, they could not successfully navigate the gel, likely due to advanced aging and physical limitations. See Table 1.

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[0087] Table 1: Two-way Anova, Dunnetts’ multiple comparison test

[0088] Example 4: Assessment of neuromuscular health of transgenic C. elegans - Burrowing assay

[0089] The burrowing assay utilizes a Pluronic gel, a transparent, semi-solid medium that solidifies at room temperature but remains liquid at 4°C. This assay measures the ability of transgenic C. elegans to travel through the gel toward a chemoattractant over a 120-minute period, which reflects their neuromuscular strength. Age-synchronized young adult worms were either fed a probiotic (selected from B94, R0052, RO 175 and the probiotic composition in accordance with an embodiment of the present disclosure (Bifidobacterium animalis ssp. lactis LAFTI® B94 50% weight vol + Lactobacillus helveticus Rosell-52 45% weight vol + Bifidobacterium longum Rosell-175 5% weight vol, referred to as MIX)) or a control diet (E. coli Op50) and were submitted to an exercise regimen for 40 mins in a standard buffer solution M9 at 20°C at days 2, 3, 4, and 7 of their adulthood. For transgenic (RW1596) C. elegans, the neuromuscular assay was performed only up to day 7, as worms at day 11 and 14 were unable to move into the gel matrix or reach the top. Plates were prepared for each time point as the worms cannot be recovered after the burrowing assay. The burrowing assay was performed as previously described and the number of worms that appear on top over a 2-hour period were counted and analyzed.

[0090] As shown in Figure 5, the burrowing assay measured neuromuscular strength in transgenic worms on various diets with an added exercise regimen. Transgenic worms fed the probioticcomposition in accordance with an embodiment of the present disclosure (Bifidobacterium animalis ssp. lactis LAFTI® B94 50% weight vol + Lactobacillus helveticus Rosell®-52 45% weight vol + Bifidobacterium longum Rosell®-1755% weight vol) displayed significantly better burrowing efficiency (p < 0.0001) toward the chemoattractant from the young adult stage (Day 2) through Day 4 compared to those on the control diet, and significantly better burrowing efficiency (p < 0.01) on Day 7 compared to those on the control diet. Statistical analysis was performed using Two-way ANOVA followed by Dunnett' s multiple comparisons test to assess the significance of the diets relative to the control. By Day 11, although worms remained alive in the probiotic diets, they could not successfully navigate the gel. Without being bound to theory, this is likely due to advanced aging and physical limitations. See Table 2 summarizing the results of the statistical analysis.

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[0105] Table 2: Two-way Anova, Dunnetts’ multiple comparison test

[0106] Example 5: Effect of a probiotic composition in accordance with an embodiment of the present disclosure on intestinal barrier integrity of wildtype Caenorhabditis elegans The objective of this study was to evaluate the effect of a probiotic composition in accordance with an embodiment of the present disclosure on intestinal barrier integrity of wildtype C. elegans. Without being bound to theory, maintaining intestinal barrier integrity is associated with delayed onset of age-associated decline in muscle function. Herein and throughout this disclosure, intestinal barrier integrity is also referred to as gut integrity.

[0107] Experiments were carried out with the wildtype strain N2 of C. elegans. Worms were age synchronized by isolating eggs from gravid adults, collecting the eggs in M9 buffer, hatching the eggs overnight in NGM plates and sterilely isolating LI -stage worms with M9 buffer. The LI -stage worms were transferred to NGM (Nematode Growth Medium) plates inoculated with Escherichia coh strain OP50 and incubated for three days at 20°C to reach adulthood.

[0108] To evaluate intestinal barrier integrity, the Smurf assay was performed on Days 2, 7, 12 and 19 of adulthood. Smurf assay was performed to quantify intestinal barrier dysfunction in N2 worms fed E. coli OP50 (control diet) or probiotic strains (B94, R0052, R0175, or the probiotic composition in accordance with an embodiment of the present disclosure (Bifidobacterium animalis ssp. lactis LAFTI® B94 50% weight vol + Lactobacillus helveticus Rosell-52 45% weight vol + Bifidobacterium longum Resell- 1755% weight vol, referred to as MIX)). F or each condition, 30-50 worms were picked and incubated in M9 buffer for 40 minutes at 20 °C to simulate endurance exercise conditions (swimming). Following this pre-treatment, worms were transferred to plates containing a dye-bacteria mix composed of 2.5% (w / v) Brilliant Blue FCF in E. coli OP50. This concentration was chosen based on method validation experiments comparing different dye: bacteria:M9 buffer ratios to maximize reproducibility and intestinal dye uptake. Worms were incubated in 12-well plates with the dye-bacteria mixture for 2 hours at 20 °C in the dark to prevent photodegradation. After incubation, worms were washed gentlywith M9 buffer to remove excess dye and then visualized under a stereomicroscope. Worms showing dye outside the intestinal lumen were scored as “leaky gut phenotype” -positive indicative of compromised intestinal barrier integrity and function.

[0109] Figure 6 illustrates the results of an intestinal barrier integrity assay, also referred to as a “Smurf assay” with wildtype (N2) C. elegans. Data represent the percentage of worms exhibiting dye leakage into the body cavity, which as thus “leaky gut phenotype”-positive. At Day 2 (Figure 6A), only B94-fed C. elegans showed significantly reduced leakage compared to OP50-fed controls. At Day 7 (Figure 6B), no significant differences were observed among dietary groups, with gut integrity largely preserved across all conditions. By Day 12 (Figure 6C), probiotic supplementation significantly improved gut integrity, with B94, R0052, R0175, and MIX-fed worms showing markedly reduced leakage relative to OP50 controls. By Day 19 (Figure 6D), age-associated decline in barrier function was most pronounced in OP50-fed worms, while all probiotic diets conferred significant protection, with the MIX diet producing the strongest effect. Data represents the mean of three assays. Analysis was made through 2-way ANOVA followed by Dunnett’s multiple comparisons test, (vs OP50); **** p < 0.001.

[0110] Example 6: Effect of a probiotic composition in accordance with an embodiment of the present disclosure on intestinal barrier integrity of transgenic C. elegans

[0111] The objective of this study was to evaluate the effect of a probiotic composition in accordance with an embodiment of the present disclosure on intestinal barrier integrity of transgenic C. elegans. Without being bound to theory, maintaining intestinal barrier integrity is associated with delayed onset of age-associated decline in muscle function. Herein and throughout this disclosure, intestinal barrier integrity is also referred to as gut integrity.

[0112] Experiments were carried out with the transgenic strain (RW 1596) of C. elegans. The RW 1596 strain carries a mutation in the myo-3 gene, this gene is involved in muscle function, and the mutation affects muscle integrity and health. Additionally, RW1596 contains an extrachromosomal array stEx30, which includes a myo-3p::GFP::myo-3 construct that allows for visualization of muscle cells. It carries a genetic modification that causes the worms to move in a distinctive rolling motion. This is due to the presence of the rol-6 (sul006) gene in the strain. The rolling movement is a result of changes in the worm’s muscle structure, which affects how it moves.Worms were age synchronized by isolating eggs from gravid adults, collecting the eggs in M9 buffer, hatching the eggs overnight in NGM plates and sterilely isolating LI -stage worms with M9 buffer. The LI -stage worms were transferred to NGM (Nematode Growth Medium) plates inoculated with Escherichia coh strain OP50 and incubated for three days at 20°C to reach adulthood. Upon reaching adulthood, worms were divided into separate, age-matched cohorts and maintained on NGM plates seeded with either E. coli OP50 (control diet) or the indicated probiotic strains.

[0113] To evaluate intestinal barrier integrity, the Smurf assay was performed on Days 2, 7, 12 and 19 of adulthood. Smurf assay was performed to quantify intestinal barrier dysfunction in transgenic (RW1596) worms fed E. coli OP50 (control diet) or probiotic strains (B94, R0052, R0175, or the probiotic composition in accordance with an embodiment of the present disclosure (Bifidobacterium animalis ssp. lactis LAFTI® B9450% weight vol + Lactobacillus helveticus Rosell-5245% weight vol + Bifidobacterium longum Rosell-175 5% weight vol, referred to as MIX)). For each condition, 30-50 worms were picked and incubated in M9 buffer for 40 minutes at 20 °C to simulate endurance exercise conditions (swimming). Following this pretreatment, worms were transferred to plates containing a dye-bacteria mix composed of 2.5% (w / v) Brilliant Blue FCF in E. coli OP50. This concentration was chosen based on method validation experiments comparing different dye: bacteria:M9 buffer ratios to maximize reproducibility and intestinal dye uptake. Worms were incubated in 12-well plates with the dyebacteria mixture for 2 hours at 20 °C in the dark to prevent photodegradation. After incubation, worms were washed gently with M9 buffer to remove excess dye and then visualized under a stereomicroscope. Worms showing dye outside the intestinal lumen were scored as “leaky gut phenotype”-positive indicative of compromised intestinal barrier integrity and function.

[0114] Figure 7 illustrates the results of an intestinal barrier integrity assay, also referred to as a “Smurf assay” with transgenic (RW1596) C. elegans. Data represent the percentage of C. elegans exhibiting dye leakage into the body cavity, which as thus “leaky gut phenotype”-positive. At Day 2 (Figure 7A), no significant differences in leakage rates were observed across dietary groups, consistent with preserved gut integrity early in life. By Day 7 (Figure 7B), probiotic supplementation significantly reduced intestinal leakage compared to OP50, with B94 and MIX providing the strongest effects. At Day 12 (Figure 7C), gut barrier deterioration was evident in OP50-fed worms, while B94 and MIX-fed groups exhibited markedly lower rates of leaky guts.By Day 19 (Figure 7D), age-associated decline in barrier function was most pronounced in OP50-fed worms, whereas B94, R0175, and particularly the MIX diet significantly attenuated gut permeability. Data represents the mean of three assays. Analysis was made through 2-way ANOVA followed by Dunnett’s multiple comparisons test, (vs OP50); **** p < 0.0001.

[0115] Example 7: Comparing the effects of a probiotic composition in accordance with an embodiment of the present disclosure on intestinal barrier integrity of wildtype and transgenic C. elegans.

[0116] At Day 2, it can be seen that B94 supplementation in wildtype N2 worms significantly reduced intestinal permeability relative to OP50-fed controls (p = 0.0131) (Figure 6A). No significant effects were observed in N2 worms fed R0052, R0175, or the MIX diet. Across all dietary conditions, including OP50, intestinal leakage remained low (-10%), indicating N2 worms preserved gut integrity early in adulthood. In contrast, RW1596 worms did not show significant differences among dietary groups at this stage (Figure 7A).

[0117] At Day 7, probiotic supplementation in wildtype N2 worms did not alter gut integrity compared to OP50-fed controls (Figure 6B). In contrast, RW1596 worms demonstrated pronounced reductions in intestinal permeability relative to controls at Day 7 upon probiotic feeding. In particular, B94 and MIX-fed RW1596 worms showed marked reductions in intestinal permeability (p = 0.0081 and p = 0.0013, respectively), whereas R0052 and R0175 feeding conferred modest but significant protection (p = 0.0425) (Figure 7B). These findings indicate that RW 1596 worms exhibit a heightened mid-life responsiveness of the gut barrier to probiotic intervention compared to N2 worms.

[0118] During mid-adulthood (at Day 12), all probiotic diets significantly improved intestinal integrity in wildtype N2 worms relative to OP50 (Figure 6C). B94 and R0052 produced the strongest effects (p = 0.0002), while R0175 provided moderate protection (p = 0.0182) and the MIX diet conferred robust benefit (p = 0.0011). In RW1596 worms (Figure 7C), OP50-fed animals exhibited 40% intestinal leakage at Day 12, whereas B94 (20%, p=0.001) and MIX (15%, p=<0.0001) diets maintained substantially lower leakage rates. R0052 (30%) and R0175 (35%) provided intermediate protection.

[0119] During late adulthood (at Day 19), age-associated decline in gut integrity was most pronounced in OP50-fed wildtype N2 worms (60% leakage) (Figure 6D). Probiotic supplementationmarkedly attenuated this deterioration. R0052, R0175, and MIX-fed worms showed strong protection (p < 0.0001), while B94-fed worms also remained significantly protected (p = 0.0009). In the RW1596 worms (Figure 7D) leakage rates were reduced to 33% in B94-fed worms (p=<0.001), and 48% in R0175-fed worms (p =0.0005). Notably, the MIX diet provided the greatest protection, with only 23% of animals exhibiting a leaky gut phenotype (p<0.0001)

[0120] Example 8: Effect of a probiotic composition in accordance with an embodiment of the present disclosure on pharyngeal pumping rate of wildtype C. elegans

[0121] The objective of this study was to evaluate the effect of a probiotic composition in accordance with an embodiment of the present disclosure on pharyngeal pumping rates of wildtype C. elegans.

[0122] Experiments were carried out with the wildtype strain N2 of C. elegans. Worms were age synchronized by isolating eggs from gravid adults, collecting the eggs in M9 buffer, hatching the eggs overnight in NGM plates and sterilely isolating LI -stage worms with M9 buffer. The LI -stage worms were transferred to NGM (Nematode Growth Medium) plates inoculated with Escherichia coh strain OP50 and incubated for three days at 20°C to reach adulthood.

[0123] The pharyngeal pumping assay was performed on Days 2, 5, 10, 15, and 20 of adulthood. For each condition, 30-50 worms were picked and incubated in M9 buffer for 40 minutes at 20 °C to simulate endurance exercise conditions. Following this pre-treatment, about 10 adult worms were transferred to the edge of the 60mm NGM plates (i.e. away from food) either containing probiotics (B94, R0175, R0052, or the probiotic composition in accordance with an embodiment of the present disclosure (Bifidobacterium animalis ssp. lactis LAFTI® B94 50% weight vol + Lactobacillus helveticus Rosell-52 45% weight vol + Bifidobacterium longum Rosell-175 5% weight vol, referred to as MIX)) or a control diet (E. coli OP50). Plates were incubated agar side up at 20 °C for 1 hour. This allowed the worms to crawl to the bacterial lawn and start foraging again. Working on one plate at a time, a 60mm plate was placed upon a dissecting Stereo microscope at the highest magnification and the worms / bacterial lawns were brought to focus. Working on a single worm at a time, the grinder (in the terminal bulb of the head) of each worm was identified and put clearly in focus. Using a tally counter, the grinder movements of a worm were counted over a 20-second period. This was repeated 3 times perworm and recorded as an average of the three counts. Going clockwise around the bacterial lawn, the steps above were repeated for each worm on the 60mm plate and then repeated for each 60mm plate remaining in the incubator. Pharyngeal pumps per minute were calculated by multiplying averages over 20 seconds by three (pumps per minute =pumps per 20 seconds *3). Figure 8 illustrates the results of a pharyngeal pumping rate assay with wildtype (N2) C. elegans. Pharyngeal pumping rates (pumps / min) were quantified in N2 worms fed E. coli OP50 (control diet) or probiotic strains (B94, RO 175, R0052, or the probiotic composition in accordance with an embodiment of the present disclosure (Bifidobacterium animalis ssp. lactis LAFTI® B94 50% weight vol + Lactobacillus helveticus Rosell-52 45% weight vol + Bifidobacterium longum Rosell-175 5% weight vol, referred to as MIX)) at Day 2, Day 5, Day 10, Day 15, and Day 20 of adulthood. At early time points Day 2 and Day 5, no significant differences were observed across dietary groups. By Day 10, probiotic-fed worms showed significantly higher pumping rates compared to OP50-fed controls, with R0175, R0052, and MIX providing the strongest stimulation. This trend persisted at Day 15 and Day 20, where worms fed with probiotic diets consistently maintained superior pharyngeal activity, while E. coli OP50-fed worms exhibited age-related decline. Data represent mean ± SEM from three biological replicates. The statistical significance was determined using two-way Anova with Dunnett’s multiple comparison test.

[0124] Example 9: Effect of a probiotic composition in accordance with an embodiment of the present disclosure on pharyngeal pumping rate of transgenic C. elegans

[0125] The objective of this study was to evaluate the effect of a probiotic composition in accordance with an embodiment of the present disclosure on pharyngeal pumping rates of transgenic C. elegans.

[0126] Experiments were carried out with the transgenic strain (RW 1596) of C. elegans. The RW 1596 strain carries a mutation in the myo-3 gene, this gene is involved in muscle function, and the mutation affects muscle integrity and health. Additionally, RW1596 contains an extrachromosomal array stEx30, which includes a myo-3p::GFP::myo-3 construct that allows for visualization of muscle cells. It carries a genetic modification that causes the worms to move in a distinctive rolling motion. This is due to the presence of the rol-6 (sul006) gene in the strain. The rolling movement is a result of changes in the worm’s muscle structure, which affects how it moves.Worms were age synchronized by isolating eggs from gravid adults, collecting the eggs in M9 buffer, hatching the eggs overnight in NGM plates and sterilely isolating LI -stage worms with M9 buffer. The LI -stage worms were transferred to NGM (Nematode Growth Medium) plates inoculated with Escherichia coh strain OP50 and incubated for three days at 20°C to reach adulthood.

[0127] The pharyngeal pumping assay was performed on Days 2, 5, 10, 15, and 20 of adulthood. For each condition, 30-50 worms were picked and incubated in M9 buffer for 40 minutes at 20 °C to simulate endurance exercise conditions. Following this pre-treatment, about 10 adult worms were transferred to the edge of the 60mm NGM plates (away from food) containing probiotics (B94, R0175, R0052, or the probiotic composition in accordance with an embodiment of the present disclosure (Bifidobacterium animalis ssp. lactis LAFTI® B94 50% weight vol + Lactobacillus helveticus Rosell-5245% weight vol + Bifidobacterium longum Resell- 175 5% weight vol, referred to as MIX)) or a control diet (E. coli OP50). Plates were incubated agar side up at 20 °C for 1 hour. This allowed the worms to crawl to the bacterial lawn and start foraging again. Working on one plate at a time, a 60mm plate was placed upon a dissecting Stereo microscope at the highest magnification and the worms / bacterial lawns were brought to focus. Working on a single worm at a time, the grinder (in the terminal bulb of the head) of each worm was identified and put clearly in focus. Using a tally counter, the grinder movements of a worm were counted over a 20-second period. This was repeated 3 times per worm and recorded as an average of the three counts. Going clockwise around the bacterial lawn, the steps above were repeated for each worm on the 60mm plate and then repeated for each 60mm plate remaining in the incubator. Pharyngeal pumps per minute were calculated by multiplying averages over 20 seconds by three (pumps per minute = pumps per 20 seconds *3).

[0128] Figure 9 illustrates the results of a pharyngeal pumping rate assay with transgenic (RW1596) C. elegans. Pharyngeal pumping rates (pumps / min) were quantified in N2 worms fed E. coli OP50 (control diet) or probiotic strains (B94, R0175, R0052, or the probiotic composition in accordance with an embodiment of the present disclosure (Bifidobacterium animalis ssp. lactis LAFTI® B94 50% weight vol + Lactobacillus helveticus Rosell-52 45% weight vol + Bifidobacterium longum Rosell-175 5% weight vol, referred to as MIX)) at Day 2, Day 5, Day 10, Day 15, and Day 20 of adulthood. At Day 2 and Day 5 no significant difference was observed in the pharyngeal pumping rates. On Day 10 probiotic fed worms specifically R0052and MIX exhibited significantly higher pumping rates than OP50 fed controls. Day 15 (D) all the groups fed with probiotic diets showed a significant increase in pharyngeal pumping rates compared to worms fed E. coli OP50. Furthermore, on Day 20, worms on the MIX diet exhibited markedly higher pharyngeal pumping rates compared to worms fed with A", coli OP50. Results are presented as the mean ± SEM from three biological replicates. The significance was assessed using Dunnett’s multiple comparison test.

[0129] Example 10 Comparing the effects of a probiotic composition in accordance with an embodiment of the present disclosure on intestinal barrier integrity of wildtype and transgenic C. elegans.

[0130] In wildtype (N2) worms, all worms fed probiotic diets displayed significantly increased pumping rates at Day 10 compared to E. coli OP50, with R0175 (p= 0.0036) and MIX (p=0.0028) showing the most pronounced difference (Figure 8C). In RW1596 mutants (Figure 9C), R0052 (p=0.0081) and MIX (p=0.0016) supplementation each significantly increased pharyngeal activity at Day 10 while other diets showed no significant change.

[0131] In wildtype (N2) worms, pharyngeal pumping rates declined markedly in OP50-fed worms at Day 15, whereas probiotic supplementation significantly preserved feeding activity (Figure 8D). Worms fed B94 (p = 0.0309), R0175 (p = 0.0324), R0052 (p =0.0047), and MIX (p =0.0058) all exhibited higher pumping rates compared to OP50 controls, with the MIX diet providing the strongest protection. For RW1596 worms, those fed with the B94 (p=0.0148), R0175 (p=0.0206), R0052 (p=0.0242), or MIX (p=0.0359) diets all exhibited significantly higher pharyngeal pumping rates at Day 15 compared to the E. coli OP50 control group (Figure 9D).

[0132] In wildtype (N2) worms, those fed a diet of R0175, R0052 or MIX (p < 0.0001) maintained significantly greater pharyngeal pumping rates at Day 20 relative to worms fed with E. coli OP50 (Figure 8E). Additionally, in RW1596 worms, a surprising effect is observable worms at Day 20, where the MIX diet exhibited the strongest improvement with highly significant effects (*** p < 0.0001), followed by R0175 andR0052 (** p < 0.005), whereas B94 no longer showed any significant benefit relative to the control diet of E. coli OP50 (p = 0.0546) (Figure 9E). The results discussed above and exemplified in the Examples demonstrate that probiotic supplementation, particularly the probiotic composition in accordance with an embodiment ofthe present disclosure, extends lifespan and improves locomotion, muscle endurance, and neuromuscular health in both wildtype and transgenic C. elegans. These findings support the potential of probiotics as an intervention for sarcopenia by enhancing muscle function and mitigating age-related declines as well as preventing muscle loss and increasing muscle strength in individuals that suffered from muscle loss due to immobility.

[0133] Additional Embodiments of the Invention

[0134] 1. A probiotic composition comprising at least one Lactobacillus helveticus, at least one Bifidobacterium longum, and at least one Bifidobacterium animalis.

[0135] 2. The probiotic composition of embodiment 1, wherein the Lactobacillus helveticus is Lactobacillus helveticus Rosell®-52.

[0136] 3. The probiotic composition of embodiment 1 or 2, wherein the Bifidobacterium longum is Bifidobacterium longum Rosell®-175.

[0137] 4. The probiotic composition of any one of embodiments 1 to 3, wherein the Bifidobacterium animalis is Bifidobacterium animalis ssp. lactis LAFTI® B94.

[0138] 5. The probiotic composition of any one of embodiments 1 to 4, wherein the least one Lactobacillus helveticus, the at least one Bifidobacterium longum, and the at least one Bifidobacterium animalis are each individually present in an amount ranging from 0,5 to 99,5 dry weight percent based on the total weight of the probiotic composition.

[0139] 6. The probiotic composition of any one of embodiments 1 to 5, wherein the probiotic composition is formulated as a food composition, a dietary supplement, a nutritional composition, a nutraceutical, a powdered nutritional product to be reconstituted in a liquid, such as water or milk, before consumption, a food additive, a medicament or a drink.7. Use of a probiotic composition as defined in any one embodiments 1 to 6 for: preventing or reducing the loss of muscle morphology in an individual; increasing or maintaining muscle strength in an individual; increasing muscle morphology, stimulating muscle strength in an individual; preventing locomotion syndrome in an individual; and / or preventing or reducing symptoms associated with sarcopenia in an individual.

[0140] 8. The use of embodiment 7 which is a non-therapeutic use.

[0141] 9. A method for preventing or reducing the loss of muscle morphology, for increasing muscle morphology and / or for treating and / or preventing muscle atrophy, e.g. improving muscle recovery after muscle atrophy, wherein said method comprises administering a probiotic composition as defined in any one of embodiments 1 to 6 to an individual in need thereof.

[0142] 10. A method for preventing or alleviating symptoms associated with sarcopenia comprising administering a probiotic composition as defined in any one of embodiments 1 to 6 to an individual in need thereof.

[0143] 11. A method for preventing locomotive syndrome comprising administering a probiotic composition as defined in any one of embodiments 1 to 6 to an individual in need thereof.

[0144] 12. A probiotic composition as defined in any one of embodiments 1 to 6 for use in: preventing or reducing the loss of muscle morphology in an individual; increasing or maintaining muscle strength in an individual; stimulating muscle strength in an individual; preventing locomotion syndrome in an individual; preventing or reducing symptoms associated with sarcopenia in an individual; increasing muscle morphology in an individual; treating and / or preventing muscle atrophy in an individual, e.g. improving muscle recovery after muscleatrophy; preventing or alleviating symptoms associated with sarcopenia in an individual; and / or preventing locomotive syndrome.

[0145] 13. The use of embodiment 7 or 8, the method of any one of embodiments 9 to 11 or the probiotic composition for use according to embodiment 12, wherein the individual is an aging or elderly individual, a performance athlete or an individual suffering from muscle loss due to immobility.

Claims

CLAIMS1. A probiotic composition comprising at least one Lactobacillus helveticus, at least one Bifidobacterium longum, and at least one Bifidobacterium animalis, wherein the at least one Lactobacillus helveticus is individually present in an amount greater than 40,0 dry weight percent based on the total weight of the probiotic composition.

2. The probiotic composition of claim 1, wherein the Lactobacillus helveticus is Lactobacillus helveticus Rosell®-52.

3. The probiotic composition of claim 1 or 2, wherein the Bifidobacterium longum is Bifidobacterium longum Rosell®-175.

4. The probiotic composition of any one of claims 1 to 3, wherein the Bifidobacterium animalis is Bifidobacterium animalis ssp. lactis LAFTI® B94.

5. The probiotic composition of any one of claims 1 to 4, wherein the at least one Bifidobacterium animalis is individually present in an amount greater than 40,0 dry weight percent based on the total weight of the probiotic composition.

6. The probiotic composition of any one of claims 1 to 5, wherein the probiotic composition consists essentially of or consists of the at least one Lactobacillus helveticus, the at least one Bifidobacterium longum, and the at least one Bifidobacterium animalis.

7. The probiotic composition of any one of claims 1 to 6, wherein the probiotic composition is formulated as a food composition, a dietary supplement, a nutritional composition, a nutraceutical, a powdered nutritional product to be reconstituted in a liquid, such as water or milk, before consumption, a food additive, a medicament or a drink.

8. Use of a probiotic composition as defined in any one claims 1 to 7 for: preventing or reducing the loss of muscle morphology in an individual; increasing or maintainingmuscle strength in an individual; increasing muscle morphology, stimulating muscle strength in an individual.

9. The use of claim 8 which is a non-therapeutic use.

10. A method for preventing or reducing the loss of muscle morphology, for increasing muscle morphology and / or for treating and / or preventing muscle atrophy, wherein said method comprises administering a probiotic composition as defined in any one of claims 1 to 7 to an individual in need thereof.

11. A method for preventing or alleviating symptoms associated with sarcopenia comprising administering a probiotic composition as defined in any one of claims 1 to 7 to an individual in need thereof.

12. A method for preventing locomotive syndrome comprising administering a probiotic composition as defined in any one of claims 1 to 7 to an individual in need thereof.

13. A method for preventing or reducing the loss of intestinal barrier integrity and / or for reducing intestinal permeability, wherein said method comprises administering a probiotic composition as defined in any one of claims 1 to 7 to an individual in need thereof.

14. A probiotic composition as defined in any one of claims 1 to 7 for use in:(a) preventing or alleviating symptoms associated with sarcopenia in an individual; (b) preventing locomotive syndrome; and / or(c) preventing or reducing the loss of intestinal barrier integrity.

15. The use of claim 8 or 9, the method of any one of claims 10 to 13 or the probiotic composition for use according to claim 14, wherein the individual is an aging or elderly individual, a performance athlete or an individual suffering from muscle loss due to immobility.

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