Akkermansia muciniphila strains DSM 35203 and DSM 35204 for use in preventing and / or delaying aging and conditions related thereto

Abstract

The present invention relates to a mixture comprising or, alternatively, consisting of at least one strain of bacteria belonging to the species Akkermansia muciniphila selected from the group comprising or, alternatively, consisting of : i) Akkermansia muciniphila AKM9, deposited by SynBalance SRL on 08.10.2024 at the Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSMZ) and having accession number DSM 35203; and ii) Akkermansia muciniphila AKM22, deposited by SynBalance SRL on 08.10.2024 at the Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSMZ) and having accession number DSM 35204, and iii) mixtures thereof. Furthermore, the present invention relates to compositions comprising said mixture, for example, probiotic compositions, postbiotic compositions, food, nutraceutical and / or pharmaceutical compositions comprising said mixtures and, optionally, food or pharmaceutical grade additive and / or excipients, and to uses thereof in a method, therapeutic or non-therapeutic, for preventing and / or delaying aging and / or one or more diseases and / or conditions related thereto. Finally, the present invention also relates to a non-therapeutic use of said at least one strain of bacteria, and mixtures and compositions thereof, for example as a food.

Description

[0001] AKKERMANSIA MUCINIPHILA STRAINS DSM 35203 AND DSM 35204 FOR USE IN PREVENTING AND / OR DELAYING AGING AND CONDITIONS RELATED THERETO

[0002] DESCRIPTION

[0003] The present invention relates to a mixture comprising or, alternatively, consisting of at least one strain of bacteria belonging to the species Akkermansia muciniphila selected from the group comprising or, alternatively, consisting of:

[0004] I) Akkermansia muciniphila AKM9, deposited by SynBalance SRL on 08.10.2024 at the Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSMZ) and having accession number DSM 35203; and ii) Akkermansia muciniphila AKM22, deposited by SynBalance SRL on 08.10.2024 at the Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSMZ) and having accession number DSM 35204, and ill) mixtures thereof.

[0005] Furthermore, the present invention relates to a composition comprising said mixture and, optionally, food or pharmaceutical grade additive and / or excipients, and uses thereof in a therapeutic method for preventing and / or delaying aging and / or one or more diseases and / or conditions related thereto.

[0006] Finally, the present invention also relates to a non-therapeutic use of said at least one strain of bacteria, and mixtures and compositions thereof, for example as a food.

[0007] BACKGROUND OF THE INVENTION

[0008] It is well known that aging is a stage of biological development in which various physical and biological mechanisms are implicated— mechanisms that lead cells to stop reproducing, organs to become less efficient, tissues to atrophy, and muscles to lose tone and, consequently, strength— and during which there is a progressive worsening of many bodily functions, with an increase in the risk of diseases that, over time, lead the individual to a physical decline and ultimately to death.

[0009] At present, it is possible to say that aging is due to a series of natural, genetic, environmental and social causes. From a strictly scientific viewpoint, aging is a genetically programmed event that occurs through changes in the functioning of the nervous, immune and endocrine systems.

[0010] It is well known that oxidative stress may bring about an anomalous acceleration of the aging process and, consequently, may lead to the onset of various diseases.

[0011] The term "oxidative stress” indicates an altered condition of the physiological balance between the free radicals produced by cellular metabolic reactions (cellular respiration) and the substances used by the body to counter the effects thereof, i.e. antioxidants.

[0012] In other words, oxidative stress occurs when the level of reactive oxygen species (ROS) exceeds the capacity of neutralisation by antioxidant systems, leading to a condition where the antioxidant defences of tissues are overwhelmed by the reactivity of free radicals. Oxidative stress seems to be implicated in the process of cell death in various degenerative diseases affecting the central nervous system, but not only, and moreover the majority of genetic mutations affecting our DNA are strictly caused by oxidative stress.

[0013] Another aspect involving aging is the loss of muscle mass and thus loss of strength associated with it. Muscle loss (sarcopenia) is a process that begins at around 40-50 years (gradually) and then progresses more rapidly from 60- 70 years of age. In this process, the amount of muscle tissue, as well as the number and size of muscle fibres, gradually decrease, and the loss of muscle strength increases stress on some joints (such as the knee) and may predispose a person to arthritis or falls.

[0014] Furthermore, one of the key phenomena associated with aging is inflammaging, i.e. a state of systemic, asymptomatic, low-grade chronic inflammation that takes place at an advanced age in the absence of infection and results in the occurrence of tissue and organ damage, with the consequent onset of chronic diseases.

[0015] Moreover, one indicator of the aging process consists in the shortening of telomeres, which are highly repeated DNA sequences located in the terminal regions of chromosomes and are responsible for maintaining the integrity of genetic information with every cell division.

[0016] Various studies correlate progressive telomere shortening at every replication cycle with cellular aging. In fact, it is known that, over time, the length of telomeres progressively decreases to enable the cell to divide, inducing senescence and ultimately cell death.

[0017] To date various attempts have been made by science to develop new therapies and new methods aimed at slowing the general aging of the body and the conditions associated therewith, such as, for example, musculoskeletal impairment, and at extending the average lifespan while simultaneously improving the quality of life.

[0018] Over the years, several strategies have been developed to combat aging; however, they mainly act on the skin and do not favour the wellbeing of the body as a whole. Therefore, the products associated with said treatments do not make it possible to improve the health of an individual's organs or to prevent and / or delay aging and / or one or more diseases and / or conditions related thereto.

[0019] Therefore, it is desirable to be able to have therapies and products which are easy to prepare and free of side effects and comprise natural ingredients that are not harmful for the body, and which make it possible to improve the health of an individual's organs and to prevent and / or delay aging and / or one or more diseases and / or conditions related thereto.

[0020] These aims and still others, which will emerge clearly from the detailed description that follows, are achieved by specific selected strains of bacteria belonging to the species Akkermansia muciniphila, and the mixtures containing said strains and compositions comprising said mixtures (mixtures and / or compositions of the invention) which have the technical features claimed in the appended claims.

[0021] SUMMARY OF THE INVENTION

[0022] Following an intense research activity, the Applicant selected specific strains of bacteria belonging to the species Akkermansia muciniphila, whose properties are capable of preventing and / or delaying aging of an organism, increasing the longevity thereof and improving quality of life. According to the present invention, said organism is preferably a human body.

[0023] A first aspect of the present invention relates to a mixture comprising or, alternatively, consisting of at least one strain of bacteria belonging to the species Akkermansia muciniphila selected from the group comprising or, alternatively, consisting of:

[0024] I) Akkermansia muciniphila AKM9, deposited by SynBalance SRL on 08.10.2024 at the Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSMZ) and having accession number DSM 35203; and ii) Akkermansia muciniphila AKM22, deposited by SynBalance SRL on 08.10.2024 at the Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSMZ) and having accession number DSM 35204, and ill) mixtures thereof.

[0025] Said strains of bacteria were deposited by the Applicant, as Depositor, under the provisions of the Budapest Treaty, at the Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSMZ) Institute, InhoffenstraBe 7B, 38124 Braunschweig, Germany.

[0026] The strains Akkermansia muciniphila AKM9 (DSM 35203) and Akkermansia muciniphila AKM22 (DSM 35204) were identified and selected by SynBalance SRL and represent isolated strains of bacteria.

[0027] Said at least one strain of bacteria Akkermansia muciniphila AKM9 (DSM 35203) and / or Akkermansia muciniphila AKM22 (DSM 35204) can be advantageously used as a medicament.

[0028] As shown in the experimental section, these strains express a marked antioxidant activity and, for this reason, are useful for treating aging-related disorders characterized by cellular oxidative stress.

[0029] Furthermore, said strains of bacteria also show to have an anti-inflammatory activity against the chronic inflammation that characterizes the physiological condition of an individual at an advanced age. By virtue of its antioxidant and anti-inflammatory activity, said at least one strain belonging to the species Akkermansia muciniphila, AKM9 (DSM 35203) and / or AKM22 (DSM 35204), can preferably be used to prevent and / or delay aging and / or one or more diseases and / or conditions related thereto.

[0030] The generation of oxygen free radicals (ROS) increases with age due to the progressive deregulation of cellular metabolism, thus generating a status of homeostasis that is compromised in favour of progressive cell oxidation. Decreasing the ROS levels and improving antioxidant mechanisms increases the probability of healthy aging and a longer life. Oxidative stress may also contribute to the development of chronic inflammation.

[0031] The Applicant assessed the antioxidant activity of the inactivated strains of the invention by administration in C. elegans. A reduction in ROS was observed for both AKM9 and AKM22.

[0032] It was observed that the strain AKM9 has a greater antioxidant activity, as it is capable of more greatly reducing ROS production in C. elegans. Therefore, the mixture comprising at least one strain of Akkermansia muciniphila as defined in the present invention is preferably used in a method for preventing and / or delaying aging, more preferably aging of the body caused by cellular oxidative stress, and / or one or more diseases and / or conditions related thereto. More preferably, said mixture comprises at least the strain belonging to the species Akkermansia muciniphila AKM9 (DSM 35203). More preferably, said strain AKM9 is used for the treatment, therapeutic or non-therapeutic and preventive and / or curative, of one or more diseases or conditions characterized by high oxidative stress and / or for the treatment of aging of the body caused by cellular oxidative stress.

[0033] Furthermore, considering that oxidative stress contributes to the development of chronic inflammation, the strains AKM9 and AKM22 are for use in the treatment, therapeutic or non-therapeutic and preventive and / or curative, of one or more diseases characterized by chronic inflammation. Said one or more diseases characterized by chronic inflammation are preferably characterized by high levels of oxidative stress.

[0034] More preferably, the strain AKM9 is used for the treatment, therapeutic or non-therapeutic and preventive and / or curative, of one or more diseases characterized by chronic inflammation.

[0035] In the context of the present invention, the term "aging” is understood as the physiological process that leads the human body to a progressive transformation due to degenerative changes occurring as a result of the deterioration of bodily structures and functions with age, e.g. the reduction in the weight of each tissue and body weight deriving from a decrease in the number of parenchymal cells, changes in connective tissues, a variation in body composition, a reduction in the elasticity of blood vessels or of skin, a decline in the functions of each organ, a reduction in the ability to recover from diseases due, for example, to a weakening of the immune system, a decline in the functions of sense organs and a deterioration of memory, as well as alterations in the functioning of the musculoskeletal system.

[0036] In the context of the present invention, the expression "one or more diseases and / or conditions related thereto” relates to age-related diseases generally due to cellular aging, and more in particular, to muscle aging, skin aging, aging of eyesight, aging of hearing, aging of digestive organs, immunosenescence, aging of urogenital system organs, aging of the body caused by cellular oxidative stress, aging due to a state of chronic inflammation (inflammaging), and cognitive decline.

[0037] The subject matter of the present invention relates to a mixture comprising or, alternatively, consisting of said at least one strain of bacteria belonging to the species Akkermansia muciniphila selected from the group comprising or, alternatively, consisting of: i) Akkermansia muciniphila AKM9, deposited by SynBalance SRL on 08.10.2024 at the Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSMZ) and having accession number DSM 35203; and / or ii) Akkermansia muciniphila AKM22, deposited by SynBalance SRL on 08.10.2024 at the Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSMZ) and having accession number DSM 35204, and iii) mixtures thereof.

[0038] In the context of the present invention, the term "mixture” indicates the set of active components that exert a beneficial effect, preferably a biological effect.

[0039] Preferably, the mixture of the invention comprises at least one of the strains of bacteria Akkermansia muciniphila AKM9 (DSM 35203) and / or Akkermansia muciniphila AKM22 (DSM 35204).

[0040] Preferably, said mixture is advantageously used in a method, therapeutic or non-therapeutic, for preventing and / or delaying aging and / or one or more diseases and / or conditions related thereto.

[0041] Another aspect of the present invention relates to a food, nutraceutical and / or pharmaceutical composition comprising the mixture of the invention together with at least one acceptable pharmaceutical or food grade additive and / or excipient.

[0042] In the context of the present invention, the term "composition” is understood to indicate the whole set of active components present in the mixture and additives and / or excipients and / or further components that are not active components.

[0043] Even where not expressly stated, the term "composition" in the present description includes pharmaceutical compositions, nutraceutical compositions, probiotic compositions, postbiotic compositions, food compositions, or compositions for medical devices as per Regulation (EU) 2017 / 745 (briefly, the composition(s) of the present invention) or compositions for supplements and foods for special medical purposes (FSMPs).

[0044] Unless otherwise specified, the indication that a composition "comprises” one or more components means that other components may also be present in addition to the one or ones specifically indicated, even where they have not necessarily been expressly indicated, i.e. the composition may also contain exclusively those components, and the indication that a composition "consists” of certain components means that the presence of other components is excluded.

[0045] Preferably, the strain of Akkermansia muciniphila AKM9 (DSM 35203) can be present in the composition in a percentage by weight out of the total weight of the composition comprised from 0.1 % to 99%, preferably from 20% to 70%, more preferably from 30% to 50%, for example 50%.

[0046] Preferably, the strain of Akkermansia muciniphila AKM22 (DSM 35204) can be present in the composition in a percentage by weight out of the total weight of the composition comprised from 0.1 % to 99%, preferably from 20% to 70%, more preferably from 30% to 50%, for example 50%.

[0047] Unless otherwise specified, within the scope of the present invention, with respect to ranges of numerical values for a certain feature, the indication "from X to Y” comprises the endpoints, i.e. X and Y, in addition to all the possible intermediate numerical values.

[0048] According to the invention, said composition can be used as a food.

[0049] The composition according to the invention can be used as a medicament, preferably in a method for preventing and / or delaying aging and / or one or more diseases and / or conditions related thereto.

[0050] Preferably, said at least one strain of bacteria Akkermansia muciniphila AKM9 (DSM 35203) and / or Akkermansia muciniphila AKM22 (DSM 35204) is in a non-viable, inactivated, tyndallized, dead but metabol ically active or dead form, in pasteurized form; more preferably, said at least one strain of bacteria is in pasteurized form.

[0051] The subject matter of the present invention relates to at least one strain of bacteria belonging to the species Akkermansia muciniphila selected from the group consisting of:

[0052] I) Akkermansia muciniphila AKM9, deposited by SynBalance SRL on 08.10.2024 at the Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSMZ) and having accession number DSM 35203; and ii) Akkermansia muciniphila AKM22, deposited by SynBalance SRL on 08.10.2024 at the Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSMZ) and having accession number DSM 35204, and iii) mixtures thereof.

[0053] DESCRIPTION OF THE FIGURES

[0054] Figure 1 : activation of human TLR2 following incubation of HEK-Blue cells expressing human TLR2 with the inactivated strains at a MOI (multiplicity of infection) of 5. Two-way Anova + Dunnett's multiple comparisons, AKM9 vs BAA-835 ****p<0.0001 ;

[0055] Figure 2: activation of human TLR2 following incubation of HEK-Blue cells expressing human TLR2 with the inactivated strains at a MOI (multiplicity of infection) of 2.5. Two-way Anova + Dunnett's multiple comparisons, AKM9 vs BAA-835 ****p<0.0001;

[0056] Figure 3: assessment of the antioxidant power of the bacterial supernatant via the DPPH test;

[0057] Figure 4: survival of the nematode C. elegans fed every 2 days with the inactivated strains indicated in the Figure; Figure 5: comparison: survival of the nematode C. elegans fed every 2 days with the inactivated strains 5a) AKM9 and OP50 and 5b) AKM22 and OP50;

[0058] Figure 6: count of bends in the characteristic sinusoidal movement of the nematode at the 11th day of life (beginning of adulthood); the nematode was fed every 2 days with the inactivated strains. The graph shows the means ± SD. One-way Anova + Dunnett's multiple comparison. BAA-835 vs E. coll OP50 *p<0.05, AKM22 vs E. coll OP50 ****p<0.0001, AKM9 vs E. coll OP50 ****p<0.0001, AKM22 vs BAA-835 ***p<0.001, AKM9 vs BAA-835 ****p<0.0001;

[0059] Figure 7: ATP assay in HaCaT cells (immortalized human keratinocytes) treated with the inactivated strains (106CFU / ml). One-way Anova, Dunnett's multiple comparison test, **p<0.01 AKM9 vs BAA-835.

[0060] Figure 8: number of nematodes that maintain movement after treatment with Aldicarb and the strains indicated in the Figure. Two-way Anova, Dunnett's multiple comparison test, at 2 h KM9 vs control *p<0.05, at 3 h AKM22 and BAA-835 vs control ***p<0.001, at 3 h AKM9 vs control ****p<0.0001 .

[0061] Figure 9: nematode survival after treatment with Levamisole and the strains indicated in the Figure.

[0062] DETAILED DESCRIPTION OF THE INVENTION

[0063] The subject matter of the present invention relates to at least one strain of bacteria belonging to the species Akkermansia muciniphila selected from the group consisting of:

[0064] I) Akkermansia muciniphila AKM9, deposited by SynBalance SRL on 08.10.2024 at the Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSMZ) and having accession number DSM 35203; and ii) Akkermansia muciniphila AKM22, deposited by SynBalance SRL on 08.10.2024 at the Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSMZ) and having accession number DSM 35204, and iii) mixtures thereof.

[0065] Said strains of bacteria are obligate anaerobic bacteria (i.e. microorganisms for which the absence of molecular oxygen is an essential condition for existence) belonging to the genus Akkermansia, species Akkermansia muciniphila.

[0066] Preferably, the strain Akkermansia muciniphila AKM9 (DSM 35203) can be cultured in BHI culture medium supplemented with mucin and cysteine and incubated for 48 hours at a temperature of 37 °C.

[0067] Preferably, the strain Akkermansia muciniphila AKM22 (DSM 35204) can be cultured in BHI bacterial culture medium supplemented with mucin and cysteine and incubated for 48 hours at a temperature of 37 °C.

[0068] According to the invention, said strains Akkermansia muciniphila AKM9 (DSM 35203) and Akkermansia muciniphila AKM22 (DSM 35204) can be used, individually or in combination, live or inactivated, in a method for preventing and / or delaying aging and / or one or more diseases and / or conditions related thereto.

[0069] The term "aging” means the physiological process that leads the human body to a progressive transformation due to degenerative changes occurring as a result of the deterioration of bodily structures and functions with age.

[0070] For the purposes of the present invention, the term "aging” includes cellular aging, and more specifically muscle aging, skin aging, aging of eyesight, aging of hearing, aging of digestive organs, immunosenescence, aging of urogenital organs, aging of the body caused by cellular oxidative stress, and aging due to a state of chronic inflammation (inflammaging).

[0071] In the context of the present invention, "cellular aging” is understood to indicate an irreversible process in which cells stop dividing and undergo distinctive alterations, in both their appearance and activity. Such cells, called senescent, have very precise characteristics that make them easily recognizable; they produce specific molecules, contributing to the state of aging of the whole body. Cellular senescence is due to different factors, including, mainly, the presence of DNA damage. In particular, replicative senescence is linked to "telomere shortening”, a process that leads to chromosome instability and favours the onset of cancer.

[0072] In the context of the present invention, "muscle aging” is understood to indicate the muscular decline that accompanies aging, e.g. the deterioration of muscle functions (muscle strength, muscle resistance, instantaneous muscle power, etc.). This multifactorial situation takes the name of sarcopenia and leads to a progressive reduction of muscle mass due to a decrease in muscle tissue and the reduced contractility of muscle cells, thus contributing to increase the risk of falls and fractures.

[0073] In the context of the present invention, "skin aging” is understood to indicate the physiological process characterized by structural modifications the skin undergoes with the advancing of age. The term "skin aging” comprises the aging that occurs naturally with the passing of time, and the photoaging that occurs in skin due to the exposure to ultraviolet light. Skin aging is characterized by a reduced amount of synthesis of collagen, hyaluronic acid, elastin, proteoglycan, fibronectin and / or the precursors thereof, an increase in the expression of degrading enzymes and a reduction in the expression of enzymes synthesizing the components of epithelial cells.

[0074] In the context of the present invention, "aging of eyesight” is understood to indicate the impairment of eyesight that accompanies aging due to various causes, for example the reduction in the elasticity of the crystalline lens with age, aging of the retina, reduction in the elasticity of ciliary muscle fibres and sclerosis of the cornea.

[0075] In the present invention, "aging of hearing” refers to the gradual hearing loss that accompanies aging and may be caused by a reduction in the number of neurons connected to the inner ear, middle ear and brain and the decline in their functions, and it may be accompanied by tinnitus, hearing loss or the like.

[0076] In the present invention, "aging of digestive organs” is understood to indicate the changes that occur in the oral cavity, oesophagus and gastrointestinal system and are accompanied by symptoms such as, for example, a reduced secretion of stomach acid and pancreatic juice, reduced motility of the gastrointestinal tract or a significant reduction in the rate of absorption of ingested nutrients. Furthermore, with age there is an increase in the predisposition to the development of diverticulosis and intestinal alterations, e.g. constipation, as a side effect of the use of certain drugs, or faecal incontinence due, in women, to the weakness of the pelvic floor.

[0077] "Immunosenescence” means the progressive degenerative change which occurs in the immune system with the advancing of age and is characterized by a reduction in immune function, chronic inflammation, and change in the composition of immune cells: for example, a decrease in the number of naive (non-specialized) T cells and an increase in the number of memory (specialized) T cells may occur, which can limit the immune system's ability to recognise new pathogenic agents.

[0078] "Aging of urogenital organs” is understood to include urination disorders, for example due to a weakening of bladder muscles. The aging of urogenital organs as per the present invention comprises the symptoms caused by the variations, occurring with age, in intraabdominal pressure, the muscular strength of the pelvis, urethra and bladder and thickening of urethral mucosa and may be accompanied by pathological conditions such as an overactive bladder, urinary incontinence, prostate enlargement, lower urinary tract symptoms, glomerulonephritis and chronic kidney disease. Furthermore, signs of aging tied to hormonal changes can also be found at the level of the vaginal mucosa.

[0079] Aging of the body caused by cellular oxidative stress is understood to include conditions and / or diseases characterized by a dysregulation of cellular metabolism with the generation of oxygen free radicals (ROS) which lead to a homeostasis that is compromised in favour of progressive cell oxidation. Oxidative stress can also contribute to the development of chronic inflammation and vice versa.

[0080] "Aging due to a state of chronic inflammation” is understood to indicate systemic, asymptomatic, low-grade chronic inflammation which, in the long term, has harmful effects on health and contributes to the development of age- related diseases.

[0081] Preferably, said at least one or more strains of bacteria as defined in the present invention are preferably used in a method, therapeutic or non-therapeutic, for preventing and / or delaying muscle aging (e.g. due to loss of muscle strength or deterioration of the skeletal system, for example, due to a reduction in energy metabolism) and / or aging of the body caused by cellular oxidative stress, and / or aging due to chronic inflammation, and / or for increasing the longevity of an individual; and / or for preventing and / or delaying aging-related conditions preferably selected from the group that comprises or, alternatively, consists of sarcopenia, asthenia, tiredness, cognitive decline, decreased immune defences, and loss of overall energy and strength.

[0082] The Applicant has advantageously observed that the strains Akkermansia muciniphila AKM9 (DSM 35203) and Akkermansia muciniphila AKM22 (DSM 35204) according to the invention are capable of increasing the motility of the nematode C. elegans at the 11th day of life (beginning of adulthood) when fed every 2 days with the inactivated strains, compared to the reference strain BAA-835. Furthermore, in the presence of the inactivated strains AKM9 (DSM 35203) and AKM22 (DSM 35204), HaCaT cells (commonly used in vitro model of immortalized human keratinocytes) demonstrate an increased production of ATP compared to the reference strain BAA-835. Therefore, said at least one strain of bacteria, preferably AKM9 (DSM 35203), is capable of countering the loss of energy and muscle strength, and the deterioration of the skeletal system occurring in an individual at an advanced age.

[0083] Furthermore, as shown in the experimental section, AKM9 and AKM22, more preferably AKM9, improve the functionality of the neuromuscular junction. Therefore, AKM9 and / or AKM22, more preferably AKM9, are particularly useful in a method, therapeutic or non-therapeutic, for preventing and / or delaying muscle aging (e.g. due to loss of muscle strength or deterioration of the skeletal system, for example, due to a reduction in energy metabolism).

[0084] Furthermore, thanks to the effect of improving the functionality of the neuromuscular junction, AKM9 and / or AKM22, more preferably AKM9, are particularly useful in a method, therapeutic or non-therapeutic, for the treatment of sarcopenia.

[0085] The Applicant has advantageously observed that said at least one strain of bacteria Akkermansia muciniphila AKM9 (DSM 35203) and / or Akkermansia muciniphila AKM22 (DSM 35204) according to the invention are capable of extending the lifespan of C. elegans— a nematode used as an animal model and possessing a marked genetic homology with humans— fed by administering the strains every 2 days from time=0 (chronic treatment starting from adulthood), as shown in the experimental section. Therefore, said at least one strain of bacteria, preferably AKM22 (DSM 35204), is capable of extending lifespan and improving the quality of life.

[0086] Therefore, said at least one strain of bacteria AKM9 (DSM 35203) and / or AKM22 can be advantageously used to delay general aging of the body and to extend the lifespan of an individual.

[0087] Preferably, said at least one strain of bacteria Akkermansia muciniphila AKM9 (DSM 35203) and / or Akkermansia muciniphila AKM22 (DSM 35204) can be used both in a viable form and in a viable but non-replicating form, or a non-viable, inactivated, tyndallized, or dead but metabolically active or dead form.

[0088] In the context of the present invention, said at least one "non-viable", "inactivated", "dead” or “postbiotic” strain of bacteria Akkermansia muciniphila AKM9 (DSM 35203) and Akkermansia muciniphila AKM22 (DSM 35204) refers to a microorganism that is no longer capable, temporarily or definitively, to form colonies in a culture. "Non-viable”, "inactivated”, "dead” or "postbiotic” microorganisms may have intact or broken cell membranes. Therefore, the term "non-viable", "inactivated”, "tyndallized”, "dead” or "postbiotic” also indicates fragments and derivatives of microorganisms.

[0089] In the context of the present invention, the term "postbiotic” may also indicate solely derivatives of microorganisms. "Derivatives” is understood to include, by way of non-limiting example, supernatants and metabolites, e.g. a supernatant produced by fermentation of said at least one strain of bacteria Akkermansia muciniphila AKM9 (DSM 35203) and / or Akkermansia muciniphila AKM22 (DSM 35204) or one or more metabolites produced by said at least one strain of bacteria Akkermansia muciniphila AKM9 (DSM 35203) and / or Akkermansia muciniphila AKM22 (DSM 35204).

[0090] A particular type of "inactivated” microorganisms are "viable but non-replicating” or "dead but metabol ically active” microorganisms, which are not capable of replicating, that is, of forming colonies in a culture, but retain a metabolic activity characteristic of live, non-inactivated microorganisms. Inactivated microorganisms can be produced with any method known to the person skilled in the art, such as, purely by way of non-limiting illustration, irradiation (with gamma rays, X rays, exposure to UV radiation), exposure to mechanical forces (pressure, sonication, etc.), exposure to heat (sterilization, UHT treatment, tyndallization, pasteurization). The type of treatment, intensity, dose and exposure time are controlled by the person skilled in the art based on the amount and nature of the probiotic microorganisms to be inactivated.

[0091] Preferably, said strain of bacteria of Akkermansia muciniphila AKM9 (DSM 35203) is in pasteurized form. Preferably, said strain of bacteria of Akkermansia muciniphila AKM22 (DSM 35204) is in pasteurized form. Preferably, a mixture of said strain of bacteria of Akkermansia muciniphila AKM9 (DSM 35203) and Akkermansia muciniphila AKM22 (DSM 35204) is in pasteurized form.

[0092] Pasteurization is a thermal sanitization process applied to some foods with the aim of minimizing health risks due to heat-sensitive pathogenic microorganisms, such as bacteria in vegetative form, fungi and yeast, with a minimal alteration of the chemical, physical and organoleptic characteristics of the food.

[0093] Said at least one strain of bacteria Akkermansia muciniphila AKM9 (DSM 35203) and / or Akkermansia muciniphila AKM22 (DSM 35204) is subjected to pasteurization using methods and apparatus known in the art. When both strains Akkermansia muciniphila AKM9 (DSM 35203) and Akkermansia muciniphila AKM22 (DSM 35204) are present in the mixture or composition of the invention, both are preferably in pasteurized form and are obtained using methods and apparatus known in the art.

[0094] For example, the pasteurization of said at least one strain of bacteria Akkermansia muciniphila AKM9 (DSM 35203) and / or Akkermansia muciniphila AKM22 (DSM 35204) can be carried out with a heat treatment, preferably at temperatures comprised from 60°C to 90°C for a time comprised from 1 second to 3600 seconds, preferably from 10 seconds to 2700 seconds, even more preferably from 60 seconds to 1200 seconds.

[0095] The subject matter of the present invention relates to a mixture comprising or, alternatively, consisting of at least one strain of bacteria belonging to the species Akkermansia muciniphila selected from the group comprising or, alternatively, consisting of:

[0096] I) Akkermansia muciniphila AKM9, deposited by SynBalance SRL on 08.10.2024 at the Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSMZ) and having accession number DSM 35203; and / or ii) Akkermansia muciniphila AKM22, deposited by SynBalance SRL on 08.10.2024 at the Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSMZ) and having accession number DSM 35204, and ill) or mixtures thereof. Preferably, said strain of bacteria Akkermansia muciniphila AKM9 (DSM 35203) preferably has a concentration comprised from 1 x106CFU / g to 1 x1012CFU / g, more preferably comprised from 1x108CFU / g to 1 x1010CFU / g, for example 1 x109CFU / g, wherein the measurement in CFU / g refers to the plate count of live cells.

[0097] Preferably, said strain of bacteria Akkermansia muciniphila AKM9 (DSM 35203) has a concentration comprised from 1 x108CFU / g to 1x1011CFU / g, for example 1 x1010CFU / g, wherein the measurement in CFU / g refers to the plate count of live cells.

[0098] Furthermore, said strain of bacteria Akkermansia muciniphila AKM9 (DSM 35203) preferably has a concentration comprised from 1 x106CFU / dose to 1 x1012CFU / dose, more preferably comprised from 1 x108CFU / dose to 1 x1011CFU / dose, for example 1x1010CFU / dose, wherein the measurement in CFU / dose refers to the plate count of live cells.

[0099] The CFU parameter refers to the colony-forming units counted on the plate before any inactivation of one or more of said strains of bacteria.

[0100] In the mixture according to the invention, said bacterial strain Akkermansia muciniphila AKM9 (DSM 35203) and / or said bacterial strain Akkermansia muciniphila AKM22 (DSM 35204) are in a viable form, in a viable but nonreplicating form, a non-viable, inactivated, tyndallized, dead but metabolically active or dead form, in pasteurized form; wherein said non-viable, inactivated, tyndallized or dead form preferably comprises fragments or derivatives of said at least one strain of bacteria belonging to the species Akkermansia muciniphila AKM9 (DSM 35203) and / or AKM22 (DSM 35204), more preferably said derivatives being selected from a supernatant and / or metabolites.

[0101] Preferably, said bacterial strain Akkermansia muciniphila AKM9 (DSM 35203) and / or said bacterial strain Akkermansia muciniphila AKM22 (DSM 35204) in the mixture according to the invention are in pasteurized form.

[0102] Preferably, said strain of bacteria Akkermansia muciniphila AKM22 (DSM 35204) preferably has a concentration comprised from 1 x106CFU / g to 1 x1012CFU / g, more preferably comprised from 1x108CFU / g to 1 x1010CFU / g, for example 1 x109CFU / g, wherein the measurement in CFU / g refers to the plate count of live cells.

[0103] More preferably, said strain of bacteria Akkermansia muciniphila AKM22 (DSM 35204) has a concentration comprised from 1x108CFU / g to 1x1011CFU / g, for example 1x1010CFU / g, wherein the measurement in CFU / g refers to the plate count of live cells.

[0104] Preferably, said strain of bacteria Akkermansia muciniphila AKM22 (DSM 35204) preferably has a concentration comprised from 1 x106CFU / dose to 1 x1012CFU / dose, more preferably comprised from 1 x108CFU / dose to 1 x1011CFU / dose, for example 1x1010CFU / dose, wherein the measurement in CFU / dose refers to the plate count of live cells.

[0105] The CFU parameter refers to the colony-forming units counted on the plate before any inactivation of one or more of said strains of bacteria.

[0106] In the context of the present invention, the method of counting bacterial cells, live or dead, can also be carried out, for example, by flow cytometry. According to the invention, the mixture comprises or consists of the bacterial strain Akkermansia muciniphila AKM9 (DSM 35203).

[0107] According to the invention, the mixture comprises or consists of the bacterial strain Akkermansia muciniphila AKM22 (DSM 35204).

[0108] Furthermore, the mixture according to the invention can comprise or consist of both strains, Akkermansia muciniphila AKM9 (DSM 35203) and Akkermansia muciniphila AKM22 (DSM 35204).

[0109] Preferably, the mixture according to the invention comprises Akkermansia muciniphila AKM9 (DSM 35203) and Akkermansia muciniphila AKM22 (DSM 35204), preferably each at a concentration comprised from 1x106CFU / g to 1 x1012CFU / g, more preferably comprised from 1 x108CFU / g to 1 x1010CFU / g, for example 1 x109CFU / g, wherein said CFU refer to the colony-forming units counted on the plate; preferably, said count is carried out before any inactivation of one or more of said strains.

[0110] Preferably, the mixture according to the invention comprises Akkermansia muciniphila AKM9 (DSM 35203) and Akkermansia muciniphila AKM22 (DSM 35204) in a concentration comprised from 1 x108CFU / g to 1 x1011CFU / g, for example 1x1010CFU / g, wherein the measurement in CFU / g refers to the colony-forming units counted on the plate; preferably, said count is carried out before any inactivation of one or more of said strains.

[0111] More preferably, the mixture according to the invention comprises Akkermansia muciniphila AKM9 (DSM 35203) and Akkermansia muciniphila AKM22 (DSM 35204), preferably each at a concentration comprised from 1x106CFU / g to 1x1012CFU / dose, more preferably comprised from 1x108CFU / dose to 1x1011CFU / dose, for example 1 x1010CFU / dose, wherein said CFU refer to the colony-forming units counted on the plate; preferably, said count is carried out before any inactivation of one or more of said strains.

[0112] Preferably, said strains of bacteria Akkermansia muciniphila AKM9 (DSM 35203) and Akkermansia muciniphila AKM22 (DSM 35204), when both are present in the mixture, are preferably in a weight ratio comprised from 1 :10 to 10:1 , more preferably comprised from 1 :5 to 5:1 , even more preferably comprised from 1 :3 to 3:1 , for example 1 : 1.

[0113] The mixture as described above is advantageously used as a food.

[0114] The mixture as described above is advantageously used as a medicament.

[0115] Preferably, said mixture comprising at least one strain selected from AKM9 (DSM 35203) and AKM22 (DSM 35204), or both the strains AKM9 (DSM 35203) and AKM22 (DSM 35204), is advantageously used in a method, therapeutic or non-therapeutic, for preventing and / or delaying aging and / or one or more diseases and / or conditions related thereto, as described above.

[0116] Said mixture used in a method, therapeutic or non-therapeutic, for preventing and / or delaying aging and / or one or more diseases and / or conditions related thereto, as described above, may comprise only the strain AKM9 (DSM 35203), or only the strain AKM22 (DSM 35204), or both strains.

[0117] Preferably, the mixture further comprises one or more further active components. For example, said further active components can be one or more further strains of bacteria belonging to different species and / or molecules having antioxidant or anti-inflammatory action.

[0118] The mixture can comprise, in addition to AKM9 and / or AKM22, as further active components, one or more strains of bacteria belonging to one of the following genera of bacteria: Lactobacillus, Bifidobacterium and Streptococcus. Preferably, the mixture can comprise, in addition to AKM9 and / or AKM22, as further active components, one or more strains of bacteria belonging to one of the genera of bacteria: Lactobacillus, Bifidobacterium and Streptococcus, for example one or more of the strains of bacteria specified below, or mixtures thereof.

[0119] Preferably, the mixture can comprise, in addition to AKM9 and / or AKM22, a strain of bacteria belonging to the species Lactobacillus acidophilus, e.g. Lactobacillus acidophilus PBS066 (DSM 24936) deposited by Principium Europe S.r.l. at Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSMZ) on 17.06.2011 with accession number DSM 24936. The Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSMZ) is located in InhoffenstraBe 7B, 38124 Braunschweig, Germany.

[0120] Preferably, the mixture can comprise, in addition to AKM9 and / or AKM22, a strain of bacteria belonging to the species Lactiplantibacillus plantarum, e.g. Lactiplantibacillus plantarum PBS067 (DSM 24937) deposited by Principium Europe S.r.l. at Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSMZ) on 17.06.2011 with accession number DSM 24937.

[0121] Preferably, the mixture can comprise, in addition to AKM9 and / or AKM22, a strain of bacteria belonging to the species Limosilactobacillus reuteri, e.g. Limosilactobacillus reuteri PBS072 (DSM 25175), deposited by Principium Europe S.r.l. at Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSMZ) on 14.09.2011 with accession number DSM 25175.

[0122] Preferably, the mixture can comprise, in addition to AKM9 and / or AKM22, a strain of bacteria belonging to the species Lacticaseibacillus rhamnosus, e.g. Lacticaseibacillus rhamnosus LRH020 (DSM 25568), deposited by Principium Europe S.r.l. at Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSMZ) on 17.01.2012 with accession number DSM 25568.

[0123] Preferably, the mixture can comprise, in addition to AKM9 and / or AKM22, a strain of bacteria belonging to the species Limosilactobacillus fermentum, e.g. Limosilactobacillus fermentum PBS073 (DSM 25176), deposited by Principium Europe S.r.l. at Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSMZ) on 14.09.2011 with accession number DSM 25176.

[0124] Preferably, the mixture can comprise, in addition to AKM9 and / or AKM22, a strain of bacteria belonging to the species Lacticaseibacillus casei, e.g. Lacticaseibacillus casei LC010 (DSM 25569), deposited by Principium Europe S.r.l. at Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSMZ) on 17.01 .2012 with accession number DSM 25569.

[0125] Preferably, the mixture can comprise, in addition to AKM9 and / or AKM22, a strain of bacteria belonging to the species Lactobacillus crispatus, e.g. Lactobacillus crispatus LCR030 (LMG P-31003) deposited by Roelmi HPC srl at the Belgian Coordinated Collections of Micro-organisms (BCCM) on 21.09.2018 with accession number LMG P- 31003. The BCCM is located at Universiteit Gent K.L. Ledeganckstraat 35 9000 Gent, Belgium.

[0126] Preferably, the mixture can comprise, in addition to AKM9 and / or AKM22, a strain of bacteria belonging to the species Lactobacillus gassed, e.g. Lactobacillus gassed LG050 (LMG P-29638), deposited by Principium Europe S.r.l. at the Belgian Coordinated Collections of Micro-organisms (BCCM) on 24.05.2016 with accession number LMG P-29638.

[0127] Preferably, the mixture can comprise, in addition to AKM9 and / or AKM22, a strain of bacteria belonging to the species Lactobacillus delbrueckii subsp. bulgadcus e.g. Lactobacillus delbrueckii subsp. bulgadcus LB025 (DSM 25567), deposited by Principium Europe S.r.l. at Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSMZ) on 17.01.2012 with accession number DSM 25567.

[0128] Preferably, the mixture can comprise, in addition to AKM9 and / or AKM22, a strain of bacteria belonging to the species Lactobacillus helveticus, e.g. Lactobacillus helveticus LH060 (LMG P-31392), deposited by Roelmi HPC srl at the Belgian Coordinated Collections of Micro-organisms (BCCM) on 09.04.2019 with accession number LMG P-31392.

[0129] Preferably, the mixture can comprise, in addition to AKM9 and / or AKM22, a strain of bacteria belonging to the species Lacticaseibacillus paracasei, e.g., Lacticaseibacillus paracasei LPC1082 (DSM 34557), deposited by Synbalance srl at Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSMZ) on 23.03.2023 with accession number DSM 34557, Lacticaseibacillus paracasei LPC1101 (DSM 34558) deposited by Synbalance srl. at Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSMZ) on 23.03.2023 with accession number DSM 34558, Lacticaseibacillus paracasei LPC1114 (DSM 34559) deposited by Synbalance srl at Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSMZ) on 23.03.2023 with accession number DSM 34559.

[0130] Preferably, the mixture can comprise, in addition to AKM9 and / or AKM22, a strain of bacteria belonging to the species Bifidobacterium animalis subsp. lactis, e.g. Bifidobacterium animalis subsp. lactis BL050 (DSM 25566), deposited by Principium Europe S.r.l. at Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSMZ) on 17.01.2012 with accession number DSM 25566.

[0131] Preferably, the mixture can comprise, in addition to AKM9 and / or AKM22, a strain of bacteria belonging to the species Bifidobacterium bifidum, e.g. Bifidobacterium bifidum BBF210 (LMG P-29508), deposited by Principium Europe S.r.l. at the Belgian Coordinated Collections of Micro-organisms (BCCM) on 08.04.2016 with accession number LMG P-29508.

[0132] Preferably, the mixture can comprise, in addition to AKM9 and / or AKM22, a strain of bacteria belonging to the species Bifidobacterium longum subsp. longum, e.g. Bifidobacterium longum subsp. longum BLG240 (LMG P- 29511), deposited by Principium Europe S.r.l. at the Belgian Coordinated Collections of Micro-organisms (BCCM) on 08.04.2016 with accession number LMG P-29511. Preferably, the mixture can comprise, in addition to AKM9 and / or AKM22, a strain of bacteria belonging to the species Bifidobacterium longum subsp. infantis, e.g. Bifidobacterium longum subsp. infantis BI221 (LMG P-29639), deposited by Principium Europe S.r.l. at the Belgian Coordinated Collections of Micro-organisms (BCCM) on 24.05.2016 with accession number LMG P-29639.

[0133] Preferably, the mixture can comprise, in addition to AKM9 and / or AKM22, a strain of bacteria belonging to the species Bifidobacterium breve, e.g. Bifidobacterium breve BB077 (LMG P-30157), deposited by Roelmi HPC srl at the Belgian Coordinated Collections of Micro-organisms (BCCM) on 09.06.2017 with accession number LMG P- 30157.

[0134] Preferably, the mixture can comprise, in addition to AKM9 and / or AKM22, a strain of bacteria belonging to the species Streptococcus thermophilus, e.g. Streptococcus thermophilus ST075 (DSM 26721), deposited by Principium Europe S.r.l. at Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSMZ) on 13.12.2012 with accession number DSM 26721. Said mixture can be advantageously used in a method of treatment, therapeutic or non-therapeutic, for preventing and / or delaying muscle aging (e.g. due to loss of muscle strength or deterioration of the skeletal system, for example, due to a reduction in energy metabolism) and / or aging of the body caused by cellular oxidative stress, and / or aging due to chronic inflammation, and / or for increasing the longevity of an individual; and / or for preventing and / or delaying the conditions related to aging, preferably selected from the group that comprises or, alternatively, consists of sarcopenia, asthenia, tiredness, cognitive decline, decreased immune defences, and loss of overall energy and strength.

[0135] Said mixture for use as defined above may comprise only the strain AKM9 (DSM 35203), or only the strain AKM22 (DSM 35204), or both strains.

[0136] The subject matter of the present invention further relates to a food, nutraceutical and / or pharmaceutical composition comprising the mixture according to the invention and, optionally, at least one acceptable pharmaceutical or food grade additive and / or excipient.

[0137] The composition according to the invention is, for example, a pharmaceutical composition or a nutraceutical composition or a composition for supplements or a food composition or a composition for medical devices, for example according to Regulation (EU) 2017 / 745, probiotic compositions, postbiotic compositions, or a composition for foods for special medical purposes, for example according to Regulation (EU) 609 / 2013 (briefly, the compositions of the invention).

[0138] In the context of the present invention, the expression "at least one pharmaceutical or food grade additive and / or excipient” means a substance devoid of therapeutic activity that is suitable for pharmaceutical use or food use. In the context of the present invention, the acceptable additives and / or excipients for pharmaceutical or food use include all the auxiliary substances known to the person skilled in the art for the preparation of compositions in solid, semisolid or liquid form, e.g. emulsifiers, diluents, solvents, solubilizers, acidifiers, thickening agents, sweeteners, flavour enhancers, colourants, lubricants, surfactants, preservatives, pH stabilizing buffers and mixtures thereof. The composition of the invention can be administered orally or topically (by way of non-exhaustive example, by nasal, buccal, auricular or vaginal administration).

[0139] According to the invention, said composition is formulated for oral administration, preferably as a tablet, capsule, lozenge, chewing gum, functional food such as ice-cream or yogurt, granular powder, orodispersible stick, hardshell capsule, suppository, orodispersible granules, sachet or pill, softgel, suspensions (e.g. drinkable vial) or solutions (monophasic or biphasic).

[0140] The at least one strain of bacteria Akkermansia muciniphila AKM9 (DSM 35203) and Akkermansia muciniphila AKM22 (DSM 35204) in the composition is pasteurized.

[0141] Preferably, the composition comprises the strain Akkermansia muciniphila AKM9 (DSM 35203).

[0142] Preferably, the composition comprises the strain Akkermansia muciniphila AKM22 (DSM 35204).

[0143] Preferably, the composition comprises both the strain Akkermansia muciniphila AKM9 (DSM 35203) and the strain Akkermansia muciniphila AKM22 (DSM 35204).

[0144] Preferably, one or more of the strains Akkermansia muciniphila AKM9 (DSM 35203) and Akkermansia muciniphila AKM22 (DSM 35204) present in the composition are pasteurized.

[0145] Preferably, the strain of Akkermansia muciniphila AKM9 (DSM 35203) can be present in the composition in a percentage by weight, out of the total weight of the composition, comprised from 0.1 % to 99%, preferably from 20% to 70%, more preferably from 30% to 50%, for example 50%, and in a concentration comprised from 1 x106CFU / g to 1 x1012CFU / g, more preferably comprised from 1 x108CFU / g to 1 x1010CFU / g, even more preferably comprised from 1x108CFU / g to 1x1011CFU / g, for example 1x109CFU / g or 1x1010CFU / g, wherein said CFU refer to the colony-forming units counted before any inactivation of one or more of said strains.

[0146] More preferably, the strain of Akkermansia muciniphila AKM9 (DSM 35203) can be present in the composition in a percentage by weight, out of the total weight of the composition, comprised from 0.1 % to 99%, preferably from 20% to 70%, more preferably from 30% to 50%, for example 50%, and in a concentration comprised from 1x106CFU / dose to 1 x1012CFU / dose, more preferably comprised from 1 x108CFU / dose to 1 x1011CFU / dose, for example 1x1010CFU / dose, wherein said CFU refer to the colony-forming units counted before any inactivation of one or more of said strains.

[0147] Preferably, the strain of Akkermansia muciniphila AKM22 (DSM 35204) can be present in the composition in a percentage by weight out of the total weight of the composition comprised from 0.1 % to 99%, preferably from 20% to 70%, more preferably from 30% to 50%, for example 50%, and in a concentration comprised from 1 x106CFU / g to 1 x1012CFU / g, more preferably comprised from 1 x108CFU / g to 1 x1010CFU / g, even more preferably comprised from 1x108CFU / g to 1x1011CFU / g, for example 1x109CFU / g or 1x101° CFU / g, wherein said CFU refer to the colony-forming units preferably counted before any inactivation of one or more of said strains.

[0148] More preferably, the strain of Akkermansia muciniphila AKM22 (DSM 35204) can be present in the composition in a percentage by weight out of the total weight of the composition comprised from 0.1 % to 99%, preferably from 20% to 70%, more preferably from 30% to 50%, for example 50%, and in a concentration comprised from 1x106 CFU / dose to 1 x1012CFU / dose, more preferably comprised from 1 x108CFU / dose to 1 x1011CFU / dose, for example 1 x1010CFU / dose, wherein said CFU refer to the colony -forming units preferably counted before any inactivation of one or more of said strains.

[0149] The composition as described in the present invention is a food composition which can advantageously be used as a food.

[0150] Furthermore, according to the invention, the food, nutraceutical and / or pharmaceutical composition comprising said mixture can be advantageously used as a dietary supplement in a non-therapeutic method for preventing and / or delaying aging.

[0151] The Applicant has found that the mixture of the invention and / or the pharmaceutical or nutraceutical compositions containing it are particularly useful in delaying the body's physiological aging process, preferably for preventing and / or delaying muscle aging (e.g. due to loss of muscle strength or deterioration of the skeletal system, for example, due to a reduction in energy metabolism) and / or aging of the body caused by cellular oxidative stress, and / or aging due to inflammaging and / or for increasing the longevity of an individual.

[0152] Therefore, said composition can be advantageously used as a medicament.

[0153] Preferably, at least one of the strains of bacteria of the invention and / or the mixture and / or the composition as described above are used in a therapeutic method for the treatment of one or more diseases and / or conditions related to aging of the body.

[0154] Preferably, said one or more aging-related diseases are diseases of the musculoskeletal system, for example osteoarthritis, diseases of the musculoskeletal system characterized by loss of muscle strength or deterioration of the skeletal system, or ataxia.

[0155] Preferably, said conditions related to the aging of the body are sarcopenia, asthenia, tiredness, cognitive decline, decreased immune defences, and loss of overall energy and strength.

[0156] Therefore, the mixture of the invention and / or the pharmaceutical or nutraceutical compositions according to the invention can advantageously be administered to an individual who wishes to delay the aging process in an amount that is effective for that purpose.

[0157] The daily dosage of the composition or mixture according to the invention depends on the patient's state of health, weight, sex and age. In general, the compositions of the invention are well tolerated and can be administered once or several times a day, preferably once or twice a day, even for an extended period of time.

[0158] The mixture according to the invention, the composition comprising it or the individual strains AKM9 and AKM22 can be advantageously used for cosmetic purposes, to prevent and / or delay aging as defined in the present invention.

[0159] Both the mixture of the invention and the compositions comprising it are easy to prepare and economical, considering that they can be prepared using the apparatus and procedures for preparing nutritional supplements and foods which are known to the person skilled in the art and in pharmaceutical practice.

[0160] The Applicant has observed in vitro and in vivo in C. elegans (chronic treatment in adulthood) the effect of the strains of bacteria AKM9 (DSM 35203) and AKM22 (DSM 35204) from the standpoint of reducing inflammation, increasing energy, increasing lifespan, and increasing motility, and the ability to improve the functionality of the neuromuscular junction.

[0161] The following examples further illustrate the invention.

[0162] EXAMPLES

[0163] Example 1.

[0164] A composition was prepared in the form of a capsule containing:

[0165] Table 1 a

[0166] Table 1b

[0167] Example 2

[0168] A composition was prepared in the form of a capsule containing:

[0169] Table 2 a

[0170] Table 2 b

[0171] Example 3 A composition was prepared in the form of a capsule containing:

[0172] Table 3 a Example 4

[0173] A composition was prepared in the form of an orodispersible stick containing :

[0174] Table 4

[0175] Example 5

[0176] A composition was prepared in the form of an orodispersible stick containing :

[0177] Table 5

[0178] Example 6

[0179] A composition was prepared in the form of a water-soluble stick containing:

[0180] Example 7

[0181] A composition was prepared in the form of a sachet containing :

[0182] Table 7

[0183] Example 8

[0184] A composition was prepared in the form of a 3-phase vial containing:

[0185] Table 8

[0186] Example 9

[0187] A composition was prepared in the form of a 3-phase vial containing:

[0188] Example 10

[0189] A composition was prepared in the form of a tablet containing :

[0190] Table 10

[0191] Example 11

[0192] A composition was prepared in the form of an oil containing :

[0193] Table 11

[0194] Example 12 - / 'n vitro experiments

[0195] Strain inactivation

[0196] After being cultured under anaerobic conditions at 37 °C, Akkermansia muciniphila AKM9 (DSM 35203), Akkermansia muciniphila AKM22 (DSM 35204), and the strain of Akkermansia muciniphila BAA-835 were centrifuged and the bacterial pellets washed twice with PBS.

[0197] They were then resuspended in PBS and heat inactivated, specifically with a 30-minute heat treatment at 70 °C. After cooling, 10% glycerol was added and the samples were aliquoted and stored at -80 °C until the time the experiments were conducted.

[0198] The strain of Akkermansia muciniphila BAA-835 was used as a reference strain, to which the activity of the strains of the invention was compared.

[0199] In particular, the strain of Akkermansia muciniphila BAA-835 was deposited in the American Type Culture Collection under ATCC BAA-835 and at Deutsche Sammlung von Mikroorganismen und Zel Ikulturen GmbH (DSMZ) with the accession number DSM 22959.

[0200] Said strain BAA-835 is an Akkermansia muciniphila type strain, used in the present experiments as a reference.

[0201] Said strain is freely accessible to the public for research purposes, available at the following ATCC link: https: / / www.atcc.org / products / baa- 835?matchtype=&network=x&device=c&adposition=&keyword=&gad_source=1 &gad_campaignid=17725151935 &gbraid=OAAAAADR6fppL1 bakNOKYvrG41tVJOOUuJ&gclid=CjwKCAjwgeLHBhBuEiwAL5gNEbT3lcOTxSNTeM mdXpLlw_cGveOIGeKtThJ4e8js6W506o7XpviYsRoCHbMQAvD_BwE.

[0202] For the test with Caenorhabditis elegans, the control strain Escherichia coli OP50 was inactivated with the same method as described for the other strains.

[0203] Anti-inflammatory activity

[0204] In the elderly, chronic inflammation is tied to an increase in TNF-o and a reduction in the activity of the immune system in general.

[0205] The supporting tests were set up using HEK-Blue cells expressing human TLR2.

[0206] In particular, HEK-Blue hTLR2 cells stably co-express the human toll-like receptor 2 and a reporter gene called SEAP, which may be induced through NK-kB. The levels of SEAP, starting from the stimulation of TLR2, are determined in real time thanks to the use of the HEK-Blue™ Detection culture medium, with a consequent colour change of the medium from fuchsia to purple / blue, quantifiable by spectrophotometric analysis.

[0207] The inactivated strains of AKM9, AKM22 and BAA-835 at two different concentrations (MOI 2.5 and 5) were seeded in a 96-well multiwell plate. HEK-Blue hTLR2 cells were subsequently plated at a density of 105cells / well in HEK- Blue™ Detection medium. After suitable incubation at 37 °C with 5% CO2, the absorbance was read at 650 nm.

[0208] All the strains are capable of activating the TLR2 pathway, but the strains of the invention demonstrate a more marked activity, which means that they promote cellular activation to a greater degree and may potentially increase the anti-inflammatory immune response, thus countering the effect of inflammaging (see Tables 12-13 and Figures 1-2).

[0209] Table 12: MOI 5

[0210] Table 13: MOI 2.5

[0211] Antioxidant activity (DPPH) The antioxidant activity of the bacterial supernatants was evaluated in vitro by using the free radical 2,2-diphenyl- 1-picrylhydrazyl (DPPH). Antioxidant compounds capable of transferring a hydrogen atom to the DPPH radical cause a fading of the colour of the solution, which is proportional to the antioxidant load present in the sample and quantifiable by spectrophotometry.

[0212] After being cultured under suitable conditions, the strains AKM9, AKM22 and BAA-835 were centrifuged to separate the bacterial pellet from the supernatant. The latter was filtered with 0.22 pm filters before the experiment took place. The test was conducted by mixing the same amount of bacterial supernatant and the DPPH molecule (300 pM in ethanol) and incubating at room temperature in darkness for 30 minutes. Subsequently, a reading was taken with a spectrophotometer at 517 nm.

[0213] The antioxidant activity of the bacterial strains of the present invention was observed to be greater than the antioxidant activity of the reference strain BAA-835 (Table 14 and Figure 3).

[0214] Table 14: evaluation of the antioxidant power of the bacterial supernatant by means of the DPPH test

[0215] Longevity

[0216] In order to assess the effect that the inactivated strains can have in extending life expectancy, an extensively studied animal model was used, namely the nematode C. elegans.

[0217] C. elegans is a nematode widely used as a preclinical animal model. It lives on a plate, feeds on bacteria and can be easily observed using a stereomicroscope. Its life cycle is about 4 weeks long and its genome shows a surprising homology with respect to the human genome. It is thus a useful model for rapidly obtaining results regarding aging / longevity mechanisms which can be easily translatable to humans.

[0218] The test supporting longevity provides for a determination of the days of life of adult nematodes fed the inactivated bacteria AKM22 and AKM9, with administration of the strains every 2 days starting from time t=0 (chronic treatment from adulthood).

[0219] 200 pL of an inactivated bacterial culture were plated on the surface of Nematode Growth Medium (NGM) and left to dry overnight. Subsequently, 60 nematodes for every condition were moved onto the plates and allowed to grow at 20 °C. The inactivated strains were administered every 2 days starting from the first day of adulthood and the live nematodes were selected and moved onto new plates, until the death of all the nematodes.

[0220] Though without statistically significant differences, the survival curves for the strains that were the subject of the study showed in many stages to have values above the curve of the control strain E. coll OP50 and always above the curve of the reference strain BAA-835, indicating a greater survival of the nematode, in terms of days, in different stages of its life (see figures 4, 5 a) and 5 b)).

[0221] AKM22 is capable of increasing the survival time of C. elegans to over 30 days, with a trend similar to that of AKM9.

[0222] Analysis of the motility of C. elegans

[0223] The age-related reduction in musculoskeletal function may contribute to a substantial degree to a decrease in mobility. For this reason, the reduction in the movement of C. elegans was evaluated as an aging parameter. The nematode's movements are measured by counting the number of typical sinusoidal movements (defined as body bends) recorded in 1 minute.

[0224] At day 11 of adulthood, the number of the movements of 20 nematodes per condition fed the inactivated strains of AKM9, AKM22 and BAA-835 was recorded. The strain Escherichia coli OP50 was used as a control. In this case it was surprisingly observed that the nematodes fed the inactivated strains AKM22 and AKM9 had a larger number of body bends compared both to the reference strain BAA-835 and to the control OP50 (see Table 15 and Figure 6). In particular, the C. elegans fed the strain AKM9 had a larger number of body bends, with a trend similar to that of AKM22.

[0225] Table 15: number of movements per minute

[0226] ATP assay in HaCaT cells (immortalized human keratinocytes) treated with inactivated strains (106CFU / ml)

[0227] With aging, muscles and the skeletal system undergo deterioration, leading to the onset, for example, of sarcopenia and osteoporosis. This physical deterioration is due to a reduction in energy metabolism, since mitochondria, upon aging, show a reduced ability to synthetize ATP, the primary energy source of the body's cells.

[0228] The aim of this test is to determine a strain's ability to increase, in vitro, the synthesis of adenosine triphosphate (ATP) in a cell culture of human keratinocytes. ATP in fact represents the main form for the storage of energy that is immediately available for the cells of the human body and is thus necessary for carrying out any biological activity. The levels of ATP in the cell extracts of each experimental group were measured using an indirect colorimetric method, exploiting the enzymatic reaction catalysed by creatine kinase, which converts ATP and creatine into phosphocreatine. The reaction product can be measured by adding a chromogen (phosphomolybdic acid), which produces a colour quantifiable with spectrophotometric measurement. The higher the level of the reaction product, and thus of the colour, the greater will be the amount of ATP present in the analysed sample. After 24 hours of treatment with the inactivated bacterial strains under examination at a concentration of 106cells / ml, the human keratinocytes (HaCaT cells) were lysed and centrifuged. The cellular protein extract was subjected to analysis by mixing it with a specific working solution. The reaction mixture was incubated at 37 °C for 30 minutes and a chromogenic agent was subsequently added for 2 minutes, which allowed for the measurement of the colour. The reaction was blocked by adding a stop solution and the absorbance was read at 636 nm.

[0229] The test performed made it possible to observe an increased production of ATP for the strain AKM9 compared to the reference one, BAA-835, whilst a similar trend was noted in the strain test for the strain AKM22 (see Table 16 and Figure 7).

[0230] Table 16: ATP assay in HaCaT cells treated with the inactivated strains

[0231] Therefore, AKM9 is preferably for use in a method, therapeutic or non-therapeutic, for preventing and / or delaying preferably muscle aging (e.g. due to loss of muscle strength or deterioration of the skeletal system, for example, due to a reduction in energy metabolism)

[0232] Further studies

[0233] The Applicant has further studies underway, aimed at assessing the effect of the one or more strains of bacteria of the invention on the accumulation of lipofuscin.

[0234] Lipofuscin is a waste product made up mainly of lipids and oxidated proteins that accumulate at the cellular level in the form of granules, and it cannot be eliminated either enzymatically or by exocytosis. Its accumulation is thus considered a marker of aging.

[0235] The accumulation of lipofuscin is determined by exploiting its autofluorescence, which enables both a quantitative and a qualitative analysis to be obtained. It is therefore possible to assess the effect of the individual bacterial strain on the accumulation of lipofuscin and hence its anti-aging activity.

[0236] Further studies will also be conducted to test the effect of the one or more strains AKM9 and AKM22 on telomere shortening, with the aim of assessing the reduction of cellular senescence, and on an ataxic model of C. elegans, which expresses the pathological version of human ataxin-3.

[0237] Functionality of the neuromuscular junction

[0238] The neuromuscular junction is the point of contact between a neuron and muscle. The transmission of the nerve impulse that causes muscle contraction takes place here. In practical terms, it is the mechanism that enables the brain to control the movement of muscles. The action of 2 drugs, Aldicarb and Levamisole, was tested in C. elegans fed a strain selected from: AKM22, AKM9, BAA-835 and E. coli OP50. Aldicarb and Levamisole act with different mechanisms of action, respectively at the pre- and postsynaptic levels of the neuromuscular junction, both inducing paralysis in the nematode.

[0239] Aldicarb is an inhibitor of acetylcholinesterase, which brings about an accumulation of acetylcholine in the junction space.

[0240] The observed reduction in the number of nematodes showing movement is a positive sign, as it is indicative of a good functioning of the neuromuscular junction in "older” nematodes, which, despite their advanced age, are more responsive.

[0241] Therefore, a larger number of paralysed animals (tendency towards zero) means a better functioning of the neuromuscular junction (Table 17 and Figure 8).

[0242] Table 17: Mean number of nematodes that maintain movement after treatment with Aldicarb

[0243] In parallel, in the test with Levamisole, an acetylcholine receptor agonist at the muscle level, one observes a surprisingly prolonged survival of nematodes.

[0244] Levamisole causes muscle hypercontraction because it is capable of binding to the cholinergic receptors present on the membrane of muscle cells, thus mimicking the action of the acetylcholine. On the eleventh day, nematodes fed the pasteurized strains that were the subject of the study were transferred to plates containing Levamisole. Thereafter, the nematodes were analysed every 10 minutes, and a positive score was attributed in the presence of movement or contraction of the nematodes after a light touch.

[0245] A more rapid hypercontraction compared to the control indicates better functioning of the muscle receptors, suggesting that, despite their advanced age, neuromuscular transmission is maintained in the treated nematodes, ultimately resulting in an increase in survival (Figure 9).

[0246] The experiments carried out show that AKM9 and AKM22, AKM9 to a larger degree, improve the functionality of the neuromuscular junction. Therefore, AKM9 and / or AKM22, more preferably AKM9, are particularly useful in a method, therapeutic or non-therapeutic, for preventing and / or delaying muscle aging (e.g. due to loss of muscle strength or deterioration of the skeletal system, for example, due to a reduction in energy metabolism). Furthermore, thanks to the effect of improving the functionality of the neuromuscular junction, AKM9 and / or AKM22, more preferably AKM9, are particularly useful in a method, therapeutic or non-therapeutic, for the treatment of sarcopenia.

Claims

CLAIMS1. A mixture comprising or, alternatively, consisting of at least one strain of bacteria belonging to the species Akkermansia muciniphila selected from the group comprising or, alternatively, consisting of:I) Akkermansia muciniphila AKM9, deposited by SynBalance SRL on 08.10.2024 at the Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSMZ) and having accession number DSM 35203; and / or ii) Akkermansia muciniphila AKM22, deposited by SynBalance SRL on 08.10.2024 at the Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSMZ) and having accession number DSM 35204, and ill) mixtures thereof.

2. The mixture according to claim 1, wherein said strain of bacteria Akkermansia muciniphila AKM9 (DSM 35203) and / or said Akkermansia muciniphila AKM22 (DSM 35204) is in a viable form, in a viable but non-replicating form, a non-viable, inactivated, tyndallized, dead but metabol ically active or dead form, in pasteurized form, preferably in pasteurized form; wherein said non-viable, inactivated, tyndallized or dead form preferably comprises fragments or derivatives of said at least one strain of bacteria belonging to the species Akkermansia muciniphila AKM9 (DSM 35203) and / or AKM22 (DSM 35204), more preferably said derivatives being selected from a supernatant and / or metabolites.

3. The mixture according to claim 1 or 2, wherein said mixture comprises both strains of bacteria, Akkermansia muciniphila AKM9 (DSM 35203) and Akkermansia muciniphila AKM22 (DSM 35204), preferably in a weight ratio comprised from 1 :10 to 10:1, more preferably comprised from 1 :5 to 5:1, even more preferably comprised from 1 :3 to 3:1, for example 1 :1.

4. Use of a mixture according to any one of claims 1 to 3, as a food.

5. Non-therapeutic use of a mixture according to any one of claims 1 to 4, in preventing and / or delaying aging and / or one or more diseases and / or conditions related thereto.

6. The mixture according to any one of claims 1 to 4, for use as a medicament, preferably for use in a therapeutic method for preventing and / or delaying aging and / or one or more diseases and / or conditions related thereto.

7. The mixture for use according to claim 5 or 6, for use in a method, therapeutic or non-therapeutic, for preventing and / or delaying muscle aging (e.g. due to loss of muscle strength or deterioration of the skeletal system, for example, due to a reduction in energy metabolism) and / or aging of the body caused by cellular oxidative stress, and / or aging due to chronic inflammation, and / or for increasing the longevity of an individual; and wherein said conditions related to the aging of the body are preferably selected from the group that comprises or, alternatively, consists of sarcopenia, asthenia, tiredness, cognitive decline, decreased immune defences, and loss of overall energy and strength.

8. A food, nutraceutical and / or pharmaceutical composition comprising a mixture according to any one of claims 1 to 7, together with at least one acceptable pharmaceutical or food grade additive and / or excipient.

9. The composition according to claim 8, wherein said composition comprises said strain of bacteria of Akkermansia muciniphila AKM9 (DSM 35203) preferably in an amount by weight out of the total weight of the compositioncomprised from 0.1 % to 99%, preferably from 20% to 70%, more preferably from 30% to 50%, in a concentration comprised from 1 x106CFU / dose to 1 x1012CFU / dose, more preferably comprised from 1 x108CFU / dose to 1 x1010CFU / dose, for example 1 x1010CFU / dose determined before the inactivation treatment; and / or said strain of bacteria of Akkermansia muciniphila AKM22 (DSM 35204) preferably in an amount by weight out of the total weight of the composition comprised from 0.1 % to 99%, preferably from 20% to 70%, more preferably from 30% to 50%, in a concentration comprised from 1x106CFU / dose to 1x1012CFU / dose, more preferably comprised from 1x108CFU / dose to 1 x1010CFU / dose determined before the inactivation treatment.

10. Use of a composition according to claim 7 or 8, as a food.11 . The composition according to claims 8 to 10 for use as a medicament, preferably for use in a therapeutic method for preventing and / or delaying aging and / or one or more diseases and / or conditions related thereto.

12. The composition according to claim 11 , for use in a method, therapeutic or non-therapeutic, for preventing and / or delaying muscle aging (e.g. due to loss of muscle strength or deterioration of the skeletal system, for example, due to a reduction in energy metabolism) and / or aging of the body caused by cellular oxidative stress, and / or aging due to chronic inflammation, and / or for increasing the longevity of an individual; and wherein said conditions related to the aging of the body are preferably selected from the group that comprises or, alternatively, consists of sarcopenia, asthenia, tiredness, cognitive decline, decreased immune defences, and loss of overall energy and strength.

13. The composition for use according to claims 8 to 12, wherein the composition is administered orally or topically.

14. At least one strain of bacteria belonging to the species Akkermansia muciniphila selected from the group comprising or, alternatively, consisting of:I) Akkermansia muciniphila AKM9, deposited by SynBalance SRL on 08.10.2024 at the Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSMZ) and having accession number DSM 35203; and ii) Akkermansia muciniphila AKM22, deposited by SynBalance SRL on 08.10.2024 at the Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSMZ) and having accession number DSM 35204. ill) mixtures thereof.

15. The at least one strain of bacteria according to claim 14, wherein said at least one strain of bacteria is in a viable form, in a viable but non-replicating form, a non-viable, inactivated, tyndallized, dead but metabolically active or dead form, in pasteurized form, preferably in pasteurized form, wherein said non-viable, inactivated, tyndallized or dead form preferably comprises fragments or derivatives of said at least one strain of bacteria belonging to the species Akkermansia muciniphila AKM9 (DSM 35203) and / or AKM22 (DSM 35204), more preferably said derivatives being selected from a supernatant and / or metabolites.

16. The at least one strain of bacteria according to claim 14 or 15, wherein said strain of bacteria is Akkermansia muciniphila AKM9 (DSM 35203), and is for use as a medicament.

17. The at least one strain of bacteria according to claim 14 or 15, wherein said strain of bacteria is Akkermansia muciniphila AKM22 (DSM 35204), and is for use as a medicament.

18. The at least one strain of bacteria according to any one of claims 14 to 17, for use in a method, therapeutic or non-therapeutic, for preventing and / or delaying aging and / or one or more diseases and / or conditions related thereto.

19. The at least one strain of bacteria for use according to claim 18, for use in a method, therapeutic or non- therapeutic, for preventing and / or delaying cellular aging, muscle aging (e.g. due to loss of muscle strength or deterioration of the skeletal system, for example, due to a reduction in energy metabolism) and / or aging of the body caused by cellular oxidative stress, and / or aging due to chronic inflammation, and / or for increasing the longevity of an individual; and wherein said conditions related to the aging of the body are preferably selected from the group that comprises or, alternatively, consists of sarcopenia, asthenia, tiredness, cognitive decline, decreased immune defences, and loss of overall energy and strength.

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