Cell regeneration method

Abstract

The present invention relates to a composition for inducing at least one cell renewal, said composition comprising:-at least one probiotic Bacillus subtilis strain, and-at least one amino acid or peptide wherein said amino acid is selected from glutamine, glutamic acid or salts thereof and conjugated glutamine, and the peptide is an oligopeptide having 2 to 10 amino acid units in length, the amino acid units being natural amino acids, and at least one amino acid unit being a glutamine or glutamic acid unit.

Description

Field of the Invention

[0001] The present invention relates to a composition for inducing cell renewal and / or reducing the biological age of at least one cell or a subject. The present invention also relates to a method for inducing cell renewal and / or reducing the biological age of a subject by administering the composition to the subject. In particular, the composition is a synbiotic composition comprising a probiotic strain of Bacillus subtilis and at least one amino acid. Background of the Invention

[0003] Aging is known to involve a gradual loss of function at the molecular, cellular, tissue, and organismal levels. Aging of individuals and tissues leads to the onset of a variety of diseases. In particular, senescent cells are frequently observed in inflamed tissues such as those found in rheumatoid arthritis, osteoarthritis, hepatitis, chronic skin lesions, and arteriosclerosis. When these senescent cells accumulate, damaged tissues cannot properly recover because they cannot divide properly.

[0004] At the chromatin level, aging is associated with the progressive accumulation of epigenetic errors, which ultimately lead to aberrant gene regulation, stem cell exhaustion, aging, and dysregulated cell / tissue homeostasis. In particular, Steve Horvath (2013) supported the role of epigenetics in aging by showing that DNA methylation correlates with chronological age (Horvath S. (2013) Genome Biol.; 14(10): R115). Epigenetic age is highly correlated with biological age and responds to environmental factors that accelerate or slow the aging process, resulting in considerable deviations from "true" biological age. Thus, environmental confounders or cues lead to accelerated or decelerated aging, while accelerated aging is matched by negative confounders / factors, such as disease, stress, malnutrition, negative environmental conditions, etc. Conversely, well-being and healthy nutrition are both associated with decelerated aging.

[0005] Epigenetic age acceleration (epigenetic age > chronological age) indicates that the underlying tissue is aging faster than expected based on chronological age, while negative values ​​(epigenetic age < chronological age, decelerated aging) indicate that the tissue is aging slower than expected. Epigenetic age acceleration is associated with many age-related conditions and diseases, such as inflammatory processes.

[0006] As mentioned above, healthy nutrition is known to reduce epigenetic age. Therefore, there is a need in the art for simple and reliable food supplements that can modulate cellular aging and, in the long term, prevent disease. In particular, there is a need in the art for food supplements that can be used to regenerate senescent cells into young, healthy cells.

[0007] Description of the Invention

[0008] The present invention attempts to solve the above problems by providing a food supplement that can delay cell aging or restore cells to their original / earlier state and thus reduce the biological age of the cells or subjects. As a food supplement, it is not an important component of the diet of the subject. However, it has the advantage of reducing the biological age of cells or subjects and / or inducing cell regeneration in the subject. In addition, as a food supplement, compared to diet and / or physical activity, it is also a very convenient way / tool ​​that can be used to influence the biological age of cells or subjects. In particular, the food supplement is a synbiotic. More particularly, the food supplement is not only a synbiotic (i.e., including prebiotics and probiotics), but also contains other components that increase the benefits of the food supplement according to any aspect of the present invention.

[0009] The term "food supplement" as used herein is defined as a concentrated source of nutrients or other substances having a nutritional or physiological effect, which is intended to supplement the normal diet (www.efsa.europa.eu / en / topics / topic / food-supplements).

[0010] As used herein, the term "synbiotic" refers to a food ingredient or dietary supplement that combines probiotics and prebiotics in a synergistic manner. Therefore, the synbiotic composition according to the present invention is a composition comprising a probiotic strain and an amino acid, dipeptide or oligopeptide as a prebiotic. In particular, prebiotics and probiotics can be combined to support the survival and metabolic activity of the latter, and the resulting product belongs to the synbiotic category. The updated definition of the term prebiotic is: "a substrate that is selectively utilized by host microorganisms and brings health benefits" (Gibson GR, Nat Rev Gastroenterol Hepatol 2017, 14 (8): 491-502), and therefore refers not only to certain carbohydrates, but also to, for example, amino acids and peptides (such as dipeptides or oligopeptides). Prebiotics can also include other meaningful bioactive ingredients, such as plant extracts, vitamins, etc.

[0011] According to one aspect of the present invention, there is provided a use of a composition for inducing regeneration of at least one cell, the composition comprising:

[0012] - at least one probiotic strain of Bacillus subtilis, and

[0013] - at least one dipeptide selected from the group consisting of glycine-glutamine, glycine-glutamic acid, alanine-glutamine, alanine-glutamic acid and acetylated forms thereof.

[0014] As used herein, the phrase "rejuvenation of at least one / a type of cell" may refer to restoring senescent cells to young, healthy cells. In particular, "rejuvenated cell(s)" refers to senescent cells that have been treated with one or more cell reprogramming factors or transiently reprogrammed such that the cells have transcriptomic characteristics of younger cells while still retaining one or more cell identity markers.

[0015] As used herein, the term "cell" refers to a naturally occurring or modified intact living cell. The cell can be isolated from other cells, mixed with other cells in culture, or in a tissue (partial or complete) or organism. In particular, the term "mammalian cell" refers to any cell derived from a mammalian subject. The cell can also be a cell derived from the culture and expansion of cells obtained from a subject. The cell can also be genetically modified to express recombinant proteins and / or nucleic acids. In particular, the mammalian cell can be an intestinal cell.

[0016] According to a further aspect of the present invention, there is provided use of a composition for reducing the epigenetic age of at least one subject, the composition comprising:

[0017] - at least one probiotic strain of Bacillus subtilis, and

[0018] - at least one dipeptide selected from the group consisting of glycine-glutamine, glycine-glutamic acid, alanine-glutamine, alanine-glutamic acid and acetylated forms thereof.

[0019] The composition according to any aspect of the present invention may also include other components. These other components may include one or more plant extracts. In particular, the plant extract may be selected from turmeric extract, green tea extract and combinations thereof. More particularly, the composition may also include other components, such as zinc, vitamin B6, D-biotin, vitamin B12, vitamin D, pantothenic acid, etc. More particularly, the composition according to any aspect of the present invention also includes turmeric extract, green tea extract, zinc, vitamin B6, D-biotin, vitamin B12, vitamin D and pantothenic acid in addition to components (a) and (b).

[0020] The biological age depends on the biological state or condition of the individual or population and takes into account the living environment (e.g. stress, nutrition, etc.). The terms "epigenetic age", "methylation age" and "biological age" have the same meaning and can be used interchangeably in the context of this application.

[0021] The term "chronological age" refers to the calendar time elapsed from birth / hatching.

[0022] As used herein, the term "epigenetic clock" refers to a biochemical assay that can estimate the DNA methylation age / epigenetic age in a specific tissue or independently of tissue and can predict mortality and time remaining to death (Horvath 2013).

[0023] The composition according to any aspect of the present invention can cause post-translational modification (PTM) of histones, which is a key step in gene epigenetic regulation. PTM can lead to changes in the expression of the gene.

[0024] The phrase "post-translational modification of histones" is used interchangeably with the phrase "histone modification" as used herein and refers to the covalent modification of the N-terminal tail of a histone. Examples of histone modifications include, for example, methylation, acetylation, phosphorylation, ubiquitination, and ADP-ribosylation. Histone modifications and other epigenetic mechanisms are crucial for regulating gene activity and cellular processes. Different histone modifications regulate different processes, such as transcription, DNA replication, and DNA repair. Any imbalance in these modifications can alter the balance of gene expression, leading to abnormal epigenetic patterns and cellular abnormalities. In one example, the one or more histone modifications can be methylation. In particular, the methylation can occur at, for example, H3-K4, H3-K9, H4-K20, H3-K27, H3-K36, and / or H3-K79.

[0025] The composition according to any aspect of the present invention can be used to alter histone PTMs, thereby altering gene expression, and to treat diseases associated with histone PTMs.

[0026] As used herein, the terms "subject" and "individual" can be used interchangeably and can be any living organism. For example, the subject according to any aspect of the present invention can be any kind of plant or animal, preferably, a farmed animal (or farmed livestock) or livestock, which can be a vertebrate or an invertebrate. Typical examples of invertebrates that can be used as the subject according to any aspect of the present invention can be shrimp or crabs, such as marbled crayfish. Typical examples of vertebrates that can be used as the subject according to any aspect of the present invention can be fish or terrestrial animals, such as chickens or other farmed livestock. The subject used herein can also refer to any vertebrate subject, including but not limited to humans and other primates, including non-human primates (such as chimpanzees and other apes and monkey species); farm animals such as cattle, sheep, pigs, goats and horses; domestic mammals such as dogs and cats; rodents such as mice, rats, rabbits, hamsters and guinea pigs; birds, including poultry, wild birds and game birds, such as chickens, turkeys and other quail and gallinaceous birds, ducks, geese, etc. In particular, the subject is a mammal. More particularly, the mammal is selected from the group consisting of mouse, rat, guinea pig, dog, miniature pig, human, cow, sheep, pig, goat, horse, donkey and mule.

[0027] The probiotic strain in the composition according to any aspect of the present invention can be selected from the group consisting of Bacillus subtilis DSM 32315, Bacillus subtilis DSM 32540, Bacillus subtilis DSM 32592 and mixtures thereof. In particular, the probiotic strain can be Bacillus subtilis DSM 32315.

[0028] In the context of the present invention, the expression "natural amino acid" should be understood to include the L- and D-forms of the known 20 amino acids. In particular, the L-forms of the amino acids can be used in the compositions according to any aspect of the present invention. In one embodiment, the term "amino acid" also includes analogs or derivatives of those amino acids.

[0029] As used herein, the term "free amino acid" is understood to mean an amino acid having its amino group and its (α-) carboxyl functional group in free form, i.e., not covalently bound to other molecules, e.g., not forming a peptide bond. Free amino acids may also exist in the form of salts or hydrates. When an amino acid is mentioned as part of or in an oligopeptide, it is understood to be that part of the corresponding oligopeptide structure derived from the corresponding amino acid according to known biochemistry and peptide biosynthesis mechanisms.

[0030] A "peptide" is understood as a molecule comprising at least two amino acids covalently coupled to each other via peptide bonds (R1-CO-NH-R2).

[0031] The amino acid or peptide in the composition according to any aspect of the present invention may be an oligopeptide having at least two amino acids. In particular, the oligopeptide may comprise alanine or glycine. More particularly, the oligopeptide may be selected from the group consisting of glycine-glutamine, glycine-glutamic acid, alanine-glutamine, alanine-glutamic acid, and acetylated forms thereof.

[0032] The composition according to any aspect of the present invention may be in the form of a pill, capsule, tablet, powder mix, granules or liquid.

[0033] In one example, the composition according to any aspect of the present invention can be in the form of a capsule, and the capsule contains 1×10 8 to 2×10 10 CFU of said probiotic strain and 50 mg to 800 mg of said dipeptide.

[0034] In particular, the total amount of the probiotic strain and amino acids or peptides is at least 40 wt%, in particular at least 50 wt%, more in particular at least 60 wt%, even more in particular at least 70 wt% of the total weight of the composition.

[0035] In particular, the total amount of plant extracts in the composition according to any aspect of the present invention is at least 10 wt%, in particular at least 20 wt%, more in particular 20 to 40 wt% of the total weight of the composition.

[0036] In one example, the composition according to any aspect of the present invention comprises an enteric coating, wherein the enteric coating comprises one or more of the following: methyl acrylate-methacrylic acid copolymer, cellulose acetate phthalate (CAP), cellulose acetate succinate, hydroxypropyl methylcellulose phthalate, hydroxypropyl methylcellulose acetate succinate (hydroxypropyl methylcellulose acetate succinate), polyvinyl acetate phthalate (PVAP), methyl methacrylate-methacrylic acid copolymer, shellac, cellulose acetate trimellitate, sodium alginate, zein, and the like.

[0037] According to another aspect of the present invention, there is provided a composition for inducing at least one cell renewal, the composition comprising:

[0038] - at least one probiotic strain of Bacillus subtilis, and

[0039] - at least one dipeptide selected from the group consisting of glycine-glutamine, glycine-glutamic acid, alanine-glutamine, alanine-glutamic acid and acetylated forms thereof.

[0040] The use according to any aspect of the present invention may be a non-therapeutic use, in particular a non-therapeutic food use. In particular, the composition according to any aspect of the present invention may be a food supplement and is not considered a medicament.

[0041] According to another aspect of the present invention, there is provided a composition for reducing the epigenetic age of at least one subject, the composition comprising:

[0042] - at least one probiotic strain of Bacillus subtilis, and

[0043] - at least one dipeptide selected from the group consisting of glycine-glutamine, glycine-glutamic acid, alanine-glutamine, alanine-glutamic acid and acetylated forms thereof.

[0044] In one example, the composition may also be used as a "concomitant therapy" or "adjuvant therapy." In such cases, the composition according to any aspect of the present invention may be used for preventive, prophylactic and / or therapeutic purposes.

[0045] The composition according to any aspect of the present invention further comprises one or more plant extracts selected from turmeric extract and green tea extract.

[0046] According to another aspect of the present invention, there is provided a method of reducing the epigenetic age of at least one subject, the method comprising administering to a subject in need thereof a composition comprising:

[0047] - at least one probiotic strain of Bacillus subtilis, and

[0048] - at least one dipeptide selected from the group consisting of glycine-glutamine, glycine-glutamic acid, alanine-glutamine, alanine-glutamic acid and acetylated forms thereof.

[0049] According to another aspect of the present invention, there is provided a method for inducing cell proliferation in at least one cell, the method comprising administering to a subject in need thereof a composition comprising:

[0050] - at least one probiotic strain of Bacillus subtilis, and

[0051] - at least one dipeptide selected from the group consisting of glycine-glutamine, glycine-glutamic acid, alanine-glutamine, alanine-glutamic acid and acetylated forms thereof.

[0052] The method according to any aspect of the present invention, wherein the probiotic strain is selected from the group consisting of Bacillus subtilis DSM 32315, Bacillus subtilis DSM 32540, Bacillus subtilis DSM 32592 and mixtures thereof, and / or wherein the composition further comprises one or more plant extracts selected from turmeric extract and green tea extract.

[0053] A therapeutically effective dose or therapeutically effective amount of the composition according to any aspect of the present invention can be administered to a subject in need thereof. A "therapeutically effective dose or therapeutically effective amount" refers to an amount that is capable of inducing the regeneration of at least one cell and, in the long term, can result in a positive therapeutic response in a subject in need of tissue repair or regeneration, such as restoration of function and / or an amount that results in the generation of new tissue at the treatment site. Thus, for example, a "positive therapeutic response" would be an improvement in an aging-related disease or condition associated with the treatment, and / or an improvement in one or more symptoms of an aging-related disease or condition associated with the treatment, such as restored tissue function, reduced pain, improved endurance, increased strength, increased mobility, and / or improved cognitive function. The exact dosage required will vary from subject to subject, depending on the species, age, and general condition of the subject, the severity of the condition being treated, the mode of administration, and the like. In any individual case, an appropriate "effective" amount can be determined by one of ordinary skill in the art using routine experimentation based on the information provided herein.

[0054] BRIEF DESCRIPTION OF THE DRAWINGS

[0055] Figure 1 is a graph showing changes in methylation of the 18 subjects, demonstrating a decrease in biological age of the subjects after taking a food supplement according to any aspect of the present invention. Example

[0056] While the foregoing describes preferred embodiments, as will be appreciated by those skilled in the art, changes or modifications may be made in design, construction or operation without departing from the scope of the claims. For example, the changes are intended to be covered by the scope of the claims.

[0057] Example 1

[0058] A proof-of-concept study to evaluate the efficacy of a dietary supplement ( Effects of FORCE (Bacillus subtilis DSM 32315, L-alanyl-L-glutamine, turmeric extract, green tea extract, zinc, vitamin B6, D-biotin, vitamin B12, vitamin D, pantothenic acid) on the production of short-chain fatty acids and the microbiome in healthy subjects

[0059] Proof-of-concept study to investigate food supplements The effects of Force (consisting of probiotics, dipeptides, plant extracts, and vitamins and minerals) on changes in the gut microbiota and short-chain fatty acid production were evaluated. Furthermore, fecal and blood biomarkers related to the gut barrier and GI health were assessed. Furthermore, tolerability and parameters of intestinal function (defecation frequency, stool consistency, and GI symptoms) were assessed during the study intervention period.

[0060] Test products, dosages and administration methods:

[0061] FORCE contains 2 billion colony-forming units (CFU) of Bacillus subtilis DSM 32315, 290 mg L-alanyl-L-glutamine, 90 mg turmeric extract (approximately 70-80% curcuminoids), 90 mg green tea extract (approximately 50% EGCG), 5 mg zinc, 0.56 mg vitamin B6, 20 μg D-biotin, 0.75 μg vitamin B12, 4 μg vitamin D, and 2.4 mg pantothenic acid.

[0062] Dosage: 1 capsule in the morning (flexible, can be taken with or without breakfast) and 1 capsule in the evening (flexible, can be taken with or without dinner), without chewing, with water.

[0063] Intervention duration: 2-week run-in period, followed by 4-week intervention period and 2-week follow-up period.

[0064] Evaluation criteria:

[0065] The main focus of the said study is to determine The effects of Force on changes in short-chain fatty acid (SCFA) production in feces and blood during a 4-week intake phase were investigated. In addition, the gut microbiota, assessed by 16S-rRNA, was analyzed.

[0066] Statistical methods:

[0067] This study was conducted as an exploratory proof-of-concept study. Changes over time were investigated. All data obtained in the study, as well as data recorded in the eCRF, questionnaires, and diaries, were presented and summarized using descriptive statistics or frequency tables, as appropriate. All statistical tests were two-sided. A p-value of less than 5% was considered statistically significant. Sequencing companies applied bioinformatics to microbiome analysis.

[0068] Results and Conclusions:

[0069] A significant increase in the SCFA butyrate was observed during the study (baseline vs. 2 weeks, p=0.0278, baseline vs. 4 weeks, p=0.0728). To what extent this can be attributed primarily to the metabolism of the probiotic Bacillus subtilis and the dipeptide provided, or whether this is due to further changes in the microbiota associated with an increase in amino acid converters, cannot be concluded from the data. However, the data clearly demonstrate an effect of the supplement on the microbiome, which also resulted in changes in gut biomarkers, but not in stool frequency and consistency. A significant increase in secretory IgA was determined (baseline vs. 4 weeks, p=0.0151 (n=17)), suggesting an immunostimulatory effect.

[0070] Possibly due to a rather unhealthy diet characterized by low fiber and low intake of fruits and vegetables, relatively high levels of biomarkers of leaky gut and calprotectin were identified in the study cohort despite the absence of symptomatic clinical signs or a history of any gastrointestinal disorder. In addition, significant reductions in total cholesterol (baseline vs. 4 weeks, p=0.0037) and LDL cholesterol (baseline vs. 4 weeks, p=0.0313) were observed. Possibly due to Whether FORCE components affect the microbiome or other direct modes of action, highly significant reductions in fasting total GLP-1 and PYY were observed (baseline vs. week 4, p < 0.001).

[0071] in conclusion:

[0072] In short, in use During the 4-week FORCE intervention, the exploratory studies demonstrated a significant increase in the SCFA butyrate in fecal samples, accompanied by interesting changes in the microbiome, with elevations in known butyrate producers such as Faecalibacterium prausnitzii, as well as metabolic biomarkers and gut hormones.

[0073] Example 2

[0074] Determine biological age differences (start and end) using blood samples

[0075] Accepted from 18 DNA was extracted from whole blood samples obtained from subjects taking SAMANAFORCE (as described in Example 1). Specifically, 18 male subjects with poor dietary habits were included in the study. The methylation patterns of their blood cells were analyzed before and after 4 weeks of SAMANAFORCE supplementation. This resulted in 36 samples analyzed for this trial. DNA methylation analysis was performed by Elysium Health Inc. using the APEX platform, which uses the Horvath Clock to detect blood samples. The results are shown in Figure 2. Figure 1 As shown. Additives such as butyrate-forming synbiotics and curcumin appear to show a trend towards slowing aging. For all 18 subjects, biological age was determined by their DNA methylation profiles at two time points. Table 1 shows the difference in biological age between subject V1 (starting point) and V3 (end point): Age difference = V1-V3; if the value is positive, aging is slowing down because V1>V3; if it is negative, aging is accelerating and the subject is older at V3 than at the beginning of the study (V1<V3). * = Subject 108 had a fever at time point V3 and was therefore invalid.

[0076] Table 1 shows the biological age, assigned genotype ID, and predicted sex measured using the Index for all samples that passed QC.

[0077]

[0078]

Claims

1. A composition for inducing at least one cell regeneration, the composition comprising: - at least one probiotic strain of Bacillus subtilis, and - at least one dipeptide selected from the group consisting of glycine-glutamine, glycine-glutamic acid, alanine-glutamine, alanine-glutamic acid and acetylated forms thereof.

2. Use of a composition for reducing the epigenetic age of at least one subject, the composition comprising: - at least one probiotic strain of Bacillus subtilis, and - at least one dipeptide selected from the group consisting of glycine-glutamine, glycine-glutamic acid, alanine-glutamine, alanine-glutamic acid and acetylated forms thereof.

3. The use according to claim 2, wherein the subject is a mammal, preferably, the mammal is selected from the group consisting of mice, rats, guinea pigs, dogs, mini pigs, humans, cows, sheep, pigs, goats, horses, donkeys and mules.

4. The use according to claim 1, wherein the cell is a mammalian cell, preferably a human cell.

5. Use according to any one of the preceding claims, wherein the composition further comprises one or more plant extracts selected from turmeric extract and green tea extract.

6. Use according to any one of the preceding claims, wherein the probiotic strain is selected from the group consisting of Bacillus subtilis DSM 32315, Bacillus subtilis DSM 32540, Bacillus subtilis DSM 32592 and mixtures thereof.

7. Use according to any one of the preceding claims, wherein the total amount of the probiotic strain and the dipeptide is at least 40% by weight of the composition and / or the total amount of the plant extract is at least 10% by weight of the total weight of the composition.

8. The use according to any of the preceding claims, wherein the composition comprises an enteric coating, wherein the enteric coating comprises at least one of: methyl acrylate-methacrylic acid copolymer, cellulose acetate phthalate (CAP), cellulose acetate succinate, hydroxypropyl methylcellulose phthalate, hydroxypropyl methylcellulose acetate succinate (hydroxypropyl methylcellulose acetate succinate), polyvinyl acetate phthalate (PVAP), methyl methacrylate-methacrylic acid copolymer, shellac, cellulose acetate trimellitate, sodium alginate and zein.

9. A composition for inducing at least one cell regeneration, the composition comprising: - at least one probiotic strain of Bacillus subtilis, and - at least one dipeptide selected from the group consisting of glycine-glutamine, glycine-glutamic acid, alanine-glutamine, alanine-glutamic acid and acetylated forms thereof.

10. A composition for reducing the epigenetic age of at least one subject, the composition comprising: - at least one probiotic strain of Bacillus subtilis, and - at least one dipeptide selected from the group consisting of glycine-glutamine, glycine-glutamic acid, alanine-glutamine, alanine-glutamic acid and acetylated forms thereof.

11. The composition according to claim 9 or 10, wherein the composition further comprises one or more plant extracts selected from turmeric extract and green tea extract.

12. The composition according to any one of claims 9 to 11, wherein the probiotic strain is selected from the group consisting of Bacillus subtilis DSM 32315, Bacillus subtilis DSM 32540 and Bacillus subtilis DSM 32592.

13. A method of reducing the epigenetic age of at least one subject, the method comprising administering to a subject in need thereof a composition comprising: - at least one probiotic strain of Bacillus subtilis, and - at least one dipeptide selected from the group consisting of glycine-glutamine, glycine-glutamic acid, alanine-glutamine, alanine-glutamic acid and acetylated forms thereof.

14. A method of inducing cell regeneration in at least one cell, the method comprising administering to a subject in need thereof a composition comprising: - at least one probiotic strain of Bacillus subtilis, and - at least one dipeptide selected from the group consisting of glycine-glutamine, glycine-glutamic acid, alanine-glutamine, alanine-glutamic acid and acetylated forms thereof.

15. The method according to claim 13 or 14, wherein the probiotic strain is selected from the group consisting of Bacillus subtilis DSM 32315, Bacillus subtilis DSM 32540 and Bacillus subtilis DSM 32592.

16. The method according to any one of claims 13 to 15, wherein the composition further comprises one or more plant extracts selected from turmeric extract and green tea extract.

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