Orally administered compositions obtained from lysates of probiotic microorganisms for use as anti-aging agents - Patent Application 20070122993

Abstract

1. An orally administered composition for use in the prevention and / or treatment of oxidative stress associated with ageing, comprising a lysate of probiotic microorganisms of the genera Bacillus, Bifidobacterium, Lactobacillus, Saccharomyces, and Streptococcus in the form of a dry powder.

Description

[Technical Field]

[0001] The present invention describes a composition containing a lysate of probiotic microorganisms in the form of a dry powder as an anti-aging agent, which is highly safe for long-term consumption, and is used as a food supplement for preventing and delaying human aging.

[0002] The present invention therefore falls within the field of therapy aimed at delaying or preventing the onset of energy loss and changes in appearance in humans and animals due to aging. [Background technology]

[0003] According to the World Health Organization (2018), life expectancy has increased rapidly in recent decades, reflecting the positive developments that society has experienced. While this increase is undoubtedly positive, aging presents new challenges as it is directly linked to cognitive, biological, and physical degeneration, increasing the risk of contracting certain diseases. Age-related diseases are posing the greatest threat to health in the 21st century.

[0004] Aging is a universal, multifactorial, progressive, endogenous process characterized by degeneration and progressive loss of function. According to the World Health Organization (WHO), a person's functional capacity begins to decline after reaching adulthood, after which aging, defined as a biological process, occurs. Living organisms undergo a series of structural and functional changes that occur over time, not as a result of disease or accident. As we age, we experience joint problems that affect flexibility, muscle loss that leads to fatigue, and bone mineral density that increases the susceptibility to fractures. The rate at which a person's functional capacity declines appears to be determined, at least in part, by their behavior and the environmental living conditions they are exposed to throughout their life. Diet, physical activity, smoking, harmful alcohol consumption, and exposure to harmful substances are influential factors.

[0005] One of the most frequent recommendations to mitigate, slow or delay the effects of aging as much as possible and extend healthy lifespan is for people to get enough physical activity and eat a healthy diet based on vegetables, fruits, whole grains, foods high in dietary fiber, and lean protein sources such as fish that contain natural antioxidants and are low in saturated fat and salt.

[0006] Another theory explaining the aging process is the accumulation of reactive oxygen species (ROS), which accumulate within cells and damage proteins, lipids, and DNA. When the efficiency of the endogenous antioxidant system declines, the presence of antioxidants alone is no longer enough to compensate for the increased ROS, resulting in oxidative stress, which plays a key role in aging and the development of age-related diseases such as certain cancers, diabetes, and heart failure, as well as digestive disorders. Numerous studies have shown that resistance to oxidative stress is crucial for maintaining health and mitigating the adverse effects of aging. Therefore, nutritional interventions using food-grade antioxidant compounds and foods are considered as a useful option for improving the health and quality of life of older adults. However, currently, no treatments have been proven to be truly effective in slowing the aging process.

[0007] Human aging appears to involve a large number of extrinsic and intrinsic factors interacting with each other to a great extent, which raises the problem of finding easily administered products that will mitigate or slow the aging process by extending human healthspan.

[0008] There are many developments in the literature on the dynamics of the gut microbiota and its variations over time, as well as other factors that may alter it and contribute to human aging.

[0009] As a result, the field is actively working to confront the technical challenges posed by current treatments and develop orally administered treatments and compositions to slow the effects of aging, as evidenced by the following patent documents:

[0010] Patent No. US8492353B2 provides an anti-aging composition that contains at least one member selected from the group consisting of ascorbic acid, ascorbic acid derivatives and their salts (A), and purine nucleic acid-related substances (B), and that can effectively delay skin aging, particularly alleviating skin pigmentation. The present invention also provides a method for enhancing the anti-aging effect of ascorbic acids. The present invention provides an anti-aging composition.

[0011] Patent application US2020345694A1 relates to an anti-aging composition containing cytochalasin D or SAG and a method for selecting an anti-aging substance, and provides a novel anti-aging substance and a method for discovering a novel anti-aging substance.

[0012] Patent application JP2003155234A describes an anti-aging composition containing a methyl group donor compound, particularly betaine, and optionally further containing one or more substances selected from the group consisting of choline, methionine, and carnitine. This composition, whether administered orally or transdermally, inhibits an increase in blood homocysteine ​​and prevents aging in the human body caused by an increase in homocysteine.

[0013] Nevertheless, none of the prior art documents disclose a stable composition that functions to prevent and delay aging upon oral ingestion, as described herein, which is composed of a dried bacterial lysate.

[0014] Furthermore, Spanish patent applications P201930242 and P201930280 (patent applications of the present inventor, the contents of which are incorporated herein by reference) describe a composition for modulating the human intestinal microflora obtained from a lysate of probiotic microorganisms, a method for obtaining the same, and a food supplement containing the composition, which is useful for the prevention and treatment of disorders caused by, or at least promoted by, an abnormality in the human intestinal microflora. Summary of the Invention [Problem to be solved by the invention]

[0015] The present invention therefore aims to solve the problems of the prior art by using an orally administered composition obtained from a lysate of a probiotic microorganism for the prevention and retardation of ageing in humans. [Means for solving the problem]

[0016] Brief description of the invention In a first aspect, the present invention provides an orally administered composition for preventing and delaying aging, comprising, by weight percentage relative to the total, lysates of probiotic microorganisms in the form of a dry powder: - 5% to 30% of bacterial lysates of the genus Bacillus, - 3% to 25% of bacterial lysate of the genus Bifidobacterium, - 15% to 35% of bacterial lysates of the genus Lactobacillus, - 30% to 65% of bacterial lysates of the genus Saccharomyces; - 1.5% to 10% of bacterial lysates of the genus Streptococcus, and for use in the prevention and / or treatment of oxidative stress associated with aging.

[0017] In a further aspect, the present invention relates to the use of the composition of the present invention as a food supplement for the prevention and delay of aging in animal or human cells.

[0018] In a further aspect, the present invention relates to a pharmaceutical composition comprising an effective pharmaceutical amount of a composition according to the first aspect of the invention and a pharmaceutically acceptable excipient.

[0019] In a further aspect, the present invention relates to the use of an orally administered composition for preventing and delaying aging, which composition comprises a lysate of probiotic microorganisms in the form of a dry powder in the following amounts, expressed as percentages by weight relative to the total: - 5% to 30% of bacterial lysates of the genus Bacillus, - 3% to 25% of bacterial lysate of the genus Bifidobacterium, - 15% to 35% of bacterial lysates of the genus Lactobacillus, - 30% to 65% of bacterial lysates of the genus Saccharomyces; - 1.5% to 10% of bacterial lysates of Streptococcus spp.

[0020] To complement this specification and for a better understanding of the present invention, attached as an integral part of this specification are figures that, for illustrative and non-limiting purposes, show graphs of the experimental results obtained in Examples 1 to 4 collected herein. [Brief explanation of the drawings]

[0021] [Figure 1] Figure 1 shows the standard curve of HeLa cell protein Ct values ​​versus the logarithm of protein (ng). The cycle number at the threshold (Ct value) for each sample was interpolated on the curve to calculate the relative activity (RTA) of telomerase. [Figure 2] Figure 2 shows a bar chart (RTA) of Q-TRAP results for in vitro treatments in primary fibroblast cultures. Data were pooled by condition and treatment time. [Figure 3] Figure 3 shows the growth curves of untreated adult primary human fibroblasts (control - DMSO) and cells treated with IG (IG_1, IG_2, IG_3) under standard conditions. Each point on the population curve represents the average of triplicates for each cell passage. [Figure 4] Figure 4 shows the growth curves of untreated adult primary human fibroblasts (control-MSO) and treated IGs (IG_1, IG_2, IG_3) under oxidizing conditions (10 μM H2O2). Each point on the population curve represents the mean value of triplicates for each cell step. [Figure 5a]In Figure 5, a histogram shows the distribution of telomere lengths for a representative sample. The bars represent the relative frequency of each specific normalized fluorescence intensity (X-axis). The 20th percentile (red bar) indicates the length at which 20% of the telomeres were observed. The median telomere length (MTL) and average telomere length (ATL) are also shown in the histogram. This histogram also allows for analysis of telomere length variation. [Figure 5b] In Figure 5, a histogram shows the distribution of telomere lengths for a representative sample. The bars represent the relative frequency of each specific normalized fluorescence intensity (X-axis). The 20th percentile (red bar) indicates the length at which 20% of the telomeres were observed. The median telomere length (MTL) and average telomere length (ATL) are also shown in the histogram. This histogram also allows for analysis of telomere length variation. [Figure 6] Figure 6 shows both DAPI (blue) and fluorescence indicating telomere speckles (pink and white) within each nucleus. [Figure 7a] Figure 7 shows bar graphs of TAT results. Figure 7a shows median telomere length. Figure 7b shows 20th percentile. Figure 7c shows % of telomeres <3 kbp. [Figure 7b] Figure 7 shows bar graphs of TAT results. Figure 7a shows median telomere length. Figure 7b shows 20th percentile. Figure 7c shows % of telomeres <3 kbp. [Figure 8a] Figure 8 shows bar graphs of TAT results. Figure 8a shows median telomere length. Figure 8b shows 20th percentile. Figure 8c shows % of telomeres <3 kbp. [Figure 8b] Figure 8 shows bar graphs of TAT results. Figure 8a shows median telomere length. Figure 8b shows 20th percentile. Figure 8c shows % of telomeres <3 kbp. [Figure 8c] Figure 8 shows bar graphs of TAT results. Figure 8a shows median telomere length. Figure 8b shows 20th percentile. Figure 8c shows % of telomeres <3 kbp. [Figure 9] Figure 9 shows a bar graph of telomere shortening rates under standard conditions and with different time and treatments. [Figure 10]FIG. 10 is a bar graph showing the shortening of telomere ratios under oxidative stress conditions depending on the treatment and time. [Figure 11] FIG. 11 is a graph showing the effect of a composition according to the present invention at low doses on the lifespan of C. elegans (N2). [Figure 12] FIG. 12 is a graph showing the effect of a composition according to the present invention at intermediate doses on the lifespan of C. elegans (N2). [Figure 13] FIG. 13 is a graph showing the effect of a high dose of a composition according to the present invention on the lifespan of C. elegans (N2). [Figure 14] FIG. 14 shows the survival curve of C. elegans (N2) obtained from a population treated with a composition according to the invention (1 mg / mL), using the computer program GraphPad Prism. [Figure 15] Figure 15 shows the average mobility of populations of C. elegans treated with a composition according to the invention at different doses. [Figure 16] FIG. 16 shows the average mobility of populations of C. elegans treated with a composition according to the invention at doses of 0.1, 0.5 and 1 mg / mL. [Figure 17] FIG. 17 shows the bending frequency of populations of C. elegans treated with a composition according to the invention at doses of 0.1, 0.5 and 1 mg / mL. [Figure 18] FIG. 18 shows the degree of dispersion of the nematode C. elegans treated with a composition according to the invention at doses of 0.1, 0.5 and 1 mg / mL. [Figure 19] Figure 19 shows the antioxidant activity of a composition according to the present invention evaluated at different doses (10 to 400 μg / mL) in C. elegans N2. The survival rate after oxidative stress with H2O2 is shown. Vitamin C (10 μg / mL) was added as a positive control. Data are from a single experiment. [Figure 20]Figure 20 shows the antioxidant activity of a composition according to the present invention evaluated at different doses in C. elegans N2. The survival rate after oxidative stress with H2O2 is shown. Vitamin C (10 μg / mL) was added as a positive control. Data are the average of two independent experiments. [Figure 21] FIG. 21 shows the relative increase in survival rate of C. elegans when an effective amount of the composition of the present invention was administered compared to the control condition, showing an increase of 88.5%. DETAILED DESCRIPTION OF THE INVENTION

[0022] Detailed Description of the Invention As indicated in the previous section, the present inventors have recognized a need to develop compositions that can be administered as anti-aging agents, that are easy to administer, and that do not exhibit toxicity.

[0023] Based on this, they developed an orally administered composition containing a lysate of the microorganism along with other ingredients for the composition, which proved to be extremely beneficial for humans and animals, as it exhibited antioxidant properties, was proven to be a promoter of cell growth, and was a highly effective anti-aging agent.

[0024] Thus, in a first aspect, the present invention relates to an orally administered composition for preventing and retarding aging, which comprises a lysate of a probiotic microorganism in the form of a dry powder, in percentages by weight relative to the total: - 5% to 30% of bacterial lysates of the genus Bacillus, - 3% to 25% of bacterial lysate of the genus Bifidobacterium, - 15% to 35% of bacterial lysates of the genus Lactobacillus, - 30% to 65% of bacterial lysates of the genus Saccharomyces; - 1.5% to 10% of bacterial lysates of Streptococcus spp. and is used to prevent and / or treat oxidative stress associated with aging.

[0025] According to the invention, the composition may comprise 20% to 99.5% by weight of bacterial lysate, in particular 25% to 95% by weight of bacterial lysate, more in particular 50%±10% by weight of bacterial lysate.

[0026] In the context of the present invention, bacterial lysate is understood as the product obtained after a step of culturing and subsequent mechanical or chemical disruption of said bacterial cells in order to obtain a product containing microbial fragments and all the components contained therein. Likewise, in this specification, these lysates are indicated as dried bacterial lysates, since, due to the method of their acquisition, they are subsequently subjected to drying techniques, resulting in dried bacterial lysates in powder form.

[0027] In a preferred embodiment of the present invention, the bacterial lysate of the genus Bacillus is selected from the group consisting of Bacillus clausii, Bacillus coagulans, Bacillus licheniformis, Bacillus pumilus, Bacillus mesentericus, Bacillus subtilis, and combinations thereof, or at least Bacillus coagulans, Bacillus licheniformis, Bacillus mesentericus, Bacillus subtilis, and optionally Bacillus clausii. clausii).

[0028] In other embodiments, the bacterial lysate of the genus Bifidobacterium is selected from the group consisting of Bifidobacterium animalis subsp lactis, Bifidobacterium bifidum, Bifidobacterium breve, Bifidobacterium lactis, Bifidobacterium longum, Bifidobacterium infantis, Bifidobacterium animalis, Bacillus paralicheniformis, and combinations thereof, or at least Bifidobacterium breve. breve, and Bifidobacterium lactis lactis, and optionally Bifidobacterium bifidum.

[0029] In another embodiment, the bacterial lysate of the genus Lactobacillus is selected from the group consisting of Lactobacillus acidophilus, Lactobacillus bulgaricus, Lactobacillus casei, Lactobacillus fermentum, Lactobacillus paracasei, Lactobacillus plantarum, Lactobacillus reuteri, Lactobacillus rhamnosus, Lactobacillus salivarius, Lactobacillus helveticus, Lactobacillus gasseri, Lactobacillus The bacterial strains may be selected from the group consisting of Lactobacillus gasseri, Lactobacillus johnsonii, Lactobacillus brevis, Lactobacillus kefiri, and combinations thereof, or at least Lactobacillus acidophilus, Lactobacillus bulgaricus, Lactobacillus casei, Lactobacillus reuteri, and Lactobacillus rhamnosus, and optionally Lactobacillus fermentum.

[0030] In other specific embodiments of the present invention, the bacterial lysate is selected from the group consisting of Saccharomyces cerevisiae, Saccharomyces boulardii, and combinations thereof.

[0031] In other specific embodiments of the present invention, the bacterial lysate of the genus Streptococcus is of the species Streptococcus thermophilus, Streptococcus salivarius, and combinations thereof.

[0032] In a preferred embodiment of the present invention, the composition of the present invention comprises a Bacillus bacterial lysate in an amount of 5% to 30%, preferably 8% to 25%, and more preferably 10% to 18% by weight. In preferred embodiments, the amount of Bacillus bacterial lysate can be 5%, 6%, 7%, 8%, 9%, or 10%. In other preferred embodiments, the amount of Bacillus bacterial lysate can be 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, or 30%.

[0033] In a preferred embodiment of the present invention, the composition of the present invention comprises a Bifidobacterium lysate in an amount by weight of 3% to 25%, preferably 5% to 20%, and more preferably 8% to 15%. In preferred embodiments, the amount of Bifidobacterium lysate can be 3%, 4%, 5%, 6%, 7%, 8%, 9%, or 10%. In other preferred embodiments, the amount of Bifidobacterium lysate can be 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, or 25%.

[0034] In a preferred embodiment of the present invention, the composition of the present invention comprises a weight percentage of Lactobacillus lysate between 15% and 35%, preferably between 15% and 30%, more preferably between 20% and 25%.In a preferred embodiment, the amount of Lactobacillus lysate can be 15%, 16%, 17%, 18%, 19% or 20%.In another preferred embodiment, the amount of Lactobacillus lysate can be 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34% or 35%.

[0035] In a preferred embodiment of the present invention, the composition comprises a bacterial lysate of the genus Saccharomyces in an amount by weight percentage between 30% and 65%, preferably between 50% and 58%, more preferably between 35% and 60%. In a preferred embodiment, the amount of bacterial lysate of the genus Saccharomyces may be 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48% or 49%. In other preferred embodiments, the amount of bacterial lysate of the genus Saccharomyces may be 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64% or 65%.

[0036] In a preferred embodiment of the present invention, the composition comprises a bacterial lysate of the genus Streptococcus in an amount of 1.5% to 10%, preferably 2% to 7%, more preferably 3% to 6% by weight. In a preferred embodiment, the amount of bacterial lysate of the genus Streptococcus can be 1.5%, 1.6%, 1.7%, 1.8%, 1.9%, 2%, 2.1%, 2.2%, 2.3%, 2.4%, 2.5%, 2.6%, 2.7%, 2.8%, 2.9% or 3%. In other preferred embodiments, the amount of bacterial lysate of the genus Streptococcus may be 3.5%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5% or 10%.

[0037] In a particular embodiment of the invention, the composition for use comprises a lysate of a probiotic microorganism in an amount as a percentage by weight of the total lysate of the composition: - approximately 10% to 25% of bacterial lysates of the genus Bacillus; - approximately 4% to 20% of bacterial lysates of the genus Bifidobacterium, - approximately 17% to 30% of bacterial lysates of the genus Lactobacillus; -about 35% to 60% of yeast lysates of the genus Saccharomyces, - Approximately 2% to 8% of bacterial lysates of Streptococcus spp.

[0038] This composition is referred to as composition "A."

[0039] In a particular embodiment of the invention, the composition for use comprises a lysate of a probiotic microorganism in an amount as a percentage by weight of the total lysate of the composition: - Approximately 14% to 20% of bacterial lysates of the genus Bacillus, - approximately 6% to 15% of bacterial lysates of the genus Bifidobacterium, - approximately 18% to 25% of bacterial lysates of the genus Lactobacillus; - approximately 45% to 55% of yeast lysates of the genus Saccharomyces; - Approximately 2% to 5% of bacterial lysates of Streptococcus spp.

[0040] This composition is referred to as composition "B."

[0041] In a particular embodiment of the invention, the composition for use comprises a lysate of a probiotic microorganism in an amount as a percentage by weight of the total lysate of the composition: - 16% of bacterial lysates of Bacillus spp. - 8% of Bifidobacterium genus bacterial lysate, - 21% of bacterial lysates of Lactobacillus spp. - 52% of bacterial lysates of Saccharomyces spp. - 3% of bacterial lysate of Streptococcus spp.

[0042] This composition is referred to as composition "C."

[0043] In a particular embodiment of the invention, the composition for use comprises a lysate of a probiotic microorganism in an amount as a percentage by weight of the total lysate of the composition: - 5% to 30% bacterial lysate of Bacillus coagulans, Bacillus licheniformis, Bacillus mesentericus, Bacillus subtilis, and optionally Bacillus clausii, - 3% to 25% bacterial lysate of Bifidobacterium breve, and Bifidobacterium lactis, and optionally Bifidobacterium bifidum, - 15% to 35% bacterial lysate of Lactobacillus acidophilus, Lactobacillus bulgaricus, Lactobacillus casei, Lactobacillus reuteri, and Lactobacillus rhamnosus, and optionally Lactobacillus fermentum, - 30% to 65% of a bacterial lysate of Saccharomyces cerevisiae, -1.5% to 10% of Streptococcus thermophilus lysate.

[0044] This composition is referred to as composition "D."

[0045] In a particular embodiment of the invention, the composition for use comprises the following lysates of probiotic microorganisms in amounts as a percentage by weight of the total lysate of the composition: - 10% to 25% of a bacterial lysate of Bacillus coagulans, Bacillus licheniformis, Bacillus mesentericus, Bacillus subtilis, and, optionally, Bacillus clausii, - 4% to 20% bacterial lysate of Bifidobacterium breve, Bifidobacterium lactis and, optionally, Bifidobacterium bifidum, - 17% to 30% bacterial lysate of Lactobacillus acidophilus, Lactobacillus bulgaricus, Lactobacillus casei, Lactobacillus reuteri, and Lactobacillus rhamnosus, and optionally Lactobacillus fermentum, -35% to 60% of bacterial lysate of Saccharomyces cerevisiae, -2% to 8% of Streptococcus thermophilus bacterial lysate.

[0046] This composition is referred to as composition "E."

[0047] In a particular embodiment of the invention, the composition for use comprises the following lysates of probiotic microorganisms in amounts as a percentage by weight of the total lysate of the composition: - 14% to 20% of a bacterial lysate of Bacillus coagulans, Bacillus licheniformis, Bacillus mesentericus, Bacillus subtilis, and, optionally, Bacillus clausii, 6% to 15% bacterial lysate of Bifidobacterium breve, Bifidobacterium lactis and, optionally, Bifidobacterium bifidum, - 18% to 25% bacterial lysate of Lactobacillus acidophilus, Lactobacillus bulgaricus, Lactobacillus casei, Lactobacillus reuteri, Lactobacillus rhamnosus, and, optionally, Lactobacillus fermentum, - 45% to 55% of bacterial lysate of Saccharomyces cerevisiae, -2% to 5% of Streptococcus thermophilus bacterial lysate.

[0048] This composition is designated as composition "F."

[0049] In a particular embodiment of the invention, the composition for use comprises the following lysates of probiotic microorganisms in amounts as a percentage by weight of the total lysate of the composition: - 0.1% to 10% of Bacillus coagulans, 1% to 12% of Bacillus licheniformis, 1% to 12% of Bacillus mesentericus, 0.1% to 10% of Bacillus subtilis, and optionally 0.1% to 5% of Bacillus clausii, - 0.1% to 12% Bifidobacterium breve, 0.1% to 10% Bifidobacterium lactis, and optionally 0.5% to 8% Bifidobacterium bifidum, - 0.3% to 15% of Lactobacillus acidophilus, 0.3% to 15% of Lactobacillus bulgaricus, 0.3% to 15% of Lactobacillus casei, 0.3% to 12% of Lactobacillus reuteri, 0.5% to 10% of Lactobacillus rhamnosus, and optionally 0.5% to 10% of Lactobacillus fermentum, - Saccharomyces cerevisiae 35% to 60% -Streptococcus thermophilus, 2% to 8%

[0050] This composition is referred to as composition "G."

[0051] In a particular embodiment of the invention, the composition for use comprises the following lysates of probiotic microorganisms in amounts as a percentage by weight of the total lysate of the composition: - 0.1% to 6% of Bacillus coagulans, 2% to 10% of Bacillus licheniformis, 2% to 10% of Bacillus mesentericus, 0.3% to 8% of Bacillus subtilis, and optionally 0.1% to 4% of Bacillus clausii, - Bifidobacterium breve 1% to 10%, Bifidobacterium lactis 1% to 8%, and optionally Bifidobacterium bifidum 1% to 6%, - Lactobacillus acidophilus 1% to 12%, Lactobacillus bulgaricus 1% to 12%, Lactobacillus casei 1% to 10%, Lactobacillus reuteri 1% to 7%, Lactobacillus rhamnosus 0.3% to 7%, and optionally Lactobacillus fermentum 1% to 7%, -Saccharomyces cerevisiae 45% to 55% -Streptococcus thermophilus: 2% to 5%

[0052] This composition is designated as composition "H."

[0053] In a particular embodiment of the invention, the composition for use comprises the following lysates of probiotic microorganisms in amounts as a percentage by weight of the total lysate of the composition: - 0.1% to 4% of Bacillus coagulans, 3% to 8% of Bacillus licheniformis, 3% to 8% of Bacillus mesentericus, 0.1% to 10% of Bacillus subtilis, and optionally 0.5% to 7% of Bacillus clausii, - 1.5% to 8% Bifidobacterium breve and 1.5% to 5% Bifidobacterium lactis, and optionally 1.5% to 4% Bifidobacterium bifidum, - 2% to 10% Lactobacillus acidophilus, 2% to 10% Lactobacillus bulgaricus, 2% to 8% Lactobacillus casei, 1.5% to 5% Lactobacillus reuteri, 0.5% to 5% Lactobacillus rhamnosus, and optionally 2% to 5% Lactobacillus fermentum, - Saccharomyces cerevisiae 47% to 56% -Streptococcus thermophilus, 2% to 4%

[0054] This composition is referred to as composition "I."

[0055] In certain embodiments of the present invention, the composition for use comprises a lysate of a probiotic microorganism in the form of a dry powder in an amount as a percentage by weight of the total as disclosed in composition "A", "B", "C", "D", "E", "F", "G", "H" or "I".

[0056] In other specific embodiments of the present invention, the compositions of the present invention may comprise another additional ingredient or additive selected from the group consisting of riboflavin, vitamin C, manganese, selenium, zinc, vitamin D, and magnesium.

[0057] Riboflavin, vitamin C, magnesium, selenium, and zinc protect cells from oxidative damage. Vitamin D, manganese, and zinc contribute to the maintenance of normal bones. Vitamin D and magnesium contribute to the normal formation of collagen for normal bone function. Vitamin D, vitamin C, zinc, and selenium contribute to the normal functioning of the immune system.

[0058] The following provides the amount that may be included of each of the additional components or additives that may optionally comprise the composition according to the first aspect of the present invention.

[0059] Table 1. A list of various additional ingredients that compositions subject of the present invention may contain, and the amounts in which these ingredients are found in each of the specific preferred embodiments of the present invention.

[0060] [Table 1]

[0061] In a particular embodiment of the present invention, the composition object of the present invention is obtained by a method comprising the following steps: - culturing the microbial strain selected for the preparation of the composition using standard conditions established for the microbial species described in this document; - filtering or centrifuging the culture until a biomass of the selected bacterial lysate is obtained; - Repeated freeze-thawing and sonication of the resulting biomass until at least 90%, preferably at least 95%, more preferably at least 99% bacterial lysis is achieved; The biomass of the bacterial lysate is dried by atomization or freeze-drying to obtain a powdered product containing all the bacterial strains selected to prepare the object of the composition of the invention.

[0062] Therefore, the compositions of the present invention are provided in powder form.

[0063] Due to the fact that the subject of the composition of the present invention is in powder form, the composition according to the first aspect of the present invention may itself be presented in a sealed sachet as is customary in the food and pharmaceutical industries.

[0064] Another form of food supplement according to the present invention is in the form of a capsule, such as a conventional gelatin capsule, containing the composition in powder form.

[0065] In a further aspect, the present invention relates to the use of the composition of the present invention as a food supplement for the prevention and delay of aging in animal or human cells.

[0066] In a further aspect, the present invention relates to a composition as defined herein for use as a medicament.

[0067] In a further aspect, the present invention relates to a composition as defined herein for use as an anti-ageing agent.

[0068] In a further aspect, the present invention relates to a pharmaceutical composition comprising an effective pharmaceutical amount of a composition according to the first aspect of the invention and a pharmaceutically acceptable excipient.

[0069] In the context of the present invention, what are known as anti-aging agents, which can also be called anti-maturation agents, are compounds that have the ability to combat aging, their effect being to slow down the effects of aging and the ability of cells to proliferate.

[0070] In the context of the present invention, the expression "pharmaceutical composition" refers to a formulation adapted to deliver a predetermined amount of one or more useful therapeutic agents to a cell, group of cells, organ or tissue.

[0071] The term "effective pharmaceutical amount" as used herein is understood as an amount that can provide a therapeutic effect and can be determined by commonly used means by those skilled in the art.

[0072] Also, in the context of the present invention, "pharmaceutically acceptable excipient" means a therapeutically inactive substance used to incorporate an active ingredient, which is acceptable to patients from a pharmacological / toxicological point of view and acceptable to the manufacturing pharmaceutical chemist from a physical / chemical point of view in terms of composition, formulation, stability, patient acceptability and bioavailability.

[0073] In the context of the present invention, food supplements are understood as foods intended to supplement the normal diet and consisting of concentrated sources of nutrients or other substances with a nutritional or physiological effect, in simple or complex form, sold in dosage forms, i.e. liquid and powder capsules, pastilles, tablets, pills and other similar forms, powder sachets, liquid ampoules, dropper bottles and other similar forms to be taken in small quantities, as defined in Directive 2002 / 46 / EC of the European Parliament.

[0074] In a particular embodiment of the present invention, the use of the composition as a food supplement involves oral administration of between 20 mg and 200 mg of the composition two to six times daily. Thus, the use of the composition as an anti-aging agent is precisely the object of the present invention, particularly for the prevention and / or treatment of oxidative stress associated with aging.

[0075] In certain embodiments of the present invention, the use of composition as food supplement comprises that dosage is orally administered to individual at least twice a day.And, in order to be effective in treatment and achieve desired effect, this dosage should be administered to patient at least twice a day, and at most 6 times a day.Preferably, administration to patient is 3, 4 or 5 times a day, most preferably 3 times a day.

[0076] In the context of the present invention, a dose is defined as the amount of a pharmaceutical agent containing the correct active ingredient that is efficient, effective, safe for the patient, and solves the indicated health problem.

[0077] Therefore, in the context of the present invention, a dose is determined to have an amount of the composition object of the present invention between 20 mg and 200 mg. In a preferred embodiment, a dose may contain 80 mg to 150 mg of the composition. In certain embodiments of the present invention, a dose may contain 20 mg, 30 mg, 40 mg, 50 mg, 60 mg, 70 mg, 80 mg, 90 mg, 20 mg, 100 mg, 110 mg, 120 mg, 130 mg, 140 mg, 150 mg, 160 mg, 170 mg, 180 mg, 190 mg, or 200 mg of the composition object of the present invention.

[0078] In another specific embodiment, the dose is determined to have an amount of the composition of the present invention between 1 μg and 20 mg. In a preferred embodiment, the dose can contain 5 μg to 20 mg of the composition. In a specific embodiment of the present invention, the dose can contain 20 μg, 30 μg, 40 μg, 50 μg, 60 μg, 70 μg, 80 μg, 90 μg, 20 μg, 100 μg, 110 μg, 120 μg, 130 μg, 140 μg, 150 μg, 160 μg, 170 μg, 180 μg, 190 μg, or 200 μg, 500 μg, 1 mg, 2 mg, 3 mg, 4 mg, 5 mg, 6 mg, 7 mg, 8 mg, 9 mg, or 10 mg. .

[0079] In another specific embodiment, the composition for use comprises administering to a subject an effective amount of the composition. The amount of the compound for preventing and / or treating aging-related oxidative stress can be from a low amount of about 0.1 μg / ml, about 0.2 μg / ml, or about 0.5 μg / ml to a high amount of about 100 μg / ml, about 200 μg / ml, or about 500 μg / ml. For example, the amount of the compound that retards aging may be from about 0.1 μg / ml to about 100 μg / ml, from about 0.2 μg / ml to about 200 μg / ml, from about 0.5 μg / ml to about 500 μg / ml, from about 0.1 μg / ml to about 10 μg / ml, from about 0.1 μg / ml to about 20 μg / ml, from about 0.1 μg / ml to about 50 μg / ml, from about 0.5 μg / ml to about 20 μg / ml, from about 0.5 μg / ml to about 50 μg / ml, from about 0.5 μg / ml to about 100 μg / ml , about 0.5 to about 200 μg / ml, about 0.5 μg / ml to about 500 μg / ml, about 15 μg / ml to about 25 μg / ml, about 15 μg / ml to about 50 μg / ml, about 18 μg / ml to about 30 μg / ml, about 18 μg / ml to about 50 μg / ml, or about 1 μg / ml to about 10 μg / ml, or about 2 μg / ml to about 8 μg / ml, or about 10 μg / ml to about 30 mg / ml, or about 50 μg / ml to about 30 mg / ml. These amounts can be administered daily, once a week, once a week or more, or two to six times a day. For example, a daily dose of between 50 μg / ml and 30 mg / ml.

[0080] As used herein, the term "preventing" refers to administering a compound prior to the onset of clinical symptoms of a disease or condition, such that the physical manifestations of abnormalities associated with the disease or condition are prevented. In the context of aging-associated oxidative stress, the term "preventing" refers to administering a compound prior to the onset of clinical symptoms of aging-associated oxidative stress, such that the physical manifestations of abnormalities associated with aging-associated oxidative stress are prevented.

[0081] As used herein, the terms "treatment" and "treating" refer to the medical management of a subject with the intent to cure, ameliorate, stabilize, or prevent a disease, pathological condition, or disorder. The term includes active treatment, i.e., treatment specifically directed at ameliorating a disease, pathological condition, or disorder, and also includes causal treatment, i.e., treatment directed at eliminating the cause of an associated disease, pathological condition, or disorder. Additionally, the term includes palliative treatment, i.e., treatment aimed at alleviating symptoms rather than curing a disease, pathological condition, or disorder; prophylactic treatment, i.e., treatment aimed at minimizing or partially or completely suppressing the onset of an associated disease, pathological condition, or disorder; and supportive treatment, i.e., treatment used to complement another specific therapy aimed at ameliorating an associated disease, pathological condition, or disorder. It is understood that while a treatment is intended to cure, ameliorate, stabilize, or prevent a disease, pathological condition, or disorder, it need not actually result in a cure, amelioration, stabilization, or prevention. The effectiveness of treatment can be measured or assessed as described herein and as known in the art, as appropriate for the disease, pathological condition, or disorder involved. Such measurements and assessments can be made in qualitative and / or quantitative terms. Thus, for example, the characteristics or features of a disease, pathological condition, or disorder and / or the symptoms of a disease, pathological condition, or disorder can be reduced to any effect or any amount. In the context of a subject suffering from aging-associated oxidative stress, the terms "treatment" and "treating" refer to the medical management of the subject with the intent of curing, ameliorating, or stabilizing aging-associated oxidative stress. In the context of a subject at risk for developing aging-associated oxidative stress, the terms "treatment" and "treating" refer to the medical management of the subject with the intent of preventing aging-associated oxidative stress.

[0082] In certain embodiments in which the composition is provided in a watertight sachet, the composition can be administered to a patient dissolved or suspended in a liquid, preferably an aqueous liquid, most preferably a beverage such as fruit juice, milk, water, etc. It can also be mixed with foods such as yogurt, liquid yogurt, soup, puree, cream, or porridge. These foods must be at an optimal temperature for consumption and should never be heated after the composition of the present invention has been added.

[0083] The composition may comprise at least 85% of the proteins of a Saccharomyces culture lysate relative to total microbial protein. Similarly, the composition may comprise at least 5 to 0.02% of the proteins of a culture lysate of each of the following microorganisms: Bacillus, Lactobacillus, Streptococcus, Saccharomyces, and Bifidobacterium.

[0084] Functional analysis based on KEGG orthology (KO) was performed to confirm the protein functions in different metabolic pathways defined by KEGG and COG. Histone-like proteins with DNA binding function were found to be of note in the protein profile K03530 at a relative abundance of 0.5 to 3%. In a preferred embodiment, they are found at a relative abundance of 0.6 to 2%, more preferably 0.7 to 1%, and even more preferably 0.7%. These proteins play an important role in epigenetic regulation through various covalent bonds. The total level of histone proteins declines during aging, and their overexpression dramatically extends lifespan and promotes host health.

[0085] The KO (KEGG Orthologies) category is based on the Kyoto Encyclopedia of Genes and Genomes (KEGG). It corresponds to the classification of the genome sequence of the genome ...

[0086] In a preferred embodiment of the present invention, the composition may comprise proteins from a bacterial lysate of the genus Bacillus present in an amount of 1.50 to 9%, preferably 2 to 7%, and more preferably 4.45 to 6.35% by weight of total microbial proteins. In a preferred embodiment, the relative abundance of proteins from a bacterial lysate of the genus Bacillus may be 2%, 2.50%, 3%, 3.50%, 4%, 4.50%, 5%, 5.50%, 6%, 6.50%, 7%, 7.50%, 8%, 8.5%, or 9%.

[0087] In a preferred embodiment of the present invention, the proteins identified from bacterial lysates of the genus Bacillus are selected from the group consisting of Q65HF3, P54944, Q5WEC7, and combinations thereof.

[0088] In a preferred embodiment of the present invention, the composition may comprise a protein from a bacterial lysate of the genus Bifidobacterium present in an amount of 0.01% to 1.20%, preferably 0.07% to 1.10%, by weight relative to the total amount of microbial protein. In a preferred embodiment, the relative abundance of the protein from the bacterial lysate of the genus Bifidobacterium is 0.01%, 0.05%, 0.10%, 0.15%, 0.20%, 0.25%, 0.30%, 0.35%, 0.40%, 0.45%, 0.50%, 0.55%, 0.60%, 0.65%, 0.70%, 0.75%, 0.80%, 0.85%, 0.90%, 0.95%, 0.95%, 1.10%, 1.15% or 1.20%.

[0089] In a preferred embodiment of the present invention, the proteins identified from bacterial lysates of the genus Bifidobacterium are selected from the group consisting of B8DTX9, B8DSQ4, and combinations thereof.

[0090] In a preferred embodiment of the present invention, the composition may comprise at least 2% to 8% of proteins from a bacterial lysate of the genus Lactobacillus, with the amount of proteins present in the total microbial protein content being preferably between 3.40% and 7.55%, more preferably between 4% and 5.45% by weight. In a preferred embodiment, the relative abundance of proteins from a bacterial lysate of the genus Lactobacillus may be 2%, 2.50%, 3%, 3.50%, 4%, 4.50%, 5%, 5.50%, 6%, 6.50%, 7%, 7.50% or 8%.

[0091] In a preferred embodiment of the present invention, the proteins identified from bacterial lysates of the genus Lactobacillus are selected from the group consisting of P35829, A0A0H0YNJ3, A5VJ92, Q1G910, and combinations thereof.

[0092] In a preferred embodiment of the present invention, the composition may comprise a protein from a bacterial lysate of the genus Saccharomyces that is present in a relative abundance of 49% to 99.00%, preferably 65% ​​to 90%, and more preferably 75% to 85% of the total microbial protein by weight percentage. In a preferred embodiment, the relative abundance of the protein from a bacterial lysate of the genus Saccharomyces may be 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, or 70%. In other preferred embodiments, the relative abundance of the protein from a bacterial lysate of the genus Saccharomyces may be 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%.

[0093] In a preferred embodiment of the present invention, the proteins identified from bacterial lysates of the genus Saccharomyces are selected from the group consisting of E7NI79, E7LV64, AOAOL8VTA7, E7Q2B5, E7NEQ3, A0A0L8VV91, A0A0L8VIG1, A0A0L8VIS1, and combinations thereof.

[0094] In a preferred embodiment of the present invention, the composition may comprise proteins from a bacterial lysate of the genus Streptococcus present in an amount of 0.15% to 0.90%, preferably 0.17% to 89%, and more preferably 0.33% to 66% by weight of total microbial proteins. In a preferred embodiment, the relative abundance of proteins from a bacterial lysate of the genus Streptococcus may be 0.15%, 0.20%, 0.25%, 0.30%, 0.35%, 0.40%, 0.45%, 0.50%, 0.55%, 0.60%, 0.65%, 0.70%, 0.75%, 0.80%, 0.85%, or 0.90%.

[0095] In a preferred embodiment of the present invention, the proteins identified from bacterial lysates of the genus Streptococcus are selected from the group consisting of Q5M5J3, Q5M2M7, and combinations thereof.

[0096] In a specific embodiment, the subject of the composition of the present invention is obtained from a culture of probiotic microorganisms of the genera of microorganisms described herein, containing a certain amount of colony-forming units (in English: CFU). In the context of the present invention, colony-forming units is a microbiological term and is an indicator of the amount of live microorganisms present in a culture medium.

[0097] In a particular embodiment of the present invention, the starting bacterial population is Bacillus, comprising between 3.49% and 20.94% CFU, most preferably between 10.98% and 15.75% CFU of the total CFU of the composition.

[0098] In a particular embodiment of the present invention, the starting bacterial count is Lactobacillus, comprising 28.64% to 66.82% CFU, most preferably 40.66% to 57.36% CFU of the total CFU of the composition.

[0099] In a particular embodiment of the present invention, the starting bacterial count is Streptococcus, comprising between 5.72% and 38.15% CFU, most preferably between 10.98% and 20.75% CFU, relative to the total CFU of the composition.

[0100] In a particular embodiment of the invention, the starting bacterial count for the genus Saccharomyces comprises between 11.44% and 24.79% CFU, most preferably between 19.04% and 21.75% CFU for the total culture.

[0101] In a particular embodiment of the invention, the starting bacterial count for the whole culture is Bifidobacterium, comprising between 10.73% and 53.64% CFU, most preferably between 15.99% and 18.75% CFU.

[0102] These probiotic bacteria were cultivated under standard conditions as described in the culture protocols published by CECT (Colection Espanola de Cultivos Tipo) and are indicated for each bacterial species described in this paper.

[0103] The cultured microorganisms are then subjected to a lysis process. The process for obtaining the lysate involves a combination of non-mechanical and mechanical methods. First, the microbial cells undergo a heat treatment. Each batch of live cell culture undergoes a sterilization cycle in an autoclave at 121°C for 20 to 30 minutes. This temperature denatures, inactivates, and coagulates proteins. It also causes membrane damage, ribosome aggregation, DNA strand breaks, and enzyme inactivation. Once cooled, the probiotic batch is subjected to cell disruption by sonication for 15 to 20 minutes at 450 to 550 W, amplitude 38 to 43%, and pauses of 8 to 15 seconds (Qsonica, Q500). This disrupts intermolecular interactions and fragments DNA. The final solution is then freeze-dried and milled to obtain the probiotic microbial lysate in powder form. The powder should be stored in a cool, protected environment.

[0104] The composition according to the invention is the result of a combination of probiotic bacteria that, after a process of proliferation and lysis, produce an extract consisting of a set of metabolic products, proteins, DNA fragments and other components such as peptidoglycans, which, when administered efficiently, is able to alter and modify in a significant way the host microbiota through several mechanisms, resulting in an activation of the immune system that reverses dysbiosis and significantly enhances the delay of cellular aging.

[0105] In a further aspect, the present invention relates to the use of an orally administered composition for preventing and delaying aging, which composition comprises a lysate of probiotic microorganisms in the form of a dry powder in the following amounts, expressed as percentages by weight relative to the total: - 5% to 30% of bacterial lysates of the genus Bacillus, - 3% to 25% of bacterial lysate of the genus Bifidobacterium, - 15% to 35% of bacterial lysates of the genus Lactobacillus, - 30% to 65% of bacterial lysates of the genus Saccharomyces, - 1.5% to 10% of bacterial lysates of Streptococcus spp.

[0106] In certain embodiments, the compositions find use in preventing and delaying aging in animal or human cells.

[0107] In another specific embodiment of the present invention, the composition comprises a lysate of a probiotic microorganism in the form of a dry powder in an amount as a percentage by weight of the total as disclosed in composition "A", "B", "C", "D", "E", "F", "G", "H" or "I".

[0108] In another particular embodiment, the use consists of orally administering the composition in an amount between 20 mg and 200 mg, two to six times daily.

[0109] In another specific embodiment, the use comprises administering to a subject an effective amount of the composition, wherein the amount of the aging-delaying compound can be from a low amount of about 0.1 μg / ml, about 0.2 μg / ml, or about 0.5 μg / ml, to a high amount of about 100 μg / ml, about 200 μg / ml, or about 500 μg / ml. For example, the amount of the compound that retards aging may be from about 0.1 μg / ml to about 100 μg / ml, from about 0.2 μg / ml to about 200 μg / ml, from about 0.5 μg / ml to about 500 μg / ml, from about 0.1 μg / ml to about 10 μg / ml, from about 0.1 μg / ml to about 20 μg / ml, from about 0.1 μg / ml to about 50 μg / ml, from about 0.5 μg / ml to about 20 μg / ml, from about 0.5 μg / ml to about 50 μg / ml, from about 0.5 μg / ml to about 100 μg / ml , about 0.5 to about 200 μg / ml, about 0.5 μg / ml to about 500 μg / ml, about 15 μg / ml to about 25 μg / ml, about 15 μg / ml to about 50 μg / ml, about 18 μg / ml to about 30 μg / ml, about 18 μg / ml to about 50 μg / ml, or about 1 μg / ml to about 10 μg / ml, or about 2 μg / ml to about 8 μg / ml, or about 10 μg / ml to about 30 mg / ml, or about 50 μg / ml to about 30 mg / ml. These amounts can be administered daily, once a week, once or more times a day, or two to six times a day. For example, a daily dose of between 50 μg / ml and 30 mg / ml.

[0110] The technical effect obtained from the composition of the present invention is that it acts directly on the human microbiome.Thanks to this repairing and regulating effect, the inventors have surprisingly found that by intervening in the human microbiome, it is possible to significantly improve human health, and in particular to slow down the aging process, which is embodied in the slowing down and prevention of the appearance changes and energy loss caused by such aging in humans and animals, as demonstrated by the experimental results described herein.

[0111] In this way, the inventors were able to confirm that oral administration of the compositions of the invention is effective in modulating and reprogramming the microbiota present in the human intestinal tract, and that this is not just a local effect, but a systemic effect, since the entire organism benefits from this microbiota modulation.

[0112] The present invention also relates to the following clauses:

[0113] 1. An orally administered composition for preventing and delaying aging, characterized in that it contains a lysate of probiotic microorganisms in the form of a dry powder, the composition containing the following amounts, expressed as total weight percentages: - 5% to 30% of bacterial lysates of the genus Bacillus, - 3% to 25% of bacterial lysate of the genus Bifidobacterium, - 15% to 35% of bacterial lysates of the genus Lactobacillus, - 30% to 65% of bacterial lysates of the genus Saccharomyces, - 1.5% to 10% of bacterial lysates of Streptococcus spp.

[0114] 2. The composition of clause 1, wherein the Bacillus bacterial lysate is of a species selected from the group consisting of Bacillus clausii, Bacillus coagulans, Bacillus licheniformis, Bacillus pumilus, Bacillus subtilis, Bacillus paralicheniformis, and combinations thereof.

[0115] 3. The composition of clause 1 or 2, wherein the bacterial lysate of the genus Bifidobacterium is of a species selected from the group consisting of Bifidobacterium animalis subsp lactis, Bifidobacterium bifidum, Bifidobacterium breve, Bifidobacterium lactis, Bifidobacterium longum, Bifidobacterium infantis, Bifidobacterium animalis, and combinations thereof.

[0116] 4. The bacterial lysate of the genus Lactobacillus is selected from the group consisting of Lactobacillus acidophilus, Lactobacillus bulgaricus, Lactobacillus casei, Lactobacillus fermentum, Lactobacillus paracasei, Lactobacillus plantarum, Lactobacillus reuteri, Lactobacillus rhamnosus, Lactobacillus salivarius, Lactobacillus helveticus, and Lactobacillus gasseri. 4. The composition of any of clauses 1 to 3, wherein the bacterium is of a species selected from the group consisting of Lactobacillus gasseri, Lactobacillus johnsonii, Lactobacillus brevis, Lactobacillus kefiri, and combinations thereof.

[0117] 5. The composition of any of clauses 1 to 4, wherein the bacterial lysate of the genus Saccharomyces is selected from the group consisting of Saccharomyces cerevisiae, Saccharomyces boulardi, and combinations thereof.

[0118] 6. The composition of any of clauses 1 to 5, wherein the bacterial lysate of the genus Streptococcus is a species of Streptococcus thermophilus, Streptococcus salivarius, and combinations thereof.

[0119] 7. The composition of any of clauses 1 to 6, comprising at least one additional ingredient selected from the group consisting of riboflavin, vitamin C, manganese, selenium, zinc, vitamin D and magnesium.

[0120] 8. The composition according to any of clauses 1 to 7, wherein the composition is comprised between the relative amounts present, as a weight percentage: - 1.50 to 9% of the protein obtained from bacterial lysates of the genus Bacillus; - 0.01% to 1.20% of the protein content of a bacterial lysate of the genus Bifidobacterium; - 2% to 8% of proteins from bacterial lysates of Lactobacillus spp. - 49% to 99.00% of proteins from bacterial lysates of the genus Saccharomyces, - 0.15% to 0.90% of the protein in bacterial lysates of Streptococcus spp.

[0121] 9. Use of a composition according to any of clauses 1 to 8 as a food supplement for the prevention and delay of aging in animal or human cells.

[0122] 10. The use of the composition according to clause 9, wherein the composition is administered orally in an amount of between 20 mg and 200 mg of the composition 2 to 6 times daily.

[0123] 11. Use of the composition according to clause 9 or 10, wherein the composition is presented in the form of a dry powder in a watertight sachet or enclosed in a gelatin capsule.

[0124] 12. A composition according to any one of clauses 1 to 8 for use as a medicament.

[0125] 13. The composition of claim 12 for use in preventing and delaying aging in animal or human cells.

[0126] 14. Use of the composition according to clause 12 or 13, wherein the composition is administered orally 2 to 6 times daily in an amount between 20 mg and 200 mg of the composition.

[0127] 15. A pharmaceutical composition comprising an effective pharmaceutical amount of the composition according to any one of clauses 1 to 8 and a pharmaceutically acceptable excipient.

[0128] Thus, the main advantages resulting from the objective of the composition of the present invention are: - supplementing and / or complementing the physiological functions of the human and animal microbiota; - the reversal of changes that cause dysbiosis and affect oxidative and aging processes; - the oral administration of the product makes the composition and its significant effects a highly advantageous alternative compared to anti-aging treatments currently used in medicine; - The composition contains lysates of microorganisms, i.e. it does not present living organisms, and therefore presents itself as a safe alternative, since it avoids the possible dangers posed by colonizing organisms and, moreover, does not present the toxicity that conventional drugs may present.

[0129] The terms "comprising," "consisting essentially of," and "consisting of" can each be replaced with either of the other two terms. The terms "a" or "an" can refer to one or more of the elements it modifies, unless the context clearly indicates that any element or elements are being described (e.g., "a reagent" can mean one or more reagents). As used herein, the term "about" refers to a value within 10% of the underlying parameter (i.e., plus or minus 10%; e.g., a weight of "about 100 grams" can include weights between 90 grams and 110 grams). The use of the word "about" at the beginning of a list of values ​​modifies each value (e.g., "about 1, 2, and 3" refers to "about 1, about 2, and about 3"). When a list of values ​​is stated, the list includes all intermediate values ​​and all fractional values ​​thereof (e.g., the list of values ​​"80%, 85%, or 90%" includes the intermediate value 86% and the fractional value 86.4%). When a list of values ​​is followed by the term "or more," the term "or more" applies to each of the listed values ​​(e.g., the list "80%, 90%, 95% or more" or "80%, 90%, 95% or more" or "80%, 90%, or 95% or more" refers to "80% or more, 90% or more, or 95% or more"). When a list of values ​​is stated, the list includes all ranges between any two of the listed values ​​(e.g., the list "80%, 90% or 95%" includes the ranges "80% to 90%, "80% to 95%, and "90% to 95%"). Specific examples of the present technology are provided below.

[0130] Preferred Embodiments of the Invention For the purpose of providing a better understanding of the present invention and in accordance with its practical realization, preferred embodiments of the present invention are attached as an integral part of this specification.

[0131] Example 1 - Preparation of a lysate composition of probiotic microorganisms

[0132] The process involves obtaining a dried microbial lysate mixture of different strains of Bacillus, Lactobacillus, Bifidobacterium, Streptococcus, and Saccharomyces. To achieve this, they are grown by fermentation, concentrated, digested, and finally the cellular components are dried. The resulting postbiotic formulation is a lysate mixture that can be ingested with food ingredients in the form of a capsule or powder supplement.

[0133] For growth, prepare an inoculum in a flask of culture medium for each microorganism, with a volume and amount sufficient to inoculate the fermenter with the respective cultured inoculum. To do this, immerse the microorganisms stored in vials with 20% glycerol at -80°C in a 30°C water bath to quickly and completely thaw each microorganism.

[0134] Once the inoculum for each microorganism was obtained, it was inoculated separately directly into the fermenter, with the inoculation rates being 3% for Bacillus, 5% for Bifidobacterium, Lactobacillus, Streptococcus, and Saccharomyces respectively.

[0135] After inoculating the fermentor with the corresponding microbial inoculum, the fermentation step for each microbial species is carried out to generate microbial biomass, and growth conditions for each species are established.

[0136] Bacillus bacteria were cultured in nutrient broth (NB: Culture in Nutrient Broth (Glucose H2O 25g / L, Yeast extract 5g / L, K2HPO4 2.5g / L, MgSO4-7H2O 5g / L, Tween 80) for 15 hours at 250 rpm. The fermentation medium, consisting of 1 g / L, is incubated at 30°C ± 1°C throughout the entire process, with a pH of 6.8 ± 0.1, using automated aseptic addition of 25% NH4OH or 35% H3PO4. Dissolved oxygen is maintained at 40% or higher by agitation (150-250 rpm) at 0.5 atmospheres and aeration (1-1.5 vvm). After approximately 20-24 hours, base consumption (NH4OH) is stopped, the temperature is lowered to 4-8°C to stop cell metabolism, and the broth is harvested.

[0137] For Streptococcus, the inoculum is grown in tryptic soy broth (TSB) at 37°C with 150 rpm agitation for 24 hours. The fermentation medium consists of 25 g / L glucose, 8 g / L milk powder, 5 g / L yeast extract, 2 g / L KHPO, 2 g / L KHPO, 0.5 g / L MgSO7H2O, and 0.5 g / L (NH)SO. The growth temperature is 37°C ± 1°C, and the pH is maintained at 6.8 ± 0.1 throughout the process by aseptically adding 25% NHOH or 35% HPO, with agitation at 150 rpm. After fermentation is complete, approximately 20 to 24 hours later, the temperature is lowered to 4 to 8°C to stop cell metabolism, and the broth is harvested.

[0138] The growth of Bifidobacterium and Lactobacillus inocula was Fermentation was carried out in Man, Rogosa, Sharpe Broth (MRS Broth) at 37°C for 24 hours. Fermentation was carried out at 37°C ± 1°C, pH 6.2 ± 0.1 and 6.4 ± 0.1, with the automatic aseptic addition of 25% NH4OH or 35% H3PO4, at 50 rpm (the minimum to ensure accurate homogenization of the medium components). The medium consisted of glucose HO 20 g / l, yeast extract 20 g / l, and ethanol. 5g / l, K2HPO42g / l, casein peptone 10g / l, milk powder 8g / l, sodium acetate 5g / l, diammonium citrate 2g / l, Mn SO4- The medium consists of 0.05g / L H2O, 0.2g / L MgSO4 7H2O, and 1g / L Tween 80. Nitrogen is bubbled in initially until the dissolved oxygen concentration reaches 0%, and this process is repeated as necessary to maintain O2 at that level throughout the process. At the end of fermentation (approximately 20 to 24 hours), the temperature is lowered to 4 to 8°C to stop cell metabolism, and the broth is harvested.

[0139] S. cerevisiae was grown in YPD for 24 hours. The fermenter temperature was maintained at 30°C, pH 5 at 150 rpm, and 1.5 vvm. Fermentation was stopped in the fermenter, and the temperature was lowered to 4-8°C.

[0140] The biomass obtained from the fermentation of each microorganism is recovered by centrifugation or microfiltration to obtain a wet recovered biomass, which is then concentrated as many times as necessary to obtain a CFU / ml concentration of 1.00E+09 to 1.00E+11 for each microorganism.

[0141] The cells of the wet harvested biomass are then ruptured or lysed by heat treatment and ultrasonic treatment to obtain the respective bacterial lysates, which are then dried separately in a freeze dryer or sprayer, and the dried bacterial lysates are mixed in a mixer to obtain the adjusted composition.

[0142] Each batch of live cell culture undergoes an autoclave sterilization cycle at 121°C for 20 minutes. It is then sonicated for 20 minutes at 500W, 40% amplitude, and a 10-second pause to obtain the lysed probiotic cell mass (Qsonica, Q500). The final solution is freeze-dried and pulverized to obtain the powdered probiotic microbial lysate. The powder is stored in a cool environment, away from heat.

[0143] For lysate analysis, 0.5g of powder from each batch is taken and dissolved in 10ml of distilled water. A 1:8 dilution is prepared from this suspension (100µl sample, 700µl water) and centrifuged at 5000 rpm for 5 minutes. The supernatant is collected, and 1.5µl of the sample is placed in a cuvette. The absorbance is measured at 260nm, 230nm, and 280nm using a Nanodrop Tecan Spark 10M with distilled water as the target. Measurements are repeated at least twice, usually four times or more.

[0144] The maximum absorption peak for DNA, a nucleic acid, occurs at a wavelength of 260 nm, so the absorption at that wavelength is proportional to the DNA concentration. (ng / ul DNA: A260nm x dilution factor x 50 (conversion factor)

[0145] The amount of DNA contained in each lysate is as follows:

[0146] Table 2. DNA concentrations of dried lysates of each bacterial strain prepared according to the procedure.

[0147] [Table 2]

[0148] Samples of the compositions of the present invention were prepared using the following dried bacterial lysates in the following proportions:

[0149] Table 3. Sample composition 1 of the compositions of the present invention. The weight percentage of each genus described herein is shown.

[0150] [Table 3]

[0151] Example 2: Measurement of telomerase activity by MTT assay and QTRAP

[0152] 2.1 MTT assay Throughout this study, toxicity was measured by MTT assay and the results were obtained from Igen Biolabs (Igen Biolabs) as shown in Table 4. We evaluated the effect of a probiotic extract from Biolab on telomerase activity by Q-TRAP in primary human fibroblast cultures.

[0153] Table 4. Composition of one sample of the composition of the present invention. The weight percentage of each bacterial species that makes up the dried bacterial lysate described herein is shown. The table also shows the number of colony forming units.

[0154] [Table 4]

[0155] 2.1.1 Methodology Description The MTT toxicity assay uses the orange tetrazolium dye MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium It is a colorimetric assay that measures cellular metabolic activity by reducing NAD(P)H-dependent mitochondrial metabolic activity via NAD(P)H-dependent cellular oxidoreductases, and serves as a surrogate marker of cell viability.

[0156] 2.1.2 Cells and culture conditions Primary culture stocks of adult human fibroblasts (passage 3) were established under standard culture conditions. Cells were cultured in fibroblast medium (Innoprot kit) at 3 x 10 3 cells / cm 2 The seeds were sown in.

[0157] Fibroblast Medium (FM) is a complete medium designed for optimal growth of human fibroblasts in vitro. It is a sterile liquid medium containing essential and non-essential amino acids, vitamins, organic and inorganic compounds, hormones, growth factors, trace minerals, and a low concentration of fetal bovine serum (2%). The medium is HEPES and bicarbonate buffered, with a pH of 7.4 after equilibration in a 5% CO2 / 95% CO2 atmosphere incubator. This medium is formulated (quantitatively and qualitatively) to provide a defined, optimally balanced nutritional environment that selectively promotes the growth and proliferation of normal human fibroblasts in vitro.

[0158] 0.5 × 10 for the 72-hour and 1-week treatment periods, respectively. 4 cells / plate and 0.35 x 10 4 Cells were seeded at 1000 cells / plate in a 96-well plate (Nunc). This concentration showed an optimal window for the MTT assay, with good sensitivity and low variability.

[0159] Compounds were supplied as dry powders and kept under optimal conditions until use. 18 mg of compound (powder) was dissolved in 18 ml of 20% DMSO (1 mg / ml) at room temperature. An 8-point curve was generated and diluted 1 / 2 (highest concentration 1 mg / ml). The final DMSO concentration was 0.5% (this concentration has no effect on cells).

[0160] 24 hours after the seeding process, cells were washed once with PBS and treated with compounds in their respective cell culture media (fibroblast medium kit). Each condition was analyzed in triplicate with or without 10 μM HO. Positive and negative controls included 8 mM methyl methanesulfonate (MMS) and 100% DMSO, respectively.

[0161] After compound addition, the plates were cultured for 72 hours and one week, with compound-containing medium being replaced every other day. After the treatment period, the cells were washed twice with PBS and the medium was replaced with 0.5 ml of phenol red-free DMEM. The medium was replaced with 100 mg / ml MTT reagent. The plate was gently shaken and incubated for 4 hours. After incubation, the medium was removed and replaced with DMSO. The plate was gently shaken to solubilize the formazan crystals.

[0162] Absorbance was measured at a wavelength of 570 nm using an Envision multiplate reader.

[0163] 2.1.3 Results Fibroblast toxicity test (MTT) for 72 hours and 1 week.

[0164] Eight serial dilutions (50 μg / ml - 16.7 μg / ml - 5.6 μg / ml - 1.9 μg / ml - 0.6 μg / ml - 0.2 μg / ml - 0.1 μg / ml - 0.02 μg / ml) were prepared and the compounds were evaluated.

[0165] Compounds were evaluated by preparing triplicate concentrations and testing under standard and oxidative stress conditions.

[0166] If the MTT results in cell death of 20% or more, it is considered that there is a significant toxic effect.

[0167] Table 5. Results of the MTT assay expressed as percentage of cell death after 72 h of treatment under standard conditions.

[0168] [Table 5]

[0169] Table 6. Results of the MTT assay expressed as percentage of cell death after 72 h of oxidative stress treatment.

[0170] [Table 6]

[0171] Table 7. Results of the MTT assay expressed as percentage of cell death after 1 week of treatment under standard conditions.

[0172] [Table 7]

[0173] Table 8. Results of the MTT assay expressed as percentage of cell death after treatment after 1 week for oxidative stress.

[0174] [Table 8]

[0175] 2.1.4 Results and Observations No precipitation of the compound was observed at the concentrations tested. Results showed that the concentrations of the composition of the present invention selected for the Q-TRAP and TAT assays were 50, 16.7, and 5.6 μg / ml.

[0176] 2.2. Measurement of telomerase activity by Q-TRAP The assay measures Q-TRAP-mediated telomerase activity in primary cultures of adult human fibroblasts after treatment with the above concentrations for 6, 24, 48, and 72 hours.

[0177] 2.2.1 Methodology Description Q-TRAP is a quantitative real-time PCR (Q-TRAP) based method for measuring relative telomerase activity. This method offers advantages over the standard TRAP assay: greater sensitivity, speed, and a high-throughput format. It assesses the activity of the enzyme telomerase in lysates of whole cells from cell cultures.

[0178] The general mechanism of the Q-TRAP method involves lysing cell granules to extract proteins, which are then quantified and stored under specific conditions to prevent degradation. Proteins obtained from this step are used within 24 hours, and samples are stored at 4°C. Telomerase protein extracts are then incubated with specific oligonucleotide substrates to allow enzymatic addition of telomeric DNA repeats by endogenous telomerase.

[0179] After the enzymatic reaction, telomerase extension products are amplified and quantified by real-time qPCR. In qPCR, a positive reaction is detected by the accumulation of a fluorescent signal. The Ct (cycle threshold) is defined as the number of cycles required for fluorescence to exceed a threshold (i.e., above background levels). A telomerase-positive standard dilution series is plotted as a standard curve of Ct values ​​versus telomere protein concentration (r2 > 0.9).

[0180] To ensure data reproducibility and quantitation, tests are performed in triplicate. The mean and standard deviation (SD) of each triplicate is calculated, including both the positive control (standard lymphocyte cell line curve) and the negative control (heat inactivation).

[0181] Data are reported as RTA (relative telomerase activity) and the overall workflow scheme is as follows:

[0182] 2.2.2 Results Quality Control Parameters Prior to seeding and performing the Q-TRAP protocol, samples were evaluated as follows: - Protein Concentration: Protein quantification is performed on each sample using the Biorad protein assay. To ensure consistent results, a minimum protein concentration of 0.3 μg / μl is required for sample analysis to proceed. - Regression curve: An internal control is included and a regression analysis is performed for each cycle / plate. The regression curve is repeated for plates with an R2 of less than 0.9. - Replicas: Samples with less than two valid replicas are discarded. - Amplification cycle: Signals from cycle #35 onwards are considered to be non-specific amplification.

[0183] Table 9. Abbreviations used in this report.

[0184] [Table 9]

[0185] Table 10 (Tables 10A-10B). Protein concentration results for samples analyzed by Q-TRAP. Each row represents the concentration of each triplicate.

[0186] [Table 10A]

[0187] [Table 10B]

[0188] 2.2.3 Results of telomerase activity Only samples containing sufficient protein (>0.3 mg / ml) were analyzed. Standard curve results: Generated by graphing the threshold cycle (Ct value) of the HeLa cell line standard versus the registry.

[0189] Measurements were performed in triplicate to calculate the coefficient of variation and the average amplification signal.

[0190] The table below summarizes the average data from triplicates. The relative telomerase activity (RTA) of each sample is reported as the mean and standard deviation (SD) after normalization to the Hela control.

[0191] Table 11. RTA values ​​for each sample analyzed.

[0192] [Table 11]

[0193] 2.2.4 Statistical analysis Q-TRAP analysis of primary adult human fibroblast cultures was compared at different time points. Data were pooled by treatment condition and treatment time point. Statistical analysis of pooled data compared with the control condition was performed using T-Student's test.

[0194] Table 12. T-Student analysis shows whether the QTRAP results are significantly different compared to the control group. The significant differences are shown in the "Significant Difference" column. From low to high: No: Not significant; Yes (*): p<0.05; Yes(**): p<0.01; Yes(***): p<0.001; Yes(****): p<0.0001.

[0195] [Table 12]

[0196] 2.2.5 Observations and Conclusions Quality control results - Protein extraction yields from all samples produced sufficient amounts of protein (>0.3 μg / μl) to perform the Q-TRAP assay. - The coefficient of determination of the regression curve (HeLa cells) was greater than 0.9 (R2 = 0.99).

[0197] 2.2.5. Results of telomerase activity - The IG_1 group had higher RTA than the control group at all time points. Although the difference was not statistically significant, the effect maintained on activation at all points showed a consistent positive effect in the IG_1 group. - IG_2 group showed telomerase activation at 12, 24 and 72 hours. - The IG_3 group showed higher RTA at 12 and 72 hours compared to the untreated control.

[0198] Example 3: Proliferation analysis in cell culture and telomere length measurement by TAT®

[0199] 3.1. Proliferation analysis in cell culture 3.1.1. Technique Description Adult human fibroblasts were cultured in primary culture at 5x10 3 cells / cm 2 The cells were seeded in a Fibroblast Medium Kit (Innoprot). Fibroblast Medium (FM) is a complete medium designed for optimal in vitro growth of normal human fibroblasts. It is a sterile liquid medium containing essential and non-essential amino acids, vitamins, organic and inorganic compounds, hormones, growth factors, trace minerals, and a low concentration of fetal bovine serum (2%). The medium contains HEPES and buffered bicarbonate, and has a pH of 7.4 when equilibrated in a 5% CO2 / 95% air incubator. This medium is formulated (quantitatively and qualitatively) to provide a defined, balanced nutritional environment that selectively promotes the proliferation and growth of normal human fibroblasts in vitro.

[0200] The medium was refreshed every 2–3 days, and cells were passaged every 7 days at subconfluence (70–80%). Compounds and treatments were added to the cells during culture. Cell proliferation was monitored for each condition by counting the number of cells at each step using a Countess® (Invitrogen) cell counter. Population doublings (PD) were calculated using the formula: PD = 3.322 (Log(Cf)–Log(Ci)) + x (Cf: final concentration; Ci: initial concentration; X: PD final passage). PD corresponds to one round of cell replication.

[0201] Materials and Methods Treatment method The different treatments were evaluated in a proliferation assay:

[0202] Table 13. Abbreviations used in the report.

[0203] [Table 13]

[0204] Cells were grown under standard conditions and under oxidative stress conditions (H2O2) for 8 weeks.

[0205] 3.1.3.Results Table 14. Cumulative population doubling per passage under standard conditions (average of three replicates).

[0206] [Table 14]

[0207] Table 15. Cumulative population doubling per passage under oxidative stress conditions (average of three replicates).

[0208] [Table 15]

[0209] 3.1.4. Observations and Conclusions After 8 weeks of treatment at the prescribed strength: Under standard conditions - No significant differences were observed in the proliferation of cells treated with different concentrations of IG_1, IG_2, and IG_3 over the 8-week growth period.

[0210] Under oxidative stress conditions: - No significant differences were observed in the proliferation of cells treated with different concentrations of IG_1, IG_2, and IG_3 over the 8-week growth period.

[0211] 3.2. Telomere Length Measurement by TAT® 3.2.1 Methodology Description To measure median telomere length in any cell line, Life Length uses a high-throughput (HT) Q-FISH method. This method is based on in-situ hybridization, a quantitative fluorescence in situ hybridization technique modified for interphase cells. Briefly, telomeres are probed with a fluorescent peptide nucleic acid (PNA) probe (sequence: Alexa488-OO-CCCTAACCCTAACCCTAAA, ) that recognizes three telomere repeats. The nuclei and telomeres are hybridized with a PNA probe (Panagene). Images of the nuclei and telomeres are captured using a high-content display system (see below). The fluorescent signal intensity of the telomere PNA probe hybridized to each telomere is proportional to the length of that telomere. Fluorescence intensity is converted to base pairs through a standard regression curve generated using a control cell line with known telomere length.

[0212] Sample preparation and HT Q-FISH: On the day of processing, liquid nitrogen-frozen samples and control cell lines are thawed at 37°C for cell count and viability determination. Aliquots with viability below 60% are considered below our quality control standards and are not further analyzed. Cells are seeded at a density of 15,000 cells / well in 384-well plates, with five replicates for each sample and eight replicates for each control cell line. Two identical, independent plates are prepared for each sample set. Cells are fixed in methanol / acetic acid (3 / 1, vol / vol). Once fixed in the plate, they are treated with pepsin to digest the cytoplasm, and nuclei are processed for in-situ hybridization with PNA probes. After several washing steps and incubation with standard DAPI for DNA staining, the wells are filled with mounting medium, and the plates are stored at 4°C overnight.

[0213] HT microscopy: Quantitative image acquisition and analysis were performed using the Opera Phenix high-content screening system (Perkin Elmer) with Columbus software, version 2.9 (Perkin Elmer). Images were captured using a 40x 0.95NA water-immersion lens. UV excitation and 488 nm wavelengths were used to detect DAPI and A488 signals, respectively. Fifteen independent images were taken at different locations in each well at a fixed exposure setting. Next, nuclear images were used to define regions of interest for each cell, and the telomere fluorescence intensity was measured in all A488 images. The intensity results for each region of interest were exported to Columbus 2.4 (Perkin Elmer) software. The distribution and median telomere length were calculated using Life Length's proprietary software. Statistical analysis of the data was performed using the Student t-test.

[0214] 3.2.2 Technical Verification The TAT technology has been validated for the following parameters: Accuracy: Agreement between TAT fluorescence intensity values ​​and telomere length measurements is achieved by performing TRF (terminal restriction fragmentation) on six human lymphocyte cell lines (calibration / method comparison). The same sample set is analyzed by both the TAT and TRF (systematic error TAT definition) reference methods.

[0215] Validation data shows a correlation of 0.99.

[0216] Analyze median telomere length in human lymphocyte samples of different lineages, days, and plate positions to define parameters of random error TAT (standard deviation, variance).

[0217] Validation data indicates that the standard deviation of the TAT is 454 base pairs.

[0218] Detection limits and specificity: Define the minimum intensity and image analysis algorithms and protocol configurations that prevent interference from nonspecific fluorescent signals.

[0219] Validation data defined the detection limit at 800 base pairs, demonstrating very high specificity.

[0220] Median reportable range: Analysis of median telomere lengths for six cell lines was performed to cover the reportable range, defining its lower and upper limits.

[0221] In the validation data, the lower limit level is set to 4,700 base pairs and the upper limit level is set to 14,400 base pairs.

[0222] Analysis of reference range median telomere lengths has been evaluated in hundreds of human samples to define reference ranges for TAT and their percentiles (5th, 10th, 25th, 50th, 75th, and 95th) by age.

[0223] For the validation data, population curves for ages 18 to 85 years old - normal population curves were established and extrapolated to create a report.

[0224] 3.2.3 Results Quality Control Parameters During the TAT protocol, samples were analyzed for the following: - Cell count: An automated cell counter is used to measure the total number of cells in the vial. - Cell viability using a proprietary trypan blue method - Regression curve: Regression curve: An internal control is included and a regression analysis is performed for each sample / plate. Regression curves are repeated for plates where the R2 of the regression curve is less than 0.92. Replicates: TAT seeding and testing were performed: Samples must have a CV of 10% or less. At the end of the analysis, samples with fewer than three replicates are discarded. The number of analytical spots per sample must be at least 10,000.

[0225] Table 16. (Tables 16A-16B) Sample concentrations and viability after thawing triplicate samples under standard conditions.

[0226] [Table 16A]

[0227] [Table 16B]

[0228] Table 17. (Tables 17A-17B) Concentration and survival rate after thawing triplicate samples under oxidative stress.

[0229] [Table 17A]

[0230] [Table 17B]

[0231] Telomere length results by TAT The table below shows the median and 20th percentile telomere lengths (both in base pairs, or bp) for each sample, as well as the percentage of short telomeres, defined as the percentage of telomeres less than 3 Kbp in length (<3 Kbp). All measurements were performed in quintuplicate.

[0232] Table 18. Telomere length results of TAT assay under standard conditions.

[0233] [Table 18]

[0234] Table 19. Telomere length results of TAT assay under oxidative stress conditions.

[0235] [Table 19]

[0236] 3.2.4 Analysis of results Standard Growth Conditions Because cell replication is one of the major causes of telomere shortening, measured telomere lengths were normalized by population doublings for each condition and time.

[0237] Table 20. Telomere shortening ratio (median telomere length (initial - final) / population doubling) treatment and time defined under standard conditions.

[0238] [Table 20]

[0239] Table 21. T-Student analysis shows whether there was a significant difference in the rate of telomere shortening between each treatment and time observation. The significant differences are indicated in the "Significant Difference" column. From least significant to most significant: No: Not significant; Yes (*): p<0.05; Yes (**): p<0.01; Yes (***): p<0.001; Yes (****): p<0.0001

[0240] [Table 21]

[0241] Table 22. Telomere shortening ratio (median telomere length (initial-final) / population doubling) defined by treatment and time under oxidative stress conditions.

[0242] [Table 22]

[0243] Table 23. T-Student analysis shows whether there was a significant difference in the rate of telomere shortening between each treatment and time observation. The significant differences are indicated in the "Significant Difference" column. From least significant to most significant: No: Not significant; Yes (*): p<0.05; yes (**): p<0.01; yes (***): p<0.001; yes (****): p<0.0001.

[0244] [Table 23]

[0245] 3.2.5 Observations and conclusions Various variables of telomere length (median, 20 th After determining percentiles and %<3kbp) and normalizing the data by population doubling, the following observations were made: - Standard conditions After 2 weeks of treatment, IG_2 and IG_3 showed a reduction in the rate of telomere shortening compared to the control group. This effect was not evident at week 4, but a positive effect was observed again at week 6 (for IG_2) and week 8 (for IG_2 and IG_3), suggesting a protective effect on telomeres. The reduction in shortening at week 8 was statistically significant. - Oxidative stress state After 4 weeks of treatment, the IG_1, IG_2, and IG_3 groups showed improvement in the reduction of telomere shortening rate. At 6 weeks, the IG_1 and IG_3 groups maintained this effect, with the IG_1 group showing a statistically significant difference.

[0246] Example 4: In vitro (in vivo) testing of the anti-aging effects of the compositions analyzed in Example 2 on C. elegans

[0247] The anti-aging effect of a sample of the composition according to the present invention corresponding to the composition analyzed in Example 2 was investigated using an in vitro (in vivo) model of the nematode Caenorhabditis elegans (worms Studies were conducted to characterize the effects of the samples on lifespan, motor performance, and antioxidant capacity in Caenorhabditis elegans.

[0248] Materials and Methods sample A sample of a composition corresponding to the composition analyzed in Example 2 was evaluated according to the present invention (hereinafter also referred to as "sample").

[0249] First, a stock solution was prepared in distilled water and serially diluted to different final concentrations in Nematode Growth Medium (NGM), the medium used for culturing C. elegans. The solution was then applied to the surface of an agar plate.

[0250] 4.2. Life expectancy testing The experiment was carried out using a wild-type strain of C. elegans (N2). Age-synchronized populations were cultured and embryos were collected in different culture dishes: -NGM medium (control) - NGM medium + sample (0.05, 0.1, 0.5, 1, 5, 10, 25, 50, 100, 200, 400 μg / mL; 1, 10, 20 mg / mL)

[0251] Plates were incubated at 20°C, and the number of viable C. elegans cells was counted. The test population was periodically transferred to fresh medium every two days. During this period, viable counts were measured, ultimately yielding survival curves under each condition. The test was conducted twice.

[0252] 4.3. Mobility Research The experiment was performed using a wild-type strain of C. elegans (N2). Age-synchronized nematodes were collected from pregnant adults and plated under the following different conditions: -NGM medium (control) -NGM medium + sample (0.5, 1, 5, 10, 100 μg / mL; and 0.5 and 1 mg / mL).

[0253] C. elegans were cultured at 20°C until they reached adulthood. At this point, samples of nematodes (25 worms per condition and assay) were harvested and their motility measured. Motility was determined by quantifying the number of sinusoidal bends over a 40-second period. To measure nematode dispersion, a total of 15 worms per condition were analyzed. Five worms were simultaneously placed in the center of the NGM plate and their positions determined after 2 minutes (zone 1 was closest to the center, and zones 2 and 3 were furthest from the center).

[0254] 4.4. Antioxidant activity of C. elegans The experiment was performed using a wild-type strain of C. elegans (N2). Age-synchronized nematodes were obtained by culturing gravid adults on corresponding culture plates. Embryos were cultured at 20°C under different culture conditions: NGM (control) and NGM supplemented with different doses of the sample: - NGM medium (control) - NGM + Vitamin C medium (10 μg / mL) (positive control) - NGM + sample medium (0.05, 0.1, 0.5, 1, 5, 10, 25, 50, 100, 200, 400 μg / mL).

[0255] Nematodes were cultured under different conditions: in NGM + vitamin C medium, NGB medium (control), and in NGB + sample medium at 20°C. Five-day-old adults were then transferred to media plates containing hydrogen peroxide and cultured at 20°C. Finally, the survival rate of rotifers under each condition was measured.

[0256] 4.5.Statistical analysis Comparison of survival and mobility results between the NGM control condition and the different treatments was performed using one-way Anova statistical test with Tukey's post hoc test (Tukey's multiple comparison test).

[0257] In the analysis of survival curves, conditions that gave positive results in the log-rank test were statistically analyzed.

[0258] All of these analyses were performed using the statistical software GraphPad Prism version 7.0 from GraphPad Software, Inc., USA.

[0259] 4.6.Results Life expectancy research A lifespan test was performed to evaluate the effectiveness of this sample on the lifespan of C. elegans.

[0260] First, a series of doses was analyzed: 0.05, 1, 0.5, 1, and 5 μg / mL. Figure 11 shows the results obtained across this dose range. None of the doses was able to increase survival compared to the NGM control condition.

[0261] Next, we performed new lifespan studies using higher doses of the sample (10, 25, 50, 100, 200, and 400 μg / mL). Again, we observed that none of the doses tested affected the lifespan of C. elegans (Figure 12).

[0262] Finally, high-dose samples of 1, 10, and 20 mg / mL were evaluated in duplicate. The results showed a significant effect of the product on C. elegans lifespan at a dose of 1 mg / mL (P value < 0.05) (Figures 13 and 14, Table 2). The activity of the tested sample extended the average lifespan by 2 days (a 16.6% increase) (Table 2). At higher doses of 10 mg / mL and 20 mg / mL, some negative effect on nematode lifespan was observed, as nematode survival decreased with increasing product concentration.

[0263] Figure 13. C. elegans N2 The effect of high dose samples on the life span of control (NGM) and 1 The mean lifespan (the time 50% of the population survives) of this sample at 1 mg / mL is shown by the dashed line. Figure 14 shows the survival curve of C. elegans (N2) obtained from the population treated with this sample (1 mg / mL) using GraphPad Prism software. Meanwhile, Table 24 shows the statistical data of the survival curve of C. elegans after treatment with this sample at a dose of 1 mg / mL.

[0264] Table 24. Statistical data on the survival curve of C. elegans after treatment with samples at a dose of 1 mg / mL

[0265] [Table 24]

[0266] Mobility and Dispersion Research We investigated the motility of C. elegans after treatment with different doses of the sample. We collected adult populations from different culture conditions and quantified the average number of sinusoidal movements over a 40-second period.

[0267] Doses of 0.5 μg / mL, 1 μg / mL, 5 μg / mL, and 10 μg / mL were initially tested. Figure 15 shows the average sinusoidal curves for each of the different doses. As can be seen in Figure 15, treatment did not improve the motility of C. elegans. This shows the average motility of the C. elegans population at different doses. Data are for a single test.

[0268] Subsequently, higher doses of the sample (0.1, 0.5, and 1 mg / mL) were tested in two independent experiments. As can be seen in Figure 16, which shows the average mobility of C. elegans populations treated with this sample, in this case, the product exhibited a significant effect at the three doses evaluated (0.1, 0.5, and 1 mg / mL). At 1 mg / mL, 1 mg / mL significantly increased the mean mobility of C. elegans, with the 1 mg / mL dose showing the greatest increase (P ≤ 0.01) relative to the control (NGM) (15.3% increase in mobility vs. control). Doses of 0.1 mg / mL and 0.5 mg / mL showed a 10.3% and 8.9% increase in mobility, respectively, compared to the control condition (P ≤ 0.05).

[0269] In the next step, the frequency of bending was analyzed for each of the three doses (0.1, 0.5, and 1 mg / mL). To do this, we grouped the C. elegans based on the number of sine waves per 40 seconds. Figure 17 shows the distribution of C. elegans movement in each of the analyzed conditions. As can be seen, in the 1 mg / mL dose, only worms with ≥25 waves were observed. In the remaining conditions (0.1 and 0.5 mg / mL), worms with 23 to 24 bending waves were observed, and this percentage increased with increasing product concentration in the plates. At the effective dose of 1 mg / mL, the number of worms with 23 to 24 bending waves was the highest. This result reinforces previous observations that this product positively affects C. elegans motility.

[0270] Next, we analyzed whether treatment with this product increased nematode dispersion compared to the control condition by placing nematodes in the center of the plate and measuring their position (zone 1, 2, or 3) after 2 minutes, as described in Materials and Methods.

[0271] Figure 18 shows the dispersion of nematodes treated with this sample at 0.1, 0.5 and 1 Data are shown for adults treated with 1 mg / mL doses. At the 1 mg / mL dose, nematodes were more concentrated in zone 3 (the most distal of the origin), indicating a higher degree of nematode dispersion in this condition. Furthermore, with increasing concentrations, more nematodes were observed to concentrate in zone 3.

[0272] Thus, these results indicate that treatment with the sample increases the locomotion and dispersal ability of C. elegans.

[0273] 4.7. Antioxidant Activity in C. elegans This study was conducted to investigate whether administration of the sample could improve the sensitivity of C. elegans to acute oxidative stress.

[0274] Initially, doses of 10, 25, 50, 100, 200, and 400 μg / mL of this agent were evaluated. The results obtained at these doses are shown in Figure 19, which represents the percentage of surviving nematodes after acute oxidative stress for each condition. Figure 20 shows the antioxidant activity of samples evaluated at different doses (10 to 400 μg / mL) in C. elegans N2. The survival rate after oxidative stress with H2O2 is shown. Vitamin C (10 μg / mL) was added as a positive control. Data are from a single experiment.

[0275] At these doses, some protection by the samples was observed (slight increase in survival rate compared to the NGM control condition). However, results were similar among the different doses analyzed, except that the 400 μg / mL dose showed lower survival rates of rotifers than the control. This result may be due to a lack of optimal dose range, so lower doses of the samples were evaluated: 0.05, 0.1, 0.5, 1, and 5 μg / mL.

[0276] When C. elegans treated with low doses of the sample were subjected to acute oxidative stress, a significant improvement in C. elegans survival was observed in populations treated with 0.1 μg / mL and 0.5 μg / mL samples. Figure 20 shows the antioxidant activity of this sample evaluated at different doses in C. elegans N2. The survival rate after oxidative stress with H2O2 is shown. Vitamin C (10 μg / mL) was added as a positive control. Data are the average of two independent experiments (*** significant at P ≤ 0.001; ** significant at P ≤ 0.01; NS: not significant).

[0277] Table 25 shows the percentage increase in viability of each sample condition relative to the NG control condition and the resulting P-values ​​(ANOVA test).

[0278] Table 25. Percent increase in survival rate of each condition of samples relative to the NG control condition and the resulting P values ​​(ANOVA test).

[0279] [Table 25]

[0280] The most effective dose was 0.5 μg / mL (23% increase in survival rate over control conditions; P value ≦0.001) (Table 23). A dose of 0.1 μg / mL resulted in a 9% increase in survival rate over control (P value ≦0.01) (Table 23).

[0281] These results indicate that this product can improve resistance to acute oxidative stress in vivo, and demonstrates strong antioxidant activity at low doses.

[0282] Figure 21 shows the relative increase in viability of C. elegans with an effective dose of the sample (0.5 μg / mL) compared to the control condition, which shows an increase of 88.5%.

[0283] 4.8. Summary of test results This example provides an overview of the functional activity of the analyzed samples in C. elegans. It shows that the samples were functionally characterized in preclinical models of C. elegans, exhibiting a small effect on lifespan (at high doses), a significant effect on motility, and high antioxidant activity (at very low doses).

[0284] Example 5. Testing of the novel compositions of the present invention

[0285] The novel compositions were prepared according to the method described in Example 1, and the components of the novel compositions are listed in Table 26.

[0286] Table 26. Composition of Sample 2 of the composition of the present invention. Shows the weight percent of each bacterial species that makes up the dried bacterial lysate described herein.

[0287] [Table 26]

[0288] In Examples 3 and 4, the oxidative stress activity and anti-aging activity were evaluated under the same conditions as in Sample 1, and the results obtained were very similar.

[0289] These results reinforce the anti-aging and anti-oxidative stress effects of the orally administered composition of the present invention.

Claims

1. 1. An orally administered composition for preventing and delaying aging, comprising a lysate of a probiotic microorganism in dry powder form, in an amount as a percentage by weight of the total: 16% of bacterial lysates of the genus Bacillus, - 8% of Bifidobacterium genus bacterial lysate, - 21% of bacterial lysate of the genus Lactobacillus, - 52% of bacterial lysates of the genus Saccharomyces, - 3% of bacterial lysate of the genus Streptococcus, 10. An orally administered composition for use in the prevention and / or treatment of oxidative stress associated with aging, comprising:

2. 10. A composition for use according to any preceding claim, wherein the bacterial lysate of the genus Bacillus is a species selected from the group consisting of Bacillus clausiai, Bacillus coagulans, Bacillus licheniformis, Bacillus pumilus, Bacillus mesentericus, Bacillus subtilis, Bacillus paralicheniformis, and combinations thereof.

3. 10. A composition for use according to any of the preceding claims, wherein the bacterial lysate of the genus Bifidobacterium is a species selected from the group consisting of Bifidobacterium animalis subsp. lactis, Bifidobacterium bifidum, Bifidobacterium breve, Bifidobacterium lactis, Bifidobacterium longum, Bifidobacterium infantis, Bifidobacterium animalis, and combinations thereof.

4. 10. A composition for use according to any of the preceding claims, wherein the bacterial lysate of the Lactobacillus genus is a species selected from the group consisting of Lactobacillus acidophilus, Lactobacillus bulgaricus, Lactobacillus casei, Lactobacillus fermentum, Lactobacillus paracasei, Lactobacillus plantarum, Lactobacillus reuteri, Lactobacillus rhamnosus, Lactobacillus salivarius, Lactobacillus helveticus, Lactobacillus gasseri, Lactobacillus johnsonii, Lactobacillus brevis, Lactobacillus kefiri, and combinations thereof.

5. 10. A composition for use according to any preceding claim, wherein the bacterial lysate of the genus Saccharomyces is a species selected from the group consisting of Saccharomyces cerevisiae, Saccharomyces boulardii, and combinations thereof.

6. 10. A composition for use according to any preceding claim, wherein the bacterial lysate of the genus Streptococcus is a species selected from the group consisting of Streptococcus thermophilus, Streptococcus salivarius, and combinations thereof.

7. 10. A composition for use according to any preceding claim, comprising at least one additional ingredient selected from the group consisting of riboflavin, vitamin C, manganese, selenium, zinc, vitamin D, and magnesium.

8. A composition for use according to any of the preceding claims, comprising, as percentages by weight: - 1.50% to 9% of proteins from the lysate of bacteria of the genus Bacillus; - 0.01% to 1.20% of proteins from the bacterial lysate of the Bifidobacterium genus; - 2% to 8% of proteins from the bacterial lysate of the Lactobacillus genus, - 49% to 99.00% of the protein from the bacterial lysate of said Saccharomyces genus; - 0.15% to 0.90% of protein from the bacterial lysate of said Streptococcus genus; The composition includes a relative content in the range of:

9. 10. A composition for use as claimed in any preceding claim, as a food supplement.

10. 10. A composition for use according to any preceding claim, to be administered orally in an amount of 0.1 μg to 200 mg two to six times per day.

11. 10. A composition for use according to any of the preceding claims, wherein the composition is provided in the form of a dry powder in a watertight sachet or enclosed in a gelatin capsule.

12. 12. A pharmaceutical composition comprising an effective pharmaceutical amount of the composition of any of claims 1 to 11 and a pharmaceutically acceptable excipient for use in the prevention and / or treatment of oxidative stress associated with ageing.

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