Bifidobacterium longum with anti-aging function and application thereof

Abstract

This invention discloses *Bifidobacterium longum* with anti-aging functions and its applications, belonging to the field of microbial technology. The *Bifidobacterium longum* BBS 02 provided by this invention can promote cell vitality and proliferation, inhibit the high activity of β-galactosidase in senescent cells, and inhibit the high expression of the cyclin-dependent kinase inhibitor gene Cdkn1a, thereby achieving an anti-aging effect. The *Bifidobacterium longum* BBS 02 can be used to prepare edible nutrients or skin nutrients.

Description

Technical Field

[0001] This invention relates to Bifidobacterium longum with anti-aging function and its applications, belonging to the field of microbial technology. Background Technology

[0002] Bifidobacterium is a Gram-positive, non-motile, rod-shaped, sometimes forked, strictly anaerobic bacterium widely found in the digestive tract, vagina, and oral cavity of humans and animals. As an important component of the gut microbiota in humans and animals, Bifidobacterium not only plays a variety of physiological roles in human health, including biological barrier function, nutritional support, anti-tumor activity, immune enhancement, and improvement of gastrointestinal function, but also exhibits significant anti-aging effects.

[0003] Bifidobacterium longum, an important branch of the Bifidobacterium genus, is a type of probiotic commonly found in the human gut and is closely related to human health.

[0004] First, Bifidobacterium longum can regulate the gut microbiota and maintain its balance. As we age, the number of Bifidobacteria in the body gradually decreases, which is related to intestinal aging. Supplementing with Bifidobacterium longum may help slow this process, thus combating aging. Furthermore, Bifidobacterium longum can form a biofilm on intestinal epithelial cells, activate the immune system, and produce appropriate amounts of cytokines, such as tumor necrosis factor (TNF) and interleukins. These factors can induce programmed cell death in aging or mutated cells, reducing the accumulation of senescent cells and thus exerting an anti-aging effect.

[0005] Secondly, Bifidobacterium longum has antioxidant properties. It can induce the synthesis of superoxide dismutase (SOD), which delays aging, and reduce the damage of free radicals to cells through mechanisms such as chelating metal ions, activating the antioxidant system, and scavenging reactive oxygen species, thus significantly reducing the aging process.

[0006] Furthermore, *Bifidobacterium longum* may exert its anti-aging effects by influencing the host's physiological activities. For example, as cited in reference 1, a population genome analysis of 418 *Bifidobacterium longum* strains revealed a strong positive correlation between host age and strain genotype. Specific *Bifidobacterium longum* genotypes may exert different protective effects on D-galactose-induced aging mouse models by altering gut microbial metabolism, thereby preventing host aging.

[0007] Reference 2 discloses the protective effect of the combination of *Bifidobacterium longum* and *Bifidobacterium animalis* against age-related cognitive decline. The study found that these probiotics could reverse anxiety-like behavior, incoordination, and cognitive decline in d-galactose-treated mice. Furthermore, they reduced the expression of inflammatory factors in the hippocampus and increased the activity of antioxidant enzymes. The study concludes that the combination of *Bifidobacterium longum* and *Bifidobacterium animalis* may improve age-related cognitive deficits by upregulating Sirt 1 expression, thereby inhibiting NF-κB / TLR4-induced neuroinflammation and oxidative stress.

[0008] Therefore, Bifidobacterium longum has demonstrated its anti-aging potential through multiple mechanisms, including regulating gut microbiota, enhancing immune function, anti-oxidation, and improving cognitive function. However, the exact mechanisms of action are still not fully understood, and current research is relatively limited. More effective anti-aging Bifidobacterium longum formulations are still needed.

[0009] References

[0010] Cited literature 1: Xiao Y, Yang C, Yu L, Tian F, Wu Y, Zhao J, Zhang H, Yang R, ChenW, Hill C, Cui Y, Zhai Q.Human gut-derived B.longum subsp.longum strains protectagainst aging in a D-galactose-induced aging mouse model.Microbiome.2021Sep1;9(1):180.

[0011] Cited literature 2: Xia C, Cao X, Cui L, et al. Anti-aging effect of the combination of Bifidobacterium longum and B. animalis in a D-galactose-treatedmice[J]. Journal of Functional Foods, 2020, 69: 103938. Summary of the Invention

[0012] The problem the invention aims to solve

[0013] To address the shortcomings of existing technologies, such as poor anti-aging effects, this invention provides a strain of Bifidobacterium longum BBS 02 with anti-aging effects, which can promote cell vitality and proliferation, inhibit the high activity of β-galactosidase, and inhibit the high expression of the cyclin-dependent kinase inhibitor gene Cdkn1a. Furthermore, this strain can also achieve a high survival rate in simulated digestion experiments, allowing it to exert its effects more effectively in vivo.

[0014] Solution for solving the problem

[0015] [1]. Bifidobacterium longum has been deposited at the China General Microbiological Culture Collection Center, with accession number CGMCC No.30652.

[0016] [2]. A metagene derived from Bifidobacterium longum as described in [1].

[0017] [3]. The metagene according to [2], wherein the metagene is selected from inactivated bacterial cells, fermentation supernatant and bacterial cell lysate, or powder prepared by any drying method thereof.

[0018] [4]. The metagenes according to [2] or [3], wherein the metagenes are obtained by culturing Bifidobacterium longum in a culture system as described in [1], and then processing the culture to obtain the metagenes.

[0019] [5]. A composition comprising Bifidobacterium longum as described in [1] and / or metabiotics as described in any one of [2] to [4].

[0020] [6]. The use of Bifidobacterium longum as described in [1], metabiotics as described in any one of [2] to [4], or compositions as described in [5] in the preparation of products for preventing and / or improving skin aging;

[0021] Optionally, the prevention of skin aging and / or improvement of skin aging includes at least one of the following (a) to (c):

[0022] (a) Enhances cell vitality and proliferation capacity;

[0023] (b) Inhibits β-galactosidase activity;

[0024] (c) Inhibit the high expression of Cdkn1a, the gene encoding cyclin-dependent kinase inhibitor.

[0025] [7]. According to the application described in [6], wherein the product includes skin nutrients or edible nutrients.

[0026] [8]. A skin nutrient, wherein the nutrient comprises or uses the composition as described in [5].

[0027] [9]. An edible nutrient, wherein the edible composition comprises or uses the composition described in [5].

[0028]

[10] . According to the edible nutrient described in [9], wherein the content of Bifidobacterium longum in the edible nutrient is not less than 10%. 8 cfu / g or 10 8 cfu / mL.

[0029]

[11] . The nutrient according to any one of [8] to

[10] , wherein the nutrient exists in solid, semi-solid or liquid form.

[0030] The effects of the invention

[0031] By implementing the above technical solutions, the present invention achieves the following technical effects:

[0032] The *Bifidobacterium longum* BBS 02 provided by this invention can significantly enhance cell viability and proliferation, inhibit the high activity of β-galactosidase, and inhibit the high expression of the cyclin-dependent kinase inhibitor gene Cdkn1a, thereby playing an anti-aging role. Furthermore, *Bifidobacterium longum* BBS 02 exhibits good tolerance to saliva, gastric juice, and intestinal fluid, allowing it to exert its full effect in vivo.

[0033] Information on the preservation of biological materials

[0034] The present invention, Bifidobacterium longum BBS 02, classified and named Bifidobacterium longum, was deposited on May 16, 2024, at the China General Microbiological Culture Collection Center, Institute of Microbiology, Chinese Academy of Sciences, No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC No. 30652. Attached Figure Description

[0035] Figure 1 The construction and experimental treatment process of the aging model are shown.

[0036] Figure 2 The results show cell viability and proliferation capacity in an aging model after different treatments; where ns represents p>0.05 and **** represents p<0.0001.

[0037] Figures 3-4The β-galactosidase activity in aging models after different treatments is shown; where ns represents p>0.05 and **** represents p<0.0001.

[0038] Figure 5 The expression levels of the Cdkn1a gene in aging models after different treatments are shown; where ns represents p>0.05.

[0039] Figure 6 The growth status of the strain in a simulated in vitro digestion experiment is shown. Detailed Implementation

[0040] Various exemplary embodiments, features, and aspects of the present invention will be described in detail below. The term "exemplary" as used herein means "serving as an example, embodiment, or illustration." Any embodiment described herein as "exemplary" is not necessarily to be construed as superior to or better than other embodiments.

[0041] Furthermore, to better illustrate the present invention, numerous specific details are set forth in the following detailed embodiments. Those skilled in the art should understand that the present invention can be practiced without certain specific details. In other instances, methods, means, apparatus, and steps well known to those skilled in the art have not been described in detail in order to highlight the spirit of the present invention.

[0042] Unless otherwise stated, all units used in this specification are international standard units, and all numerical values ​​and ranges appearing in this invention should be understood to include systematic errors that are unavoidable in industrial production.

[0043] In this specification, the word "may" has two meanings: to perform a certain process and not to perform a certain process.

[0044] In this specification, references to "some specific / preferred embodiments," "other specific / preferred embodiments," "implementation," etc., refer to specific elements (e.g., features, structures, properties, and / or characteristics) related to that embodiment, which are included in at least one of the embodiments described herein and may or may not be present in other embodiments. Furthermore, it should be understood that these elements may be combined in any suitable manner in various embodiments.

[0045] In this specification, "optional" and "optionally" mean that the events or circumstances described below may or may not occur, and the description includes both cases where the events or circumstances occur and cases where the events or circumstances do not occur.

[0046] In this specification, the range of values ​​referred to as "value A to value B" refers to the range including the endpoint values ​​A and B.

[0047] In addition, unless otherwise defined, other technical and scientific terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0048] This invention is mainly based on the following insights:

[0049] This invention screened and obtained a strain of Bifidobacterium longum BBS 02. This strain, whether in fermentation supernatant or cell lysate, can effectively promote cell regeneration and delay aging in an aging state. For example, it can promote the activity and proliferation of skin cells, regulate β-galactosidase activity, and inhibit the high expression of the Cdkn1a gene. Compared with existing strains, it was verified that Bifidobacterium longum BBS 02 has better anti-aging and skin aging improvement effects than existing probiotics. At the same time, Bifidobacterium longum BBS 02 also has good tolerance to saliva, gastric juice and intestinal juice, and can maintain good activity in the digestive system to exert its effects.

[0050] The technical solution of the present invention will be further described in detail below:

[0051] <strain>

[0052] This invention provides a strain of *Bifidobacterium longum* BBS 01, which was deposited at the China General Microbiological Culture Collection Center (CGMCC) on May 16, 2024, with accession number CGMCC No. 30652. This strain can promote cell activity and proliferation, and inhibit the high expression of β-galactosidase and Cdkn1a genes in the aging state, thereby achieving the effects of preventing aging and improving skin aging.

[0053] In some embodiments, the Bifidobacterium longum BBS 02 is anaerobically cultured in a suitable culture medium. When it reaches a stable growth phase, the microorganism can be collected by conventional isolation techniques (e.g., membrane filtration or centrifugation) and resuspended in sterile physiological saline.

[0054] <Post-birth>

[0055] The present invention provides an epigenetic agent derived from the Bifidobacterium longum BBS 02.

[0056] In some specific embodiments, the postbiotic composition of the present invention is prepared according to a process comprising: (a) preparing a seed culture of Bifidobacterium longum BBS 02; (b) inoculating the seed culture of Bifidobacterium longum BBS 02 into a culture medium; (c) culturing the Bifidobacterium longum BBS 02 for a predetermined time to produce a culture; and (d) subjecting the culture to operations such as heat treatment, physical treatment, freeze drying, etc., to obtain the postbiotic composition.

[0057] In some optional embodiments, the postbiotic composition after treatment (e.g., heat treatment, physical treatment, spray drying, or freeze drying) contains non-viable bacteria and their metabolite components, including cell components and metabolites.

[0058] In some optional embodiments, the treated (e.g., heat treatment, physical treatment, spray drying, or freeze drying) metabiotic composition contains low levels of viable or live bacteria. In embodiments where the metabiotic composition is administered to an aging subject, the metabiotic may contain viable or non-viable bacteria. In some optional embodiments, the bacterial survival rate in the treated (e.g., heat treatment, physical treatment, spray drying, or freeze drying) metabiotic is up to 10%. In some optional embodiments, the survival rate of bacteria in the metabiotic after treatment (e.g., heat treatment, physical treatment, spray drying, or freeze drying) is at most 0.01%, 0.02%, 0.03%, 0.04%, 0.05%, 0.06%, 0.07%, 0.08%, 0.09%, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, or 10%.

[0059] In some embodiments, the probiotic cell components in the metabiotic of the present invention include cell components and cell metabolites (e.g., fermentation supernatant). The cell components include not only intact cells (e.g., non-viable bacteria as described above), but also components resulting from cell lysis (i.e., cell lysates), such as peptidoglycan, teichoic acid, lipoteichoic acid, phospholipids, cell membrane proteins, etc.

[0060] In some embodiments, the metabolites of Bifidobacterium longum BBS 02 in the metabiotic of the present invention include: vitamins, lipids, enzymes, proteins, peptides, organic acids, short-chain fatty acids, intracellular polysaccharides, extracellular polysaccharides, etc.

[0061] The metabiotic provided in this invention has the function of promoting cell viability and proliferation and inhibiting the high expression of aging factors. In particular, the fermentation supernatant and cell lysate of Bifidobacterium longum BBS O2 contained in the metabiotic have even better functions of promoting cell viability and proliferation and inhibiting the high expression of aging factors.

[0062] <Composition>

[0063] The present invention also provides compositions comprising the aforementioned *Bifidobacterium longum* BBS O2 and / or metabiotics, wherein the *Bifidobacterium longum* BBS O2 and / or metabiotics contained in the compositions can be dried by freeze-drying and may include excipients used when drying common strains or compositions derived from said strains. Excipients for freeze-drying may be selected from gluconic acid, alginate, sodium alginate, hydroxypropyl cellulose, hydroxypropyl methylcellulose, sodium carboxymethyl cellulose, methylcellulose, carbomer, hyaluronic acid, astragalus gum, guizitan gum, water-soluble starch, pectin, gelatin, polyvinyl alcohol, polyvinylpyrrolidone, oligosaccharides, sugar alcohols, calcium gluconate, calcium lactate, polymethyl methacrylate, wheat protein, soy protein, and methylcellulose.

[0064] In some alternative embodiments, the composition comprises the aforementioned Bifidobacterium longum BBS 02, fermentation supernatant of Bifidobacterium longum BBS 02, and / or cell lysate. The composition can be dried using conventional freeze-drying methods.

[0065] <Application of strains, metageners, or compositions>

[0066] This invention provides the application of the aforementioned Bifidobacterium longum BBS O2, the aforementioned metabiotic, or the aforementioned composition in the preparation of products for preventing and / or improving skin aging. Specifically, the aforementioned Bifidobacterium longum BBS O2, the aforementioned metabiotic, or the aforementioned composition have the effects of promoting cell vitality and proliferation, and inhibiting the high expression of aging factors. In some embodiments, the fermentation supernatant and cell lysate of the aforementioned metabiotic Bifidobacterium longum BBS O2 can also be used in the preparation of products for preventing and / or improving skin aging. Both the fermentation supernatant and the cell lysate have the effects of promoting cell vitality and proliferation, and inhibiting the high expression of aging factors.

[0067] In some embodiments, cell replication capacity is related to the length of telomeres, which are repetitive sequences at the ends of chromosomes. These telomeres shorten after each cell division until they reach a critical length at which point cell division ceases. Therefore, cell replication capacity gradually decreases with age. In some exemplary embodiments of the present invention, the aforementioned Bifidobacterium longum BBS 02, the metabiotic, or the composition can enhance cell viability and maintain a strong proliferative capacity.

[0068] In other embodiments, the aging factors include β-galactosidase and the Cdkn1a gene.

[0069] In some embodiments, the β-galactosidase is an enzyme capable of catalyzing the hydrolysis of the β-galactosidic bond in β-galactoside compounds and simultaneously possessing transgalactosylation capabilities. During cellular senescence, the activity level of β-galactosidase increases significantly. This increased activity leads to the accumulation of senescent pigment lipofuscin within cells, a decline in intracellular waste disposal mechanisms, resulting in increased cell size and flattened morphology in senescent cells, and impairing cell division and proliferation. In some exemplary embodiments of the present invention, the aforementioned Bifidobacterium longum BBS 02, the metabiotic, or the composition can inhibit the significant increase in β-galactosidase activity caused by cellular senescence, inhibit pigment accumulation, maintain normal cell morphology, and regulate cell division and proliferation.

[0070] In other embodiments, Cdkn1a is a cyclin-dependent kinase inhibitor. Cdkn1a is closely related to aging, and its encoded p21 protein is a cyclin-dependent kinase inhibitor that blocks cell cycle progression by binding to cyclin-dependent kinases (CDKs). The accumulation of Cdkn1a and p21 is widely considered a marker and inducing factor of aging. Based on the above characteristics, the effects of the present invention can be further enhanced; that is, the aforementioned Bifidobacterium longum BBS 02, the aforementioned metabiotic, or the aforementioned composition can effectively inhibit the high expression of Cdkn1a and reduce the accumulation of Cdkn1a and p21.

[0071] In some alternative implementations, the product includes skin nutrients or edible nutrients.

[0072] <Skin Nutrients>

[0073] The present invention provides a skin nutrient, which includes or uses the composition described above.

[0074] The skin nutrients described in this invention refer to any substance that can be applied externally to the skin and may include various pharmaceutical dosage forms. For example, the skin nutrients may be solid, semi-solid, or liquid, and exemplaryly may be in the form of ointments, lotions, gels, creams, sprays, emulsions, skin emulsifiers, skin suspensions, transdermal delivery patches, medicated bandages, lotions, or combinations thereof, but are not limited thereto.

[0075] In some embodiments, the skin nutrients may further comprise a cosmetic or pharmaceutical carrier, which may include one or more preservatives, one or more antioxidants, one or more chelating agents, or a combination thereof.

[0076] Examples of preservatives include, but are not limited to, phenoxyethanol, parabens, and natural preservatives, and are known to those skilled in the art. The skin nutrients may contain about 0.01 to about 20% by weight (sometimes more preferably about 0.5 to about 5% by weight) of preservatives.

[0077] Antioxidants and / or chelating agents can also be used to extend the shelf life and stability of skin nutrients. Antioxidants can be added for formulation stabilization and bioavailability. Antioxidant compounds and their derivatives include, but are not limited to, water-soluble antioxidants such as thiol compounds and their derivatives (e.g., sodium metabisulfite and N-acetylcysteine), lipoic acid and dihydrolipoic acid, resveratrol, acetylcysteine ​​or lactoferrin, and ascorbic acid and ascorbic acid derivatives (e.g., ascorbyl palmitate and ascorbyl peptides).

[0078] Chelating agents also contribute to the stability of the composition. Examples of chelating agents include, but are not limited to, EDTA and its derivatives (e.g., disodium EDTA and dipotassium EDTA), lactoferrin, and citric acid. The skin nutrients of the present invention may contain about 0.001% to about 20% by weight (e.g., about 0.01% to about 10% by weight) of the chelating agent in the composition.

[0079] In some embodiments, the skin nutrients may further comprise functional additives and ingredients typically found in topical skin compositions. The functional additives may include ingredients selected from water-soluble vitamins, oil-soluble vitamins, polymeric peptides, polymeric polysaccharides, sphingolipids, and seaweed extracts. Ingredients typically found in topical skin compositions include oil components, moisturizers, emollients, surfactants, organic or inorganic pigments, organic powders, UV absorbers, bactericides, plant extracts, pH adjusters, alcohols, colorants, flavorings, blood circulation enhancers, coolants, antiperspirants, and purified water.

[0080] In other embodiments, the skin nutrients of the present invention further comprise a delivery system capable of retaining active catalase and / or glutathione peroxidase or catalase-related activity and delivering active ingredients (which may include active proteins) into the skin. Such delivery systems may include micro and nanoparticles, liposomes, aspartic proteases, organic gels, liposomes, transfections, patches, micro and nanoneedles, micro and nanocapsules, micro and nanosponges, films, polymers, etc.

[0081] Edible nutrients

[0082] This invention provides an edible nutrient that includes or uses the composition described above. Optionally, the content of Bifidobacterium longum BBS O2 in the edible nutrient is not less than 10%. 8 cfu / g or 10 8 cfu / mL, for example, could be 10 8 cfu / g, 10 9 cfu / g, 10 10 cfu / g, 10 8 cfu / mL, 10 9 cfu / mL, 10 10 cfu / mL, etc.

[0083] The edible nutritional composition of the present invention can have a sweet, salty, bitter, sour, or umami flavor, or a combination of two or more thereof.

[0084] This invention does not particularly limit the specific form of the edible nutrient, which may be solid, semi-solid, or liquid. Exemplarily, it may include a drinkable composition, a powder or granule composition, a gel, or a frozen or partially frozen composition. The edible nutrient may optionally be part of a capsule filler, or may optionally be part of a beverage, dairy product, non-dairy cream, sauce, or baked goods.

[0085] In some embodiments, the edible nutrient can have any texture, which can be defined by optionally adding it to the edible nutrient. For example, as a tissue and / or nutrient, one or more polypeptides of the extracellular matrix (e.g., elastin, etc.) can be added to the edible nutrient. The nutritional value may be comparable to or even superior to meat.

[0086] In some specific implementations, the edible nutrients may be powdered reconstituted foods (solid beverages, instant coffee, cereal powder, nut powder or lotus root powder, etc.), baked goods (bread, cakes or biscuits, etc.), beverages (carbonated beverages, fruit and vegetable juices, functional beverages, tea beverages, milk beverages or alcoholic beverages, etc.), candies (gel candies, hard candies, compressed candies, etc.), milk and dairy products (fresh milk, milk powder, whey powder, fermented milk, cheese or condensed milk, etc. derived from raw cow (sheep) milk), etc.

[0087] In some other specific embodiments, the edible nutrients described in this invention are oral preparations, including but not limited to tablets, pills, granules, powders, teas, capsules, or oral liquids.

[0088] In addition to the essential components described above, the edible nutrients of this invention may also include other optional ingredients, depending on the needs of the final product. Examples of such ingredients include:

[0089] Fiber, which can be any edible fiber, may also be specified as dietary fiber. Typically, in this invention, fiber is a plant-based food component that cannot be completely broken down by human digestive enzymes. For example, it may be derived from legumes, whole grains and cereals, vegetables, fruits, nuts, or seeds. For example, fiber may comprise or consist of cellulose, hemicellulose, chitin, pectin, resistant starch, or a combination of two or more thereof. Fiber may also comprise inulin, beta-glucan, raw guar gum, xanthan gum, fructan, lignin, polyuronic acid, alginic acid, or salts thereof, agar, carrageenan, raffinose, polydextrose, cyclodextrin, soybean polysaccharides, galactooligosaccharides, konjac flour, or a combination of two or more thereof. Many fibers are known to those skilled in the art and may be commercially available.

[0090] Sugars may include one or more sugars, such as sucrose, glucose, fructose, trehalose, sucrose, sorbitol, and glucan. For example, edible nutrients may contain a total amount of sugars of 0 to 80% by weight, 0 to 40% by weight, 0 to 6% by weight, 1 to 15% by weight, 5 to 15% by weight, or 10 to 40% by weight, based on the total mass of the edible nutrients.

[0091] Amino acids may include one or more naturally occurring amino acids. For example, edible nutrients may contain 0 to 80% by weight, 0 to 40% by weight, 0 to 6% by weight, 1 to 15% by weight, 5 to 15% by weight, or 10 to 40% by weight of total edible nutrients.

[0092] A polypeptide (also known as a protein) may contain one or more polypeptides of natural origin (e.g., obtained from milk, bacteria, plant-based expression systems, etc.). For example, an edible nutrient may contain 0 to 80% by weight, 0 to 40% by weight, 0 to 6% by weight, 1 to 15% by weight, 5 to 15% by weight, or 10 to 40% by weight of total edible nutrient content.

[0093] Fats (i.e., lipids) and / or fatty acids may comprise one or more naturally sourced fats and / or fatty acids (e.g., obtained from plants, fish, and / or milk). This includes essential fatty acids, unsaturated or polyunsaturated fatty acids. For example, edible nutrients may comprise a total amount of fats and / or fatty acids, calculated as 0% to 80% by weight, 0% to 40% by weight, 0% to 6% by weight, 1% to 15% by weight, 5% to 15% by weight, or 10% to 40% by weight, based on the total mass of the edible nutrients.

[0094] Electrolytes can include metal ion salts of organic acids, such as one or more of calcium citrate, L-calcium lactate, calcium hydrogen phosphate, potassium gluconate, sodium citrate, ferrous gluconate, potassium iodide, zinc gluconate, sodium selenite, copper gluconate, chromium sulfate, manganese gluconate, and magnesium gluconate. The osmolar concentration of electrolytes can be hypotonic, isotonic, or hypertonic compared to human blood (understood herein as the osmolar concentration of an aqueous solution of water and 0.9% sodium chloride). The molar osmolar concentration can be adjusted and regulated by the total content of dissolved components in edible nutrients.

[0095] Any food-grade excipients, including but not limited to solvents, antioxidants, antibacterial agents, thickeners, diluents, solubilizers, stabilizers, emulsifiers, fillers, disintegrants, lubricants, coating materials, anti-caking agents, flavoring agents, sweeteners, edible flavorings, and edible colorings. This invention does not impose a specific limitation on the specific content of any acceptable excipient in the edible nutrient. Those skilled in the art can adjust it according to actual needs. For example, the specific content of any acceptable excipient in the edible nutrient does not exceed 5% by mass, preferably not more than 3% by mass.

[0096] In some optional embodiments, the pH of the edible nutrient can be adjusted to the desired range. Preferably, the pH is substantially neutral. In one embodiment, the pH is in the range of 6-8 or 6.5-7.5. The pH can be adjusted using edible acids, bases, buffers, and / or acidity regulators.

[0097] The edible nutrients provided by this invention are suitable for all people in principle, especially for people with cellular aging and skin aging. The components in the food can also be adjusted accordingly for people with different characteristics.

[0098] Example

[0099] The embodiments of the present invention will be described in detail below with reference to examples. However, those skilled in the art will understand that the following examples are for illustrative purposes only and should not be considered as limiting the scope of the invention. Unless otherwise specified in the examples, conventional conditions or conditions recommended by the manufacturer are followed. Reagents or instruments whose manufacturers are not specified are all commercially available conventional products.

[0100] Example 1: Isolation, identification and preservation of Bifidobacterium longum BBS 02

[0101] (1) Isolation and screening: The samples were obtained from the intestines of healthy infants. After pretreatment, the samples were serially diluted and spread on MRS medium. They were anaerobically cultured at 37°C for 48 hours. Single colonies on the plates were picked and streaked to obtain pure colonies.

[0102] (2) Identification:

[0103] Gram staining of the selected strains showed that they were Gram-positive. The colonies were round, white, raised, with a smooth, moist surface and regular edges. The strains were rod-shaped with varying lengths and thicknesses.

[0104] The 16S rDNA of the strain was amplified and sequenced. The obtained 16S rDNA sequence was compared with the nucleic acid in NCBI. The results showed that the strain was Bifidobacterium longum, named Bifidobacterium longum BBS 02, and deposited in the China General Microbiological Culture Collection Center (CGMCC) with the accession number CGMCC No. 30652.

[0105] 16s rDNA sequence (SEQ ID NO:1):

[0106]

[0107] Example 2: Preparation of fermentation supernatant and lysate of Bifidobacterium longum BBS 02

[0108] Bifidobacterium longum BBS 02 was inoculated into MRS medium and cultured at 37°C for 24 h. The cultured bacterial suspension was vortexed for 15 s and centrifuged at 4500 rpm for 20 min. The clear supernatant (extracellular secretions) was collected. The lower bacterial pellet was resuspended in PBS, centrifuged at 4500 rpm for 20 min, the supernatant was discarded, and the sample was washed twice with PBS. After centrifugation at 4500 rpm for 20 min, the colony count was adjusted to 1 × 10⁻⁶ cells / mL with PBS. 9 CFU / mL. The colony count was 1×10⁻⁶. 9 The bacterial suspension of CFU / mL was sonicated in an ice bath for 30 min with the following parameters: power 35%, working time 4 s, interval 6 s. The resulting solution is the cell lysate containing cell lysates.

[0109] Example 3: Effects of Bifidobacterium longum BBS O2 on senescent dermal fibroblasts

[0110] (1) Construction of cell model

[0111] A D-galactose (D-gal)-induced human skin fibroblast senescence model, the induction process is as follows: Figure 1 As shown: FM-2 complete cell culture medium was added to a six-well plate, and human skin fibroblasts (HSF cells) were seeded into the six-well plate and cultured at 37°C and 5% CO2. When the cells grew to adhere (confluence of 70%), the original culture medium was removed, and the cells were washed twice with PBS. Then, 2 mL of D-gal at a concentration of 50 g / L was added to each well, and the cells were cultured for another 24 h.

[0112] (2) Test treatment

[0113] Using the cell lysate and fermentation supernatant prepared in Example 2, PBS was added to dilute them and cell lysates with concentrations of 12.5% ​​(v / v), 25% (v / v), and 50% (v / v) and fermentation supernatants with concentrations of 5% (v / v), 10% (v / v), and 15% (v / v) were prepared.

[0114] Add 12.5% ​​(v / v), 25% (v / v), and 50% (v / v) of the cell lysis buffer and 2 mL / well of 5% (v / v), 10% (v / v), and 15% (v / v) of fermentation supernatant to the aging model in step (1), and culture at 37°C and 5% CO2 for 24 h.

[0115] (3) Detection of cell viability and aging-related gene expression levels

[0116] ① Cell viability assay: After the culture is completed, discard the original culture medium in the wells, add freshly prepared complete culture medium containing 1 / 10 of the final volume of CCK-8 solution to each well, and incubate the plate at 37℃ and 5% CO2 for 4 hours. Measure the absorbance at OD450nm.

[0117] ② β-galactosidase activity assay: The β-galactosidase activity in the aging model supplemented with 50% (v / v) cell lysate and 15% (v / v) fermentation supernatant was measured.

[0118] The Solarbio β-galactosidase staining kit (catalog number: G1580) was used. After culture, the original culture medium in the wells was discarded, and the cells were washed once with PBS. 1 mL of β-Gal fixative was added, and the cells were fixed at room temperature for 15 min. The cell fixative was removed, and the cells were washed twice with 1×PBS for 3 min each time. Before adding the staining working solution, the cells were washed and incubated using reagent (E):β-Gal staining solution C instead of 1×PBS. The staining working solution was prepared according to the ratio of reagents B:C:D:E = 5:1:1:93. The β-Gal washing solution was removed, and 1 mL of the staining working solution was added to each well. The 6-well plate was sealed with plastic wrap to prevent evaporation and incubated overnight at 37°C. The staining was observed under a regular optical microscope.

[0119] ③ Detection of aging-related gene expression levels: The expression levels of aging-related genes in the aging model above, which was supplemented with 50% (v / v) cell lysate and 15% (v / v) fermentation supernatant, were detected.

[0120] After culture, RNA was extracted from the cells, and the RNA was reverse transcribed and then subjected to RT-PCR to detect the expression levels of aging-related genes.

[0121] The cell viability is calculated using the formula: Cell viability = [(Experimental group absorbance - Blank group absorbance) / (Control group absorbance - Blank group absorbance)] × 100%.

[0122] Untreated human skin fibroblasts were used as the control group, 50 g / L D-gal was added as the model control group, and 50 μg / ml vitamin C was added as the positive control group.

[0123] Cell viability test results as follows Figure 2 As shown, cell lysate and fermentation supernatant of Bifidobacterium longum BBS 02 significantly promoted cell proliferation in a cell senescence model.

[0124] β-galactosidase activity results are as follows Figure 3 and Figure 4 As shown, cell lysates of Bifidobacterium longum BBS 02 significantly reduced the activity of β-galactosidase.

[0125] The expression levels of the aging-related gene Cdkn1a are as follows: Figure 5 As shown: Cell lysate and fermentation supernatant of Bifidobacterium longum BBS 02 inhibited the high expression of the gene Cdkn1a, which encodes a cyclin-dependent kinase inhibitor.

[0126] Example 4: Stability test of Bifidobacterium longum BBS O2

[0127] To test the stability of Bifidobacterium longum BBS 02, this embodiment simulated the digestion process in vitro, using artificial saliva, artificial gastric juice, and artificial intestinal juice.

[0128] Artificial saliva composition (pH=6.8): 3.0 g / L α-amylase, 1.60 g / L sodium chloride, 0.35 g / L ammonium nitrate, 0.65 g / L potassium dihydrogen phosphate, 0.30 g / L potassium chloride, 0.40 g / L trisodium citrate dihydrate, 0.15 g / L sodium lactate.

[0129] Artificial gastric juice composition (pH=2.0): 3.2 g / L pepsin, 3.0 g / L sodium chloride, 0.27 g / L sodium dihydrogen phosphate, 1.0 g / L potassium chloride, 0.02 g / L ascorbic acid, 1.0 g / L glucose.

[0130] Artificial intestinal fluid composition (pH=7.0): 24.0 g / L trypsin, 24.0 g / L lipase, 198.0 g / L sodium chloride, 40.0 g / L calcium chloride, 5.6 g / L sodium bicarbonate, 54 g / L bile salts.

[0131] The specific steps are as follows:

[0132] (1) Inoculate Bifidobacterium longum BBS 02 into MRS medium and culture for 24 h. Inoculate artificial saliva at a rate of 10% (v / v) and culture at 37°C for 5 min. Take a sample to determine the number of viable bacteria and the survival rate.

[0133] (2) The strain that had been cultured in artificial saliva for 5 min was added to artificial gastric juice and cultured at 37°C for 2 h. Samples were taken at 30 min, 60 min and 120 min to determine the number of viable bacteria and the survival rate of the strain.

[0134] (3) The strain that had been cultured in artificial gastric fluid for 120 min was added to artificial intestinal fluid and cultured at 37°C for 120 min. Samples were taken at 60 min and 120 min to determine the number of viable bacteria and the survival rate of the strain.

[0135] Survival rate (%) = (Number of viable bacteria measured / Initial number of viable bacteria) × 100%

[0136] The results are as follows Figure 6 As shown, the survival rate of Bifidobacterium longum BBS 02 after 5 min of culture in saliva was 96%. The survival rates after 30 min, 60 min and 120 min of culture in gastric juice were 77%, 65% and 58% respectively. Finally, the survival rates after 60 min and 120 min of culture in intestinal juice were 49% and 45% respectively. Therefore, Bifidobacterium longum BBS 02 can tolerate the environment of saliva and gastrointestinal tract and can grow well in the digestive environment.

[0137] Comparative Example 1

[0138] While screening for Bifidobacterium longum BBS 02, three strains of Bifidobacterium longum, L-22, L-37, and L-113, were obtained. After culturing these three strains according to the method in Example 2, fermentation supernatant and cell lysate were obtained according to the method in Example 2. After treatment according to the method in Example 3, cell viability, β-galactosidase activity, and expression levels of aging-related genes were detected.

[0139] The results are as follows Figures 2-5 As shown, the fermentation supernatant and cell lysate of L-22, L-37, and L-113 did not promote cell proliferation in the cell senescence model, or their promoting effect on cell proliferation was smaller compared to that of Bifidobacterium longum BBS 02. Figure 2 The inhibitory effects of L-22, L-37, and L-113 on the fermentation supernatant and cell lysate of β-galactosidase activity were not significant. Figure 3 and Figure 4 Similarly, the fermentation supernatant and cell lysate of L-22, L-37, and L-113 did not significantly inhibit the high expression of the cyclin-dependent kinase inhibitor gene Cdkn1a. Figure 5 ).

[0140] Comparative Example 2

[0141] The *Bifidobacterium pseudomallei* obtained during the screening of *Bifidobacterium longum* BBS 02 was treated according to the method in Example 4, and the viable count was tested. The results are as follows: Figure 6 As shown in b, Bifidobacterum pseudomallei has relatively poor tolerance to saliva and the digestive environment of the gastrointestinal tract.

Claims

1. Bifidobacterium longum, which has been deposited at the China General Microbiological Culture Collection Center, with accession number CGMCC No. 30652.

2. A metabiotic derived from Bifidobacterium longum as described in claim 1.

3. The postbiotic of claim 2, wherein, The post-biotic is selected from inactivated bacterial cells, fermentation supernatant and bacterial cell lysate, or powders prepared by any drying method thereof.

4. The postbiotic of claim 2 or 3, wherein, The metabiotic is obtained by culturing Bifidobacterium longum in a culture system as described in claim 1, and then processing the culture to obtain the metabiotic.

5. A composition characterized in that, The composition comprises Bifidobacterium longum as described in claim 1 and / or metabiotics as described in any one of claims 2 to 4.

6. The use of the Bifidobacterium longum as described in claim 1, the metabiotic as described in any one of claims 2 to 4, or the composition as described in claim 5 in the preparation of products for preventing and / or improving skin aging; Optionally, the prevention of skin aging and / or improvement of skin aging includes at least one of the following (a) to (c): (a) Enhances cell vitality and proliferation capacity; (b) Inhibits β-galactosidase activity; (c) Inhibit the high expression of Cdkn1a, the gene encoding cyclin-dependent kinase inhibitor.

7. The application according to claim 6, characterized in that, The products include skin nutrients or edible nutrients.

8. A skin nutrient, characterized in that, The nutrients include or use the composition according to claim 5.

9. An edible nutrient, characterized in that, The edible composition includes or uses the composition according to claim 5.

10. The edible nutrient according to claim 9, characterized in that, The content of the Bifidobacterium longum in the edible nutrient is not less than 10 8 cfu / g or 10 8 cfu / mL.

11. The nutrient according to any one of claims 8 to 10, characterized in that, The nutrients exist in solid, semi-solid, or liquid form.

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