Bifidobacterium adolescentis (BA)-3 and use thereof in resisting aging, resisting oxidation, and resisting inflammation

Abstract

Provided are bifidobacterium adolescentis (BA)-3 and a use thereof in resisting aging, resisting oxidation, and resisting inflammation. The BA-3 can break a vicious circle of radicals, oxidative stress, inflammation, oxidative stress, and radicals, and can realize comprehensive improvement in multiple aspects, so that the BA-3 has the performance advantage significantly superior to that of other strains when being used for resisting aging, resisting oxidation, and resisting inflammation.

Description

Bifidobacterium adolescentis BA-3 and its application in anti-aging, anti-oxidation and anti-inflammation

[0001] Cross-reference information

[0002] This application claims priority to Chinese patent application No. 2023111480463 filed on September 6, 2023, the entire contents of which are incorporated herein by reference. Technical Field

[0003] The present invention belongs to the field of microorganisms, and in particular relates to Bifidobacterium adolescentis BA-3 and its application in anti-aging, anti-oxidation and anti-inflammation. Background Art

[0004] Aging is a natural and unavoidable physiological process within organisms. It's generally believed that the fundamental purpose of anti-aging efforts is to extend lifespan as much as possible and, on this basis, enhance vitality. However, there are currently no recognized effective interventions to delay aging. Therefore, in-depth research into the mechanisms of aging and the development of safe and effective anti-aging products are of great significance.

[0005] Aging is often accompanied by chronic, low-grade, systemic inflammation, involving the activation of inflammatory networks and the release of related cytokines, including key pro-inflammatory mediators such as TNF-α, IL-6, and IL-1β. This process is a key driver of aging and age-related diseases. Aging is also inextricably linked to oxidative stress. In 1956, Harman of the United States proposed the famous free radical theory. This theory posits that under normal circumstances, the body maintains a balance between the generation and scavenging of oxygen free radicals. However, with aging, the body's ability to scavenge free radicals decreases, disrupting this balance. Oxygen free radical production exceeds the cellular antioxidant capacity, leading to oxidative stress and aging. Inflammation is also closely linked to oxidative stress. Oxidative stress caused by free radicals and inflammation interact through transcriptional regulation. Oxidative stress exacerbates the inflammatory response, and inflammation, through inflammatory mediators, promotes oxidative stress, creating a vicious cycle of free radicals, oxidative stress, inflammation, oxidative stress, and free radicals.

[0006] From birth to aging and death, the intestinal flora is always in a dynamic process of change. Studies on different population cohorts at home and abroad have confirmed that there are significant differences in the composition and abundance of intestinal flora in different age groups. Studies have shown that as the body gradually ages and enters old age, the number and abundance of Bifidobacteria and Bacteroides decrease significantly, the number of facultative anaerobic bacteria such as Fusobacterium increases significantly, the diversity of intestinal microbiota decreases, and the changes in differences between individuals are greater than those in young people. At the strain level, more studies have shown that the abundance of adolescent Bifidobacteria in the intestines will decrease significantly with age.

[0007] Research results published in "Nature Aging" in 2021 showed that dietary supplementation of the model strain Bifidobacterium adolescentis ATCC15703 may extend the healthspan and lifespan of various animal models by upregulating the expression and activity of the host's catalase (CAT).

[0008] Another report in "Gut Microbes" shows that Bifidobacterium adolescentis can alleviate DSS-induced chronic colitis by inducing protective Th2 / Treg responses and reshaping intestinal microbiota, suggesting that Bifidobacterium adolescentis may improve the clinical treatment effect of inflammatory bowel disease.

[0009] Summary of the Invention

[0010] The present invention screened three superior candidate strains of Bifidobacterium adolescentis (BA-3, BA-4, and BA-11) from healthy young women. Using an LPS (lipopolysaccharide)-induced mouse inflammation model, the researchers tested the pro-inflammatory cytokines TNF-α, IL-6, and IL-1β, SOD activity, and MDA levels, confirming that BA-3 possessed significantly superior anti-inflammatory and antioxidant effects. Subsequently, in vivo anti-aging experiments in Drosophila were conducted, comparing the strains with the known anti-aging model strain ATCC15703. This demonstrated that BA-3 significantly outperformed ATCC15703 in delaying aging.

[0011] Based on the above findings, the present invention first provides Bifidobacterium adolescentis BA-3, which was deposited on June 14, 2023 in the General Microbiology Center of China Culture Collection Committee (CGMCC for short, address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, Postal Code 100101), and was classified and named Bifidobacterium adolescentis, and the collection number is CGMCC NO.27625.

[0012] Furthermore, the present invention also provides a bacterial agent containing the Bifidobacterium adolescentis BA-3.

[0013] In some embodiments, the bacterial agent may be a liquid bacterial agent or a solid bacterial agent.

[0014] In some embodiments, the bacterial agent further contains other functional bacteria.

[0015] In some embodiments, the bacterial agent further contains excipients permitted in the field of microbial preparations.

[0016] In the specific implementation scheme, the bacterial agent can be prepared by conventional technical means.

[0017] Furthermore, the present invention also provides a fermentation product or fermentation extract, which is obtained by fermenting the Bifidobacterium adolescentis BA-3.

[0018] In the present invention, the fermentation supernatant and bacterial cells obtained after fermentation of Bifidobacterium adolescentis BA-3 have positive effects in anti-aging, anti-oxidation and anti-inflammatory.

[0019] In the present invention, the fermentation product is the fermentation supernatant and / or bacterial cells obtained after fermenting the Bifidobacterium adolescentis BA-3.

[0020] In the present invention, the fermentation extract is an organic solvent extract of the fermentation broth supernatant and / or bacterial cells obtained after fermentation of the Bifidobacterium adolescentis BA-3.

[0021] Furthermore, the present invention also provides the use of the Bifidobacterium adolescentis BA-3 or the bacterial agent or the fermentation product or fermentation extract in at least one of the following aspects: 1) anti-aging; 2) anti-oxidation; 3) anti-inflammatory.

[0022] The term "anti-aging" as used herein may also be understood to mean "delaying aging," "preventing or slowing signs of aging," "extending lifespan," and similar meanings. Similarly, the term "antioxidant" as used herein may also be understood to mean "preventing or reducing peroxidative damage," "eliminating excessive oxidative free radicals," and similar meanings. The term "anti-inflammatory" as used herein may also be understood to mean "preventing or reducing inflammation," and similar meanings. The use of the strains, bacterial agents, or fermented products or fermented extracts thereof in the aforementioned aspects also falls within the scope of protection of the present invention.

[0023] Furthermore, the present invention also provides the use of the Bifidobacterium adolescentis BA-3 or the bacterial agent or the fermentation product or fermentation extract in the preparation of a composition, and the composition is used for at least one of the following aspects: 1) anti-aging; 2) anti-oxidation; 3) anti-inflammatory.

[0024] In some embodiments, the Bifidobacterium adolescentis BA-3 or the bacterial agent or the fermentation product or fermentation extract is used to reduce the level of pro-inflammatory factors.

[0025] Preferably, the pro-inflammatory factors include one or more of TNF-α (tumor necrosis factor-α), IL-1β (interleukin-1β), and IL-6 (interleukin-6). TNF-α, IL-1β, and IL-6 are all pro-inflammatory factors that play an important role in inflammation in the body, and changes in their levels can reflect the inflammatory status in the body.

[0026] In some embodiments, the Bifidobacterium adolescentis BA-3 or the bacterial agent or the fermentation product or fermentation extract is used for at least one of the following purposes: a) increasing the activity of antioxidant enzymes; b) reducing the level of peroxidation products.

[0027] Preferably, the antioxidant enzyme includes SOD (superoxide dismutase). SOD is a class of enzymes that can catalyze the superoxide anion free radical (O2 - ) into H2O2 and O2, which has antioxidant and anti-aging effects. Its mechanism of action is mainly to remove superoxide anion free radicals (O2 - ).

[0028] Preferably, the peroxidation product includes MDA (malondialdehyde). MDA is a product of intracellular lipid peroxidation and is also an important oxidative stress indicator.

[0029] In some embodiments, the subjects of the application of the present invention include humans and animals other than humans. The animals other than humans mentioned herein can include primates such as monkeys, orangutans, gibbons, gorillas, etc.; livestock such as pigs, cattle, sheep, horses, camels, cats, dogs, etc.; poultry such as chickens, ducks, geese, quails, etc.; and the like.

[0030] In some embodiments, the composition is a food composition, a feed composition, a skin topical composition, or a pharmaceutical composition.

[0031] The present invention also provides a food composition, which contains the Bifidobacterium adolescentis BA-3 or the bacterial agent or the fermentation product or fermentation extract.

[0032] In some embodiments, the food composition is a health functional food.

[0033] The present invention is not particularly limited to the type of food composition, and can be in the form of oral preparations such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, or can be added to ordinary foods such as sugar, biscuits, chewing gum, ice cream, noodles, bread, beverages, etc.

[0034] The food composition of the present invention can be prepared by conventional methods using one or more of fillers, extenders, binders, wetting agents, disintegrants, sweeteners, flavorings, preservatives, surfactants, lubricants, excipients, etc., depending on the form.

[0035] The present invention also provides a feed composition, which contains the Bifidobacterium adolescentis BA-3 or the bacterial agent or the fermentation product or fermentation extract.

[0036] In some embodiments, the feed composition may be a carbohydrate feed, a protein feed, a green feed, or a mineral feed, etc. Meanwhile, those skilled in the art can determine the specific components of the above feeds based on common sense. For example, the carbohydrate feed may contain corn, bran, wheat, barley, sorghum, and rice bran, etc.

[0037] The present invention also provides a skin external composition, which contains the Bifidobacterium adolescentis BA-3 or the bacterial agent or the fermentation product or fermentation extract.

[0038] The skin external composition of the present invention refers to any substance that can be applied externally to the skin and can include various pharmaceutical dosage forms. For example, the composition can be in the form of an ointment, lotion, gel, cream, spray, suspension, emulsion, patch, etc., but is not limited thereto.

[0039] In some embodiments, the skin topical composition may further include functional additives and ingredients included in general skin topical compositions. Wherein, the functional additives may include ingredients selected from water-soluble vitamins, oil-soluble vitamins, polymer peptides, polymer polysaccharides, sphingolipids and seaweed extracts. The ingredients included in general skin topical compositions include oil components, moisturizers, emollients, surfactants, organic or inorganic pigments, organic powders, ultraviolet absorbers, preservatives, bactericides, antioxidants, plant extracts, pH regulators, alcohols, colorants, flavorings, blood circulation promoters, coolants, antiperspirants and purified water.

[0040] The present invention also provides a pharmaceutical composition containing the Bifidobacterium adolescentis BA-3 or the bacterial agent or the fermentation product or fermentation extract.

[0041] The pharmaceutical composition of the present invention can be administered orally (e.g., by oral administration or inhalation) or parenterally (e.g., by injection, percutaneous absorption, or rectal administration). Injection can be, for example, intravenous, subcutaneous, intramuscular, or intraperitoneal.

[0042] The pharmaceutical composition of the present invention can be formulated into tablets, capsules, granules, fine subtilae, powders, sublingual tablets, suppositories, ointments, injections, emulsions, suspensions, syrups, sprays, etc., depending on the route of administration. The pharmaceutical composition of the present invention in various forms can be prepared using pharmaceutical carriers commonly used in various dosage forms and by known techniques. Examples of pharmaceutically acceptable carriers include excipients, binders, disintegrating agents, lubricants, preservatives, antioxidants, isotonic agents, buffers, coating agents, sweeteners, solubilizers, bases, dispersants, wetting agents, suspending agents, stabilizers, colorants, etc.

[0043] The terms "and / or", "or / and", and "and / or" used in the present invention include any one of two or more related listed items, and also include any and all combinations of the related listed items, wherein the arbitrary and all combinations include any combination of two related listed items, any more related listed items, or all related listed items. It should be noted that when at least three items are connected by at least two conjunctions selected from "and / or", "or / and", and "and / or", it should be understood that in this application, the technical solution undoubtedly includes technical solutions connected by "logical and" and also undoubtedly includes technical solutions connected by "logical or". For example, "A and / or B" includes three parallel solutions of A, B and A+B. For example, the technical solution of "A, and / or, B, and / or, C, and / or, D" includes any one of A, B, C, and D (that is, the technical solution of all being connected by "logical OR"), and also includes any and all combinations of A, B, C, and D, that is, the combination of any two or any three of A, B, C, and D, and also includes the four-item combination of A, B, C, and D (that is, the technical solution of all being connected by "logical AND").

[0044] As used herein, the terms "comprising," "including," and "comprising" are synonymous and are inclusive or open-ended and do not exclude additional, unrecited members, elements, or method steps.

[0045] In the present invention, descriptions such as "plurality" and "multiple" refer to quantities greater than or equal to 2 unless otherwise specified.

[0046] In the present invention, the technical features described in an open manner include closed technical solutions composed of the listed features, and also include open technical solutions containing the listed features.

[0047] In the present invention, "preferably", "better", "more preferably", and "suitably" are merely descriptions of preferred implementation methods or examples, and should be understood to not limit the scope of protection of the present invention. In the present invention, "optionally", "optional", and "optional" refer to being optional, that is, to being selected from either of the two parallel options of "with" or "without". If multiple "options" appear in a technical solution, unless otherwise specified and without contradiction or mutual restriction, each "optional" is independent.

[0048] The Bifidobacterium adolescentis BA-3 of the present invention can break the vicious cycle of free radicals-oxidative stress-inflammation-oxidative stress-free radicals and achieve comprehensive improvements in many aspects. Therefore, it has performance advantages that are significantly superior to other strains of the same species when used for anti-aging, anti-oxidation and anti-inflammation. BRIEF DESCRIPTION OF THE DRAWINGS

[0049] In order to more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the specific embodiments or the description of the prior art. Obviously, the drawings described below are some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0050] Figure 1 shows the effects of different Bifidobacterium adolescentis interventions on the body weight, food intake and water intake of mice in an embodiment of the present invention; in the figure, A: changes in body weight of mice continuously gavaged with different Bifidobacterium adolescentis for 14 days; B: food intake of mice; C: water intake of mice.

[0051] Figure 2 shows the effects of different Bifidobacterium adolescentis interventions on inflammatory factors in the serum of mice with LPS inflammation model in an embodiment of the present invention; in the figure, A: serum TNF-α level; B: serum IL-1β level; C: serum IL-6 level; *P<0.05, **P<0.01, ***P<0.001vs LPS group; #P<0.05, ##P<0.01, ###P<0.001vs Control Group.

[0052] Figure 3 shows the effects of different Bifidobacterium adolescentis interventions on oxidative stress factors in the serum of mice with LPS inflammation model in an embodiment of the present invention; in the figure, A: SOD activity in serum; B: MDA level in serum; *P<0.05, **P<0.01, ***P<0.001 vs LPS group; #P<0.05, ##P<0.01, ###P<0.001 vs Control Group.

[0053] Figure 4 shows the effect of exogenously added BA-3 on the wild-type Drosophila w 1118 In the figure, A and B are: wild-type female and male fruit flies w 1118 Survival curves of wild-type female and male fruit flies; C and D are respectively 1118 Comparison of average lifespan, median lifespan and maximum lifespan; *P<0.05, **P<0.01, ***P<0.001 vs Placebo group; #P<0.05, ##P<0.01, ###P<0.001 vs ATCC15703 group.

[0054] Figure 5 shows the effect of exogenously added BA-3 on the wild-type Drosophila w 1118 The effect of gravity on climbing ability; In the figure, A and B are: wild-type female and male fruit flies w 1118 Comparison of anti-gravity crawling ability on the 30th day; *P<0.05, **P<0.01, ***P<0.001 vs Placebo group; #P<0.05, ##P<0.01, ###P<0.001 vs ATCC15703 group. DETAILED DESCRIPTION

[0055] The following describes specific embodiments of the present invention in detail. It should be understood that the specific embodiments described herein are intended only to illustrate and explain the present invention and are not intended to limit the present invention. Those skilled in the art may make various modifications and variations to the present invention without departing from the scope or spirit of the present invention. For example, features illustrated or described as part of one embodiment may be used in another embodiment to produce further embodiments.

[0056] Unless otherwise indicated, all terms (including technical and scientific terms) used to disclose the present invention have the same meaning as commonly understood by one of ordinary skill in the art to which the present invention belongs. By way of further guidance, the following definitions are provided to better understand the teachings of the present invention. The terms used herein in the specification of the present invention are only for the purpose of describing specific embodiments and are not intended to limit the present invention.

[0057] The embodiments of the present invention will be described in detail below with reference to the examples. It should be understood that these examples are intended to illustrate the present invention only and are not intended to limit the scope of the invention. For experimental methods in the following examples where specific conditions are not specified, reference is made to the guidance provided in the present invention, and may also be made to experimental manuals or conventional conditions in the art, other experimental methods known in the art, or conditions recommended by the manufacturer.

[0058] The Bifidobacterium adolescentis BA-4 and Bifidobacterium adolescentis BA-11 used in the following examples are other dominant strains of Bifidobacterium adolescentis obtained during the screening of dominant strains in the present invention. The Bifidobacterium adolescentis ATCC15703 used in the following examples was obtained from a commercial source.

[0059] In the following specific examples, the measured parameters of raw material components may have slight deviations within the range of weighing accuracy unless otherwise specified. For temperature and time parameters, acceptable deviations caused by instrument testing accuracy or operational accuracy are allowed.

[0060] Example 1 Isolation, screening and identification of Bifidobacterium adolescentis BA-3

[0061] 1. Isolation and screening of Bifidobacterium adolescentis BA-3

[0062] Several healthy young women without inflammation or any other intestinal diseases were recruited to provide samples. Participants underwent health checkups at a medical examination center and provided information about their age (20-25 years), menstrual cycle, and other health behaviors via a questionnaire. Within six months prior to sample collection, no antibiotics, probiotics, or inflammatory diseases were used.

[0063] The collected fecal sample was diluted and spread on GAM blood agar medium, incubated anaerobically at 36℃~38℃ for 48~72h, and a single colony was picked and streaked on a new GAM blood agar plate to obtain purified colonies. After isolation, activity screening and identification, Bifidobacterium adolescentis BA-3 was obtained.

[0064] 2. Physiological characteristics of Bifidobacterium adolescentis BA-3

[0065] After anaerobic culture of Bifidobacterium adolescentis BA-3 on GAM blood agar medium for 48 hours, the colonies were round and raised with neat edges and a moist and smooth surface; Gram staining was positive, with no spores, no capsules, no flagella, and the bacteria were straight or curved.

[0066] 3. Molecular identification of Bifidobacterium adolescentis BA-3

[0067] Bifidobacterium adolescentis BA-3 was inoculated into GAM broth medium and cultured anaerobically at 36°C to 38°C for 24 hours. The bacteria were then collected by centrifugation and DNA was extracted for whole genome sequencing.

[0068] The molecular identification results showed that the ANI (Average Nucleotide Identity) value between Bifidobacterium adolescentis BA-3 and GCF_003467335.1 (Bifidobacterium adolescentis) was 98.7072%.

[0069] Bifidobacterium adolescentis BA-3 was deposited on June 14, 2023 in the General Microbiology Center of China Culture Collection Administration (CGMCC, address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, Postal Code 100101), and classified and named Bifidobacterium adolescentis, with the preservation number CGMCC NO.27625.

[0070] 4. Whole-genome data analysis

[0071] The whole genome of Bifidobacterium adolescentis BA-3 was sequenced using a whole genome shotgun (WGS) strategy to construct libraries with different inserts. Sequencing was performed using second-generation sequencing (NGS) technology on the Illumina NovaSeq sequencing platform and third-generation single-molecule sequencing technology on the PacBio Sequel sequencing platform.

[0072] Then, combined with the sequencing results, we analyzed the drug resistance genes, virulence factors, and bioamine production potential of Bifidobacterium adolescentis BA-3. The specific analysis process and results are as follows:

[0073] 1) Analysis of drug-resistant genes of Bifidobacterium adolescentis BA-3

[0074] Analytical methods

[0075] The Comprehensive Antibiotic Resistance Database (CARD, website: card.mcmaster.ca) was used, and the whole genome sequence of BA-3 was uploaded to https: / / card.mcmaster.ca / analyze / rgi. The software RGI (v6.0.2) provided by CARD was used to compare it with the CARD 3.2.7 database. The default parameters were selected for analysis, specifically Select Data Type: DNA sequence; Select Criteria: Perfect and Strict hits only; Nudge ≥ 95% identity Loose hits to Strict: Exclude nudge; Sequence Quality: High quality / coverage.

[0076] Analysis Conclusion

[0077] By comparing with the drug-resistant gene database, no extended-spectrum β-lactamases, vancomycin-resistant genes, carbapenem-resistant genes, methicillin-resistant genes and multidrug-resistant genes were detected in the genome of Bifidobacterium adolescentis BA-3.

[0078] 2) Virulence gene analysis of Bifidobacterium adolescentis BA-3

[0079] Analytical methods

[0080] The virulence factor database (VFDB, http: / / www.mgc.ac.cn / VFs / ) was used to download the virulence factor gene database VFDB (version 2023.04.11). Diamond v2.0.15.153 software was used based on the blastx algorithm with parameters selected as identity>80% and coverage>70% (command line: --id 80 --query-cover 70) to compare the BA-3 genome nucleotide sequence with the amino acid sequence in the database.

[0081] Analysis results

[0082] By comparing with the virulence gene database, no gene in the genome of Bifidobacterium adolescentis BA-3 was annotated as a virulence gene.

[0083] 3) Analysis of the bioamine production potential of Bifidobacterium adolescentis BA-3

[0084] Analytical methods

[0085] The amino acid sequence of the whole genome gene of Bifidobacterium adolescentis BA-3 was compared with the eggNOG (evolutionary gene genealogy Nonsupervised Orthologous Groups) database using emapper-2.1.9 software.

[0086] Analysis results

[0087] By comparing with the eggNOG database and searching for bioamine-related genes, no bioamine-related genes such as histidine decarboxylase, tyrosine decarboxylase, ornithine decarboxylase and lysine decarboxylase were detected in the genome of Bifidobacterium adolescentis BA-3.

[0088] Example 2 LPS-induced mouse inflammation model

[0089] Experimental methods

[0090] 1. Grouping and modeling methods

[0091] Healthy ICR mice, all male, totaling 30, with a body weight range of 22±2g, were kept at an ambient temperature of 20-25℃, fed with standard feed, and had free access to food and water, with a diurnal cycle of every twelve hours. The mice were adaptively raised for one week and randomly divided into a blank control group (Control), a model control group (LPS), a Bifidobacterium adolescentis BA-3 intervention group (LPS+BA-3), a Bifidobacterium adolescentis BA-4 intervention group (LPS+BA-4), and a Bifidobacterium adolescentis BA-11 intervention group (LPS+BA-11), with 6 mice in each group. The intervention groups were given Bifidobacterium adolescentis (resuspended in sterile PBS to a final concentration of 10) by gavage every day. 9 CFU / mL) 200 μL. The blank control group and model control group were gavaged daily with an equal volume of sterile PBS solution for 14 consecutive days. After the last dose, mice in the model control group and the Bifidobacterium adolescentis BA-3, Bifidobacterium adolescentis BA-4, and Bifidobacterium adolescentis BA-11 intervention groups were intraperitoneally injected with LPS 5 mg / kg (prepared in normal saline) to establish an LPS mouse acute inflammation model. The blank control group was injected with an equal volume of normal saline and the mice were killed 2 hours later for blood collection.

[0092] 2. Changes in weight, food intake, and water intake

[0093] During the intervention period, the body weight, 24-hour food intake, and water intake of each group of mice were recorded every two days. GraphPad Prism 9 was used to analyze the data for significance.

[0094] 3. Determination of serum IL-1β, IL-6, and TNF-α levels

[0095] Blood samples were collected from each group of mice, incubated at room temperature for 30 minutes, and centrifuged at 3000 g for 20 minutes. Serum was carefully aspirated and used for subsequent assays. Serum concentrations of IL-6, TNF-α, and IL-1β were measured using the double antibody sandwich assay according to the instructions of the ELISA kits. Data were analyzed for significance using GraphPad Prism 9.

[0096] 4. Determination of SOD and MDA levels in mouse serum

[0097] The levels of SOD and MDA in mouse serum were determined according to the kit instructions. The absorbance was measured at 450 nm using a microplate reader. GraphPad Prism 9 was used for data significance analysis.

[0098] Experimental results

[0099] 1. Changes in weight, food intake, and water intake

[0100] All groups of mice grew normally during the intervention period, and there were no significant differences in mouse body weight between the different groups (Figure 1A). Compared with the blank group and the model group, the 24-hour food and water intake of mice in the oral gavage Bifidobacterium adolescentis BA-3, BA-4, and BA-11 intervention groups did not show significant differences (Figure 1B, C), preliminarily indicating that the Bifidobacterium adolescentis BA-3, BA-4, and BA-11 strains did not adversely affect the health of the mice.

[0101] 2. Changes in serum inflammatory factor levels

[0102] Lipopolysaccharide (LPS) can induce an inflammatory response by activating inflammatory signaling pathways and promoting the secretion of inflammatory cytokines. Among these, TNF-α, IL-1β, and IL-6 play important roles in the LPS-induced acute inflammation model. Therefore, the inflammatory status of mice can be assessed by measuring changes in proinflammatory cytokine levels in mouse serum. The levels of inflammatory cytokines in the serum of each group of mice after intraperitoneal injection of LPS are shown in Figure 2. Compared with the blank control group, the levels of TNF-α, IL-6, and IL-1β in the model control group were significantly elevated, indicating that the LPS-induced acute inflammation model was successfully established. Compared with the model control group, intervention with Bifidobacterium adolescentis BA-3, BA-4, and BA-11 reduced serum proinflammatory cytokine levels to varying degrees. BA-3 was the most effective of the three strains in reducing proinflammatory cytokines. Compared with the model control group, serum TNF-α, IL-1β, and IL-6 levels in the LPS-treated mice in the BA-3 group were significantly reduced. Compared with the model control group, the BA-11 intervention group showed a significant decrease in serum TNF-α and IL-1β levels in LBP mice, while there was no significant difference in IL-6. In the BA-4 intervention group, IL-1β levels were significantly reduced, but TNF-α and IL-6 levels were not significantly different from the model control group. These results indicate that Bifidobacterium adolescentis strains BA-3, BA-4, and BA-11 exhibited a moderate anti-inflammatory effect in ameliorating LPS-induced inflammation in mice, with the BA-3 strain exhibiting the greatest anti-inflammatory effect.

[0103] 3. Changes in serum SOD and MDA levels

[0104] Oxidative stress and inflammation are closely linked and form one of the foundations of LPS-induced inflammation. Superoxide dismutase (SOD) is the primary antioxidant enzyme in the body that scavenges free radicals. Its function is to block the primary reaction of superoxide anion radicals, thereby reducing the production of other reactive oxygen species. MDA is a product of lipid peroxidation, and its level indirectly reflects the severity of free radical attack on cells. To evaluate the alleviating effects of different strains of Bifidobacterium adolescentis on oxidative stress in LPS-induced mice, serum SOD activity and MDA levels were measured to assess the antioxidant capacity of the strains. As shown in Figure 3, two hours after LPS injection, SOD activity in the model group was significantly decreased, while MDA levels were significantly increased, compared to the blank control group. Intervention with Bifidobacterium adolescentis BA-3, BA-4, and BA-11 all increased SOD activity and reduced MDA levels to varying degrees. Compared to the model control group, BA-3 significantly increased serum SOD activity, while BA-4 and BA-11 showed no significant differences. BA-3, BA-4, and BA-11 all significantly reduced serum MDA levels compared to the model control group. The experimental results showed that Bifidobacterium adolescentis BA-3, BA-4 and BA-11 exhibited certain antioxidant effects in improving LPS-induced oxidative stress in mice.

[0105] in conclusion

[0106] In this example, Bifidobacterium adolescentis BA-3, BA-4 and BA-11 were gavaged to healthy male ICR mice for 14 consecutive days, and then an LPS acute inflammation model was established. By monitoring body weight, food intake and water intake, it was shown that Bifidobacterium adolescentis BA-3, BA-4 and BA-11 had no adverse effects on the health of the mice. The determination of pro-inflammatory cytokine levels showed that BA-3, BA-4 and BA-11 could reduce the levels of TNF-α, IL-6 and IL-1β in the serum of LPS model mice to a certain extent, among which BA-3 could significantly reduce the levels of the three pro-inflammatory cytokines at the same time. The results of the detection of oxidative stress-related factors showed that BA-3, BA-4 and BA-11 all showed a certain anti-oxidative stress effect in LPS-inflamed mice. Overall, among the three strains of Bifidobacterium adolescentis BA-3, BA-4 and BA-11, BA-3 not only had no adverse effects on the health of mice, but also showed better anti-inflammatory and antioxidant effects.

[0107] Example 3 Drosophila experiment

[0108] Experimental methods

[0109] Fruit flies, as a model organism widely used in the field of aging research, have the advantages of a short growth cycle, strong reproductive capacity, and easy differentiation of males and females. At the same time, their genes are highly conserved, and about 75% of them have genes that are homologous to human diseases. Their metabolic pathways are also very similar to those of humans.

[0110] 1. Cultivation of Drosophila

[0111] The wild-type Drosophila strain used in this example is w 1118 All cells were raised in a fruit fly incubator at a constant temperature of 25°C, a constant humidity of 65%, and a 12-hour light / 12-hour dark period. The culture medium formula is shown in Table 1. In the intervention group, Bifidobacterium adolescentis BA-3 and ATCC15703 were resuspended in sterile PBS to 10 10 CFU / mL, and the Bifidobacterium adolescentis BA-3 suspension and ATCC15703 suspension were mixed with the basal medium to a final concentration of 10 9 CFU / mL, and the same volume of sterile PBS was mixed with basal medium in the control group.

[0112] Table 1 Culture medium formula

[0113] 2. Lifespan experiment

[0114] After 48 hours of free mating, 300 male and female fruit flies were randomly selected and transferred to empty culture tubes for 2 hours. They were then randomly divided into three groups, with 100 male and female fruit flies in each group. The flies were then transferred to culture tubes containing culture medium containing Bifidobacterium adolescentis BA-3, culture medium containing ATCC15703, or basal culture medium, with 10 flies in each tube. The flies were transferred to new culture tubes every three days, and the number of dead fruit flies was observed and recorded until all fruit flies died. The survival time of the fruit flies in each tube was recorded, and a survival curve was drawn. The mean lifespan, median lifespan, and maximum lifespan (the maximum lifespan was the average lifespan of the last 10% of fruit flies in each group) of the fruit flies were statistically analyzed. GraphPad Prism 9 was used for significance analysis of the data.

[0115] 3. Anti-gravity crawling ability test

[0116] Male and female fruit flies were cultured in culture tubes containing medium containing Bifidobacterium adolescentis BA-3, medium containing ATCC15703, or basal medium for 30 days. Twenty male and female fruit flies were randomly selected from each group and transferred to separate assay tubes. After the flies had acclimated for 3 minutes, the test tubes were gently tapped to land on the bottom. A timer was then started, and the number of flies that climbed beyond the 8 cm mark within 10 seconds was measured. The experiment was repeated three times, with intervals of at least 10 minutes between each attempt, with the test tubes held horizontally. The fruit fly climbing index was calculated using the formula: a higher climbing index indicates a greater ability to climb against gravity. Data were analyzed for significance using GraphPad Prism 9.

[0117] Climbing index = (number of fruit flies that successfully climbed to 8 cm / total number of fruit flies) × 100%.

[0118] Experimental results

[0119] 1. Lifespan experiment

[0120] Compared with the ATCC15703-treated group and the control group, Bifidobacterium adolescentis BA-3 extended the lifespan of both male and female wild-type fruit flies (Figures 4A, B). Comparing the Bifidobacterium adolescentis BA-3-treated group with the control group, the mean lifespan, median lifespan, and maximum lifespan of both male and female fruit flies in the Bifidobacterium adolescentis BA-3-treated group were significantly longer than those in the control group (Figures 4C, D). Comparing the Bifidobacterium adolescentis BA-3-treated group with the ATCC15703-treated group, the mean lifespan and median lifespan of female wild-type fruit flies in the Bifidobacterium adolescentis BA-3-treated group were significantly longer than those in the ATCC15703-treated group. In male wild-type fruit flies, the median lifespan of the Bifidobacterium adolescentis BA-3-treated group was significantly different from that of the ATCC15703-treated group (Figures 4C, D).

[0121] 2. Anti-gravity crawling experiment

[0122] The ability to climb against gravity is a motor skill and an important indicator of the healthy lifespan of fruit flies. Compared with the control group, the Bifidobacterium adolescentis BA-3 group showed a significant improvement in the climbing ability of both male and female wild-type fruit flies on the 30th day. There was also a significant difference in the climbing ability of male wild-type fruit flies on the 30th day compared with the ATCC15703 group.

[0123] in conclusion

[0124] In this example, Bifidobacterium adolescentis BA-3 was added to the basal culture medium of Drosophila, and the effect of Bifidobacterium adolescentis BA-3 on delaying aging of Drosophila was investigated by detecting the lifespan index of Drosophila and the ability of Drosophila to crawl against gravity at specific time points. 11 18 The median survival time, mean lifespan, and mean maximum lifespan of the Bifidobacterium adolescentis BA-3 intervention group were significantly higher than those of the ATCC15703 and control groups, as shown in Figures 4A, 4B, 4C, and 4D. Furthermore, on day 30, the anti-gravity crawling ability of the Bifidobacterium adolescentis BA-3 intervention group was significantly better than that of the ATCC15703 and control groups, as shown in Figures 5A and 5B.

[0125] In summary, this example demonstrates that exogenous supplementation with Bifidobacterium adolescentis BA-3 can increase the lifespan of wild-type fruit flies of different sexes and improve the health lifespan indicators of wild-type fruit flies, and the effect is better than the model strain ATCC15703 that has been reported to have anti-aging function.

[0126] The above-described embodiments merely illustrate several implementations of the present invention. While the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the present invention. It should be noted that a person skilled in the art would be able to make numerous variations and improvements without departing from the spirit of the present invention, and all such variations and improvements fall within the scope of protection of the present invention.

Claims

1. Bifidobacterium adolescentis BA-3, which is deposited in the General Microbiological Center of China Microbiological Culture Collection Administration, with the deposit number CGMCC NO.27625.

2. A bacterial agent comprising Bifidobacterium adolescentis BA-3 as described in claim 1.

3. A fermentation product or fermentation extract obtained by fermentation of Bifidobacterium adolescentis BA-3 according to claim 1.

4. Use of the Bifidobacterium adolescentis BA-3 according to claim 1 or the bacterial agent according to claim 2 or the fermentation product or fermentation extract according to claim 3 in at least one of the following aspects: 1) anti-aging; 2) anti-oxidation; 3) anti-inflammatory.

5. Use of the Bifidobacterium adolescentis BA-3 described in claim 1 or the bacterial agent described in claim 2 or the fermentation product or fermentation extract described in claim 3 in the preparation of a composition, wherein the composition is used for at least one of the following aspects: 1) anti-aging; 2) anti-oxidation; 3) anti-inflammatory.

6. The use according to claim 4 or 5, wherein: The Bifidobacterium adolescentis BA-3 or the bacterial agent or the fermentation product or fermentation extract is used to reduce the level of pro-inflammatory factors.

7. The use according to claim 6, wherein: The pro-inflammatory factors include one or more of TNF-α, IL-1β and IL-6.

8. The use according to claim 4 or 5, wherein: The Bifidobacterium adolescentis BA-3 or the bacterial agent or the fermentation product or fermentation extract is used for at least one of the following purposes: a) increasing the activity of antioxidant enzymes; the antioxidant enzymes include SOD; b) reducing the level of peroxidation products; the peroxidation products include MDA.

9. The use according to claim 4 or 5, wherein: The subjects of the application include humans and animals other than humans.

10. The use according to claim 5, wherein: The composition is a food composition, a feed composition, a skin external composition or a pharmaceutical composition.

11. A food composition comprising Bifidobacterium adolescentis BA-3 according to claim 1 or the bacterial agent according to claim 2 or the fermentation product or fermentation extract according to claim 3.

12. A feed composition comprising Bifidobacterium adolescentis BA-3 according to claim 1 or the bacterial agent according to claim 2 or the fermentation product or fermentation extract according to claim 3.

13. A composition for external use on the skin, comprising Bifidobacterium adolescentis BA-3 according to claim 1 or the bacterial agent according to claim 2 or the fermentation product or fermentation extract according to claim 3.

14. A pharmaceutical composition comprising Bifidobacterium adolescentis BA-3 according to claim 1 or the bacterial agent according to claim 2 or the fermentation product or fermentation extract according to claim 3.

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