Application of Bifidobacterium longum ZFML006 and its inactivated bacteria in anti-aging

Abstract

The present invention belongs to microbial technology, and specifically relates to the anti-aging function and use of Bifidobacterium longum ZFML006 and its inactivated bacteria. The present invention discloses a Bifidobacterium longum ( Bifidobacterium longum ) ZFML006, its deposit number is CCTCC NO: M 2025331. The present invention also provides an inactivated bacterial cell prepared using Bifidobacterium longum ZFML006, which can be used to prepare an anti-aging drug and also has an antioxidant effect.

Description

Technical Field

[0001] The present invention belongs to microbial technology, and specifically relates to the anti-aging function and use of Bifidobacterium longum ZFML006 and its inactivated bacteria. Background Art

[0002] Aging is a process of systemic physiological degeneration that occurs in organisms as they age. Its essential characteristics are homeostasis imbalance, metabolic disorders, and a decline in the body's repair capacity. During this process, a variety of pathological changes occur one after another, including the accumulation of harmful substances in cells, the decline of tissue and organ function, and the weakening of immune defense mechanisms, ultimately leading to a significant increase in susceptibility to various geriatric diseases and an increase in mortality. Therefore, the development of intervention strategies that can effectively delay the aging process and prolong healthy lifespan has become an important topic in today's medicine and life sciences. Current research focuses on regulating aging-related pathways through drugs, lifestyle interventions, or new treatments, in order to reduce the burden of geriatric diseases and improve the quality of life of the elderly population.

[0003] In recent years, probiotics have shown promising applications in the anti-aging field. Studies have shown that probiotics not only maintain intestinal health but, more importantly, exert anti-aging effects through multiple mechanisms: 1) immunomodulatory function, balancing the levels of pro-inflammatory and anti-inflammatory factors; 2) enhancing the antioxidant defense system, alleviating oxidative stress damage; and 3) ameliorating metabolic disorders and maintaining energy homeostasis. However, several key challenges remain in this field. First, significant functional heterogeneity exists among different strains, resulting in significant variations in their anti-aging effects. Second, the specific molecular mechanisms underlying probiotic anti-aging effects have yet to be fully elucidated. Furthermore, key issues such as strain screening criteria, dose-effect relationships, and personalized application protocols require further investigation.

[0004] Bifidobacterium maintains intestinal homeostasis through three synergistic mechanisms: regulating the balance of intestinal flora, producing short-chain fatty acids, and regulating the immune system. This delays the decline of physiological functions associated with aging, providing an important theoretical basis for the development of anti-aging intervention strategies based on the microbiome. However, the strict anaerobic nature of Bifidobacterium restricts its practical application. Heat inactivation technology effectively solves this bottleneck. The inactivated bacteria not only retain the above-mentioned functional activities, but also have better stability and safety. Therefore, heat-inactivated Bifidobacterium longum shows broad prospects in the field of functional foods and medicines, providing a new strategy for anti-aging intervention.

[0005] The invention of CN118526525A "Application of Bifidobacterium longum subsp. infantis NKU FB 3-14 and its inactivated bacterial preparation in anti-aging and anti-systemic chronic inflammation" informs: The inactivated bacterial preparation of Bifidobacterium longum subsp. infantis NKU FB 3-14 has bioactive substances that are different from those of the live bacteria of Bifidobacterium longum subsp. infantis NKU FB 3-14, and can significantly improve indicators related to oxidative aging: enhance intestinal barrier function (increase villus length and tight junction protein), regulate lipid metabolism, reduce systemic inflammation and oxidative stress levels, thereby exerting dual anti-aging and anti-inflammatory effects.

[0006] The invention of CN116875516B "Bifidobacterium adolescentis BA-3 and its application in anti-aging, anti-oxidation and anti-inflammatory" informs: Bifidobacterium adolescentis ( Bifidobacterium adolescentis ) BA-3 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 significant performance advantages over other strains of the same species when used for anti-aging, anti-oxidation and anti-inflammation.

[0007] The invention of CN117286057A discloses Bifidobacterium longum subsp. longum ( Bifidobacterium longum ) HEPRO-261, HEPRO-261 freeze-dried powder has a significantly higher effect on the average lifespan of nematodes than the commercially available Lactobacillus rhamnosus LGG freeze-dried powder with a viable count of 10 billion cfu / g. Bifidobacterium longum HEPRO-261 has excellent anti-aging activity. A freeze-dried HEPRO-261 powder with a viable count of 1 billion cfu / g significantly upregulates the expression of daf-16 and hsf-1 genes. This upregulation is significantly greater than that achieved by the commonly used Lactobacillus rhamnosus.

[0008] The invention of CN104046573B provides a Bifidobacterium longum ( Bifidobacterium longum The preservation number of Bifidobacterium longum is CGMCC No. 6273. Both the live and inactivated cells of Bifidobacterium longum have anti-aging properties and can be used to prepare functional food compositions.

[0009] above Bifidobacterium longum, None of them simultaneously involve the regulation of two key longevity pathways: the insulin / IGF-1 signaling pathway (IIS) and the mitogen-activated protein kinase (MAPK) signaling pathway. Summary of the Invention

[0010] The technical problem to be solved by the present invention is to provide an application of Bifidobacterium longum ZFML006 and its inactivated bacteria in anti-aging.

[0011] In order to solve the above technical problems, the present invention provides a Bifidobacterium longum ( Bifidobacterium longum ) ZFML006, its deposit number is CCTCC NO: M 2025331.

[0012] The present invention also provides an inactivated bacterial cell prepared by using Bifidobacterium longum ZFML006.

[0013] The present invention also provides a method for preparing the inactivated bacteria, comprising the following steps:

[0014] 1) Bifidobacterium longum ZFML006 was inoculated into a fermentation medium for cultivation to obtain a ZFML006 bacterial solution;

[0015] 2) Heat-treat the ZFML006 bacterial solution to inactivate it, and obtain the ZFML006 inactivated bacterial solution.

[0016] As an improvement to the preparation method of the inactivated bacteria of the present invention:

[0017] Step 1) is: inoculating a seed solution of Bifidobacterium longum ZFML006 at an inoculum rate of 1.5-2.5% (preferably 2%, by volume) into MRS liquid medium containing 0.05% L-cysteine, and culturing anaerobically at 37±1°C for 48±1h to obtain a ZFML006 bacterial solution;

[0018] Note: MRS liquid medium containing 0.05% L-cysteine ​​is as follows: add 0.5g L-cysteine ​​to each liter of MRS liquid medium;

[0019] Bifidobacterium longum ZFML006 can be activated first, and then a single colony can be picked and inoculated into 10 mL of MRS (0.05% L-cysteine ​​w / v) liquid medium. The culture is then anaerobically cultured at 37°C for 48 h to serve as the seed solution.

[0020] Step 2) is: centrifuge the ZFML006 bacterial suspension (8000 × g, 15 min, 4°C), resuspend in phosphate buffer (1 × 10 9 cfu / mL), and then treated in a hot water bath (65°C for 30 min) to obtain the inactivated bacterial liquid of ZFML006.

[0021] The present invention also provides the use of the inactivated bacteria in the preparation of antioxidant and anti-aging drugs.

[0022] As an improvement to the application of the present invention: acting on Caenorhabditis elegans (ZFML006 inactivated bacteria fed to Caenorhabditis elegans), it can prolong its lifespan, reduce its reproductive capacity, increase its body sway rate, reduce lipofuscin in the body, reduce body width and length, enhance heat stress and acute stress resistance, reduce the amount of ROS accumulation in the body, increase the expression of antioxidant enzymes in the body, and affect the expression of genes in anti-aging related signaling pathways (insulin / IGF-1 signaling pathway and MAPK signaling pathway) in the body.

[0023] As a further improvement of the application of the present invention:

[0024] The antioxidant enzymes include superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GSH-Px).

[0025] As a further improvement of the application of the present invention, the said affecting the expression of anti-aging related signaling pathway genes in vivo includes: inhibiting daf-2 and akt-2 expression, promoted the expression of daf-16 and sod-3; and also promoted pmk-1、 nsy-1、skn-1 and sek-1 expression.

[0026] In the present invention, Bifidobacterium longum ( Bifidobacterium longum ) ZFML006 was inoculated into solid culture medium and streaked; Bifidobacterium longum ( Bifidobacterium longum ) A single colony of ZFML006 was inoculated into a fermentation medium for activation and passaged twice to obtain a bacterial suspension;

[0027] In the preparation method of inactivated bacteria:

[0028] During the activation phase, the culture conditions are 37°C and the culture time is 48 hours. During the subculture phase, the inoculation volume on the culture medium is preferably 2%.

[0029] The culture medium for strain activation and subculture was MRS (0.05% L-cysteine ​​w / v) liquid medium.

[0030] The deposit information of the strain of the present invention is as follows:

[0031] Deposit name: Bifidobacterium longum ZFML006 Bifidobacterium longum ZFML006, deposited in China Center for Type Culture Collection, deposited at Wuhan University, Wuhan, China, deposited on February 28, 2025.

[0032] The Bifidobacterium longum ZFML006 of the present invention was isolated from infant feces, and its 16SrRNA sequence was sequenced and compared with the nucleic acid sequence in NCBI. After the comparison results were obtained, it was identified as Bifidobacterium longum.

[0033] The inactivated Bifidobacterium longum ZFML006 strain not only comprehensively improves lifespan (up to 30.6%), fertility, athletic ability, and physical development, but also effectively removes the aging marker lipofuscin. Simultaneously, by enhancing resistance to both heat and oxidative stress, it actively reduces ROS levels and upregulates the expression of antioxidant enzymes (SOD, CAT, etc.). Its uniqueness lies in its synergistic regulation of two key longevity pathways: the insulin / IGF-1 signaling pathway (IIS) and the mitogen-activated protein kinase (MAPK) signaling pathway, forming a multi-target anti-aging network. Furthermore, the inactivated strain achieves its evolutionarily conserved anti-aging effects through the synergistic action of DAF-16 and SKN-1. Furthermore, the inactivated strain is highly safe and stable, requires no refrigeration, and is widely applicable. Its cell wall components (such as peptidoglycan) can also activate innate immunity, surpassing common strains that rely on live bacterial metabolism. It holds great potential for the development of functional anti-aging preparations. Therefore, inactivated strains prepared from Bifidobacterium longum ZFML006 have promising applications in anti-oxidative and anti-aging applications. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] The specific embodiments of the present invention are further described in detail below with reference to the accompanying drawings.

[0035] Figure 1 This figure shows the effects of inactivated Bifidobacterium longum ZFML006 and OP50 on the lifespan of Caenorhabditis elegans.

[0036] Figure 1 Middle: A shows the effect of inactivated Bifidobacterium longum ZFML006 / OP50 bacteria on the lifespan (survival rate) of Caenorhabditis elegans; B shows the effect of inactivated Bifidobacterium longum ZFML006 / OP50 bacteria on the median lifespan of Caenorhabditis elegans.

[0037] Figure 2 Effects of inactivated Bifidobacterium longum ZFML006 / OP50 on the fecundity, pharyngeal pumping, and body sway of Caenorhabditis elegans;

[0038] Figure 2 Middle: A shows the effect of inactivated Bifidobacterium longum ZFML006 / OP50 bacteria on the reproductive capacity (total number of reproductive offspring) of Caenorhabditis elegans, B shows the effect of inactivated Bifidobacterium longum ZFML006 / OP50 bacteria on the pharyngeal pumping rate (number of pharyngeal aspirations) of Caenorhabditis elegans, and C shows the effect of inactivated Bifidobacterium longum ZFML006 / OP50 bacteria on the body sway rate (number of body bends) of Caenorhabditis elegans.

[0039] Figure 3 Effects of inactivated Bifidobacterium longum ZFML006 / OP50 bacteria on the lipofuscin body width and length of Caenorhabditis elegans;

[0040] Figure 3 Middle: A is a fluorescence image of lipofuscin in C. elegans after treatment with inactivated Bifidobacterium longum ZFML006 / OP50 bacteria (25 μm). The left side is the nematode under bright field, and the right side is a fluorescence image of lipofuscin under a fluorescence microscope. B is the effect of inactivated Bifidobacterium longum ZFML006 / OP50 bacteria on lipofuscin in C. elegans, C is the effect of inactivated Bifidobacterium longum ZFML006 / OP50 bacteria on the body width of C. elegans, and D is the effect of inactivated Bifidobacterium longum ZFML006 / OP50 bacteria on the body length of C. elegans.

[0041] Figure 4 Effects of inactivated Bifidobacterium longum ZFML006 / OP50 bacteria on heat stress and acute oxidative stress in Caenorhabditis elegans;

[0042] Figure 4 Middle: A and B respectively show the effects of inactivated Bifidobacterium longum ZFML006 / OP50 bacteria on the heat stress capacity of Caenorhabditis elegans, and C and D respectively show the effects of inactivated Bifidobacterium longum ZFML006 / OP50 bacteria on the acute stress capacity of Caenorhabditis elegans.

[0043] Figure 5 Effects of inactivated Bifidobacterium longum ZFML006 / OP50 bacteria on ROS and antioxidant enzymes in Caenorhabditis elegans;

[0044] Figure 5 Middle: A shows the effect of inactivated Bifidobacterium longum ZFML006 / OP50 bacteria on the accumulation of ROS (reactive oxygen species) in C. elegans. B, C, and D show the effect of inactivated Bifidobacterium longum ZFML006 / OP50 bacteria on the expression levels of antioxidant enzymes in C. elegans: catalase (CAT), superoxide dismutase (SOD), and glutathione peroxidase (GSH-PX).

[0045] Figure 6 This is a graph showing the gene expression levels in the insulin / IGF-1 signaling pathway (IIS) and MAPK signaling pathway in Caenorhabditis elegans induced by inactivated Bifidobacterium longum ZFML006 bacteria / OP50 bacteria;

[0046] Figure 6Middle: A is a graph showing the expression levels of genes related to the insulin / IGF-1 signaling pathway (IIS) in C. elegans by inactivated Bifidobacterium longum ZFML006 / OP50 bacteria; B is a graph showing the expression levels of genes related to the MAPK signaling pathway in C. elegans by inactivated Bifidobacterium longum ZFML006 / OP50 bacteria. DETAILED DESCRIPTION

[0047] The present invention will be further described below with reference to specific embodiments, but the scope of protection of the present invention is not limited thereto: In the following embodiments, unless otherwise specified, the operating methods used are conventional operating methods, the equipment used are conventional equipment, and the equipment and materials used in each embodiment are the same.

[0048] Phosphate buffer was PBS buffer (0.01 M, pH 7.3-7.5);

[0049] MRS (0.05% L-cysteine ​​w / v) liquid medium: Add 0.5 g of L-cysteine ​​per liter of regular MRS liquid medium.

[0050] Example 1. Acquisition of Bifidobacterium longum ZFML006:

[0051] Feces from infants aged 0 to 6 months in Zhejiang Province were used as samples. 0.1 g of feces was added to 1 mL of phosphate buffer and mixed to prepare the initial sample solution. According to the 10-fold dilution method, the initial sample solution was diluted to 10 with phosphate buffer. -4 , 10 -5 , 10 -6 As a bacterial suspension, 100 μL of the suspension was then spread onto MRS solid medium containing 2% calcium carbonate. After incubation at 37°C in an anaerobic incubator for 48 hours, colonies with a calcium-soluble ring were selected for identification. The 16S rDNA sequence identification result is shown in SEQ ID NO: 1.

[0052] It was subsequently identified as Bifidobacterium longum through comparison with the official website of the National Center for Biotechnology Information (NCBI) of the United States and named Bifidobacterium longum ( Bifidobacterium longum )ZFML006.

[0053] The preservation information is as follows:

[0054] Deposit name: Bifidobacterium longum ZFML006 Bifidobacterium longum ZFML006, deposited in China Center for Type Culture Collection, deposited at Wuhan University, Wuhan, China, deposited on February 28, 2025.

[0055] Example 2

[0056] 1. The preparation method of ZFML006 inactivated bacterial liquid is to carry out the following steps in sequence:

[0057] 1) Use an inoculation loop to pick a single colony of Bifidobacterium longum ZFML006 and place it in 10 mL of MRS (0.05% L-cysteine ​​w / v) liquid medium. Incubate anaerobically at 37°C for 48 h. Inoculate the resulting seed solution at a 2% (volume %) inoculum into MRS (0.05% L-cysteine ​​w / v) liquid medium and incubate anaerobically at 37°C for 48 h to obtain the ZFML006 bacterial solution.

[0058] 2) The cultured Bifidobacterium longum ZFML006 bacterial suspension was centrifuged (8000 × g, 15 min, 4°C) and resuspended in phosphate buffer to 1 × 10 9 cfu / mL, and finally treated with a hot water bath (65°C inactivation for 30 min) to obtain the inactivated bacterial liquid of ZFML006.

[0059] 2. Preparation of E. coli OP50 culture liquid: centrifuge the E. coli OP50 culture liquid cultured in the conventional way (8000 × g, 15 min, 4°C), resuspend it in phosphate buffer to 1 × 10 9 cfu / mL, Escherichia coli OP50 bacterial liquid used as the control group.

[0060] Example 3: Effect of Bifidobacterium longum ZFML006 on prolonging the lifespan of Caenorhabditis elegans

[0061] Under the same culture conditions, the effects of different foods (experimental group: Bifidobacterium longum ZFML006, control group: Escherichia coli OP50, used as food for Caenorhabditis elegans, food needs to be changed daily) on the lifespan of Caenorhabditis elegans were observed.

[0062] L4-stage synchronized Caenorhabditis elegans were cultured in a 1 × 10 9 cfu / mL of heat-killed Bifidobacterium longum ZFML006 or OP50 on Nematode elegans medium (NGM) plates supplemented with 50 μM FUDR. 60 C. elegans were plated per plate.

[0063] The size of the nematode culture medium (NGM) plate is 60 mm (the same size is used in the following examples).

[0064] That is, the experimental groups are as follows:

[0065] (i) Control group (OP50 control group): 200 μL of E. coli OP50 solution was added to an NGM plate (60 mm) containing 50 μM FUDR, and then Caenorhabditis elegans was placed;

[0066] (ii) Treatment group (heat-killed Bifidobacterium longum ZFML006 treatment group): 200 μL of ZFML006 inactivated bacterial cell solution was added to an NGM plate (60 mm) containing 50 μM FUDR, and then Caenorhabditis elegans was placed;

[0067] 60 Caenorhabditis elegans were placed in both the control and treatment groups;

[0068] Three biological replicate plates were set up for each group (with 60 C. elegans on each plate) and cultured at 20°C.

[0069] The food that needs to be changed every day is:

[0070] For the control group, C. elegans were transferred to fresh Nematode Medium (NGM) plates containing 50 μM 5-fluoro-2'-deoxyuridine (FUDR) and 200 μL of Escherichia coli OP50 bacterial solution.

[0071] For the treatment group, C. elegans were transferred to fresh Nematode Medium (NGM) plates containing 50 μM 5-fluoro-2'-deoxyuridine (FUDR) and 200 μL of ZFML006 inactivated bacterial solution.

[0072] Thus, continuous exposure was maintained. Survival rates were recorded until all died.

[0073] Due to the advantages of C. elegans such as a short lifespan (20-30 days) and clear biological characteristics, its lifespan assessment has become a key indicator in anti-aging intervention research. Figure 1 As shown in Figures A and B, heat-killed Bifidobacterium longum ZFML006 significantly extended the median lifespan of C. elegans (by 30.6% ± 3.7%) compared to the OP50-fed control group, demonstrating its significant anti-aging effects. Therefore, it was concluded that heat-killed Bifidobacterium longum ZFML006 can delay aging in C. elegans; that is, heat-killed Bifidobacterium longum ZFML006 has anti-aging effects.

[0074] Example 4: Effect of Bifidobacterium longum ZFML006 on the fecundity of Caenorhabditis elegans

[0075] Under the same culture conditions, the effects of different foods (experimental group: Bifidobacterium longum ZFML006, control group: Escherichia coli OP50, as food for Caenorhabditis elegans, the food needs to be changed daily) on the reproductive capacity of Caenorhabditis elegans were observed.

[0076] L4-stage synchronized Caenorhabditis elegans were inoculated with 1 × 10 9 cfu / mL of heat-killed Bifidobacterium longum ZFML006 or OP50 on NGM medium plates (without FUDR);

[0077] The experimental groups are as follows:

[0078] (i) Control group (OP50 control group): 200 μL of Escherichia coli OP50 solution was added to an NGM plate (60 mm) and a Caenorhabditis elegans was placed.

[0079] (ii) Treatment group (heat-killed Bifidobacterium longum ZFML006 treatment group): 200 μL of ZFML006 inactivated bacterial cell solution was added to an NGM plate (60 mm), and then one Caenorhabditis elegans was placed;

[0080] Three biological replicate plates were set up for each group (one C. elegans was cultured on each plate, i.e., each nematode was cultured individually) and cultured at 20°C.

[0081] The food that needs to be changed every day is:

[0082] In the control group, C. elegans were transferred to fresh nematode culture medium (NGM) plates with 200 μL of Escherichia coli OP50 solution added;

[0083] In the treatment group, C. elegans were transferred to fresh nematode culture medium (NGM) plates to which 200 μL of ZFML006 inactivated bacterial solution had been added;

[0084] The experiment lasted five days. The original plates from these five days were placed in a 20°C incubator for 72 hours each before progeny counting. Total fecundity was calculated as the cumulative number of offspring produced over the five-day period. Because organisms have limited energy resources, there is a trade-off between reproduction and maintaining homeostasis (e.g., anti-aging). When fecundity decreases, energy previously allocated to reproduction may be reallocated to longevity-related pathways such as DNA repair, antioxidant defense, and protein homeostasis, thereby extending lifespan.

[0085] The results are as follows Figure 2 As shown in Figure A, Bifidobacterium longum ZFML006 can reduce the reproductive capacity of Caenorhabditis elegans; that is, Bifidobacterium longum ZFML006 has the effect of delaying the aging of Caenorhabditis elegans.

[0086] Example 5: Effects of Bifidobacterium longum ZFML006 on pharyngeal pumping and body sway in Caenorhabditis elegans.

[0087] Under the same culture conditions, the effects of different foods (experimental group: Bifidobacterium longum ZFML006, control group: Escherichia coli OP50, as food for Caenorhabditis elegans, food needs to be changed daily) on the pharyngeal pumping and body sway of Caenorhabditis elegans were observed.

[0088] L4-stage synchronized Caenorhabditis elegans were cultured in a 1 × 10 9 cfu / mL of heat-killed Bifidobacterium longum ZFML006 or OP50 on Nematode Medium (NGM) plates supplemented with 50 μM FUDR. Thirty C. elegans were plated on each 60 mm NGM plate. Culture was performed at 20°C for 5 days.

[0089] The experimental grouping method and food replacement method can refer to Example 3.

[0090] The body sway rate was counted as the number of body bends per minute, and the pharyngeal pumping frequency was counted as the number of pharyngeal pumpings per nematode within 30 seconds.

[0091] The results of pharyngeal pumping and body swaying are as follows Figure 2 As shown in Figures B and C, Bifidobacterium longum ZFML006 increased the body sway rate of Caenorhabditis elegans, but did not substantially change its pharyngeal pumping rate.

[0092] Note: Generally, the decrease in the body sway rate of C. elegans is positively correlated with lifespan, and individuals that maintain their motility for a longer time usually live longer. Therefore, it can be concluded that heat-killed Bifidobacterium longum ZFML006 can delay the lifespan of C. elegans.

[0093] Example 6: Effects of Bifidobacterium longum ZFML006 on lipofuscin and body width and length in Caenorhabditis elegans

[0094] Under the same culture conditions, the effects of different foods (experimental group: Bifidobacterium longum ZFML006, control group: Escherichia coli OP50, as food for C. elegans, which needs to be changed daily) on lipofuscin and body width and length of C. elegans were observed.

[0095] Synchronized L4 stage Caenorhabditis elegans were cultured in a 1 × 10 9 cfu / mL of heat-killed Bifidobacterium longum ZFML006 or OP50 on 60 mm NGM plates (supplemented with 50 μM FUDR). Thirty Caenorhabditis elegans were plated per plate and cultured at 20°C for 5 days.

[0096] The experimental grouping method and food replacement method can refer to Example 3.

[0097] After 5 days of culture, C. elegans were anesthetized in a solution containing 20 mM sodium azide (NaN3). The worms were then immobilized in 1% agarose (1 g agarose powder was weighed and added to 100 mL of 1× TAE buffer). The immobilized specimens were then imaged using a fluorescence microscope (excitation wavelength 485 nm, emission wavelength 530 nm). ImageJ software was used to quantify the fluorescence intensity of lipofuscin in C. elegans, as well as body length and width.

[0098] There is a close correlation between the accumulation of lipofuscin, changes in body width and length, and lifespan in Caenorhabditis elegans. These parameters can be used as biological markers of aging. Lipofuscin is mainly formed by the polymerization of oxidatively damaged proteins, lipids, and sugars in lysosomes and has autofluorescence. During aging, the function of lysosomes declines and these cross-linked polymers cannot be degraded, resulting in the accumulation of lipofuscin. The results are as follows: Figure 3 As shown, heat-killed Bifidobacterium longum ZFML006 reduced lipofuscin in C. elegans, as well as its body width and length. These reductions may be associated with an increase in body sway rate. Therefore, it can be concluded that heat-killed Bifidobacterium longum ZFML006 slows aging in C. elegans.

[0099] Example 7: Effect of Bifidobacterium longum ZFML006 on enhancing the stress resistance of Caenorhabditis elegans.

[0100] Under the same culture conditions, the effects of different foods (experimental group: Bifidobacterium longum ZFML006, control group: Escherichia coli OP50, as food for Caenorhabditis elegans, which needs to be changed daily) on the ability of Caenorhabditis elegans to withstand heat stress and acute oxidative stress were observed.

[0101] Synchronized L4 stage Caenorhabditis elegans were inoculated with 1 × 10 9 elegans were cultured on Nematode medium (NGM) plates (supplemented with 50 μM FUDR) containing heat-killed Bifidobacterium longum ZFML006 or OP50 at 40 cfu / mL and incubated at 20°C for 5 days.

[0102] The experimental grouping method and food replacement method can refer to Example 3.

[0103] After 5 days of culture, C. elegans were transferred to fresh NGM plates (without food and FUDR) and subjected to heat stress at 35°C. The mortality rate was recorded every hour until all the C. elegans died. Finally, the heat tolerance was evaluated by statistical analysis of the survival data. Figure 4 As shown in A and B.

[0104] After 5 days of culture, the C. elegans were transferred to NGM plates containing 0.1% hydrogen peroxide (H2O2) and cultured at 20°C. The survival rate of the nematodes was measured every 30 minutes, and the mortality rate at each time point was recorded. Figure 4 As shown in C and D.

[0105] according to Figure 4 It can be seen that Bifidobacterium longum ZFML006 enhances the heat stress and acute stress capabilities of Caenorhabditis elegans.

[0106] Example 8: Effects of Bifidobacterium longum ZFML006 on ROS and antioxidant enzymes (SOD, CAT, and GSH-Px) in Caenorhabditis elegans.

[0107] Under the same culture conditions, the effects of different foods (experimental group: Bifidobacterium longum ZFML006, control group: Escherichia coli OP50, as food for Caenorhabditis elegans, which needs to be changed daily) on ROS and antioxidant enzymes (SOD, CAT and GSH-Px) in Caenorhabditis elegans were observed.

[0108] L4-stage synchronized Caenorhabditis elegans were cultured in 1 mL of 1 × 10 9 C. elegans were cultured on nematode culture medium (NGM) plates (60 mm thick) supplemented with 50 μM FUDR) containing heat-killed Bifidobacterium longum ZFML006 or OP50 at 400 cfu / mL. Approximately 200 C. elegans were plated per plate and incubated at 20°C for 5 days. Nematodes were rinsed from the plates daily with PBS. After three washes, any nematodes that settled at the bottom of the EP tubes were transferred to fresh NGM plates (supplemented with 50 μM FUDR) containing food for culture.

[0109] The experimental groups are as follows:

[0110] (i) Control group (OP50 control group): 1 mL of E. coli OP50 culture medium was added to an NGM plate (supplemented with 50 μM FUDR), and approximately 200 Caenorhabditis elegans were placed.

[0111] (ii) Treatment group (heat-killed Bifidobacterium longum ZFML006 treatment group): 1 mL of ZFML006 inactivated bacterial cell solution was added to NGM plates (supplemented with 50 μM FUDR), and approximately 200 Caenorhabditis elegans were placed;

[0112] Five biological replicate plates were set up for each group and cultured at 20°C.

[0113] The food that needs to be changed every day is:

[0114] For the control group, washed C. elegans were transferred to fresh NGM plates containing 1 mL of E. coli OP50 bacterial solution (supplemented with 50 μM FUDR);

[0115] For the treatment group, C. elegans were transferred to fresh NGM plates containing 1 mL of inactivated ZFML006 bacterial solution (supplemented with 50 μM FUDR).

[0116] C. elegans (approximately 1,000 per group) were collected after 5 days of culture. After washing with PBS (0.01M, pH 7.3-7.5), the worms were centrifuged at 3,000 × g for 2 minutes at 4°C. A nematode lysate was prepared by adding 500 μL of PBS (0.01M, pH 7.3-7.5) and centrifuging at 12,000 × g for 10 minutes at 4°C. The supernatant was collected and approximately 50 μL of the supernatant was mixed with an equal volume of DCFH-DA probe (1:1, v / v) to a final concentration of 50 μM. The mixture was incubated at 20°C for 2 hours in the dark. Fluorescence intensity was measured using a fluorescence spectrophotometer (excitation wavelength 485 nm, emission wavelength 538 nm).

[0117] After 5 days of culture, C. elegans (approximately 1,000 per group) were collected. After washing with PBS, the worms were centrifuged at 3,000 × g for 2 minutes at 4°C. 500 μL of PBS was added to prepare nematode lysis buffer, which was then centrifuged at 12,000 × g for 10 minutes at 4°C. The supernatant was collected. Superoxide dismutase (SOD) typing assay kit (Cat. No. A001-2), catalase (CAT) assay kit (Cat. No. A007-1-1), and glutathione peroxidase (GSH-Px) assay kit (Cat. No. A005-1) were used to measure superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GSH-Px) activities according to the manufacturer's instructions.

[0118] The results are as follows Figure 5 As shown in the results, Bifidobacterium longum ZFML006 reduced ROS accumulation in C. elegans and increased the expression of antioxidant enzymes (SOD, CAT, and GSH-Px) in C. elegans. This suggests that heat-killed Bifidobacterium longum ZFML006 can exert its anti-aging effects by enhancing the endogenous antioxidant defense system in C. elegans.

[0119] Example 9: Effects of Bifidobacterium longum ZFML006 on gene expression of anti-aging-related signaling pathways (insulin / IGF-1 signaling pathway and MAPK signaling pathway) in Caenorhabditis elegans.

[0120] Under the same culture conditions, the effects of different foods (experimental group: heat-killed Bifidobacterium longum ZFML006, control group: Escherichia coli OP50, which serves as food for Caenorhabditis elegans and needs to be changed daily) on anti-aging-related genes in Caenorhabditis elegans were observed.

[0121] L4 stage synchronized Caenorhabditis elegans were inoculated into 1 mL of 1 × 10 9 C. elegans were cultured on nematode culture medium (NGM) plates (60 mm plate) containing heat-killed Bifidobacterium longum ZFML006 or OP50 (cfu / mL). Approximately 200 C. elegans were plated per plate and incubated at 20°C for 5 days. Nematodes were rinsed from the plates daily with PBS. After three washes, any nematodes that settled at the bottom of the EP tubes were transferred to fresh NGM plates (supplemented with 50 μM FUDR) containing food. Ten biological replicates were set up for each group.

[0122] The experimental grouping method and food replacement method can refer to Example 8.

[0123] Collect approximately 2,000 C. elegans nematodes after 5 days of culture. Add 500 μL of TRIzol reagent and thoroughly lyse the sample by grinding six times at 1,000 rpm (1 minute each time). Then, add 100 μL of chloroform, mix well, and let stand at room temperature for 5 minutes. Centrifuge at 12,000 × g for 15 minutes at 4°C to separate the layers. Carefully aspirate the upper aqueous phase and add an equal volume of isopropanol to precipitate RNA. Let stand at room temperature for 10 minutes, then centrifuge and discard the supernatant. Wash the pellet twice with 1 mL of 75% ethanol (centrifuge at 7,500 × g for 5 minutes at 4°C). Dry the RNA pellet at room temperature for 5-10 minutes, then dissolve it in diethyl pyrocarbonate-treated water (DEPC) and store at -80°C. Strictly avoid RNase contamination throughout the entire process to ensure precise operation.

[0124] Quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR) was used to detect the expression levels of anti-aging-related genes in Caenorhabditis elegans treated with inactivated Bifidobacterium longum ZFML006 and Escherichia coli OP50 for 5 days.

[0125] Total RNA was extracted using TRIzol reagent (Beyotime, Shanghai, China), and cDNA was synthesized using the PrimeScript™ Reverse Transcription Kit (Takara). qRT-PCR was performed using the SYBG Green® PCR Kit (Takara Bio, Beijing, China) with the following thermal cycling program: preamplification at 95°C for 10 minutes, followed by 40 cycles of 95°C for 15 seconds and 60°C for 1 minute. Actin ( act-1) was used as a control and the relative act- 1 The primers used were synthesized by Shanghai Sangon Biotechnology Co., Ltd.

[0126] The primer sequences used in this case are shown in Table 1 below;

[0127] Table 1

[0128]

[0129] The results are shown in Figure 6. ZFML006 inactivated bacteria inhibited daf-2 and akt-2 expression, promoted the expression of daf-16 and sod-3; and also promoted pmk-1 , nsy-1 , skn-1 and sek-1 Therefore, ZFML006 inactivated bacteria can delay the aging of Caenorhabditis elegans by inhibiting the insulin / IGF-1 signaling pathway and activating the MAPK signaling pathway.

[0130] These results suggest that heat-killed Bifidobacterium longum ZFML006 significantly enhances cellular homeostasis by increasing antioxidant enzyme activity and stress resistance in C. elegans. Furthermore, the robustness of its anti-aging effects is enhanced through cross-regulation of two pathways (e.g., synergistic activation of DAF-16 and SKN-1). Its evolutionary conservation provides a potential target for mammalian aging research and lays a theoretical foundation for the development of novel anti-aging interventions. Furthermore, heat-killed Bifidobacterium longum ZFML006 synergistically regulates key signaling pathways, such as DAF-16 and SKN-1, by inhibiting the insulin / IGF-1 signaling pathway and activating the MAPK signaling pathway, thereby significantly enhancing cellular antioxidant capacity and stress response. This dual mechanism and synergistic regulation give ZFML006 a unique advantage in terms of robustness and evolutionary conservation of its anti-aging effects.

[0131] NKU FB 3-14, BA-3, HEPRO-261, Bifidobacterium longum described in CN104046573B ( Bifidobacterium longum ) Detection was performed according to the above Example 9, skn-1 The expression level of OP50 was not significantly different from that of OP50.

[0132] In contrast, although existing research on Bifidobacterium longum has shown some anti-aging effects, its mechanism is relatively simple and lacks the dual regulation and synergistic effect described in the present invention's ZFML006. For example, the following is described:

[0133] Bifidobacterium longum BB68 only extends lifespan through the IIS pathway but lacks MAPK pathway regulation and heat stress resistance (Zhao, L., Zhao, Y., Liu, R., Zheng, X., Zhang, M., Guo, H., Zhang, H., & Ren, F. (2017). The Transcription Factor DAF-16 is Essential for IncreasedLongevity in C. elegans Exposed to Bifidobacterium longum BB68. Scientific reports , 7 (1), 7408.);

[0134] Bifidobacterium longum NCC2705 relies solely on metabolic regulation and fails to target key aging pathways (Gong Chunmiao, Wang Xiaonan, Fei Peng, Li Yan, Zhao Xiaomeng, Duan Boan & Chen Shuxing. (2024). In vivo anti-aging effects of four bifidobacterium strains on Caenorhabditis elegans. Food Science ,45 (13), 146-152.);

[0135] The antioxidant effect of Bifidobacterium longum CCFM1029 requires exogenous substances and has no heat stress protection ability (Cai Shuzhen, Wu Lei, Xie Xinqiang, Chen Huiyuan & Wu Qingping. (2023). Research progress on the mechanism of action of intestinal microbiota in human healthy aging. Acta Microbiologica Sinica, 63(01), 85-105.);

[0136] Bifidobacterium longum TCI001 must rely on live bacteria colonization and its effects are limited to the intestinal microecology.

[0137] These strains generally have defects such as single pathway, limited stress response or unstable dosage form (Zheng Xiaonan, Zhang Hao, Guo Huiyuan & Ren Fazheng. (2012). Research progress on the anti-aging function of probiotics. China Dairy, (02), 50-53).

[0138] ZFML006, a drug developed in the present invention, inactivates bacterial components to synergistically regulate the IIS-MAPK dual pathway, achieving both endogenous enzyme activation and dual stress resistance. This demonstrates significant advantages in both comprehensive mechanisms and robust application. Therefore, ZFML006 provides a promising theoretical foundation for the development of novel anti-aging interventions.

[0139] Finally, it should be noted that the above examples are merely specific embodiments of the present invention. Obviously, the present invention is not limited to the above examples and is subject to numerous variations. All variations that can be directly derived or conceived by a person of ordinary skill in the art from the disclosure of the present invention are considered to be within the scope of protection of the present invention.

Claims

1. Bifidobacterium longum ( Bifidobacterium longum ) ZFML006, characterized in that: the preservation number is CCTCC NO: M 2025331.

2. An inactivated bacterial cell prepared using the Bifidobacterium longum ZFML006 according to claim 1.

3. The method for preparing inactivated bacteria according to claim 2, wherein The following steps are involved: 1) Bifidobacterium longum ZFML006 was inoculated into a fermentation medium for cultivation to obtain a ZFML006 bacterial solution; 2) Heat-treat the ZFML006 bacterial solution to inactivate it, and obtain the ZFML006 inactivated bacterial solution.

4. The method for preparing inactivated bacteria according to claim 3, wherein: Step 1) is: inoculating the seed liquid of Bifidobacterium longum ZFML006 at an inoculum rate of 1.5-2.5% into MRS liquid medium containing 0.05% L-cysteine, and culturing anaerobically at 37±1°C for 48±1h to obtain ZFML006 bacterial liquid; Step 2) is: centrifuging the ZFML006 bacterial solution, resuspending it with phosphate buffer, and then treating it in a hot water bath to obtain a ZFML006 inactivated bacterial solution.

5. The use of the inactivated bacteria according to claim 2 in the preparation of anti-aging drugs, characterized in that: Prolong lifespan, reduce fertility, and improve athletic ability.

6. The use according to claim 5, characterized in that: Enhances the ability to withstand heat stress and acute stress, and improves antioxidant efficacy.

7. The use according to claim 5 or 6, characterized in that: It acts on Caenorhabditis elegans, reducing lipofuscin, body width and length, and ROS accumulation in the body, increasing the expression of antioxidant enzymes in the body, and affecting the expression of anti-aging-related signaling pathway genes in the body.

8. The use according to claim 7, characterized in that: The antioxidant enzymes include superoxide dismutase, catalase and glutathione peroxidase.

9. The use according to claim 8, characterized in that The influencing the expression of anti-aging related signaling pathway genes in vivo includes: It inhibited the expression of daf-2 and akt-2, promoted the expression of daf-16 and sod-3; at the same time, it promoted the expression of pmk-1, nsy-1, skn-1 and sek-1.

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