Bifidobacterium longum ZFML006 and application of inactivated thallus of bifidobacterium longum ZFML006 in anti-aging

Abstract

The invention belongs to a microbial technology, and particularly relates to bifidobacterium longum ZFML006 and an anti-aging function and application of inactivated thalli of the bifidobacterium longum ZFML006. The invention discloses Bifidobacterium longum ZFML006, and the preservation number of the Bifidobacterium longum ZFML006 is CCTCC (China Center for Type Culture Collection) NO: M 2025331. The invention also provides an inactivated thallus prepared from the bifidobacterium longum ZFML006, and the inactivated thallus can be used for preparing anti-aging drugs and also has an anti-oxidation effect.

Description

Technical Field

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

[0002] Aging is a systematic physiological function degradation process that occurs as organisms age. Its essential characteristics are manifested as the imbalance of body homeostasis, metabolic disorders, and the decline of repair ability. In this process, various pathological changes occur successively, including the accumulation of harmful substances in cells, the decline of tissue and organ functions, the weakening of the immune defense mechanism, etc., ultimately leading to a significant increase in the susceptibility to various senile diseases and an increase in mortality. Therefore, developing intervention strategies that can effectively delay the aging process and extend the healthy lifespan has become an important topic in the fields of medicine and life sciences today. The current research focuses on regulating aging-related pathways through drugs, lifestyle interventions, or new treatment methods, with the aim of reducing the burden of senile diseases and improving the quality of life of the elderly population.

[0003] In recent years, probiotics have shown broad application prospects in the field of anti-aging. Research has shown that probiotics can not only maintain intestinal health, but more importantly, play an anti-aging role through the following multiple mechanisms: 1) immune regulation function, balancing the levels of pro-inflammatory and anti-inflammatory factors; 2) enhancing the antioxidant defense system and reducing oxidative stress damage; 3) improving metabolic disorders and maintaining energy homeostasis. However, there are still several key challenges in this field: First, there is significant functional heterogeneity among different strains, and their anti-aging effects vary greatly; second, the specific molecular mechanisms of probiotics' anti-aging effects have not been fully elucidated; in addition, key issues such as strain screening criteria, dose-effect relationships, and individualized application programs still need to be studied in depth.

[0004] Bifidobacterium maintains intestinal homeostasis through three mechanisms: regulating the balance of intestinal flora, producing short-chain fatty acids, and regulating the immune system, and delays the physiological function decline related to aging, providing an important theoretical basis for developing anti-aging intervention strategies based on the microbiome. However, the strict anaerobic characteristics of Bifidobacterium restrict its practical application. The heat inactivation technology effectively solves this bottleneck. The heat-inactivated cells 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 fields of functional foods and medicine, providing a new strategy for anti-aging intervention.

[0005] The invention of CN118526525, "Application of Bifidobacterium longum subsp. infantis NKU FB 3-14 and Its Heat-Killed Bacterial Cell Preparation in Anti-Aging and Anti-Systemic Chronic Inflammation", discloses that the heat-killed bacterial cell preparation of Bifidobacterium longum subsp. infantis NKU FB 3-14 has bioactive substances different from those of the live Bifidobacterium longum subsp. infantis NKU FB 3-14, and can significantly improve the indicators related to oxidative aging: enhance the intestinal barrier function (increase the villus length and tight junction proteins), regulate lipid metabolism, and reduce the levels of systemic inflammation and oxidative stress, thus exerting dual anti-aging and anti-inflammatory effects.

[0006] The invention of CN116875516B, "Bifidobacterium adolescentis BA-3 and Its Application in Anti-Aging, Antioxidation and Anti-Inflammation", discloses that Bifidobacterium adolescentis ([[]] Bifidobacterium adolescentis ) BA-3 can break the vicious cycle of free radicals-oxidative stress-inflammation-oxidative stress-free radicals, and can achieve comprehensive improvement in multiple aspects. Therefore, it has significant performance advantages over other strains of the same species when used for anti-aging, antioxidation and anti-inflammation.

[0007] The invention of CN117286057A discloses Bifidobacterium longum subsp. longum ([[]] Bifidobacterium longum ) HEPRO-261. The average lifespan of Caenorhabditis elegans treated with the freeze-dried powder of HEPRO-261 is significantly higher than that of the commercially available freeze-dried powder of Lactobacillus rhamnosus GG with a viable cell count of 10 billion cfu / g. This Bifidobacterium longum subsp. longum ([[]] Bifidobacterium longum ) HEPRO-261 has excellent anti-aging activity. The freeze-dried powder of HEPRO-261 with a viable cell count of 1 billion cfu / g can significantly up-regulate the gene expression of daf-16 and hsf-1; and its promoting effect on the gene expression of daf-16 and hsf-1 is significantly higher than that of the known and commonly used Lactobacillus rhamnosus.

[0008] The invention of CN104046573B provides a Bifidobacterium longum ([[]] Bifidobacterium longum ), and the preservation number of the Bifidobacterium longum is CGMCC No.6273. Both the live bacterial cells and the heat-killed bacterial cell substances of this Bifidobacterium longum have the effect of delaying aging and can be used to prepare functional food compositions.

[0009] The above Bifidobacterium longum, do not simultaneously involve regulating the two key longevity pathways of 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 heat-killed bacterial cells in anti-aging.

[0011] To solve the above technical problems, the present invention provides a Bifidobacterium longum ( Bifidobacterium longum ), ZFML006, with a preservation number of CCTCC NO: M 2025331.

[0012] The present invention also simultaneously provides inactivated bacteria prepared from Bifidobacterium longum ZFML006.

[0013] The present invention also simultaneously provides a preparation method of the above inactivated bacteria, including the following steps: 1). Inoculate Bifidobacterium longum ZFML006 into a fermentation medium for cultivation to obtain a ZFML006 bacterial liquid; 2). Heat-treat the ZFML006 bacterial liquid to inactivate it to obtain a ZFML006 inactivated bacteria bacterial liquid.

[0014] As an improvement to the preparation method of the inactivated bacteria of the present invention: Step 1) is: inoculate the seed liquid of Bifidobacterium longum ZFML006 at an inoculation amount of 1.5 - 2.5% (preferably 2%, v / v) into an MRS liquid medium containing 0.05% L-cysteine, and cultivate anaerobically at 37 ± 1°C for 48 ± 1 h; to obtain a ZFML006 bacterial liquid; Explanation: The MRS liquid medium containing 0.05% L-cysteine is: based on each liter of MRS liquid medium, add 0.5 g of L-cysteine; The Bifidobacterium longum ZFML006 can be first activated and then a single colony is picked and inoculated into 10 mL of MRS (0.05% L-cysteine w / v) liquid medium, and cultivated anaerobically at 37°C for 48 h to serve as the seed liquid; Step 2) is: centrifuge the ZFML006 bacterial liquid (8000 × g, 15 min, 4°C), resuspend it with phosphate buffer (1 × 10 9 cfu / mL), and then perform a hot water bath treatment (inactivate at 65°C for 30 min) to obtain a ZFML006 inactivated bacteria bacterial liquid.

[0015] The present invention also simultaneously provides the application of the above inactivated bacteria in the preparation of antioxidant and anti-aging drugs.

[0016] As an improvement to the application of the present invention: acting on Caenorhabditis elegans (feeding the ZFML006 inactivated bacteria to Caenorhabditis elegans) can extend its lifespan, reduce fecundity, increase the body swing rate, reduce lipofuscin in the body, reduce body width and body length, enhance heat stress and acute stress capabilities, reduce the accumulation amount of ROS in the body, increase the expression amount of antioxidant enzymes in the body, and affect the expression of genes related to the anti-aging signal pathways (insulin / IGF-1 signal pathway and MAPK signal pathway) in the body.

[0017] As a further improvement of the application of the present invention: The antioxidant enzymes include superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GSH-Px).

[0018] As a further improvement of the application of the present invention, the influence on the expression of genes related to the anti-aging signal pathway in the body includes: inhibiting daf-2 and the expression of akt-2, promoting the expression of daf-16 and sod-3; at the same time promoting pmk-1、 nsy-1、skn-1 and sek-1 expression.

[0019] In the present invention, Bifidobacterium longum ( Bifidobacterium longum ) ZFML006 is inoculated on a solid medium for streaking; the single colony of Bifidobacterium longum ( Bifidobacterium longum ) ZFML006 is inoculated into a fermentation medium for activation and subculture for two generations to obtain a bacterial solution; In the preparation method of inactivated bacteria: Strain activation stage: The culture conditions are 37 °C and the culture time is 48 h; the inoculation amount on the medium in the strain subculture stage is preferably 2%; The media for strain activation and subculture are both MRS (0.05% L-cysteine w / v) liquid medium.

[0020] The preservation information of the strain of the present invention is as follows: Preservation name: Bifidobacterium longum ZFML006 Bifidobacterium longum ZFML006, preservation unit: China Center for Type Culture Collection, preservation address: Wuhan University, Wuhan, China, preservation number: CCTCC NO: M2025331, preservation date: February 28, 2025.

[0021] The Bifidobacterium longum ZFML006 of the present invention is isolated from infant feces. After sequencing, its 16S rRNA sequence is compared with nucleic acid sequences in NCBI, and it is identified as Bifidobacterium longum after obtaining the comparison results.

[0022] The heat-inactivated cells of Bifidobacterium longum subsp. longum ZFML006 of the present invention not only comprehensively improve lifespan (with an increase of up to 30.6%), fecundity, locomotor ability, and body development, but also efficiently remove the senescence marker lipofuscin. At the same time, by doubly enhancing resistance to heat stress and oxidative stress, they actively reduce the ROS level and upregulate the expression of antioxidant enzymes (such as SOD, CAT, etc.). Its uniqueness lies in the coordinated regulation of two key longevity pathways, the insulin / IGF-1 signaling pathway (IIS) and the mitogen-activated protein kinase (MAPK) signaling pathway, to form a multi-target anti-aging network. And through the synergistic effect of DAF-16 and SKN-1, the evolutionary conservation of the anti-aging effect is achieved. In addition, the heat-inactivated cells have high safety and strong stability, do not require refrigeration, and are suitable for a wide range of populations. Their cell wall components (such as peptidoglycan) can also activate innate immunity, which is superior to ordinary strains that rely on the metabolism of live bacteria, and has great potential for the development of anti-aging functional preparations. Therefore, the heat-inactivated cells prepared from Bifidobacterium longum subsp. longum ZFML006 have good application prospects for antioxidant and anti-aging effects. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] The following further elaborates on the specific implementation manners of the present invention with reference to the drawings.

[0024] Figure 1 It is a graph showing the effect of heat-inactivated cells of Bifidobacterium longum subsp. longum ZFML006 / OP50 cells on the lifespan of Caenorhabditis elegans; Figure 1 In the figure: A shows the effect of heat-inactivated cells of Bifidobacterium longum subsp. longum ZFML006 / OP50 cells on the lifespan (survival rate) of Caenorhabditis elegans, and B shows the effect of heat-inactivated cells of Bifidobacterium longum subsp. longum ZFML006 / OP50 cells on the median lifespan of Caenorhabditis elegans.

[0025] Figure 2 It is the effect of heat-inactivated cells of Bifidobacterium longum subsp. longum ZFML006 / OP50 cells on the fecundity, pharyngeal pumping, and body sway of Caenorhabditis elegans; Figure 2 In the figure: A shows the effect of heat-inactivated cells of Bifidobacterium longum subsp. longum ZFML006 / OP50 cells on the fecundity (total number of offspring) of Caenorhabditis elegans, B shows the effect of heat-inactivated cells of Bifidobacterium longum subsp. longum ZFML006 / OP50 cells on the pharyngeal pumping rate (number of pharyngeal aspirations) of Caenorhabditis elegans, and C shows the effect of heat-inactivated cells of Bifidobacterium longum subsp. longum ZFML006 / OP50 cells on the body sway rate (number of body bends) of Caenorhabditis elegans.

[0026] Figure 3 It is the effect of heat-inactivated cells of Bifidobacterium longum subsp. longum ZFML006 / OP50 cells on the width and length of lipofuscin bodies in Caenorhabditis elegans; Figure 3In the figure: A is the fluorescence image of lipofuscin in Caenorhabditis elegans after treatment with heat-killed Bifidobacterium longum ZFML006 / OP50 (25 μm). The nematodes on the left are under bright field, and the fluorescence image of lipofuscin on the right is under fluorescence microscope; B shows the effect of heat-killed Bifidobacterium longum ZFML006 / OP50 on lipofuscin in Caenorhabditis elegans, C shows the effect of heat-killed Bifidobacterium longum ZFML006 / OP50 on the body width of Caenorhabditis elegans, and D shows the effect of heat-killed Bifidobacterium longum ZFML006 / OP50 on the body length of Caenorhabditis elegans.

[0027] Figure 4 Shows the effects of heat-killed Bifidobacterium longum ZFML006 / OP50 on heat stress and acute oxidative stress in Caenorhabditis elegans; Figure 4 In the figure: A and B respectively show the effects of heat-killed Bifidobacterium longum ZFML006 / OP50 on the heat stress ability of Caenorhabditis elegans, and C and D respectively show the effects of heat-killed Bifidobacterium longum ZFML006 / OP50 on the acute stress ability of Caenorhabditis elegans.

[0028] Figure 5 Shows the effects of heat-killed Bifidobacterium longum ZFML006 / OP50 on ROS and antioxidant enzymes in Caenorhabditis elegans; Figure 5 In the figure: A shows the effect of heat-killed Bifidobacterium longum ZFML006 / OP50 on the accumulation of ROS (reactive oxygen species) in Caenorhabditis elegans, and B, C and D show the effects of heat-killed Bifidobacterium longum ZFML006 / OP50 on the expression levels of antioxidant enzymes in Caenorhabditis elegans - catalase (CAT), superoxide dismutase (SOD), glutathione peroxidase (GSH-PX).

[0029] Figure 6 Shows the gene expression levels of insulin / IGF-1 signaling pathway (IIS) and MAPK signaling pathway in Caenorhabditis elegans after treatment with heat-killed Bifidobacterium longum ZFML006 / OP50; Figure 6 In the figure: A shows the gene expression levels of insulin / IGF-1 signaling pathway (IIS) related genes in Caenorhabditis elegans after treatment with heat-killed Bifidobacterium longum ZFML006 / OP50, and B shows the gene expression levels of MAPK signaling pathway related genes in Caenorhabditis elegans after treatment with heat-killed Bifidobacterium longum ZFML006 / OP50. Detailed implementation methods

[0030] The present invention will be further described below in conjunction with specific embodiments, but the protection scope of the present invention is not limited thereto: In the following embodiments, unless otherwise specified, the operation methods used are all conventional operation methods, the equipment used is all conventional equipment, and the equipment materials used in each embodiment are the same.

[0031] The phosphate buffer solution is PBS buffer solution (0.01 M, pH 7.3 - 7.5); MRS (0.05% L-cysteine w / v) liquid medium: Based on each liter of conventional MRS liquid medium, 0.5 g of L-cysteine is added.

[0032] Example 1. Obtaining of Bifidobacterium longum ZFML006: Feces from infants in Zhejiang Province aged 0 - 6 months were used as samples. 0.1 g of feces was added to 1 mL of phosphate buffer solution and mixed evenly to prepare the initial sample solution. According to the 10-fold dilution method, the initial sample solution was diluted with phosphate buffer solution to 10 -4 、10 -5 、10 -6 as bacterial suspensions. Then, 100 μL of each bacterial suspension was spread on MRS solid medium containing calcium carbonate (2%), and after incubation in an anaerobic incubator at 37°C for 48 h, the colonies with calcium dissolution halos were selected for identification. The 16S rDNA sequence identification result is as shown in SEQ ID NO:1.

[0033] Subsequently, it was confirmed to be the genus Bifidobacterium longum by comparison on the official website of the National Center for Biotechnology Information (NCBI) of the United States, and it was named Bifidobacterium longum ( Bifidobacterium longum ) ZFML006.

[0034] The preservation information is as follows: Preservation name: Bifidobacterium longum ZFML006 Bifidobacterium longum ZFML006, preservation unit: China Center for Type Culture Collection, preservation address: Wuhan University, Wuhan, China, preservation number: CCTCC NO:M2025331, preservation date: February 28, 2025.

[0035] Example 2. I. Preparation method of inactivated bacterial liquid of ZFML006, which is carried out in the following steps in sequence: 1), Pick a single colony of Bifidobacterium longum ZFML006 with an inoculation loop and incubate it anaerobically at 37°C for 48 h in 10 mL of MRS (0.05% L-cysteine w / v) liquid medium; inoculate the obtained seed liquid into MRS (0.05% L-cysteine w / v) liquid medium at an inoculation amount of 2% (volume %), and culture it anaerobically at 37°C for 48 h; obtain the ZFML006 bacterial liquid.

[0036] 2), Centrifuge the cultured Bifidobacterium longum ZFML006 bacterial liquid (8000 × g, 15 min, 4°C), resuspend it with phosphate buffer to 1 × 10 9 cfu / mL, and finally obtain the inactivated ZFML006 bacterial liquid after heat bath treatment (inactivated at 65°C for 30 min).

[0037] II. The preparation method of Escherichia coli OP50 bacterial liquid is: centrifuge the Escherichia coli OP50 bacterial liquid cultured by the conventional method (8000 × g, 15 min, 4°C), resuspend it with phosphate buffer to 1 × 10 9 cfu / mL, which is the Escherichia coli OP50 bacterial liquid used as a control group.

[0038] Example 3. The effect of Bifidobacterium longum ZFML006 on prolonging the lifespan of Caenorhabditis elegans Under the same culture conditions, observe the effect of different foods (experimental group: Bifidobacterium longum ZFML006, control group: Escherichia coli OP50, as the food of Caenorhabditis elegans, and the food needs to be changed daily) on the lifespan of Caenorhabditis elegans.

[0039] Culture the synchronized Caenorhabditis elegans at the L4 stage on a nematode medium (NGM) plate (60 mm) (added with 50 μM FUDR) supplemented with 1 × 10 9 cfu / mL of heat-inactivated Bifidobacterium longum ZFML006 or OP50. The number of Caenorhabditis elegans on each plate is 60.

[0040] The specification of the nematode medium (NGM) plate is 60 mm (the same specification in the following examples).

[0041] That is, the experimental groups are as follows: (i) Control group (OP50 control group): Add 200 μL of Escherichia coli OP50 bacterial liquid to an NGM plate (60 mm) containing 50 μM FUDR, and then place Caenorhabditis elegans; (ii)Treatment group (heat-inactivated Bifidobacterium longum ZFML006 treatment group): After adding 200 μL of heat-inactivated ZFML006 bacterial solution to an NGM plate (60 mm) containing 50 μM FUDR, Caenorhabditis elegans was placed on it; In both the control group and the treatment group, 60 Caenorhabditis elegans were placed; Three biological replicate plates were set up for each group (with 60 Caenorhabditis elegans on each plate) and cultured at 20 °C.

[0042] The food needed to be changed daily, specifically: In the control group, Caenorhabditis elegans were transferred to a fresh NGM plate containing 50 μM 5-fluoro-2'-deoxyuridine (FUDR) and added with 200 μL of Escherichia coli OP50 bacterial solution; In the treatment group, Caenorhabditis elegans were transferred to a fresh NGM plate containing 50 μM 5-fluoro-2'-deoxyuridine (FUDR) and added with 200 μL of heat-inactivated ZFML006 bacterial solution; Thus, continuous exposure was maintained. The survival rate was recorded until all died.

[0043] Due to the advantages of Caenorhabditis elegans such as short lifespan (20 - 30 days) and clear biological characteristics, its lifespan assessment has become a key indicator in anti-aging intervention research. The results of the lifespan determination experiment are as Figure 1 shown in A and B. Compared with the control group fed with OP50, heat-inactivated Bifidobacterium longum ZFML006 significantly prolonged the median lifespan of Caenorhabditis elegans (the increase was up to 30.6% ± 3.7%), indicating its significant anti-aging effect. Therefore, it can be known that: Heat-inactivated Bifidobacterium longum ZFML006 can delay the aging of Caenorhabditis elegans; that is, heat-inactivated Bifidobacterium longum ZFML006 has an anti-aging effect.

[0044] Example 4. Effect of Bifidobacterium longum ZFML006 on the fecundity of Caenorhabditis elegans 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, and the food needed to be changed daily) on the fecundity of Caenorhabditis elegans were observed.

[0045] L4-stage synchronized Caenorhabditis elegans were inoculated on NGM medium plates (without FUDR) supplemented with 1 × 10 9 cfu / mL of heat-inactivated Bifidobacterium longum ZFML006 or OP50; The experimental groups were divided as follows: (i) Control group (OP50 control group): After adding 200 μL of Escherichia coli OP50 bacterial solution to an NGM plate (60 mm), 1 Caenorhabditis elegans was placed; (ii) Treatment group (heat-inactivated Bifidobacterium longum subsp. infantis ZFML006 treatment group): After adding 200 μL of heat-inactivated ZFML006 bacterial solution to an NGM plate (60 mm), 1 Caenorhabditis elegans was placed; Three biological replicate plates were set for each group (with 1 Caenorhabditis elegans on each plate, that is, each nematode was cultured separately), and cultured at 20 °C.

[0046] The food needed to be changed daily, specifically: In the control group, the Caenorhabditis elegans were transferred to a fresh nematode medium (NGM) plate added with 200 μL of Escherichia coli OP50 bacterial solution; In the treatment group, the Caenorhabditis elegans were transferred to a fresh nematode medium (NGM) plate added with 200 μL of heat-inactivated ZFML006 bacterial solution; The experiment was set for 5 days. The original plates for these 5 days were placed in a 20 °C incubator and each was retained for 72 hours for offspring counting. The total fecundity was calculated based on the cumulative number of offspring produced within 5 days. Since the energy resources of organisms are limited, there is a trade-off between reproduction and maintaining organismal homeostasis (such as anti-aging). When fecundity decreases, the energy originally used for reproduction may be redistributed to longevity-related pathways such as DNA repair, antioxidant defense, and proteostasis, thus extending lifespan.

[0047] The results are as Figure 2 shown in A, Bifidobacterium longum subsp. infantis ZFML006 can reduce the fecundity of Caenorhabditis elegans; that is, Bifidobacterium longum subsp. infantis ZFML006 has the effect of delaying the aging of Caenorhabditis elegans.

[0048] Example 5. Effects of Bifidobacterium longum subsp. infantis ZFML006 on the pharyngeal pumping and body sway of Caenorhabditis elegans.

[0049] Under the same culture conditions, the effects of different foods (experimental group: Bifidobacterium longum subsp. infantis ZFML006, control group: Escherichia coli OP50, as food for Caenorhabditis elegans, and the food needed to be changed daily) on the pharyngeal pumping and body sway of Caenorhabditis elegans were observed.

[0050] Synchronized L4-stage Caenorhabditis elegans were cultured on nematode medium (NGM) plates (added with 50 μM FUDR) supplemented with 1 × 10 9 cfu / mL of heat-inactivated Bifidobacterium longum subsp. infantis ZFML006 or OP50. The number of Caenorhabditis elegans on each plate was 30, and the specification of the nematode medium (NGM) plate was 60 mm. They were cultured at 20 °C for 5 days.

[0051] The experimental grouping method and the method of changing food can refer to Example 3.

[0052] The body swing rate was counted as the number of body bends per minute for each nematode, and the pharyngeal pumping frequency was counted as the number of pharyngeal pumps within 30 seconds for each nematode.

[0053] The results of pharyngeal pumping and body swing are as Figure 2 shown in B and C of , Bifidobacterium longum ZFML006 increased the body swing rate of Caenorhabditis elegans, but basically did not change its pharyngeal pumping rate.

[0054] Note: Generally, the decrease in the body swing rate of Caenorhabditis elegans is positively correlated with lifespan. Individuals with a longer maintenance time of motor ability usually have a longer lifespan; therefore, it can be known that heat-inactivated Bifidobacterium longum ZFML006 can delay the lifespan of Caenorhabditis elegans.

[0055] Example 6. Effects of Bifidobacterium longum ZFML006 on lipofuscin, body width and body length of Caenorhabditis elegans 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, and the food needs to be changed daily) on lipofuscin, body width and body length of Caenorhabditis elegans were observed.

[0056] Synchronized L4-stage Caenorhabditis elegans were cultured on nematode growth medium (NGM) plates (added with 50 μM FUDR) supplemented with 1 × 10 9 cfu / mL of heat-inactivated Bifidobacterium longum ZFML006 or OP50. The specifications of the nematode growth medium (NGM) plates were 60 mm. The number of Caenorhabditis elegans on each plate was 30, and they were cultured at 20 °C for 5 days.

[0057] The experimental grouping method and the method of changing food can refer to Example 3.

[0058] After culturing for 5 days, the Caenorhabditis elegans were anesthetized in a solution containing 20 mM sodium azide (NaN3), and then the Caenorhabditis elegans were fixed on 1% agarose (weigh 1 g of agarose powder and add it to 100 mL of 1×TAE buffer). Subsequently, the fixed samples were photographed using a fluorescence microscope (excitation wavelength 485 nm, emission wavelength 530 nm). The fluorescence intensity, body length and body width of lipofuscin in Caenorhabditis elegans were quantitatively analyzed using ImageJ software.

[0059] There is a close correlation between lipofuscin accumulation, body width and body length changes in Caenorhabditis elegans and lifespan, and these parameters can all be used as biological markers of aging. Lipofuscin is mainly formed by the polymerization of oxidized damaged proteins, lipids and sugars in lysosomes and has autofluorescence. During the aging process, the function of lysosomes declines and they are unable to degrade these cross-linked polymers, resulting in the accumulation of lipofuscin. The results are as Figure 3 shown. Heat-inactivated Bifidobacterium longum ZFML006 reduced the lipofuscin in Caenorhabditis elegans, reduced the body width and body length of Caenorhabditis elegans, and the reduction in body width and body length may be related to the increase in body sway rate. Therefore, it can be known that heat-inactivated Bifidobacterium longum ZFML006 delays the aging of Caenorhabditis elegans.

[0060] Example 7. Effect of Bifidobacterium longum ZFML006 on enhancing the stress resistance of Caenorhabditis elegans.

[0061] Under the same culture conditions, observe the effects of different foods (experimental group: Bifidobacterium longum ZFML006, control group: Escherichia coli OP50, as food for Caenorhabditis elegans, and the food needs to be changed daily) on the heat stress and acute oxidative stress capabilities of Caenorhabditis elegans.

[0062] Synchronized L4-stage Caenorhabditis elegans were respectively inoculated onto nematode growth medium (NGM) plates (added with 50 μM FUDR) supplemented with 1 × 10 9 cfu / mL of heat-inactivated Bifidobacterium longum ZFML006 or OP50. The number of Caenorhabditis elegans on each plate was 30, and they were cultured at 20 °C for 5 days.

[0063] The experimental grouping method and the method of changing food can refer to Example 3.

[0064] After culturing for 5 days, transfer the Caenorhabditis elegans to fresh NGM plates (without food, without FUDR), and perform heat stress treatment at 35 °C. Record the mortality rate every hour until all are dead. Finally, evaluate its heat resistance through statistical analysis of the survival data. The results are as Figure 4 shown in A and B.

[0065] After culturing for 5 days, transfer the Caenorhabditis elegans to NGM plates containing 0.1% hydrogen peroxide (H2O2) and culture them at 20 °C. Detect the survival rate of the nematodes every 30 minutes and record the mortality rate at each time point. The results are as Figure 4 shown in C and D.

[0066] According to Figure 4 it can be known that Bifidobacterium longum ZFML006 enhanced the heat stress and acute stress capabilities of Caenorhabditis elegans.

[0067] Example 8. Effects of Bifidobacterium longum ZFML006 on ROS and antioxidant enzymes (SOD, CAT, and GSH-Px) in Caenorhabditis elegans

[0068] 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, and the food needs to be changed daily) on ROS and antioxidant enzymes (SOD, CAT, and GSH-Px) in Caenorhabditis elegans were observed

[0069] Synchronized Caenorhabditis elegans at the L4 stage were cultured on nematode growth medium (NGM) plates (60 mm in diameter, supplemented with 50 μM FUDR) containing 1 mL of heat-inactivated Bifidobacterium longum ZFML006 or OP50 at a concentration of 1 × 10 9 cfu / mL. Approximately 200 Caenorhabditis elegans were placed on each plate and cultured at 20 °C for 5 days. Every day, the nematodes on the plate were rinsed off with PBS, washed three times, and then the nematodes deposited at the bottom of the EP tube were transferred to a fresh NGM plate containing food (supplemented with 50 μM FUDR) for culture

[0070] The experimental groups were set as follows (i) Control group (OP50 control group): After adding 1 mL of Escherichia coli OP50 bacterial solution to an NGM plate (supplemented with 50 μM FUDR), approximately 200 Caenorhabditis elegans were placed on it (ii) Treatment group (heat-inactivated Bifidobacterium longum ZFML006 treatment group): After adding 1 mL of inactivated ZFML006 bacterial solution to an NGM plate (supplemented with 50 μM FUDR), approximately 200 Caenorhabditis elegans were placed on it Five biological replicate plates were set for each group and cultured at 20 °C

[0071] The specific operation of changing food daily is as follows In the control group, the washed Caenorhabditis elegans were transferred to a fresh NGM plate (supplemented with 50 μM FUDR) containing 1 mL of Escherichia coli OP50 bacterial solution In the treatment group, the Caenorhabditis elegans were transferred to a fresh NGM plate (supplemented with 50 μM FUDR) containing 1 mL of inactivated ZFML006 bacterial solution

[0072] Collect Caenorhabditis elegans after 5 days of culture (about 1,000 worms per group). After washing with PBS buffer (0.01 M, pH 7.3 - 7.5), centrifuge at 4°C and 3,000×g for 2 minutes to collect the worms. Add 500 μL of PBS buffer (0.01 M, pH 7.3 - 7.5) to prepare a worm lysate, and centrifuge at 4°C and 12,000×g for 10 minutes to obtain the supernatant. Take about 50 μL of the supernatant and mix it with an equal volume of DCFH-DA probe (1:1, v / v) until the final concentration of the DCFH-DA probe is 50 μM, and incubate at 20°C in the dark for 2 hours. Detect the fluorescence intensity using a fluorescence spectrophotometer (excitation wavelength 485 nm, emission wavelength 538 nm).

[0073] Collect Caenorhabditis elegans after 5 days of culture (about 1,000 worms per group). After washing with PBS, centrifuge at 4°C and 3,000×g for 2 minutes to collect the worms. Add 500 μL of PBS to prepare a worm lysate, and centrifuge at 4°C and 12,000×g for 10 minutes to obtain the supernatant. Use kits from Nanjing Jiancheng Bioengineering Institute: Superoxide Dismutase (SOD) Isoenzyme Assay Kit (Catalog No.: A001-2), Catalase (CAT) Assay Kit (Catalog No.: A007-1-1), Glutathione Peroxidase (GSH-PX) Assay Kit (Catalog No.: A005-1), and measure the activities of superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GSH-Px) according to the instructions.

[0074] The results are as Figure 5 shown. Bifidobacterium longum ZFML006 reduced the ROS accumulation in Caenorhabditis elegans and increased the expression levels of antioxidant enzymes (SOD, CAT, and GSH-Px) in Caenorhabditis elegans. Therefore, it can be shown that heat-inactivated Bifidobacterium longum ZFML006 can play an anti-aging role by enhancing the endogenous antioxidant defense system in Caenorhabditis elegans.

[0075] Example 9. Effects of Bifidobacterium longum ZFML006 on the gene expression of anti-aging related signaling pathways (insulin / IGF-1 signaling pathway and MAPK signaling pathway) in Caenorhabditis elegans.

[0076] Under the same culture conditions, observe the effects of different foods (experimental group: heat-inactivated Bifidobacterium longum ZFML006, control group: Escherichia coli OP50, as food for Caenorhabditis elegans, and the food needs to be changed daily) on the anti-aging related genes in Caenorhabditis elegans.

[0077] Inoculate synchronized Caenorhabditis elegans at the L4 stage into 1 mL of 1 × 10 9On nematode growth medium (NGM) plates (60 mm) of heat-inactivated Bifidobacterium longum ZFML006 or OP50 at cfu / mL (supplemented with 50 μM FUDR). The amount of Caenorhabditis elegans on each plate was approximately 200, and they were cultured at 20 °C for 5 days. Every day, the nematodes on the plate were rinsed off with PBS, washed three times, and then the nematodes deposited at the bottom of the EP tube were transferred to a fresh NGM plate containing food (supplemented with 50 μM FUDR) for culture. 10 biological replicate plates were set up for each group.

[0078] The experimental grouping method and the method of changing food can refer to Example 8.

[0079] Collect Caenorhabditis elegans (about 2000) after 5 days of culture. After adding 500 μL of TRIzol reagent, use a cryogenic grinder to grind the sample 6 times at 1000 rpm (1 minute each time) to fully lyse the sample; then add 100 μL of chloroform, mix well and let stand at room temperature for 5 minutes, centrifuge at 12000×g for 15 minutes at 4 °C to separate 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 and then centrifuge to discard the supernatant; then wash the precipitate twice with 1 mL of 75% ethanol (centrifuge at 7500×g for 5 minutes at 4 °C), dry the RNA precipitate at room temperature for 5 - 10 minutes, and finally dissolve it with diethyl pyrocarbonate-treated water (DEPC water) and store it at -80 °C. The whole process needs to strictly avoid RNase contamination to ensure accurate operation.

[0080] Use quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR) method to detect the expression levels of anti-aging related genes in Caenorhabditis elegans treated with heat-inactivated Bifidobacterium longum ZFML006 and Escherichia coli OP50 for 5 days.

[0081] Total RNA was extracted using TRIzol reagent from Beyotime (Shanghai, China), and cDNA was synthesized using PrimeScript™ Reverse Transcription Kit (Takara). qRT-PCR was performed using SYBG Green® PCR Kit (Takara Bio, Beijing, China) and followed the following thermal cycling program: pre-amplify at 95 °C for 10 minutes, then perform 40 cycles of 15 seconds at 95 °C and 1 minute at 60 °C. Actin ( act-1 ) was used as a control, and the gene expression relative to act- 1 was calculated by 2-ΔΔCT. The primers used were synthesized by Shanghai Sangon Biotech Co., Ltd.

[0082] The primer sequences used in this case are specifically as shown in Table 1 below; Table 1

[0083] The results are shown in Figure 6. The heat-inactivated ZFML006 inhibited daf-2 and the expression of akt-2, and promoted the expression of daf-16 and sod-3. At the same time, it promoted pmk-1 , nsy-1 , skn-1 and sek-1 expression. Therefore, the heat-inactivated ZFML006 can delay the aging of Caenorhabditis elegans by inhibiting the insulin / IGF-1 signaling pathway and activating the MAPK signaling pathway.

[0084] According to the above results, it can be concluded that heat-inactivated Bifidobacterium longum ZFML006 can significantly enhance the ability of cells to maintain homeostasis by increasing the activities of antioxidant enzymes and stress tolerance in Caenorhabditis elegans. And through the cross-regulation of two pathways (such as the co-activation of DAF-16 and SKN-1), it enhances the robustness of the anti-aging effect. Its evolutionary conservation also provides potential targets for mammalian aging research and lays a theoretical foundation for the development of new anti-aging intervention means. Moreover, heat-inactivated Bifidobacterium longum ZFML006 realizes the co-regulation of 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 the antioxidant capacity and stress response ability of cells. This dual mechanism and co-regulation endow ZFML006 with unique advantages in terms of the robustness and evolutionary conservation of the anti-aging effect.

[0085] Bifidobacterium longum ( Bifidobacterium longum ) described in NKU FB 3-14, BA-3, HEPRO-261, CN104046573B was detected according to Example 9 above. skn-1 The expression level was not significantly different from that of OP50.

[0086] In addition, in contrast, although existing studies on Bifidobacterium longum also have certain anti-aging effects, their mechanisms are relatively single and lack the dual regulation and synergistic effects of ZFML006 of the present invention. For example, as described below: 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 toBifidobacterium longum BB68. Scientific reports , 7 (1), 7408.); 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 strains of Bifidobacterium on Caenorhabditis elegans. Food Science ,45 (13), 146-152.); The antioxidant effect of Bifidobacterium longum CCFM1029 requires exogenous support 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.); Bifidobacterium longum TCI001 must rely on live bacterial colonization and its effects are limited to the intestinal microecology.

[0087] These strains generally have defects such as single pathway, limited stress or unstable dosage form (Zheng Xiaonan, Zhang Hao, Guo Huiyuan & Ren Fazheng. (2012). Research progress on the anti-aging function of probiotics. China Dairy Industry, (02), 50-53).

[0088] The ZFML006 of the present invention inactivates bacterial components to coordinately regulate the IIS-MAPK dual pathway, activates endogenous enzymes and has dual stress resistance, showing significant advantages in comprehensive mechanism and application stability. Therefore, ZFML006 provides a more potential theoretical basis for the development of new anti-aging intervention methods.

[0089] Finally, it should be noted that the above examples are only some specific embodiments of the present invention. Obviously, the present invention is not limited to the above embodiments, and there are many variations. All variations that can be directly derived or associated with the content disclosed by a person skilled in the art should be considered as the protection scope 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 cell mass prepared from the Bifidobacterium longum ZFML006 as claimed in claim 1.

3. The preparation method of the inactivated bacteria as described in claim 2, characterized in that It includes the following steps: 1). Inoculate Bifidobacterium longum ZFML006 into a fermentation medium for culture to obtain a ZFML006 bacterial liquid; 2). Heat-treat the ZFML006 bacterial liquid to inactivate it to obtain a ZFML006 inactivated cell mass bacterial liquid.

4. The method for preparing the inactivated cell mass as claimed in claim 3, characterized in that: Step 1) is: inoculating the seed liquid of Bifidobacterium longum ZFML006 into an MRS liquid medium containing 0.05% L-cysteine at an inoculation amount of 1.5 - 2.5%, and culturing anaerobically at 37 ± 1 °C for 48 ± 1 h; obtaining a ZFML006 bacterial liquid; Step 2) is: centrifuging the ZFML006 bacterial liquid, resuspending it with a phosphate buffer solution, and then performing a hot water bath treatment to obtain a ZFML006 inactivated cell mass bacterial liquid.

5. The application of the inactivated cell mass as claimed in claim 2 in the preparation of anti-aging drugs.

6. The application according to claim 5, wherein: It has antioxidant efficacy.

7. The application according to claim 5 or 6, characterized in that: Acting on Caenorhabditis elegans, it can extend its lifespan, reduce fertility, increase the body sway rate, reduce lipofuscin in the body, reduce body width and body length, enhance heat stress and acute stress capabilities, reduce the accumulation amount of ROS in the body, increase the expression amount of antioxidant enzymes in the body, and affect the expression of genes related to the anti-aging signaling pathway in the body.

8. The application as claimed in claim 7, characterized in that: The antioxidant enzymes include superoxide dismutase, catalase and glutathione peroxidase.

9. The application according to claim 8, wherein The affecting the expression of genes related to the anti-aging signaling pathway in the body includes: Suppressed daf-2 and the expression of akt-2, and promoted daf-16 and sod-3 the expression of; at the same time, promoted pmk-1, nsy-1, skn-1 and sek-1 the expression of.

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