Application of thermally inactivated bifidobacterium longum subsp. Infantis Y46 in promoting muscle development, delaying muscle atrophy and regulating cell functions

Abstract

The invention belongs to the technical field of microorganisms and fermentation engineering, and particularly relates to application of bifidobacterium longum subsp. Infantis Y46 in the anti-aging aspect. The bifidobacterium longum subsp. Infantis Y46 is obtained from excrement of infants. Animal experiments prove that living cell thalli (Y46) and thermally inactivated cell thalli (HKY46) obtained by culturing the bifidobacterium longum subsp. Infantis Y46 by using an MRS liquid culture medium containing L-cysteine hydrochloride (0.05%, v / v) can significantly reduce accumulation of lipofuscin in a host, and prolong the healthy life and life of the host. Therefore, the bifidobacterium longum subsp. Infantis Y46 is a promising probiotic and a promising metagen with an anti-aging function.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine and microbial application technology, specifically relating to the application of heat-inactivated Bifidobacterium longum subsp. infantis Y46 (HKY46) in promoting muscle development, delaying muscle atrophy, regulating cell differentiation (such as neural tissue, smooth muscle, and hematopoietic stem cells), and immune regulation. Furthermore, this invention also relates to methods for preparing HDY46 and its potential uses in metabolic disease intervention and drug activity regulation. Background Technology

[0002] With the increasing aging of the global population and the high incidence of metabolic diseases, muscle atrophy, muscle dysfunction, and related metabolic disorders are becoming increasingly prominent, seriously affecting patients' quality of life and increasing the medical burden. Currently, strategies to improve muscle quality and function mainly include nutritional intervention, exercise rehabilitation, and drug therapy, but these methods have significant limitations. Hormone therapy is prone to side effects (such as cardiovascular risk and hormonal imbalance), while exercise and dietary management have limited adherence and effectiveness in elderly patients or those with metabolic diseases.

[0003] In recent years, research has found that the gut microbiota can regulate the host's muscle metabolism and function through the "gut-muscle axis." Probiotics and their metabolites (such as short-chain fatty acids and extracellular polysaccharides) have been shown to improve muscle synthesis, inhibit inflammation and oxidative stress, but their mechanisms of action and targeting still need further investigation.

[0004] As one of the core probiotics of the gut, Bifidobacterium longum subsp. infantis has been reported in studies to improve metabolic health by regulating immune balance, enhancing barrier function, or indirectly affecting mitochondrial activity. However, there are no reports on the application of its inactivated bacterial cells (such as heat-inactivated forms) in muscle development, atrophy intervention, and cross-tissue differentiation regulation (such as the nervous and hematopoietic systems).

[0005] Heat-inactivated probiotics offer advantages such as high safety (no live bacteria-related risks), strong stability (good tolerance to storage and processing), and potential immunomodulatory activity, making them particularly suitable for specific populations (such as immunocompromised individuals). However, current technologies lack systematic research on heat-inactivated Bifidobacterium longum subsp. infantis Y46 (HKY46), and its mechanisms of action in promoting muscle growth, delaying atrophy, and regulating multiple tissues remain unclear.

[0006] Current technical deficiencies and needs: ① Insufficient traditional therapies, significant drug side effects, and limited effectiveness of exercise / nutrition programs; ② Limitations in probiotic research, challenges in the storage and safety of live bacteria applications, and insufficient exploration of the functions of inactivated bacteria; ③ Unclear mechanisms, and the molecular targets of gut-muscle axis and cross-tissue regulation need further clarification.

[0007] Based on the above-mentioned technological gaps, this invention reveals for the first time the groundbreaking application of heat-inactivated HKY46 in muscle health and multiple fields, providing a new strategy for developing safe and effective biological agents. Summary of the Invention

[0008] The purpose of this invention is to overcome the shortcomings of the prior art and provide an application of heat-inactivated Bifidobacterium longum subsp. infantis Y46 (HKY46) in promoting muscle development and delaying muscle atrophy.

[0009] To achieve the above objectives, the present invention is implemented using the following technical solution:

[0010] On the one hand, the present invention provides the application of a heat-inactivated Bifidobacterium longum subsp. infantis Y46 (HKY46) in promoting muscle development and delaying muscle atrophy, with heat-inactivated Bifidobacterium longum subsp. infantis 15697 (HK15697) as a control.

[0011] On the other hand, the present invention provides a method for preparing heat-inactivated Bifidobacterium longum subsp. infantis Y46 and 15697, characterized by comprising the following steps:

[0012] a. Bifidobacterium longum subsp. infantis Y46 and Bifidobacterium longum subsp. infantis 15697, with the serial number CCTCC M 20221253, were cultured in MRS liquid medium (MRSc) containing L-cysteine ​​hydrochloride (0.05%, v / v) to obtain bacterial suspensions of Bifidobacterium longum subsp. infantis Y46 and 15697.

[0013] b. Centrifuge the bacterial suspensions of Bifidobacterium longum subsp. infantum Y46 and 15697 from step a, wash the bacteria twice with sterile M9 buffer solution, and adjust the bacterial concentration to 0.2 mg / μL (wet weight) to obtain live cells of Bifidobacterium longum subsp. infantum Y46 and 15697;

[0014] c. The live cells of Bifidobacterium infantis subsp. Y46 and 15697 obtained in step b are subjected to heat inactivation treatment to obtain heat-inactivated cells of Bifidobacterium infantis subsp. Y46 (HKY46) and heat-inactivated cells of Bifidobacterium infantis subsp. 15697 (HK15697).

[0015] Furthermore, in step b, the centrifugal force is 2918 g, the temperature is 4℃, and the centrifugation time is 5 min; in step c, the inactivation temperature is 95℃, and the inactivation time is 30 min.

[0016] Furthermore, the heat-inactivated Y46 can promote muscle development and delay muscle atrophy.

[0017] Furthermore, the heat-inactivated Y46 can significantly reduce the lipofuscin content in Caenorhabditis elegans.

[0018] As can be seen from the above technical solutions, the present invention has the following beneficial effects:

[0019] The study on heat-inactivated Bifidobacterium longum subspecies Y46 (HKY46) provided by this invention shows that heat-inactivated Y46 can promote muscle development, delay muscle atrophy, and regulate cell function. Simultaneously, measurements of lipofuscin accumulation in *C. elegans* showed that heat-inactivated Y46 can significantly reduce lipofuscin accumulation in *C. elegans*. Attached Figure Description

[0020] Figure 1 Lifespan measurement results of Caenorhabditis elegans;

[0021] Figure 2 Fluorescent images of lipofuscin accumulation in *C. elegans*.

[0022] Figure 3 The relative fluorescence intensity of lipofuscin accumulation in Caenorhabditis elegans;

[0023] Figure 4 The locomotion ability of Caenorhabditis elegans;

[0024] Figure 5 Body movement data of Caenorhabditis elegans on day 5;

[0025] Figure 6 Data on head shaking of Caenorhabditis elegans on day 7.

[0026] Figure 7 Transcriptomic analysis and functional determination of Caenorhabditis elegans Figure 8 Functional analysis of Caenorhabditis elegans Detailed Implementation

[0027] The beneficial effects of the heat-inactivated Bifidobacterium longum subsp. infantis Y46 (HKY46) will be further illustrated below with specific embodiments and accompanying drawings.

[0028] The *Bifidobacterium longum* subsp. *infantis* Y46 described in this application is deposited at the China Center for Type Culture Collection, Wuhan, with accession number CCTCC M 20221253 and deposit date of August 9, 2022.

[0029] Example 1: Culture of *Bifidobacterium longum* subsp. *infantii* and *C. elegans* strains

[0030] Bifidobacterium longum subsp. infantis Y46 was provided by our laboratory. Bifidobacterium longum subsp. infantis Y46 and 15697 were inoculated into MRS liquid medium containing L-cysteine ​​hydrochloride (0.05%, v / v) and cultured anaerobically at 37°C for 24 h. Escherichia coli OP50 was provided by the Nematode Genetics Center at the University of Minnesota and cultured in LB broth at 37°C on a shaker (220 rpm) for 24 h.

[0031] *Caenorhabditis elegans* was cultured on NGM plates at 20°C using standard methods. To obtain synchronized populations, parental lysates were lysed using bleaching, and eggs were incubated overnight in M9 buffer. The transfer protocol for the worms in the experiment was as follows: synchronized L1-stage larvae were cultured on OP50 plates until they reached adulthood on day 1, and then transferred to the plates of each intervention group. The plates were changed daily for the first five days of the reproductive period, and then every two days thereafter.

[0032] Example 2: Cell preparation and experimental grouping

[0033] First, the bacterial culture was centrifuged (2918 g, 5 min, 4℃) to collect bacteria, and then washed twice with sterile M9 buffer. Subsequently, the bacterial concentration was adjusted to 0.2 mg / μL (wet weight) using sterile M9 to prepare live bacterial cells for later use. To prepare heat-inactivated bacterial cells, the live bacterial cells were heat-treated at 95℃ for 30 min to kill the bacteria.

[0034] Experimental groups: blank control group (OP50, 10 mg E. coli OP50), experimental control group (5 mg heat-inactivated 15697 cells + 5 mg E. coli OP50), and heat-inactivated Y46 cell experimental group (5 mg heat-inactivated cells + 5 mg E. coli OP50). The bacteria from each group were plated on *C. elegans* growth medium (NGM) and allowed to dry.

[0035] Example 3: Effects of Bifidobacterium longum subsp. Y46 on the motility of Caenorhabditis elegans

[0036] The lifespan of *Caenorhabditis elegans* was determined using L4 late larvae as the starting point (t=0). During the experiment, nematodes were transferred to fresh NGM plates every 24 hours during the reproductive period (approximately the first 5-6 days), and every 48 hours thereafter. Each experimental group contained at least 150 nematodes, and the number of surviving nematodes was recorded daily. The mortality criterion was no response to touch stimulation with platinum wire and cessation of the pharyngeal pump. Individuals that died due to operational errors, accidental escape, or intrauterine hatching (bursting) during the reproductive period were excluded from the final analysis.

[0037] Figure 1The results show the survival probability measurement of *C. elegans*. Compared with the OP50 control group, both HDY46 and HK15697 significantly promoted muscle development and delayed muscle atrophy in the nematodes (p<0.05). Specifically, compared with the OP50 control group, the muscle strength of *C. elegans* in the HDY46 group increased by 15.74%. This indicates that the muscle motility of *C. elegans* is significantly improved under the action of heat-inactivated *Bifidobacterium longum* subsp. *infant* Y46, suggesting that *Bifidobacterium longum* subsp. *infant* Y46 has potential application value in promoting muscle development and delaying muscle atrophy. Figure 1 ).

[0038] Example 4: Determination of the effect of Bifidobacterium longum subsp. infantis Y46 on lipofuscin accumulation in Caenorhabditis elegans.

[0039] To measure lipofuscin accumulation, *C. elegans* were transferred to sterile NGM plates and washed with M9 buffer. Subsequently, the nematodes were anesthetized with 0.1% tetraimidazole hydrochloride and fixed onto 2% agarose pads. Finally, autofluorescence images of the gut were captured using an inverted fluorescence microscope (DM 18) under blue excitation light, and the average fluorescence pixel intensity of each nematode was quantified using ImageJ software.

[0040] Figure 2 , 3 The results show the lipofuscin levels measured in *C. elegans*. Compared with the OP50 control group, the lipofuscin accumulation in the HDY46 treatment group was significantly reduced (p<0.05), a statistically significant difference. Specifically, the lipofuscin accumulation in the HDY46 treatment group was even lower, reaching 45.15% ( Figure 3 In summary, Y46 can reduce the accumulation of lipofuscin in *C. elegans*, indicating its potential for promoting muscle development and delaying muscle atrophy. This result provides a useful reference for the development of novel probiotics or functional foods.

[0041] Example 5: Effects of Bifidobacterium longum subsp. Y46 on the motility of Caenorhabditis elegans.

[0042] Methods for assessing the locomotor ability of *C. elegans*: The locomotor ability of *C. elegans* within 10-18 days after adulthood is assessed. "Grade A" indicates that the worm exhibits spontaneous sinusoidal movement without stimulation by a platinum wire picker or exhibits vigorous movement upon stimulation; "Grade B" indicates that the worm exhibits irregular or uncoordinated movement; "Grade C" indicates that the worm can only wiggle its head or tail in response to stimulation by a platinum wire picker but cannot move its body; "Grade D" indicates that the worm does not respond to the platinum wire picker at all, which is considered as death.

[0043] Figure 4The effect of HDY46 on the motility of *C. elegans* is shown in the figure. It is clear from the figure that the improvement in motility by HDY46 becomes increasingly significant over time. Specifically, on day 18, 14.8% of the nematodes in the HDY46 group reached the "Grade A" standard. In contrast, the proportion of "Grade A" nematodes in the OP50 control group on that day was 0%. This result indicates that HDY46 can improve the motility of *C. elegans*. Therefore, HDY46 has the potential as a probiotic or functional food supplement to help promote muscle development and delay muscle atrophy.

[0044] Example 6: Effects of Bifidobacterium longum subspecies Y46 on the body wiggling ability of Caenorhabditis elegans

[0045] Method for determining the body wiggling ability of Caenorhabditis elegans: Several Caenorhabditis elegans were randomly selected for testing. Using a platinum wire picker, the Caenorhabditis elegans were picked up and placed in M9 buffer solution, allowing them to swim freely for 30 seconds before measurement began. The number of body wigglings of the nematodes within 30 seconds was then counted.

[0046] Figure 5 , 6 This study observed the body wiggling behavior of *C. elegans*. Compared to the control group OP50, the experimental group (HKY46) significantly improved the body wiggling behavior of *C. elegans* on days 5 and 7 (p<0.05). By day 7, the HDY46 group showed a 49.0% improvement (…). Figure 6 These data clearly demonstrate that HKY46 can significantly improve the body twitching frequency of Caenorhabditis elegans.

[0047] Example 7: The function of *Caenorhabditis elegans* in promoting neural tissue differentiation, cardiac morphogenesis, angiogenesis, and smooth muscle differentiation.

[0048] With age or the onset of disease, muscles often experience a decline in quality, strength, and function. The results of this study suggest that HDY46 may exert the following effects by regulating the TGF-β and Wnt pathways: promoting muscle protein synthesis and inhibiting breakdown; enhancing the proliferation and differentiation of muscle satellite cells (adult stem cells); and reducing chronic inflammation and oxidative stress levels, thereby delaying muscle atrophy and promoting muscle health.

[0049] The above description is merely an embodiment of the present invention and does not limit the patent scope of the present invention. Any equivalent substitutions made using the content of the present invention specification, or direct or indirect applications in related technical fields, are similarly included within the patent protection scope of the present invention.

Claims

1. Use of heat-inactivated Bifidobacterium longum subsp. infantis Y46 (HKY46) in promoting muscle development, delaying muscle atrophy and regulating cell function, with heat-inactivated Bifidobacterium longum subsp. infantis 15697 (HK15697) as a control.

2. Use according to claim 1, characterized in that, The preparation method of heat-inactivated Bifidobacterium longum subsp. infantis Y46 (HKY46) and 15697 (HK15697) comprises the following steps: a. Bifidobacterium longum subsp. infantis Y46 and 15697 numbered CCTCCM 20221253 were cultured in MRS liquid medium (MRSc) containing L-cysteine hydrochloride (0.05%, v / v) to obtain Bifidobacterium longum subsp. infantis Y46 and 15697 bacterial solution; b. The Bifidobacterium longum subsp. infantis Y46 and 15697 bacterial solution in step a was centrifuged, the bacteria were washed twice with sterile M9 buffer solution, and the bacterial concentration was adjusted to 0.2 mg / μL (wet weight) to obtain live cell bacteria of Bifidobacterium longum subsp. infantis Y46 and 15697; c. The live cell bacteria of Bifidobacterium longum subsp. infantis Y46 and 15697 obtained in step b were heat-inactivated to obtain heat-inactivated Bifidobacterium longum subsp. infantis Y46 (HKY46) and 15697 (HK15697).

3. The use of claim 2, wherein In step b, the centrifugal force is 2918 g, the centrifugation temperature is 4°C, and the centrifugation time is 5-15 min; In step c, the inactivation temperature is 95-110°C, and the inactivation time is 5-30 min.

4. Use according to claim 2, characterized in that, The heat-inactivated Y46 can maintain cell homeostasis and protect cell structure from oxidative damage.

5. Use according to claim 2, characterized in that, The heat-inactivated Y46 can promote its differentiation into neural tissue.

6. Use according to claim 2, characterized in that, The heat-inactivated Y46 can be used to regulate smooth muscle differentiation.

7. Use according to claim 2, characterized in that, The heat-inactivated Y46 can be used for the maintenance and differentiation of hematopoietic stem cells.

8. The use according to claim 2, characterized in that, The heat-inactivated Y46 can be used for the prevention and treatment of related diseases.

9. The use according to claim 2, characterized in that, The heat-inactivated Y46 can be used as an immune enhancer to enhance immune function.

10. Use according to claim 4, characterized in that, The heat-inactivated Y46 can be used as a drug metabolism regulator to change the in vivo activity of combination drugs by affecting metabolic pathways.

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