A Lactobacillus plantarum strain for preventing and improving sarcopenia in the elderly and its application

Abstract

The present invention belongs to the technical field of fermentation and health, and discloses a Lactobacillus plantarum for preventing and improving sarcopenia in the elderly and its application. The Lactobacillus plantarum was isolated and screened from kefir grains and named Lactobacillus plantarum BFS1243. In the research of the present invention, a sarcopenia model in the elderly was established through the FMT technology, and Lactobacillus plantarum BFS1243 was used to interfere with it, which can effectively increase the muscle weight and quality of male elderly sarcopenia mice, improve the serum inflammation level, increase the anti-inflammatory ability of the body, positively regulate the intestinal flora, and increase the level of short-chain fatty acids in intestinal metabolites. It can be seen that the Lactobacillus plantarum BFS1243 provided by the present invention can effectively improve sarcopenia in elderly men, and the high-calcium and high-protein yogurt fermented with this bacterium can be used as a food to effectively prevent and control the occurrence and development of sarcopenia in elderly men, and has been well applied in improving the health of the elderly population.

Description

Technical Field

[0001] The present invention belongs to the technical field of fermentation and health, and particularly relates to a Lactobacillus plantarum for preventing and improving sarcopenia in the elderly and its application in the preparation of solid beverages. Background Art

[0002] Sarcopenia is a sports medicine term announced in 2019, also known as muscle loss, which is an age-related geriatric syndrome and one of the common geriatric syndromes that seriously affect the quality of life of the elderly. It is characterized by the gradual loss of muscle mass, strength, and function, and is an important factor leading to frailty, disability, and death in the elderly. Around 2035, the elderly population aged 60 and above in China will exceed 400 million, accounting for more than 30% of the total population, entering a stage of severe aging. Epidemiological survey data on sarcopenia in the Chinese population shows that the prevalence of sarcopenia in community-dwelling elderly is 8.9% - 38.8%. Among them, the prevalence of sarcopenia is as high as 30% in women and 68% in men, and the prevalence in men is significantly higher than that in women.

[0003] Patients with sarcopenia show obvious intestinal flora dysbiosis, and intestinal flora dysbiosis will further trigger systemic chronic inflammation, which in turn leads to a decline in muscle mass and function. The connection between the intestinal flora and muscle physiology should be the focus of research on geriatric nutrition, frailty, and sarcopenia. Some scholars fed Lactobacillus casei to SAMP8 rapidly aging mice, which could significantly delay the aging rate of the mice, increase muscle mass and endurance, and improve the intestinal flora. The patent application with the publication number CN 110833565 discloses the active substances of Lactobacillus plantarum GKM3, the composition containing the same, and its use for promoting longevity. The composition of this invention can be particularly used to increase the expression level of the Cisd2 gene, reduce mitochondrial damage, and delay aging phenomena such as neurodegeneration and muscle atrophy. The patent application with the publication number US15385786 discloses that a Lactobacillus plantarum composition can improve muscle mass and endurance, reduce lactic acid, and improve exercise ability. However, there is no report in the prior art on the specific improvement of sarcopenia in elderly men by Lactobacillus plantarum and the improvement of the body's exercise ability. Without violating ethical and moral principles, it is of great significance to construct a human-derived flora animal model based on germ-free animals to better simulate the interaction or influence between the human intestinal flora and the host. Summary of the Invention

[0004] In order to overcome the disadvantages and deficiencies existing in the prior art, the primary object of the present invention is to provide a Lactiplantibacillus plantarum for preventing and improving sarcopenia in the elderly; this Lactiplantibacillus plantarum was isolated and screened from kefir grains, named Lactiplantibacillus plantarum BFS1243, which is a probiotic that promotes muscle growth in male patients with sarcopenia. This strain was deposited at the Guangdong Provincial Culture Collection Center of Microorganisms on November 4, 2022, with the deposit number GDMCC No 62947.

[0005] Another object of the present invention is to provide the application of the above-mentioned Lactiplantibacillus plantarum for preventing and improving sarcopenia in the elderly.

[0006] A further object of the present invention is to provide a solid beverage prepared using the above-mentioned Lactiplantibacillus plantarum for preventing and improving sarcopenia in the elderly.

[0007] The object of the present invention is achieved by the following technical solutions:

[0008] A Lactiplantibacillus plantarum for preventing and improving sarcopenia in the elderly, the Lactiplantibacillus plantarum was isolated and screened from kefir grains, named Lactiplantibacillus plantarum BFS1243, and this strain was deposited at the Guangdong Provincial Culture Collection Center of Microorganisms on November 4, 2022, with the deposit number GDMCC No62947.

[0009] Preferably, the sarcopenia in the elderly is male sarcopenia.

[0010] Preferably, the solid beverage has the efficacy of preventing or improving sarcopenia in the elderly.

[0011] Preferably, the solid beverage has the efficacy of preventing or improving male sarcopenia.

[0012] Preferably, the solid beverage is a high-calcium and high-protein yogurt fermented by Lactiplantibacillus plantarum.

[0013] A solid beverage fermented by the above-mentioned Lactiplantibacillus plantarum for preventing and improving sarcopenia in the elderly.

[0014] A high-calcium and high-protein yogurt fermented by the above-mentioned Lactiplantibacillus plantarum for preventing and improving sarcopenia in the elderly.

[0015] The research of this invention first aims to screen probiotic lactic acid bacteria by improving the motor ability of the nematode model induced by Enterobacter cloacae HJ, clarify the role of Lactobacillus plantarum BSF1243 in improving nematode muscle function, and provide a research basis for the subsequent application of Lactobacillus plantarum BSF1243 in the animal model of sarcopenia in the elderly. Then, a sarcopenia model in the elderly was established through the FMT technology, that is, by fecal microbiota transplantation (FMT), the feces of sarcopenia patients in the elderly were transplanted into the intestines of male and female germ-free mice for colonization to construct a sarcopenia model, and then it was fed with Lactobacillus plantarum BFS1243 to explore the improvement and regulation effect of Lactobacillus plantarum on sarcopenia. It was found that this Lactobacillus plantarum could effectively increase the muscle weight and quality of male sarcopenia mice in the elderly, improve the serum inflammation level, increase the anti-inflammatory ability of the body, positively regulate the intestinal flora, and increase the level of short-chain fatty acids in intestinal metabolites. It can be seen that Lactobacillus plantarum BFS1243 provided by this invention can effectively improve sarcopenia in elderly men, and the subsequent high-calcium and high-protein yogurt fermented with this bacterium can be used as a food to effectively prevent and control the occurrence and development of sarcopenia in the elderly, and has been well applied in improving the health of the elderly population. This invention strives to develop new products for preventing or improving sarcopenia, and provides a research and application basis for the clinical prevention and treatment of sarcopenia in the elderly.

[0016] The present invention has the following advantages and beneficial effects compared with the prior art:

[0017] (1) Lactobacillus plantarum BFS1243 can significantly increase the muscle weight, muscle grasping force, endurance and tensile force of male sarcopenia mice in the elderly; it has no significant effect on female sarcopenia mice in the elderly.

[0018] (2) Lactobacillus plantarum BFS1243 can effectively improve the serum inflammation level of male sarcopenia mice in the elderly, promote the increase of muscle mass, and improve the sarcopenia phenotype; it has no significant effect on female sarcopenia mice in the elderly.

[0019] (3) Lactobacillus plantarum BFS1243 can significantly improve the intestinal flora diversity of male sarcopenia mice in the elderly and reduce the abundance of intestinal harmful bacteria.

[0020] (4) Lactobacillus plantarum BFS1243 can extremely significantly increase the relative abundance of short-chain fatty acids in intestinal metabolites of male sarcopenia mice in the elderly, promote skeletal muscle growth, and improve sarcopenia symptoms. Description of the Drawings

[0021] Figure 1 Effects of three strains of lactic acid bacteria on the body swing frequency of nematodes in the infected group. Different lowercase letters indicate significant differences between groups, p < 0.05. 6 - 10 nematodes were measured in each group, and at least 3 parallels were set in each group.

[0022] Figure 2 Effect of Lactobacillus plantarum BFS1243 on body sway (left) and head sway (right) of nematodes. 6-10 nematodes were measured in each group, and at least 3 replicates were set in each group.

[0023] Figure 3 Effect of Lactobacillus plantarum BFS1243 on muscles of male mice (left figure) and female mice (right figure). A: Percentage of leg weight; B: Percentage of tibialis anterior (Ta) weight; C: Percentage of gastrocnemius (Gas) weight; D: Percentage of quadriceps (Qua) weight.

[0024] Figure 4 Effect of Lactobacillus plantarum BFS1243 on behaviors of male mice (upper figure) and female mice (lower figure). A: Endurance time; B: Swimming time; C: Grip strength value.

[0025] Figure 5 Effect of Lactobacillus plantarum BFS1243 on serum indexes of male mice (left figure) and female mice (right figure). A: Interleukin 10 (IL-10); B: Tumor necrosis factor α (TNF-α); C: Prostaglandin E2 (PGE2); D: Irisin; E: C-reactive protein (CRP).

[0026] Figure 6 Effect of Lactobacillus plantarum BFS1243 on α-diversity of intestinal flora in male mice. A: Observed; B: Chao; C: ACE; D: Shannon.

[0027] Figure 7 Effect of Lactobacillus plantarum BFS1243 on dominant intestinal flora at phylum level in male mice. A: Firmicutes; B: Bacteroides; C: Verrucomicrobia; D: Proteobacteria.

[0028] Figure 8 Effect of Lactobacillus plantarum BFS1243 on dominant intestinal flora (Top10) at genus level in male mice. A: Enterocloster; B: Akkermansia; C: Parabacteroides; D: Faecalibacterium; E: Blautia; F: Phocaeicola.

[0029] Figure 9Effect of Lactobacillus plantarum BFS1243 on the dominant intestinal flora (Top10) of male mice at the species level, where A: Akkermansia muciniphila_B; B: Bacteroides intestinalis_A; C: Bacteroides thetaiotaomicron; D: Eisenbergiella massiliensi; E: Enterocloster aldenensis; F: Parabacteroides distasonis.

[0030] Figure 10 Concentrations of short-chain fatty acids (SCFAs) in three groups of samples. Detailed implementation manners

[0031] The present invention will be further described in detail below in conjunction with embodiments and the accompanying drawings, but the implementation manners of the present invention are not limited thereto.

[0032] Embodiment 1:

[0033] This embodiment aims to screen probiotic lactic acid bacteria by improving the locomotor ability of the nematode model induced by Enterobacter cloacae HJ, clarify the role of Lactobacillus plantarum BSF1243 in improving nematode muscle function, and provide a research basis for the subsequent application of Lactobacillus plantarum BSF1243 in the animal model of sarcopenia in the elderly.

[0034] Experimental grouping:

[0035] Control group: Nematodes at the L4 stage were cultured on an Escherichia coli OP50 feeding plate.

[0036] Infection group: Nematodes at the L4 stage were cultured on an E. cloacae HJ infection plate.

[0037] Protection group: Nematodes at the L4 stage were cultured on plates treated with lactic acid bacteria BFS1243, BFS0596, and BFS0309 for 24 h and then transferred to an E. cloacae HJ infection plate.

[0038] There were 20 - 30 worms per plate, and each group had three replicates. Nematodes in each group were transferred to fresh food plates every other day. Nematodes were observed under a stereomicroscope and their locomotor ability was recorded until all nematodes died.

[0039] 1 Experimental method

[0040] Caenorhabditis elegans was wild-type N2.

[0041] Nematode growth medium (NGM): 3 g / L NaCl, 2.5 g / L peptone, 20 g / L agar, 2.5 ml of 1 M KH2PO4-K2HPO4 buffer solution (pH = 6.0), 0.1 ml of 1 M MgSO4, 0.1 ml of 1 M CaCl2. Add water to make up to 1 L. After autoclaving, when the culture medium cools down to 60 °C, add 1 ml / L of cholesterol ethanol solution (5 mg / ml), mix quickly, and prepare 60 mm NGM culture medium plates.

[0042] Escherichia coli OP50: Purchased from the Caenorhabditis Genetics Center (CGC).

[0043] Enterobacter cloacae HJ: Preservation number CMCC(B) 450033, China Medical Bacteria Preservation Center.

[0044] 1.1 Isolation and identification of strains

[0045] 1.1.1 Preparation of sample diluent and bacterial isolation

[0046] Weigh 5 - 10 g of five samples, namely kefir grains, Yakult, starter culture, fermented okara, and Rodia yogurt, respectively, dissolve them in 225 mL of sterile normal saline to make the stock solution. Pipette 1 mL of the stock solution and add it to 9 mL of sterile normal saline, shake well, and label it as 10 -1 diluent. Then pipette 1 mL of the liquid from the 10 -1 diluent into fresh 9 mL of sterile normal saline, and label it as 10 -2 diluent, and so on, continuously diluting to 10 -6 . Using the plate pour plate method, take 100 μL of the diluents with dilution gradients of 10 -4 、10 -5 、10 -6 and transfer them to petri dishes, and immediately pour in the reconstituted and cooled sterile MRS agar medium at about 45 °C, and mix them evenly. Incubate the plates at 30 °C for 24 - 48 h.

[0047] 1.1.2 Strained streak isolation

[0048] Observe the colony morphology on the plates. Use a sterile inoculation loop to pick up the round or fusiform single colonies on the surface and bottom of the petri dishes, streak inoculate them in the MRS agar medium, and incubate at 30 °C for 24 - 48 h. After incubation, select the single colonies with the morphological characteristics of Lactobacillus for Gram staining, select Gram-positive bacteria, and observe the rod-shaped or spherical Lactobacillus cell morphology under an electron microscope. Continuously streak and purify them on the MRS agar medium for three generations.

[0049] 1.1.3 Collect the activated 3 strains of Lactobacillus bacteria into a 1.5 mL sterile centrifuge tube. After centrifugation to remove the supernatant, the bacterial cell precipitate at the bottom of the tube was sent to Beijing Tsingke Biotechnology Co., Ltd. for sequencing analysis. Through gene sequence alignment, it was shown that BFS1243, BFS0596, and BFS0309 were Lactobacillus plantarum, Lactobacillus plantarum, and Lactobacillus helveticus respectively, and their similarities all reached 99.9%. The following sequences were obtained by sequencing Lactobacillus plantarum BFS1243:

[0050]

[0051]

[0052] 1.2 Strain activation and nematode culture

[0053] Inoculate Lactobacillus BFS1243, BFS0596, and BFS0309 on MRS solid medium for streaking, resuscitation, and purification. Then pick single colonies and inoculate them into MRS liquid medium respectively, and place them in a constant temperature incubator at 30 °C for static culture for 24 h - 48 h; Inoculate Enterobacter cloacae HJ and Escherichia coli OP50 on LB solid medium for resuscitation and purification. Then pick single colonies and inoculate them into LB liquid medium respectively, and place them on a shaker at 37 °C and shake culture at 180 r / min for 14 - 16 h. After the above steps, the culture solutions of each strain were obtained. The obtained bacterial solutions were centrifuged, the supernatant was discarded, and then washed with sterile M9 buffer. The concentration of each bacterial solution was adjusted to 1×10 9 CFU / mL. After pipetting and mixing each bacterial solution evenly, an appropriate amount was pipetted and added to the center of the NGM plate. After drying, a thin bacterial film would be formed, which was the Lactobacillus pretreatment plate, the Enterobacter cloacae HJ infection plate, and the Escherichia coli OP50 feeding plate. The plates could be placed in a 4 °C refrigerator for storage and use. Among them, the Escherichia coli OP50 feeding plate could be used for the daily maintenance and culture of nematodes.

[0054] 1.3 Nematode synchronization

[0055] Collect nematodes in the gestational period, wash them 2 - 3 times with sterile M9 buffer to remove the excess bacteria on the surface of the nematodes. Add the mixed lysis solution (1M NaOH and NaClO stock solution mixed at 1:1), lysis solution: worm solution = 1:1, shake up and down for 2 - 3 minutes until a large number of worms are lysed. Then centrifuge at 4700 r / min for 30 s, discard the supernatant, add sterile M9 buffer to resuspend, and centrifuge at 4700 r / min for 30 s. Wash 4 - 5 times under the same conditions. Place them at 20 °C for culture. After 12 h, transfer the hatched L1 - stage larvae to the OP50 feeding plate and continue to culture for 52 - 60 h to obtain L4 - stage nematodes.

[0056] 1.4 Nematode motility assay

[0057] Monitor the motility. Briefly, if the movement direction is set as the x-axis, when the movement direction of the pharynx changes on the y-axis, calculate the number of head swings of the nematode within every 30 s; calculate the body bend frequency per minute; the reverse movement per minute is calculated as the forward and backward movement behavior of the nematode; when the body of the nematode bends 180° from head to tail, the number of omega turns per minute is calculated. Measure 6-10 nematodes in each group, with at least 3 replicates.

[0058] 2 Experimental conclusions

[0059] 2.1 Infection with Enterobacter cloacae HJ affects the body swing frequency of nematodes, leading to impaired muscle function. As Figure 1 shown, compared with the control group, the body swing frequency of nematodes on the 3rd and 4th days in the infection group was significantly decreased. Compared with the infection group, the three lactic acid bacteria protection groups could all improve the motility of nematodes on the 1st day (p<0.05). Among them, BFS1243 still had an improving effect on its motility on the 2nd, 3rd, and 4th days (p<0.05), and the motility of nematodes on the 2nd day was significantly increased compared with the control group (p<0.05). In addition, BFS0309 and BFS0596 could improve the motility of nematodes on the 3rd day, but on the 2nd or 4th day, the motility was significantly decreased compared with the infection group and the control group (p<0.05). Therefore, there were also strain differences in the ability of the three lactic acid bacteria protection groups to improve the impaired motility. In contrast, the improving ability of BFS1243 was better, which could maintain the normal muscle motility of nematodes during the infection with HJ and had a certain degree of improvement.

[0060] 2.2 The improvement of motor function is closely related to muscle activity ability. The experimental results found that BFS1243 could effectively increase the body bend frequency of nematodes on the 1st and 2nd days (p<0.05), and the head swing frequency of nematodes was significantly increased on the 2nd, 4th, 6th, and 8th days (p<0.05) ( Figure 2 ). In summary, Lactobacillus plantarum BFS1243 could comprehensively improve the motility of nematodes, which was beneficial to improving the muscle function of nematodes.

[0061] Example 2:

[0062] Select sterile male and female mice aged 18-20 weeks that have given birth to conduct fecal microbiota transplantation + single bacterium interference experiments on elderly male healthy volunteers, male sarcopenia patients, elderly female healthy volunteers, and female sarcopenia patients respectively, to explore the specific role of Lactobacillus plantarum BFS1243 in improving elderly sarcopenia by regulating the intestinal flora.

[0063] The experimental groups are as follows:

[0064] Male mice (36 in total, 12 in each group)

[0065] HM: GF + HFMT (healthy human) + PBS

[0066] SM: GF + SFMT (sarcopenia patient) + PBS

[0067] SML: GF + SFMT (sarcopenia patient) + BFS1243

[0068] Female mice (21 in total, 7 in each group)

[0069] HF: GF + XHFMT (healthy human) + PBS

[0070] SF: GF + XSFMT (sarcopenia patient) + PBS

[0071] SFL: GF + XSFMT (sarcopenia patient) + BFS1243

[0072] 1 Experimental methods

[0073] Specific operation steps:

[0074] 1. Randomly divide 36 sterile C57 male mice into three groups, with 12 mice in each group. In a sterile isolation pack, gavage one group of mice with the prepared fecal suspension of a healthy male volunteer (1 person, HFMT), 0.1 mL for each mouse, continuously for 5 days to establish a healthy group, and then gavage with 0.1 mL of PBS every day for 10 weeks; at the same time, in the sterile isolation pack, gavage the other two groups of mice with the prepared fecal suspension of male sarcopenia patients (mixed bacteria of 3 people, SFMT), 0.1 mL for each mouse, continuously for 5 days to establish a sarcopenia group, and then gavage one group of sarcopenia mice with 0.1 mL of BSF1243 bacterial solution with a concentration of 10 9 CFU / mL every day for 10 weeks, and the other group of sarcopenia mice as a control, gavage with 0.1 mL of PBS for 10 weeks continuously.

[0075] 2. Twenty-one sterile female C57 mice were randomly divided into three groups of seven mice each. In a sterile isolation package, the prepared fecal suspension of a healthy female volunteer (one person, XHFMT) was used to gavage one group of mice, 0.1 mL for each mouse, continuously for 5 days to establish a healthy group, and then each mouse was gavaged with 0.1 mL of PBS every day for 10 weeks. At the same time, in the sterile isolation package, the prepared fecal suspension of female sarcopenia patients (mixed bacteria of three people, XSFMT) was used to gavage the other two groups of mice, 0.1 mL for each mouse, continuously for 5 days to establish a sarcopenia group, and then one group of sarcopenia mice was gavaged with 0.1 mL of BSF1243 bacterial solution with a concentration of 10 9 CFU / mL every day for 10 weeks, and the other group of sarcopenia mice was used as a control, gavaged with 0.1 mL of PBS, continuously for 10 weeks.

[0076] 2 Measurement indicators

[0077] 2.1 Physiological and biochemical indicators

[0078] The weights of the body, legs, gastrocnemius muscle, tibialis anterior muscle, quadriceps femoris muscle, spleen and liver of each group of mice were measured. Then, enzyme-linked immunosorbent assay (ELISA) was used to measure irisin, prostaglandin E2 (PGE2), C-reactive protein (CRP), tumor necrosis factor α (TNFα) and interleukin 10 (IL-10) in the serum of mice.

[0079] 2.2 Tissue sections

[0080] The freshly collected leg muscles (gastrocnemius muscle, tibialis anterior muscle), spleen, colon and ileum tissues were fixed with 4% paraformaldehyde. After dehydration and paraffin embedding, the samples were cut into 5-μm thickness and stained with hematoxylin-eosin (HE). The tissue pathological analysis of the sections was performed by scanning images with a slice scanner microscope and quantitative processing with ImageJ software.

[0081] 2.3 Intestinal flora determination

[0082] Fecal samples of each group of mice at the 10th week were taken respectively. Two tubes of feces were taken from each mouse, 2-3 feces per tube, and stored at -80 °C for later use. The third-generation PacBio high-throughput technology was used to perform full-length amplicon sequencing analysis of the 16S rRNA gene for each sample. The specific operation is as follows: Using Soil DNA Kit (Omega Bio-tek, USA) was used to extract DNA from stool samples. After the genomic DNA was extracted, the extracted genomic DNA was detected by 1% agarose gel electrophoresis. Each sample was repeated three times, and the PCR products of the same sample were mixed and detected by 2% agarose gel electrophoresis. The PCR products were cut and recovered using the AxyPrepDNA gel recovery kit (AXYGEN), eluted with Tris-HCl, and detected by 2% agarose electrophoresis. Referring to the preliminary quantitative results of electrophoresis, the PCR products were quantified by QuantiFluor TM -ST blue fluorescence quantitative system (Promega) was used for detection and quantification, and then the samples were mixed in corresponding proportions according to the sequencing amount requirements of each sample, and then PacBio library construction and sequencing analysis were performed.

[0083] 2.4 Intestinal metabolite determination

[0084] Ultra-high performance liquid chromatography-tandem mass spectrometry (UPLC-MS / MS) (ACQUITY UPLC-Xevo TQ-S, Waters Corp., Milford, MA, USA) was used to quantitatively determine the functional small molecule metabolites of intestinal microorganisms. The specific operation was as follows: the fecal samples were taken out of the -80℃ freezer, thawed in an ice bath, and each sample was weighed about 5 mg and placed in 1.5mL EP tubes. 25μL of deionized water was added to each tube and homogenized for 3min. Then, 120μL of pre-cooled methanol solution (including internal standard) was added to each tube, homogenized for 3min, and centrifuged at 4℃ (18000g, 20min). 20uL of the supernatant was transferred to a 96-well plate. In the Eppendorf epMotion workstation (Eppendorf Inc., Humburg, Germany), 20μL of freshly prepared derivatization reagent was added to each well, the plate was sealed, and the derivatization was carried out at 30℃ for 60min. Then, 330 μL of 50% methanol solution in an ice bath was added to dilute the sample, followed by centrifugation at 4°C (4000 g, 30 min), 135 μL of the supernatant was aspirated and transferred to a new 96-well plate, and 10 μL of internal standard was added to each well. Serial dilutions of the derivatized standard stock solution were added to the left well, and the plate was finally sealed for LC-MS analysis.

[0085] 3 Behavioral experiments

[0086] 3.1 Forelimb grip test

[0087] In the mouse experiment, a grip strength meter (GSM Grip Strength Meter Cat.No.47200) was used to test the forelimb grip strength of male and female mice with sarcopenia to evaluate muscle strength. The operation method was to fix the end of the mouse's tail by hand, make the mouse hold the pull ring with its forelimbs, and apply force backward to the mouse's tail horizontally until the mouse's forelimbs released. The grip strength meter recorded continuously for 5 times, and the average value was taken for statistics.

[0088] 3.2 Forced swimming experiment

[0089] The mice were placed in a cylindrical container filled with clean water, with a water depth of 30 cm and a water temperature of 25 °C. The mice were forced to swim, and the video system was used to record the time when the mice swam until they were immobile. The immobile state refers to the animal giving up active struggling, with the body floating without twisting, passive floating on the water, slightly arched but in a vertical state, and the nose above the water surface.

[0090] 3.3 Wire suspension experiment

[0091] The mice were placed on a horizontal wire, making their forelimbs grip the wire tightly. By observing the time and performance of the mice hanging on the wire, the upper limb strength and motor coordination ability were evaluated. The experiment recorded the time and success rate of the animals hanging on the wire.

[0092] 4 Experimental results

[0093] 4.1 It can be seen from Figure 3 that after the interference of BFS1243, it has a significant improvement effect on the muscles of male mice ( Figure 3 left figure), and there is no statistical difference in the effect on the muscles of female mice ( Figure 3 right figure). Among them, compared with the HM group, the leg weight of the mice in the SM group decreased significantly (p<0.05); compared with the SM group, the leg weight of the mice in the SML group increased significantly ( Figure 3 A in the left figure). Further anatomical analysis of the mouse leg muscles found that compared with the HM group, the weight of the tibialis anterior muscle of the mice in the SM group showed a downward trend, but there was no statistical difference; compared with the SM group, the weight of the tibialis anterior muscle of the mice in the SML group increased significantly (p<0.05)( Figure 3 B in the left figure). At the same time, we measured the weight of the gastrocnemius muscle of the mouse leg and found that compared with the HM group, the weight of the gastrocnemius muscle of the mice in the SM group showed a downward trend, but there was no statistical difference; compared with the SM group, the weight of the gastrocnemius muscle of the mice in the SML group showed an upward trend, but there was no statistical difference( Figure 3 C in the left figure). Finally, we analyzed the weight of the quadriceps femoris muscle of the mouse leg and found that compared with the HM group, the weight of the quadriceps femoris muscle of the mice in the SM group decreased significantly (p<0.05); compared with the SM group, the weight of the quadriceps femoris muscle of the mice in the SML group increased significantly (p<0.05)( Figure 3D) of the left figure. It shows that probiotic BFS1243 can effectively increase the muscle weight of male aged sarcopenia mice and improve the sarcopenia phenotype.

[0094] 4.2 Referring to the diagnostic criteria for human sarcopenia, the sarcopenia of mice was evaluated through mouse behavioral experiments, mainly including: wire suspension test, forced swimming test, and forelimb grip strength test. Through the mouse behavioral test experiments, it can be seen that Lactobacillus plantarum BFS1243 has a significant improvement effect on the behavior of male mice ( Figure 4 upper figure above), and there is no statistical difference in the improvement effect on the behavior of female mice (except for the forced swimming test) ( Figure 4 lower figure below). Among them, compared with the HM group, the endurance time of mice in the SM group was significantly reduced (p<0.05); compared with the SM group, the endurance time of mice in the SML group was significantly increased (p<0.05) ( Figure 4 A) of the upper figure above. Through the swimming experiment, it can be seen that compared with the HM group, the swimming time of mice in the SM group was significantly reduced (p<0.05); compared with the SM group, the swimming time of mice in the SML group was significantly increased (p<0.05) ( Figure 4 B) of the upper figure above. Through the tensile instrument test, it can be seen that compared with the HM group, the grip strength value of mice in the SM group was significantly decreased (p<0.05); compared with the SM group, the grip strength value of mice in the SML group was significantly increased (p<0.05) ( Figure 4 C) of the upper figure above. It shows that Lactobacillus plantarum BFS1243 can effectively improve the motor ability of male aged sarcopenia mice and improve the sarcopenia phenotype.

[0095] 4.3 Through the determination of mouse serological indexes, it can be seen that Lactobacillus plantarum BFS1243 has a significant improvement effect on the main serological indexes of male mice ( Figure 5 left figure), and there is no statistical difference in the improvement effect on the serological indexes of female mice. Among them, compared with the HM group, the serum interleukin 10 (IL-10) and irisin of mice in the SM group were significantly decreased (p<0.05); compared with the SM group, the serum IL-10 and irisin of mice in the SML group showed an upward trend, but there was no statistical difference ( Figure 5 A and D of the left figure). Compared with the HM group, the serum tumor necrosis factor (TNF-α), prostaglandin E2 (PGE2), and C-reactive protein (CRP) of mice in the SM group were significantly increased (p<0.05); compared with the SM group, the serum TNF-α and PGE2 of mice in the SML group were significantly decreased (p<0.05), and the CRP showed a downward trend, but there was no statistical difference ( Figure 5 B, C and E of the left figure).

[0096] During the aging process, the body will experience varying degrees of inflammatory responses. Prostaglandin E2 is an inflammatory protein, and its level also increases during the aging period and in various neurodegenerative diseases. C-reactive protein is an acute-phase protein with a pentameric structure and is a non-specific marker of inflammation and tissue damage. When inflammation or tissue damage occurs in the body, C-reactive protein is promptly produced by hepatocytes and rapidly increases, causing greater harm to the body. Irisin is a protein hormone produced by muscles, which can promote the growth and metabolism of skeletal muscles, increase skeletal muscle mass, and improve bone health. The increase in muscle mass will promote the production of irisin. Therefore, Lactobacillus plantarum BFS1243 can effectively increase the serum inflammation level of male elderly sarcopenia mice, promote the increase in muscle mass, and improve the sarcopenia phenotype.

[0097] 4.4 Analysis of the effect of Lactobacillus plantarum BFS1243 on the α-diversity of the intestinal flora of male mice showed that compared with the HM group, the species abundance of intestinal microorganisms in the SM group of mice was significantly increased (p < 0.05); compared with the SM group, the species abundance of intestinal microorganisms in the SML group of mice showed an upward trend, but there was no statistical difference ( Figure 6 A). Compared with the HM group, the species diversity Chao, ACE, and Shannon indices of intestinal microorganisms in the SM group of mice showed an upward trend, but there was no statistical difference; compared with the SM group, the species diversity Chao and ACE indices of intestinal microorganisms in the SML group of mice were significantly increased (p < 0.05), and the Shannon index had no statistical difference ( Figure 6 B, C, and D).

[0098] 4.5 Analysis of different taxonomic levels of mouse intestinal microorganisms found that at the phylum level, compared with the HM group, the relative abundances of the intestinal flora Firmicutes and Verrucomicrobia in the SM and SML groups of mice were significantly decreased and significantly increased, respectively (p < 0.05); compared with the SM group, there was no statistical difference in the relative abundances of Firmicutes and Verrucomicrobia in the SML group of mice ( Figure 7 A and C); there was no statistical difference in the relative abundances of the intestinal flora Bacteroides and Proteobacteria in the three groups of mice ( Figure 7 B and D).

[0099] 4.6 Analysis of the relative abundances of the dominant intestinal bacterial genera showed that, compared with the HM group, the relative abundances of the intestinal bacterial genera Enterocloster, Akkermansia, Parabacteroides, and Phocaeicola in the SM group of mice were significantly increased (p < 0.05); compared with the SM group, the relative abundances of Enterocloster, Parabacteroides, and Phocaeicola in the intestinal tract of the SML group of mice were significantly decreased (p < 0.05), and the relative abundance of Akkermansia showed a downward trend, but there was no statistical difference ( Figure 8 A, B, C, and F). Compared with the HM group, the relative abundances of the intestinal bacterial genera Faecalibacterium and Blautia in the SM group of mice were significantly decreased (p < 0.05); compared with the SM group, the relative abundances of the intestinal bacterial genera Faecalibacterium and Blautia in the SML group of mice showed no statistical difference ( Figure 8 D and E).

[0100] 4.7 Analysis of the relative abundances at the dominant intestinal bacterial species level showed that, compared with the HM group, the relative abundances of Akkermansia muciniphila_B, Bacteroides intestinalis_A, Eisenbergiella massiliensis, and Enterocloster aldenensis at the intestinal bacterial species level in the SM group of mice were significantly increased (p < 0.05); compared with the SM group, the relative abundances of Akkermansia muciniphila_B and Enterocloster aldenensis in the intestinal tract of the SML group of mice were significantly decreased (p < 0.05), and the relative abundances of Bacteroides intestinalis_A and Eisenbergiella massiliensis showed no statistical difference ( Figure 5 A, B, D, and E). There were no statistical differences in the relative abundances of Bacteroides thetaiotaomicron and Parabacteroides distasonis at the intestinal bacterial species level among the three groups of mice ( Figure 9 C and F).

[0101] 4.8 Analysis of the intestinal metabolites of the mice showed that, compared with the HM group, the concentration of the short-chain fatty acid (SCFA) in the intestinal metabolites of the SM group of mice was significantly decreased, and compared with the SM group, the concentration of SCFA in the intestinal metabolites of the SML group of mice was significantly increased ( Figure 10)。Related research reports that SCFA can enhance the absorption of glucose by skeletal muscle, the expression of glucose intermediates, and the accumulation of intramuscular lipids, regulating insulin sensitivity; it can also act directly or indirectly on skeletal muscle through liver absorption and metabolism, enhanced phosphorylation of AMP protein kinase, enhanced expression of adiponectin receptors, etc., or through blood and hormones, indicating that Lactobacillus plantarum BFS1243 effectively improves the symptoms of male elderly sarcopenia mice by regulating the concentration of intestinal short-chain fatty acids.

[0102] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications made without departing from the spirit and principle of the present invention shall be equivalent replacement methods and are all included in the protection scope of the present invention.

Claims

1. A Lactiplantibacillus plantarum for preventing and improving sarcopenia in the elderly, characterized in that: The Lactiplantibacillus plantarum is isolated and screened from kefir grains and named Lactiplantibacillus plantarum BFS1243. This strain was deposited at the Guangdong Provincial Culture Collection Center of Microorganisms on November 04, 2022, with the deposit number GDMCC No 62947.

2. A Lactiplantibacillus plantarum for preventing and improving sarcopenia in the elderly according to claim 1, characterized in that: The sarcopenia in the elderly is male sarcopenia.

3. Use of a Lactiplantibacillus plantarum for preventing and improving sarcopenia in the elderly according to claim 1 in the preparation of a solid beverage, characterized in that: The solid beverage has the efficacy of preventing or improving sarcopenia in the elderly.

4. The use according to claim 1, characterized in that: The solid beverage has the efficacy of preventing or improving male sarcopenia in the elderly.

5. The use according to claim 1, characterized in that: The solid beverage is a high-calcium and high-protein yogurt fermented by Lactiplantibacillus plantarum.

6. A solid beverage fermented by the Lactiplantibacillus plantarum for preventing and improving sarcopenia in the elderly according to claim 1.

7. A high-calcium and high-protein yogurt fermented by the Lactiplantibacillus plantarum for preventing and improving sarcopenia in the elderly according to claim 1.

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