A compound probiotic fermented astragalus post-biotic, its preparation method and application

Abstract

This invention provides a compound probiotic fermented astragalus post-biotic, its preparation method, and its application, relating to the field of microbial technology. The six non-antagonistic probiotic strains—*Lactobacillus paracasei* IOB413, *Lactobacillus casei* IOB-P9, *Lactobacillus rhamnosus* IOB820, *Lactobacillus fermentum* IOB802, *Pediococcus lactis* IOB701, and *Lactobacillus plantarum* IOB602—are mixed in equal proportions and then used for solid-state fermentation of astragalus. The fermentation product is then inactivated, dried, and pulverized to obtain the post-biotic. This post-biotic can downregulate the expression of muscle growth inhibitors and pro-inflammatory factors, upregulate the expression of muscle growth promoters and anti-inflammatory factors, reduce skeletal muscle atrophy and fibrosis, and increase muscle volume, gastrocnemius muscle index, and motor ability in mice. The process of this invention is stable, the product contains no live bacteria, has high safety, effectively improves the shortcomings of using raw astragalus, and has good application prospects in the prevention and treatment of sarcopenia.

Description

Technical Field

[0001] This invention relates to the field of microbial technology, and in particular to a compound probiotic fermented astragalus post-generic agent, its preparation method, and its application. Background Technology

[0002] Sarcopenia is a syndrome characterized by decreased muscle mass, reduced muscle strength, and impaired physical function. This condition not only severely impairs patients' ability to live independently but also increases the risk of fractures due to reduced mobility. Furthermore, it can cause metabolic and immune dysfunction, and induce various chronic diseases such as diabetes and cardiovascular disease, making it an increasingly serious global health problem. While sarcopenia is more common in the elderly, it can also occur in young adults, especially in patients with secondary sarcopenia caused by chronic wasting diseases or prolonged bed rest. Timely intervention in the early stages of muscle loss can effectively delay or even partially reverse the condition. For high-risk groups, early management can break the vicious cycle of "disease-disability" and improve overall prognosis.

[0003] Currently, traditional interventions for sarcopenia mainly include nutritional supplementation and exercise training. However, astragalus, a traditional Chinese medicine for nutrition, can easily cause discomfort such as bloating and dry mouth in some individuals with weak gastrointestinal function. Taking it concurrently with anticoagulants may also increase bleeding tendencies, making it unsafe for individuals using multiple medications. Regarding drug treatment, although some hormonal preparations have been tested clinically, due to their potential side effects, no drug has yet been officially approved globally for the treatment of sarcopenia, and clinical intervention methods remain significantly limited. Therefore, there is an urgent need to develop new, safer, more effective, and more adherent intervention strategies.

[0004] In recent years, the core role of the gut microbiota in human health and disease has become increasingly clear, highlighting the tangible advantages of probiotics and postbiotics. Probiotics can improve digestive function and increase appetite in the elderly, thus ensuring adequate protein and energy intake. Regular probiotic supplementation in the elderly has resulted in significant improvements in muscle function indicators such as grip strength and walking speed. Postbiotics, as active metabolites produced by probiotic fermentation, do not contain live bacteria, posing no risk of infection to elderly individuals with weakened immune systems. They exhibit outstanding safety, good long-term adherence, and effectively slow muscle decline. However, single probiotics are insufficient to effectively address sarcopenia caused by multiple factors, and different strains within the same genus exhibit significant functional differences. Currently, there is a lack of specific strains that can precisely optimize human muscle function. The synergistic effect of metabolites produced by the co-fermentation of multiple strains allows for adaptation to different individual gut environments, resulting in more stable and sustained improvements in muscle function. Furthermore, multi-strain fermentation products can address concurrent issues such as indigestion and weakened immunity, leading to a more balanced improvement in the overall condition of elderly patients and those with sarcopenia caused by chronic diseases.

[0005] Therefore, developing a sarcopenic probiotic formulation for sarcopenia improvement has become an urgent need in the fields of medicine and microbiology. Clinically, there is a pressing need for a new intervention strategy to safely and effectively address sarcopenia caused by aging, disease, or metabolic stress. Summary of the Invention

[0006] In order to overcome the above-mentioned defects and deficiencies in the existing technology, this application aims to provide a compound probiotic fermented astragalus post-biotic, its preparation method and application.

[0007] Lactobacillus paracasei ( Lacticaseibacillus paracasei IOB413 was deposited on June 29, 2018, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC No. 16022.

[0008] Lactobacillus casei ( Lacticaseibacillus casei IOB-P9 was deposited on December 27, 2021, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC No. 24195.

[0009] Lactobacillus rhamnosus ( Lacticaseibacillus rhamnosus IOB820 was deposited on April 3, 2019, at the China General Microbiological Culture Collection Center (CGMCC), with accession number CGMCCNo.17522.

[0010] Fermented Lactobacillus mucinus ( Limosilactobacillus fermentum IOB802 was deposited on August 5, 2021, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC NO.23120.

[0011] Pediococcus acidilactici ( Pediococcus acidilactici IOB701 was deposited on July 10, 2018, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC No. 16077.

[0012] Lactobacillus plantarum ( Lactiplantibacillus plantarum IOB602 was deposited on June 29, 2018, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC No. 16021.

[0013] One objective of this invention is to provide a compound probiotic fermented astragalus post-biotic, the compound probiotic comprising: Lactobacillus paracasei ( Lacticaseibacillus paracasei IOB413, accession number CGMCCNo.16022; Lactobacillus casei ( Lacticaseibacillus casei IOB-P9, accession number CGMCCNo.24195; Lactobacillus rhamnosus ( Lacticaseibacillus rhamnosus IOB820, accession number CGMCCNo.17522; Fermented Lactobacillus mucinus ( Limosilactobacillus fermentum IOB802, accession number CGMCCNO.23120; Pediococcus acidilactici ( Pediococcus acidilactici IOB701, accession number CGMCC No.16077; Lactobacillus plantarum ( Lactiplantibacillus plantarum )IOB602, accession number CGMCCNo.16021.

[0014] Preferably, the volume ratio of *Lactobacillus paracasei* IOB413, *Lactobacillus casei* IOB-P9, *Lactobacillus rhamnosus* IOB820, *Lactobacillus fermentum* IOB802, *Pediococcus lactis* IOB701, and *Lactobacillus plantarum* IOB602 is 1:1:1:1:1:1.

[0015] The second objective of this invention is to provide a method for preparing a compound probiotic fermented astragalus post-biotic, comprising the following steps: Lactobacillus paracasei IOB413, Lactobacillus casei IOB-P9, Lactobacillus rhamnosus IOB820, Lactobacillus fermentum IOB802, Pediococcus lactis IOB701, and Lactobacillus plantarum IOB602 were inoculated separately onto slant culture medium for activation. Each activated strain was then individually inoculated into Astragalus membranaceus powder liquid culture medium to obtain primary seed culture for each strain. Equal volumes of the primary seed culture for each strain were then mixed and inoculated into Astragalus membranaceus powder liquid culture medium to obtain secondary seed culture. The secondary seed culture was inoculated into Astragalus powder liquid culture medium to prepare a mixed seed culture of six bacteria; Astragalus powder was mixed with water and sterilized to obtain a solid fermentation substrate for Astragalus. The six-strain mixed seed liquid was inoculated into the Astragalus solid fermentation substrate, and after fermentation, Astragalus solid fermentation product was obtained. The solid fermented Astragalus membranaceus was inactivated, dried, and pulverized to obtain the compound probiotic fermented Astragalus membranaceus post-biotic.

[0016] Preferably, the slant culture medium is composed of: yeast extract 15 g / L, glucose 18 g / L, sodium acetate 5 g / L, dipotassium hydrogen phosphate 2 g / L, magnesium sulfate 0.2 g / L, manganese sulfate 0.05 g / L, Tween-80 1 g / L, agar 15 g / L, and distilled water to a final volume of 1000 mL. Preferably, the inoculation amount of the primary seed solution is 3%; Preferably, the inoculation amount of the secondary seed solution is 3%; Preferably, the preparation method of the Astragalus solid-state fermentation substrate is as follows: dried Astragalus is pulverized to 80 mesh, and purified water is added at a material-to-liquid ratio of 1:1.0 (m / v). The mixture is then sterilized at 115℃ for 20 min and cooled to room temperature to obtain the Astragalus solid-state fermentation substrate. The inoculum amount of the six-strain mixed seed liquid inoculated into the Astragalus solid-state fermentation substrate is 8%, and the culture conditions are 37℃ for 24 h.

[0017] Preferably, the inactivation condition is inactivation at 105℃ for 30 min; the drying and pulverizing are performed by drying to a moisture content of ≤12% and then pulverizing.

[0018] The third objective of this invention is to provide an application of compound probiotic fermented astragalus post-biotic in the preparation of medicines to relieve sarcopenia.

[0019] The beneficial effects of this invention are: This invention provides a method for preparing post-biotics from Astragalus membranaceus through mixed fermentation of six strains and its application in the preparation of medicines for alleviating sarcopenia. The method uses Astragalus membranaceus as a solid culture medium and selects *Lactobacillus paracasei* IOB413, *Lactobacillus casei* IOB-P9, *Lactobacillus rhamnosus* IOB820, *Lactobacillus fermentum* IOB802, *Pediococcus lactis* IOB701, and *Lactobacillus plantarum* IOB602 for mixed fermentation. Experiments have confirmed that these six strains do not exhibit antagonistic effects and can grow synergistically in the culture medium.

[0020] This invention utilizes a compound probiotic fermented astragalus post-biotic, which, through the synergistic effect of lactic acid and γ-aminobutyric acid, and in conjunction with the energy-providing effects of polysaccharides and reducing sugars, jointly improves sarcopenia. The polysaccharides reduce muscle loss caused by inflammation, while reducing sugars quickly replenish muscle energy. Lactic acid and γ-aminobutyric acid work together to promote muscle synthesis and inhibit protein breakdown. This post-biotic reduces the expression of muscle growth inhibitor TGF-β by 41.43% and gastrocnemius muscle MSTN by 37.72%, effectively relieving muscle growth limitations. It increases the expression of muscle growth promoter IGF-I by 112.66% and HGF by 96.06%, and increases the gastrocnemius muscle index by 52.49%, effectively alleviating muscle atrophy and fibrosis. The expression of pro-inflammatory factors IL-1β and IL-6 decreases by 42.04% and 43.34%, respectively, while the expression of anti-inflammatory factor IL-10 increases by 138.76%, thus reducing the overall level of inflammation in the body, repairing muscle fibers, and improving muscle mass and athletic ability. This post-biotic does not contain live bacteria and also solves the problems of gastrointestinal irritation and medication safety associated with raw Astragalus membranaceus, making it effective for the prevention and treatment of sarcopenia. Attached Figure Description

[0021] Figure 1 This image shows the relative expression level of MyoD gene mRNA in mouse gastrocnemius muscle tissue. Figure 2 This image shows the relative expression level of Myogenin gene mRNA in mouse gastrocnemius muscle tissue. Figure 3 This image shows the relative expression level of MGF gene mRNA in mouse gastrocnemius muscle tissue. Figure 4 This image shows the relative expression level of IGF-1 gene mRNA in mouse gastrocnemius muscle tissue. Figure 5 This image shows the relative expression level of HGF gene mRNA in mouse gastrocnemius muscle tissue. Figure 6 This image shows the relative expression level of Myostatin gene mRNA in mouse gastrocnemius muscle tissue. Figure 7 This is a graph showing the relative expression level of TGF-β gene mRNA in mouse gastrocnemius muscle tissue. Figure 8 This image shows the relative expression level of IL-1β gene mRNA in mouse gastrocnemius muscle tissue. Figure 9 This image shows the relative expression level of IL-6 gene mRNA in mouse gastrocnemius muscle tissue. Figure 10 This image shows the relative expression level of IL-10 gene mRNA in mouse gastrocnemius muscle tissue. Figure 11 This is a pathological image of mouse soleus muscle stained with hematoxylin and eosin (HE). Figure 12 Histopathological image of mouse gastrocnemius muscle stained with hematoxylin and eosin (HE). Figure 13 This is a pathological image of the mouse tibialis anterior muscle stained with hematoxylin and eosin (HE). Figure 14 Masson staining image of mouse gastrocnemius muscle; Figure 15 Immunohistochemical staining of MSTN protein in mouse gastrocnemius muscle; Figure 16 Image of mouse skeletal muscle CT scan; Figure 17 A bar chart showing the statistical analysis of mouse muscle volume; Figure 18 A bar chart showing the statistical index of the gastrocnemius muscle in mice; Figure 19 A trend graph of mouse body weight; Figure 20 A bar chart showing the results of the grip strength test in mice; Figure 21 A bar chart showing the latency test results of mice suspended from a wire and falling. Figure 22 A bar chart showing the results of the time to exhaustion during swimming in mice; Figure 23 This is a bar chart showing the results of the mouse drop latency test.

[0022] Lactobacillus paracasei ( Lacticaseibacillus paracasei IOB413 was deposited on June 29, 2018, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC No. 16022.

[0023] Lactobacillus casei ( Lacticaseibacillus casei IOB-P9 was deposited on December 27, 2021, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC No. 24195.

[0024] Lactobacillus rhamnosus ( Lacticaseibacillus rhamnosus IOB820 was deposited on April 3, 2019, at the China General Microbiological Culture Collection Center (CGMCC), with accession number CGMCCNo.17522.

[0025] Fermented Lactobacillus mucinus ( Limosilactobacillus fermentumIOB802 was deposited on August 5, 2021, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC NO.23120.

[0026] Pediococcus acidilactici ( Pediococcus acidilactici IOB701 was deposited on July 10, 2018, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC No. 16077.

[0027] Lactobacillus plantarum ( Lactiplantibacillus plantarum IOB602 was deposited on June 29, 2018, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC No. 16021. Detailed Implementation

[0028] The present application will now be described in further detail with reference to embodiments. In the following description, certain specific details are included to provide a comprehensive understanding of the various disclosed embodiments. However, those skilled in the art will recognize that embodiments can be implemented without employing one or more of these specific details, but using other methods, components, materials, etc. Unless otherwise required by the present invention, the terms "comprising" and "including" should be interpreted in an open-ended, inclusive sense, meaning "including but not limited to". Throughout this specification, "an embodiment," "an embodiment," "a preferred embodiment," or "some embodiments" means that at least one embodiment includes a specific reference element, structure, or feature related to that embodiment. Therefore, the phrases "in an embodiment," "in an embodiment," "in a preferred embodiment," or "in some embodiments" appearing in different places throughout the specification do not necessarily all refer to the same embodiment. Furthermore, specific elements, structures, or features may be combined in one or more embodiments in any suitable manner.

[0029] Example 1 Antagonism Experiment The tested strains were: Lactobacillus paracasei IOB413, Lactobacillus casei IOB-P9, Lactobacillus acidophilus IOB608, Lactobacillus rhamnosus IOB820, Lactobacillus fermentum IOB802, Pediococcus lactis IOB701, Lactobacillus plantarum IOB602, and Lactobacillus johnsonii IOB801, which were numbered A, B, C, D, E, F, G, and H, respectively.

[0030] Lactobacillus acidophilus ( Lactobacillus acidophilusThe accession number for IOB608 is CGMCC No. 17521; Lactobacillus johnsonii ( Lactobacillus johnsonii The accession number for IOB801 is CGMCC No. 16824; Activation and culture of strains: The above strains were inoculated onto slant culture medium and incubated at 37°C for 24 hours. One loopful of fresh slant culture was transferred to modified MRS liquid culture medium and incubated at 37°C for 22 hours to obtain the bacterial suspension of each strain.

[0031] Experimental Method: A pairwise antagonistic experiment was conducted on MRS solid plates using the streak cross method. Strawberry A was streaked with strains B, C, D, E, and F, with the remaining strains rotated in sequence. Each experiment was repeated three times. After streaking, the plates were incubated at 37℃ for 24 hours, and the results were observed. If colonies grew continuously at the cross-streaked areas of two strains without any inhibition zone, it was determined that there was no antagonistic effect between the strains, and they could be co-cultured.

[0032] The experimental results are shown in Table 1. The results of mixed culture show that Lactobacillus paracasei IOB413, Lactobacillus casei IOB-P9, Lactobacillus rhamnosus IOB820, Lactobacillus fermentum IOB802, Pediococcus lactis IOB701 and Lactobacillus plantarum IOB602 can coexist. Lactobacillus acidophilus IOB608 cannot coexist with Lactobacillus paracasei IOB413, Lactobacillus fermentum IOB802, Pediococcus lactis IOB701, Lactobacillus plantarum IOB602, and Lactobacillus johnsonii IOB801. Lactobacillus johnsonii IOB801 cannot coexist with Lactobacillus casei IOB-P9, Lactobacillus rhamnosus IOB820, Lactobacillus fermentum IOB802, and Lactobacillus plantarum IOB602.

[0033] Therefore, six strains were selected for subsequent experiments: Lactobacillus paracasei IOB413, Lactobacillus casei IOB-P9, Lactobacillus rhamnosus IOB820, Lactobacillus fermentum IOB802, Pediococcus lactis IOB701, and Lactobacillus plantarum IOB602.

[0034] Table 1 Antagonistic Experiment Table

[0035] Note: "+" indicates that the two strains can coexist, "-" indicates that the two strains are antagonistic and cannot coexist, and "\" indicates that no corresponding experiment was conducted.

[0036] Example 2: Substrate Screening Experiment Using the six non-antagonistic mixed strains obtained in Example 1 as inoculum, parallel control experiments were conducted using Astragalus membranaceus and Lycium barbarum as fermentation substrates to screen the optimal solid-state fermentation substrate.

[0037] Strain activation and culture: *Lactobacillus paracasei* IOB413, *Lactobacillus casei* IOB-P9, *Lactobacillus rhamnosus* IOB820, *Lactobacillus fermentum* IOB802, *Pediococcus lactis* IOB701, and *Lactobacillus plantarum* IOB602 were inoculated into slant agar and incubated at 37°C for 24 h. Single slant cultures corresponding to each of the six strains were obtained.

[0038] One loopful of fresh bacterial culture was inoculated into 25 mL of Astragalus powder liquid culture medium (the content of Astragalus powder in the liquid culture medium was 2%). The culture was then incubated at 37°C in a sealed container for 22 h to obtain primary seed liquids of six strains.

[0039] Further cultivation: The primary seed cultures of six single strains were mixed in equal volumes and inoculated into 25 mL of Astragalus powder liquid medium at an inoculation rate of 3%, with a volume ratio of six strains of 1:1:1:1:1:1. The Astragalus powder content in the liquid medium was 2%. The culture was incubated at 37°C in a sealed environment for 22 h to obtain the secondary seed culture. The secondary seed culture was then transferred to 25 mL of Astragalus powder liquid medium at an inoculation rate of 3%, with the Astragalus powder content in the liquid medium being 2%. The culture was incubated at 37°C in a sealed environment for 22 h to obtain the mixed seed culture of six strains.

[0040] Preparation of Astragalus solid fermentation substrate: Dry Astragalus is pulverized to 80 mesh, and pure water is added at a material-to-liquid ratio of 1:1.0 (m / v). The mixture is stirred, sterilized at 115℃ for 20 min, and cooled to room temperature to obtain Astragalus solid fermentation substrate.

[0041] The six-strain mixed seed liquid was inoculated into the Astragalus solid fermentation substrate at an inoculation rate of 8% and cultured at 37℃ for 24 hours to obtain Astragalus solid fermentation product.

[0042] solid-state fermentation of goji berries Seed culture preparation: One loopful of fresh bacterial strain was inoculated into 25 mL of wolfberry liquid culture medium. The wolfberry powder content in the wolfberry powder liquid culture medium was 2%. The culture was incubated at 37℃ in a sealed container for 22 h to obtain primary seed cultures of six strains.

[0043] Further cultivation: The primary seed solutions of six single strains were mixed in equal volumes to obtain a volume ratio of 1:1:1:1:1:1. This mixture was then inoculated at a 3% inoculum into 25 mL of Lycium barbarum powder liquid culture medium (containing 2% Lycium barbarum powder). The mixture was incubated at 37°C in a sealed container for 22 hours to obtain a secondary seed solution. This secondary seed solution was then inoculated at a 3% inoculum into 25 mL of Lycium barbarum powder liquid culture medium (containing 2% Lycium barbarum powder). The mixture was incubated at 37°C in a sealed container for 22 hours to obtain a mixed seed solution of the six strains.

[0044] Preparation of solid-state fermentation substrate for wolfberry: Dry wolfberries are crushed to 80 mesh, and pure water is added at a material-to-liquid ratio of 1:1.0 (m / v). The mixture is stirred, sterilized at 115℃ for 20 min, and cooled to room temperature to obtain the solid-state fermentation substrate for wolfberry.

[0045] The six-strain mixed seed liquid was inoculated into the wolfberry solid fermentation substrate at an inoculation rate of 8% and cultured at 37℃ for 24 hours to obtain the wolfberry product fermented by the six-strain mixed fermentation.

[0046] Detection and judgment: The number of viable bacteria in the two groups of samples was determined by plate count method. The results are shown in Table 2. After fermentation with Astragalus membranaceus as the substrate, the total number of colonies was higher. Therefore, Astragalus membranaceus was selected as the solid-state fermentation substrate in this experiment.

[0047] Table 2. Strains activity in solid-state fermentation products of Astragalus and Lycium barbarum

[0048] Example 3: Preparation of Multi-Strain Mixed Fermentation Powder Activation of the strain: The procedure is the same as in Example 2.

[0049] Seed culture preparation: Take one loopful of fresh bacterial culture and inoculate it into the modified MRS liquid medium. Incubate at 37°C in a sealed container for 22 hours to obtain the primary seed culture of the six bacteria. Inoculate the primary seed culture of the six bacteria at a 3% inoculation rate into the modified MRS liquid medium. Incubate at 37°C in a sealed container for 22 hours to obtain the secondary seed culture of the six bacteria.

[0050] Fermentation: The mixed secondary seed culture of the six bacteria was inoculated into the modified MRS liquid medium at an inoculation rate of 3%, and cultured in a sealed container at 37°C for 24 hours to obtain the mixed fermentation broth of the six bacteria.

[0051] Preparation of bacterial powder The six-strain mixed fermentation broth was centrifuged, the bacterial sludge was collected, and then freeze-dried under vacuum to obtain the six-strain mixed fermentation powder.

[0052] The modified MRS liquid culture medium is as follows: yeast extract 15 g / L, glucose 18 g / L, sodium acetate 5 g / L, dipotassium hydrogen phosphate 2 g / L, magnesium sulfate 0.2 g / L, manganese sulfate 0.05 g / L, Tween-80 1 g / L, and distilled water is added to a final volume of 1000 mL. Example 4: Preparation of post-biotics from single-strain fermentation of Astragalus membranaceus Activation and culture of the strain: The procedure was the same as in Example 2, and a primary seed culture of a single strain was obtained.

[0053] Further cultivation: The primary seed culture of a single strain was fermented separately, following the same procedure as in Example 2, to obtain a tertiary seed culture of a single strain.

[0054] Preparation of solid-state fermentation substrate: The procedure is the same as that for the preparation of solid-state fermentation substrate for Astragalus membranaceus in Example 2.

[0055] Solid-state fermentation: The tertiary seed liquid of each single strain was inoculated into the solid-state fermentation substrate of Astragalus membranaceus at an inoculation rate of 8% and cultured at 37℃ for 24 hours to obtain single-strain solid-state fermentation products.

[0056] Preparation of post-biotic powder: The obtained single-strain solid fermentation product was inactivated at 105℃ for 30 min, dried to a moisture content of ≤12%, and then pulverized and passed through an 80-mesh sieve to obtain single-strain fermented Astragalus post-biotic powder corresponding to six strains.

[0057] Example 5: Preparation of post-biotics from Astragalus membranaceus fermented with two strains Three strain combinations were set up: Lactobacillus plantarum IOB602 and Lactobacillus paracasei IOB413, Lactobacillus paracasei IOB413 and Lactobacillus casei IOB-P9, and Lactobacillus plantarum IOB602 and Pediococcus lactis IOB701.

[0058] Activation and culture of strains: The operation was the same as in Example 2, and primary seed liquids of single strains corresponding to the six strains were prepared respectively.

[0059] Further cultivation: The primary seed liquid of the single strain was mixed in equal volumes according to the above combination method, and the volume ratio of the two strains was 1:1. The further cultivation operation was the same as in Example 2, and three groups of mixed seed liquids of two strains were prepared respectively.

[0060] Preparation of solid-state fermentation substrate: The procedure is the same as that for the preparation of solid-state fermentation substrate for Astragalus membranaceus in Example 2.

[0061] Solid-state fermentation: The seed liquid of each combination of two strains was inoculated into the solid-state fermentation substrate of Astragalus membranaceus at an inoculation rate of 8%, and cultured at 37℃ for 24 hours to obtain three solid-state fermented products of Astragalus membranaceus with two strains.

[0062] Preparation of post-biotic powder: The procedure was the same as that for the preparation of post-biotic powder from Astragalus solid fermentation in Example 4, and three groups of two-strain combination Astragalus post-biotic powder were prepared in sequence.

[0063] Example 6: Preparation of post-biotics from Astragalus membranaceus fermented with three bacteria Four strain combinations were set up: Lactobacillus plantarum IOB602, Lactobacillus paracasei IOB413 and Lactobacillus rhamnosus IOB820; Lactobacillus paracasei IOB413, Lactobacillus casei IOB-P9 and Lactobacillus rhamnosus IOB820; Lactobacillus plantarum IOB602, Lactobacillus paracasei IOB413 and Lactobacillus fermentum IOB802; Lactobacillus paracasei IOB413, Pediococcus lactis IOB701 and Lactobacillus fermentum IOB802.

[0064] Activation and culture of strains: The operation was the same as in Example 2, and primary seed liquids of single strains corresponding to the six strains were prepared respectively.

[0065] Further cultivation: The primary seed solutions of the single strains were mixed in equal volumes according to the above combination method, and the volume ratio of the three strains was 1:1:1. The further cultivation operation was the same as in Example 2, and four groups of three-strain mixed seed solutions were obtained respectively.

[0066] Preparation of solid-state fermentation substrate: The procedure is the same as that for the preparation of solid-state fermentation substrate for Astragalus membranaceus in Example 2.

[0067] Solid-state fermentation: The seed liquid of each combination of three bacteria was inoculated into the solid-state fermentation substrate of Astragalus membranaceus at an inoculation amount of 8%, and cultured at 37℃ for 24 hours to obtain four kinds of solid-state fermented products of Astragalus membranaceus with three bacteria.

[0068] Preparation of post-biotic powder: The procedure is the same as that for the preparation of post-biotic powder from Astragalus solid fermentation in Example 4, and four groups of three-strain combination Astragalus post-biotic powders are prepared in sequence.

[0069] Example 7 Preparation of Astragalus membranaceus post-fermentation by four strains Two strain combinations were set up: one combination of *Lactobacillus plantarum* IOB602, *Lactobacillus paracasei* IOB413, *Lactobacillus rhamnosus* IOB820 and *Lactobacillus fermentum* IOB802; and another combination of *Lactobacillus paracasei* IOB413, *Lactobacillus casei* IOB-P9, *Lactobacillus rhamnosus* IOB820 and *Lactobacillus plantarum* IOB602.

[0070] Activation and culture of strains: The operation was the same as in Example 2, and primary seed liquids of single strains corresponding to the six strains were prepared respectively.

[0071] Further cultivation: The primary seed solutions of the single strain were mixed in equal volumes according to the above combination method, and the volume ratio of the four strains was 1:1:1:1. The further cultivation operation was the same as in Example 2, and two groups of mixed seed solutions of four strains were obtained respectively.

[0072] Preparation of solid-state fermentation substrate: The procedure is the same as that for the preparation of solid-state fermentation substrate for Astragalus membranaceus in Example 2.

[0073] Solid-state fermentation: The seed liquid of each group of four bacteria was mixed and inoculated into the solid-state fermentation substrate of Astragalus membranaceus at an inoculation amount of 8%, and cultured at 37℃ for 24 hours to obtain two kinds of solid-state fermented Astragalus membranaceus products with four bacteria combinations.

[0074] Preparation of post-biotic powder: The procedure is the same as that for the preparation of post-biotic powder from Astragalus solid fermentation in Example 4, and two groups of four-strain Astragalus post-biotic powders are prepared in sequence.

[0075] Example 8: Preparation of post-biotics from fermented Astragalus membranaceus by five strains Three strain combinations were set up: *Lactobacillus plantarum* IOB602, *Lactobacillus paracasei* IOB413, *Lactobacillus rhamnosus* IOB820, *Lactobacillus fermentatus* IOB802, and *Pediococcus lactis* IOB701; *Lactobacillus paracasei* IOB413, *Lactobacillus rhamnosus* IOB820, *Lactobacillus fermentatus* IOB802, *Pediococcus lactis* IOB701, and *Lactobacillus casei* IOB-P9; and *Lactobacillus plantarum* IOB602, *Lactobacillus paracasei* IOB413, *Lactobacillus rhamnosus* IOB820, *Pediococcus lactis* IOB701, and *Lactobacillus casei* IOB-P9.

[0076] Activation and culture of strains: The operation was the same as in Example 2, and primary seed liquids of single strains corresponding to the six strains were prepared respectively.

[0077] Further cultivation: The primary seed solutions of the single strains were mixed in equal volumes according to the above combination method, and the volume ratio of the five strains was 1:1:1:1:1. The further cultivation operation was the same as in Example 2, and three groups of mixed seed solutions of five strains were prepared respectively.

[0078] Preparation of solid-state fermentation substrate: The procedure is the same as that for the preparation of solid-state fermentation substrate for Astragalus membranaceus in Example 2.

[0079] Solid-state fermentation: The seed liquid of each group of five bacteria was mixed and inoculated into the solid-state fermentation substrate of Astragalus membranaceus at an inoculation amount of 8%, and cultured at 37℃ for 24 hours to obtain solid-state fermented products of Astragalus membranaceus with three combinations of five bacteria.

[0080] Preparation of post-biotic powder: The procedure is the same as that for the preparation of post-biotic powder from Astragalus solid fermentation in Example 4, and three groups of five-strain Astragalus post-biotic powders are prepared in sequence.

[0081] Example 9 Preparation of post-biotics from Astragalus membranaceus fermented with six strains A combination of *Lactobacillus paracasei* IOB413, *Lactobacillus casei* IOB-P9, *Lactobacillus rhamnosus* IOB820, *Lactobacillus fermentum* IOB802, *Pediococcus lactis* IOB701, and *Lactobacillus plantarum* IOB602 was prepared.

[0082] Activation and culture of strains: The operation was the same as in Example 2, and primary seed liquids of single strains corresponding to the six strains were prepared respectively.

[0083] Further cultivation: The primary seed solutions of the single strain were mixed in equal volumes according to the above combination method, and the volume ratio of the six strains was 1:1:1:1:1:1. The further cultivation operation was the same as in Example 2, and a set of six-strain mixed seed solutions were prepared.

[0084] Preparation of solid-state fermentation substrate: The procedure is the same as that for the preparation of solid-state fermentation substrate for Astragalus membranaceus in Example 2.

[0085] Solid-state fermentation: A seed culture of six bacteria was inoculated into the Astragalus solid-state fermentation substrate at an inoculation rate of 8%, and cultured at 37°C for 24 hours to obtain the solid-state fermented product of the six bacteria Astragalus.

[0086] Preparation of post-biotic powder: The procedure is the same as that for the preparation of post-biotic powder from Astragalus solid fermentation in Example 4, to obtain the six-strain Astragalus post-biotic powder.

[0087] Example 10: Detection of Active Ingredients To obtain the optimal fermentation microbial community composition of Astragalus membranaceus, lactic acid content was used as the core screening indicator. This is because lactic acid can promote muscle protein synthesis and inhibit degradation, and lactic acid content directly reflects the connection between fermentation products and muscle synthesis.

[0088] The samples used in this experiment were all taken from the single-strain and multi-strain combination Astragalus solid fermentation products prepared in Examples 4 to 9. (1) Strains combination experiment - determination of lactic acid content This experiment used lactic acid content as the core screening indicator, and carried out solid-state fermentation of Astragalus membranaceus with single, two, three, four, five, and six strains respectively, and determined the lactic acid content of the products.

[0089] Standard configuration Standard stock solution (1 mg / mL): Accurately weigh 10 mg of lactic acid standard, place it in a 10 mL volumetric flask, dissolve it with 0.1% phosphoric acid aqueous solution and dilute to the mark, store at 4°C protected from light.

[0090] Standard working solutions: Prepare a series of standard working solutions with concentrations of 20 μg / mL, 25 μg / mL, 40 μg / mL, 50 μg / mL, 80 μg / mL, 100 μg / mL and 200 μg / mL respectively, and filter them through a 0.22 μm filter membrane for later use.

[0091] Sample processing Weigh 2.0 g of sample and add 20 mL of 0.1% phosphoric acid aqueous solution. Mix well and sonicate for 30 min. After cooling, centrifuge at 11000 r / min for 10 min, collect the supernatant, and filter through a 0.22 μm filter membrane for analysis.

[0092] Liquid chromatography conditions Chromatographic column: C18 column (4.6mm × 250mm); Mobile phase A: 0.1% aqueous phosphoric acid solution - methanol (volume ratio 97.5:2.5); Mobile phase B: Methanol; Flow rate: 1.0 mL / min; Detection wavelength: 210 nm; Injection volume: 20 μL; Column temperature: 40 °C.

[0093] The gradient method is shown in Table 3.

[0094] Table 3 Gradient elution methods

[0095] Experimental results Single bacteria screening Among the six single strains, Lactobacillus plantarum (S1) had the highest lactic acid content (13416.91 mg / kg), which was significantly higher than the other single strains. The specific results are shown in Table 4. Therefore, S1 was selected as the representative of the single strain level.

[0096] Two-strain combination screening Among all the two-strain combinations, D1 (Lactobacillus plantarum IOB602 and Lactobacillus paracasei IOB-P9) had the highest lactic acid content (19820.96 mg / kg), which was significantly better than D2 (Lactobacillus paracasei IOB413 and Lactobacillus casei IOB-P9, 16500.67 mg / kg) and D3 (Lactobacillus plantarum IOB602 and Pediococcus lactis IOB701, 17200.21 mg / kg). The specific results are shown in Table 4. The cross-strain synergistic effect was significant, so D1 was selected as the optimal two-strain combination.

[0097] Screening of three bacterial combinations Among the three-strain combinations, T1 (Lactobacillus plantarum IOB602, Lactobacillus paracasei IOB413, and Lactobacillus rhamnosus IOB820) had the highest lactic acid content (19063.59 mg / kg), which was higher than T2 (Lactobacillus paracasei IOB413, Lactobacillus casei IOB-P9, and Lactobacillus rhamnosus IOB820, 15800.07 mg / kg), T3 (Lactobacillus plantarum IOB602, Lactobacillus paracasei IOB413, and Lactobacillus fermentum IOB802, 18200.55 mg / kg) and T4 (Lactobacillus paracasei IOB413, Pediococcus lactis IOB701, and Lactobacillus fermentum IOB802, 16800.95 mg / kg). The specific results are shown in Table 4. Therefore, T1 was selected as the optimal three-strain combination.

[0098] Four-strain combination screening The lactic acid content of F1 (Lactobacillus plantarum IOB602, Lactobacillus paracasei IOB413, Lactobacillus rhamnosus IOB820, and Lactobacillus fermentum IOB802) was 18925.28 mg / kg, the highest among all four-strain combinations, and higher than F2 (Lactobacillus paracasei IOB413, Lactobacillus casei IOB-P9, Lactobacillus rhamnosus IOB820, and Lactobacillus plantarum IOB602, 17500.23 mg / kg). The specific results are shown in Table 4. Lactobacillus fermentum IOB802 can produce extracellular polysaccharides, which are beneficial to the survival and metabolic stability of the bacterial community. Therefore, F1 was selected as the optimal four-strain combination.

[0099] Five-strain combination screening The lactic acid content of V1 (Lactobacillus plantarum IOB602, Lactobacillus paracasei IOB413, Lactobacillus rhamnosus IOB820, Lactobacillus fermentum IOB802, and Pediococcus lactis IOB701) was 18602.40 mg / kg, the highest among all five strain combinations, significantly higher than V2 (Lactobacillus paracasei IOB413, Lactobacillus rhamnosus IOB820, Lactobacillus fermentum IOB802, Pediococcus lactis IOB701, and Lactobacillus casei IOB-P9, 16200.22 mg / kg) and V3 (Lactobacillus plantarum IOB602, Lactobacillus paracasei IOB413, Lactobacillus rhamnosus IOB820, Pediococcus lactis IOB701, and Lactobacillus casei IOB-P9, 17400.83 mg / kg), as shown in Table 4. Lactococcus lactis IOB701 has strong acid resistance and can optimize the microenvironment of the fermentation system. V1 was selected as the optimal combination of five bacteria.

[0100] Six-strain combination screening The lactic acid content of H1 (Lactobacillus plantarum IOB602, Lactobacillus paracasei IOB413, Lactobacillus rhamnosus IOB820, Lactobacillus fermentum IOB802, Pediococcus lactis IOB701, and Lactobacillus casei IOB-P9) was as high as 20480.39 mg / kg, which was significantly higher than all other combinations. The specific results are shown in Table 4.

[0101] Table 4 Lactic acid content

[0102] With the increase in microbial diversity, lactic acid production shows a trend of first increasing, then slightly decreasing, and then reaching a peak. The lactic acid content of unfermented Astragalus membranaceus (P0) was 9506.51 mg / kg; the highest content among single-strain groups was S1 at 13416.91 mg / kg (an increase of 41.1% compared to P0); the highest content among two-strain groups was D1 at 19820.96 mg / kg (an increase of 108.5% compared to P0); the highest content among three-strain groups was T1 at 19063.59 mg / kg (an increase of 100.5% compared to P0); the highest content among four-strain groups was F1 at 18925.28 mg / kg (an increase of 99.0% compared to P0); the highest content among five-strain groups was V1 at 18602.40 mg / kg (an increase of 95.7% compared to P0); and the highest content among six-strain groups was H1 at 20480.39 mg / kg (an increase of 115.4% compared to P0), significantly higher than all other combinations.

[0103] In summary, based on Astragalus membranaceus as the fermentation substrate and the screening results of lactic acid content in different combinations of strains, the following combinations were selected and renumbered for subsequent physicochemical index (polysaccharide, reducing sugar, γ-aminobutyric acid) detection and animal experiments: Unfermented Astragalus: P0; Optimal single strain: Lactobacillus plantarum IOB602 (P1); Optimal bacterial strains: Lactobacillus plantarum IOB602 and Lactobacillus paracasei IOB413 (P2). The three optimal bacteria were: Lactobacillus plantarum IOB602, Lactobacillus paracasei IOB413 and Lactobacillus rhamnosus IOB820 (P3). The four optimal bacteria are: Lactobacillus plantarum IOB602, Lactobacillus paracasei IOB413, Lactobacillus rhamnosus IOB820, and Lactobacillus fermentum IOB802 (P4). The five optimal bacteria are: Lactobacillus plantarum IOB602, Lactobacillus paracasei IOB413, Lactobacillus rhamnosus IOB820, Lactobacillus fermentum IOB802, and Pediococcus lactis IOB701 (P5). The six optimal bacteria are: Lactobacillus plantarum IOB602, Lactobacillus paracasei IOB413, Lactobacillus rhamnosus IOB820, Lactobacillus fermentum IOB802, Pediococcus lactis IOB701, and Lactobacillus casei IOB-P9 (P6).

[0104] (2) Polysaccharide determination experiment The total polysaccharide content in the samples was determined using the phenol-sulfuric acid method according to GB / T40632-2021. The results are shown in Table 5. The polysaccharide content of group P0 was 6674.75 mg / 100g. After fermentation with different strain combinations, the polysaccharide contents of groups P1–P5 were 4550.65 mg / 100g, 5359.11 mg / 100g, 5768.31 mg / 100g, 5808.90 mg / 100g, and 6108.36 mg / 100g, respectively, representing an overall decrease of 8.5%–31.8% compared to group P0. The polysaccharide content of group P6 reached 8368.08 mg / 100g, an increase of 25.4% compared to group P0. Under the synergistic effect of the six strains, the system can synthesize extracellular polysaccharides and simultaneously promote the enrichment of polysaccharides in Astragalus membranaceus tissue. This group of high-content polysaccharides can regulate the body's immunity, improve muscle fatigue and reduce inflammatory response, and also provide sufficient raw materials for the generation of reducing sugars in the system.

[0105] Table 5 Polysaccharide content of samples

[0106] (3) Reducing sugar determination experiment Reducing sugar extraction After pulverizing the sample, weigh 2g of the sample, add 20mL of water, and heat in a sealed 50℃ water bath for 30min. After cooling, centrifuge at 12000r / min for 5min and collect the supernatant; dilute the above solution 10 times to obtain the test solution for later use.

[0107] glucose standard curve Weigh 100 mg of anhydrous glucose, dissolve it in pure water, and dilute to 100 mL to prepare a 1.0 mg / mL glucose standard solution. Pipette 0.1 mL, 0.15 mL, 0.2 mL, 0.25 mL, 0.3 mL, 0.35 mL, 0.4 mL, 0.45 mL, and 0.5 mL of the standard solution into test tubes, add distilled water to 1.0 mL, then add 1 mL of DNS reagent and mix well. Heat in a boiling water bath in the dark for 5 min, then remove and rapidly cool under running water. Dilute to 12.0 mL with distilled water and mix well. Use the reaction system without the standard solution and the test solution, containing only distilled water and DNS reagent, as a blank control. Measure the absorbance at 520 nm and plot a standard curve.

[0108] Sample absorbance measurement Take 1.0 mL of the test solution, add 1.0 mL of DNS reagent, shake well, heat in a boiling water bath for 5 min, cool under running water, add distilled water to make up to 12.0 mL, shake well, measure the absorbance at 520 nm, and calculate the reducing sugar content of the sample according to the standard curve.

[0109] As shown in Table 6, the reducing sugar content in group P0 was 53.12 mg / g. Group P1 had 54.88 mg / g, with no significant difference compared to group P0; group P2 increased to 65.55 mg / g (a 23.3% increase from P0), and group P3 increased to 63.67 mg / g (a 19.8% increase from P0); group P4 had 54.38 mg / g, with no significant difference; group P5 increased to 92.55 mg / g (a 74.2% increase from P0); and group P6 had the highest content, reaching 96.32 mg / g (an 81.3% increase from P0), showing a significant difference.

[0110] Reducing sugars are intermediate products in the conversion of polysaccharides into lactic acid. They can be rapidly absorbed by skeletal muscle for energy, thus improving the energy metabolism disorder in sarcopenia. Group P6 had the highest reducing sugar content, and its conversion efficiency from reducing sugars to lactic acid was superior to other groups.

[0111] Table 6 Reducing sugar content of samples

[0112] (4) Experiment on determination of γ-aminobutyric acid content Gamma-aminobutyric acid extraction Extraction solution: 70% aqueous ethanol solution.

[0113] Weigh 5.0 g of sample into a 50 mL centrifuge tube, add 10 mL of extraction solution, sonicate for 30 min, vortex for 2 min, and let stand for 5 min. Centrifuge at 12000 r / min for 5 min and collect the supernatant; repeat the extraction once for the sample residue, combine the two extracts, and dilute to 20 mL with extraction solution, shake well, and wait for derivatization.

[0114] Derivatization Reagent preparation: 0.5 mol / L sodium bicarbonate solution, pH 9.0 phosphate buffer, pH 7.0 phosphate buffer, and 1% 2,4-dinitrofluorophenylacetonitrile solution.

[0115] Take 0.5 mL of sample solution, add 0.5 mL of 0.5 mol / L NaHCO3 solution (pH=9.0) and 0.5 mL of 1% 2,4-dinitrofluorophenylacetonitrile solution, mix well, and heat in a 60℃ water bath in the dark for 1 h. After cooling, add pH=7.0 phosphate buffer to a final volume of 5 mL, filter the solution through a 0.22 μm organic filter membrane, and inject 10 μL for analysis.

[0116] Chromatographic reference conditions Chromatographic column: C18 column (250mm × 4.6mm, 5μm); Mobile phase A: Acetonitrile: Water = 1:1; Mobile phase B: 20 ​​mmol / L ammonium acetate aqueous solution; Flow rate: 1.0 mL / min; detection wavelength: 360 nm; column temperature: 35 °C; injection volume: 10 μL. Gradient elution method is shown in Table 7.

[0117] Table 7 Washout Schedule

[0118] As shown in Table 8, the γ-aminobutyric acid (GABA) content in group P0 was 369.88 mg / kg. The content increased in all fermentation samples: P1 was 510.46 mg / kg (an increase of 38.0% compared to P0), P2 was 557.92 mg / kg (an increase of 50.8% compared to P0), P3 was 554.08 mg / kg (an increase of 49.8% compared to P0), P4 was 555.56 mg / kg (an increase of 50.2% compared to P0), and P5 was 542.17 mg / kg (an increase of 46.6% compared to P0). Group P6 had the highest content, reaching 650.57 mg / kg (an increase of 75.8% compared to P0). Fermentation microorganisms can convert glutamate into GABA through glutamate decarboxylase. This substance can protect the neuromuscular junction function and improve muscle contraction efficiency. Group P6 had the highest content of γ-aminobutyric acid (GABA), which synergistically interacts with lactic acid to exert anti-sarcopenia effects from multiple dimensions, including energy supply, neural regulation, and anti-inflammation.

[0119] Table 8. γ-aminobutyric acid content of samples

[0120] Experimental Analysis There were significant differences among the P0 to P6 groups in four indicators: polysaccharides, reducing sugars, lactic acid, and γ-aminobutyric acid.

[0121] Among them, the six strains, when combined, formed a stable and complementary metabolic system, effectively mitigating the adverse effects of interspecies competition and significantly improving fermentation performance. This system promotes the enrichment of Astragalus polysaccharides and the synthesis of extracellular polysaccharides; the polysaccharide content in group P6 increased by 25.4% compared to P0. This component can exert immunomodulatory, anti-fatigue, and anti-inflammatory effects, and also continuously supply raw materials for the generation of reducing sugars. On the other hand, it promotes the efficient conversion of polysaccharides into reducing sugars; the reducing sugar content in group P6 increased by 81.3% compared to P0, and the efficiency of converting reducing sugars into lactic acid was superior to other groups, ultimately resulting in a 115.4% increase in lactic acid content compared to P0. Furthermore, the complex microbial community can efficiently catalyze metabolic reactions, significantly increasing the accumulation of γ-aminobutyric acid (GABA); the content of this component in group P6 increased by 75.8% compared to P0, also superior to other groups.

[0122] The various active ingredients work together to combat sarcopenia. Studies have confirmed that astragalus polysaccharides can downregulate pro-inflammatory factors such as IL-6, improving muscle atrophy. Lactic acid can promote skeletal muscle synthesis and enhance muscle strength. Gamma-aminobutyric acid (GABA) can regulate muscle protein degradation and reduce the release of pro-inflammatory factors such as TNF-α and IL-6, thereby improving muscle strength and mass.

[0123] In summary, the six-strain combination, with its excellent symbiotic metabolic characteristics, achieves simultaneous enrichment of multiple active ingredients such as polysaccharides, reducing sugars, lactic acid, and γ-aminobutyric acid. The multiple components work synergistically from multiple dimensions such as energy metabolism, protein synthesis, and inflammation regulation to improve sarcopenia symptoms, which can support the subsequent animal experiments.

[0124] Example 11: The effect of Astragalus membranaceus post-suppressant on sarcopenia.

[0125] Grouping and Modeling Fifty-six healthy male mice were acclimatized for 7 days and then randomly divided into 6 groups: a control group (n=6), a model group, a positive control group, experimental group 1, experimental group 2, and experimental group 3 (n=10 each). After grouping, the mice were weighed and numbered. After the acclimatization period, the initial weight and baseline grip strength of the mice in each group were measured. There were no statistically significant differences in baseline data between the groups, indicating that the groups were balanced.

[0126] Except for the control group, mice in all other groups received daily subcutaneous injections of dexamethasone 10 mg / kg in the neck, while the control group received an equal dose of physiological saline. This model of sarcopenia was established for 42 consecutive days. Drug intervention was implemented concurrently with modeling, administered via gavage once daily for 6 weeks. All test samples were prepared using 0.85% physiological saline. The dosing regimens for each group are as follows: Control group and model group: administered equal volume of distilled water by gavage; Positive control group: Metformin hydrochloride was administered by gavage at a dose of 105 mg / kg / day; Experiment 1: Unfermented Astragalus powder was administered by gavage at a dose of 400 mg / kg / d; the sample corresponded to the P0 sample in Example 10 of this invention. The raw Astragalus powder was directly pulverized without microbial fermentation. After pulverization, it was passed through an 80-mesh sieve and stored in a sealed container at low temperature. Experimental Group 2: The mixed bacterial powder of six bacteria was administered by gavage at a dose of 400 mg / kg / day; the samples were prepared according to the process in Example 3 of this invention and stored in a sealed container at low temperature. Experimental Group 3: Astragalus membranaceus post-fermentation powder (P6) was administered via gavage at a dose of 400 mg / kg / d. It was prepared according to the process in Example 9 of this invention, pulverized, passed through an 80-mesh sieve, and sealed and stored at low temperature.

[0127] The mice's weight was recorded daily during the experiment, and their bedding was changed twice a week to ensure a clean environment.

[0128] As shown in Table 9, the specific grouping and processing parameters for this experiment are as follows: Table 9 Group Design in Animal Experiments

[0129] Testing items and methods 1. Detection of mRNA content in muscle tissue The mRNA expression of MyoD, Myogenin, MGF, IGF-I, HGF, Myostatin, TGF-β, IL-1β, IL-6, and IL-10 in muscle tissue was detected by qRT-PCR.

[0130] like Figures 1-10 As shown, compared with the blank group, the levels of myogenic transcription factors (MyoD, Myogenin, MGF), muscle growth promoters (IGF-I, HGF), and anti-inflammatory factor IL-10 in the model group mice were significantly decreased, while the levels of muscle growth inhibitors (Myostatin (MSTN), TGF-β) and pro-inflammatory factors (IL-6, IL-1β) were significantly increased, indicating that the dexamethasone-induced sarcopenia model was successfully constructed.

[0131] After treatment with Astragalus powder, mixed powder of six bacteria, and fermented Astragalus powder of six bacteria, the following intervention was performed: The expression levels of myogenesis-related transcription factors and muscle growth promoters were significantly increased, while the expression level of muscle growth inhibitors was significantly decreased, and the level of inflammation was significantly improved. Compared with the model group, the expression of muscle growth inhibitors TGF-β and MSTN in the six-strain fermented Astragalus post-biotic group decreased by 41.43% and 37.72%, respectively; the expression of muscle growth promoters IGF-I and HGF increased by 112.66% and 96.06%, respectively; the expression of pro-inflammatory factors IL-1β and IL-6 decreased by 42.04% and 43.34%, respectively, while the expression of anti-inflammatory factor IL-10 increased by 138.76%. The comparison of each component showed that the improvement effect of the six-strain fermented Astragalus post-biotic group was significantly better than that of the Astragalus group and the six-strain mixed mycelium powder group. The results confirmed that the six-strain fermented Astragalus post-biotic can inhibit the activity of the muscle growth inhibitor (TGF-β) signaling pathway and downregulate the expression of myostatin (MSTN), reducing MSTN expression in the gastrocnemius muscle, relieving its inhibition of muscle growth, and promoting muscle synthesis.

[0132] 2. Pathological examination HE staining After the mice were sacrificed, three types of skeletal muscles—tibialis anterior, soleus, and gastrocnemius—were harvested. After washing, the muscles were fixed in 4% paraformaldehyde solution for 24 hours, routinely embedded in paraffin, sectioned to a thickness of 4 μm, dewaxed routinely, stained with hematoxylin and eosin (HE), dehydrated with graded alcohols, cleared, and mounted. The pathological morphological changes of the mouse muscle tissue were observed under an optical microscope.

[0133] like Figures 11-13 As shown, HE staining results of the soleus, gastrocnemius, and tibialis anterior muscles of mice revealed that the muscle fibers in the blank group were plump and rounded, with uniform cross-sectional area, and tightly and regularly arranged, while the intermuscular connective tissue was sparsely and evenly distributed. In the model group, the muscle fibers were atrophied and thinned, with irregular shapes and loose arrangement, and the intermuscular spaces were significantly widened, consistent with the pathological characteristics of sarcopenia, indicating successful modeling. After intervention with Astragalus powder, mixed astragalus and Astragalus-derived post-fermentation powder, and mixed astragalus and Astragalus-derived post-fermentation powder, the muscle fibers gradually became plump, more regularly arranged, and the intermuscular spaces narrowed, with the mixed astragalus and Astragalus-derived post-fermentation group showing the best recovery effect.

[0134] MASSON staining Take the above-mentioned gastrocnemius muscle paraffin sections, dewax them routinely, stain them with Masson-aniline blue staining kit, dehydrate them stepwise with ethanol, mount them transparently, and observe the fibrosis of the gastrocnemius muscle under a microscope.

[0135] like Figure 14 As shown, Masson staining results revealed that the blank group showed only a small amount of sparsely distributed blue collagen in the intermuscular spaces, with no obvious fibrosis. The model group showed extensive blue collagen deposition in the intermuscular spaces and intramuscular tissue, with a wide range and high density of fibrosis, suggesting that sarcopenia is accompanied by the progression of interstitial fibrosis.

[0136] After intervention with the test substances in each group, the area of ​​blue collagen decreased and the degree of fibrosis was alleviated; among them, the group with six bacteria mixed fermentation of Astragalus membranaceus and post-fermentation showed the lowest degree of fibrosis and reduced the pathological changes of fibrosis in the gastrocnemius muscle.

[0137] Immunohistochemistry Paraffin sections of the gastrocnemius muscle were dewaxed and hydrated, then immersed in citrate buffer and subjected to high-pressure autoclaving to repair the antigen. Endogenous peroxidase was inactivated by incubation with 3% H₂O₂ for 10 min in the dark. The sections were then washed with PBS and blocked with normal goat serum at 37 °C for 30 min. MSTN primary antibody diluted 1:500 was added, and the sections were incubated overnight at 4 °C. Unbound antibodies were washed away with PBS, and biotin-labeled secondary antibody was added, followed by incubation at 37 °C for 1 h. After washing with PBS, the sections were developed with DAB chromogenic solution, counterstained with hematoxylin, dehydrated, mounted, and photographed under an optical microscope to assess MSTN expression levels.

[0138] like Figure 15As shown in the immunohistochemical images, the blank group exhibited a light and uniformly distributed brown signal with low positive expression in the myofiber cytoplasm. The model group showed a significantly deeper and more diffuse brown signal, with abnormally upregulated MSTN expression, consistent with the trend observed in MSTN mRNA expression. MSTN is a key factor negatively regulating skeletal muscle growth, inhibiting myofiber proliferation and hypertrophy, and reducing muscle mass. In sarcopenia, MSTN shows abnormally high expression. After drug intervention, the brown signal in all groups became lighter. The staining intensity and distribution in the group with mixed fermentation of six strains of Astragalus membranaceus and its post-fermentative agent were closest to the blank group, indicating that the mixed fermentation of six strains of Astragalus membranaceus and its post-fermentative agent can effectively inhibit MSTN expression and maximize the relief of muscle growth inhibition.

[0139] 3. CT imaging examination Experimental apparatus: A small medical animal CT scanner was used. Uniform scanning parameters were set, and images were reconstructed after scanning. Muscle volume and fat infiltration area were measured to assess the microstructure and quality of the muscle.

[0140] like Figure 16 As shown in the CT scan images, the blank control group showed uniform grayscale and full contours in the muscle area, with normal transverse and longitudinal diameters and thicknesses, clear boundaries with the bones, and no obvious fat infiltration. The model group showed shortened longitudinal diameter and thinner muscle, significantly reduced muscle volume, and extensive fat infiltration. After intervention with different test samples, the muscle fullness of all groups of mice improved, and the area of ​​fat infiltration significantly decreased.

[0141] like Figure 17 As shown in the results, the muscle volume statistics showed that the muscle volume of the model group was reduced compared with the blank group; the muscle volume of each drug administration group recovered to varying degrees, with the positive control group showing the best recovery effect, and the improvement effect of the six-strain mixed fermented Astragalus post-generating group being second only to the positive control group, both of which were significantly higher than the model group; among the test samples, the muscle volume of the six-strain mixed fermented Astragalus post-generating group was higher than that of the mycelium powder group and the six-strain mixed mycelium powder group, which showed the best improvement effect among the test samples in this experiment.

[0142] 4. Gastrocnemius muscle index Mice were fasted for 8 hours the night before testing, and their fasting weight was measured the morning of the sampling day. Mice were euthanized after anesthesia with sodium pentobarbital, and the bilateral gastrocnemius muscles were quickly dissected and accurately weighed. The gastrocnemius index is commonly used to evaluate skeletal muscle mass and can directly reflect the severity of muscle loss.

[0143] Calculation formula: Gastrocnemius index = (Total gastrocnemius muscle weight / Fasting body weight of mouse) × 100% like Figure 18As shown, the gastrocnemius muscle index statistics revealed that the gastrocnemius muscle index of the model group mice was significantly decreased compared to the blank group. After drug intervention, the gastrocnemius muscle index of each group recovered to varying degrees. Among them, the recovery levels of the gastrocnemius muscle index in the positive control group, the mixed astragalus powder group, and the mixed fermented astragalus post-biotic group were similar, all significantly higher than those in the model group. In the tested samples, the gastrocnemius muscle index of the mixed fermented astragalus post-biotic group increased by 52.49%, showing the best improvement effect, comparable to the positive control group, and significantly better than the mixed astragalus powder group. The results indicate that the mixed fermented astragalus post-biotic can promote muscle anabolic metabolism, inhibit catabolism, and improve the regenerative microenvironment, ultimately increasing the mass of the gastrocnemius muscle in sarcopenic mice.

[0144] 5. Mouse body weight and behavioral experiments weight Throughout the experiment, the mice were weighed weekly. The first weighing was conducted before the start of the experiment (day 0), and thereafter, each mouse was weighed individually every 7 days at a fixed time using an electronic balance (accuracy 0.01 g), and the data were recorded.

[0145] Grip strength test After the drug administration period ended, a grip strength test was performed on mice in each group. The mice were placed on a grip strength meter, their forepaws gripping the metal sensor. The mice's tails were gently pulled until they released the sensor, and the maximum grip strength was recorded. Each mouse was tested 5 times consecutively, and the average value was taken as one group. After a 5-10 minute rest period, the next group was tested, for a total of 3 groups and 15 tests. The final average of all test results was taken.

[0146] Steel wire suspension experiment The experiment used a wire cage with tape along the edges to prevent mice from slipping. The mice were placed on top of the cage and gently shaken to encourage them to grip the wire. The cage was then slowly tilted over, suspending the mice in mid-air. The time from when the mouse gripped the wire to when it released its grip and fell was recorded; this was called the "fall latency." To avoid injury, the fall height was controlled. Each mouse was tested three times, and the average value was taken.

[0147] Swimming Exhaustion Experiment Prepare a glass pool with a depth of at least 30 cm. Weigh the mouse and attach a lead weight equal to 5% of its body weight to the tail section. Then, place the mouse in the water. Record the time from when the mouse enters the water until its head sinks and it can no longer float on its own; this time is the swimming exhaustion time. Do not place too many mice in a single experiment to avoid them crowding each other and affecting the accuracy of the results.

[0148] Rotary bar experiment One week before the end of the drug administration cycle, mice underwent acclimatization training for 5 minutes daily to familiarize them with the rotarod system. Formal testing began after the drug administration cycle ended: mice were placed on the rotarod, and the rotarod's rotation speed was uniformly increased from 0 to 25 rpm within 0-90 seconds, then maintained at a constant speed of 25 rpm after 90 seconds. The latency time for the mouse to fall off the rotarod was recorded. Each mouse was tested three times, with test intervals of 5-10 minutes.

[0149] like Figures 19-23 As shown, in terms of five indicators—body weight, grip strength, wire suspension time, swimming exhaustion time, and spin bar drop latency—the model group mice showed significantly lower scores than the control group, demonstrating a marked decline in body weight, muscle strength, and endurance in sarcopenic mice. After drug intervention, all drug-treated groups showed varying degrees of improvement in their indicators, with the positive control group showing the best recovery. Among the tested sample groups, the six-strain fermented Astragalus membranaceus post-biotic group showed higher scores in all indicators than the Astragalus membranaceus group and the six-strain mixed mycelium powder group, indicating the best improvement among the tested samples in this experiment. This demonstrates that the six-strain fermented Astragalus membranaceus post-biotic group can effectively restore body weight in sarcopenic mice and improve muscle strength, exercise endurance, and limb coordination in these mice.

[0150] The applicant declares that the present invention is illustrated by the above embodiments, but the present invention is not limited to the above process steps, that is, it does not mean that the present invention must rely on the above process steps to be implemented. Those skilled in the art should understand that any improvements to the present invention, equivalent substitutions of the raw materials used in the present invention, addition of auxiliary components, selection of specific methods, etc., all fall within the protection scope and disclosure scope of the present invention.

Claims

1. A compound probiotic fermented astragalus post-biotic, characterized in that, The compound probiotics include: Lactobacillus paracasei ( Lacticaseibacillus paracasei IOB413, accession number CGMCCNo.16022; Lactobacillus casei ( Lacticaseibacillus casei IOB-P9, accession number CGMCC No. 24195; Lactobacillus rhamnosus ( Lacticaseibacillus rhamnosus IOB820, accession number CGMCCNo.17522; Fermented Lactobacillus mucinus ( Limosilactobacillus fermentum IOB802, accession number CGMCCNO.23120; Pediococcus acidilactici ( Pediococcus acidilactici IOB701, accession number CGMCC No.16077; Lactobacillus plantarum ( Lactiplantibacillus plantarum )IOB602, accession number CGMCCNo.16021.

2. The compound probiotic fermented astragalus post-biotic as described in claim 1, characterized in that, The volume ratio of *Lactobacillus paracasei* IOB413, *Lactobacillus casei* IOB-P9, *Lactobacillus rhamnosus* IOB820, *Lactobacillus fermentum* IOB802, *Pediococcus lactis* IOB701, and *Lactobacillus plantarum* IOB602 is 1:1:1:1:1:

1.

3. A method for preparing compound probiotic fermented astragalus post-biotic as described in any one of claims 1 to 2, characterized in that, Includes the following steps: After activating Lactobacillus paracasei IOB413, Lactobacillus casei IOB-P9, Lactobacillus rhamnosus IOB820, Lactobacillus fermentum IOB802, Pediococcus lactis IOB701, and Lactobacillus plantarum IOB602 by inoculating them onto slant culture medium, each activated strain was individually inoculated into Astragalus membranaceus powder liquid culture medium to obtain the primary seed culture of each strain. The primary seed solutions of each strain were mixed in equal volumes and then inoculated into Astragalus powder liquid culture medium to obtain secondary seed solutions. The secondary seed culture was inoculated into Astragalus powder liquid culture medium to prepare a mixed seed culture of six bacteria; Astragalus powder was mixed with water and sterilized to obtain a solid fermentation substrate for Astragalus. The six-strain mixed seed liquid was inoculated into the Astragalus solid fermentation substrate, and after fermentation, Astragalus solid fermentation product was obtained. The solid fermented Astragalus membranaceus was inactivated, dried, and pulverized to obtain the compound probiotic fermented Astragalus membranaceus post-biotic.

4. The preparation method according to claim 3, characterized in that, The inoculation amount of the six-strain mixed seed liquid into the Astragalus solid fermentation substrate was 8%, and the culture conditions were 37℃ for 24 hours.

5. The preparation method according to claim 3, characterized in that, The inactivation conditions are inactivation at 105℃ for 30 minutes; the drying and pulverizing are drying to a moisture content of ≤12% and then pulverizing.

6. The use of the compound probiotic fermented astragalus post-biotic as described in any one of claims 1 to 2 in the preparation of a medicine for relieving sarcopenia.

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