Dietary factors and combinations thereof that target gut microbiota to improve exercise performance

Abstract

This invention discloses a dietary factor and its combination that targets gut microbiota to improve exercise performance, belonging to the fields of microbial technology and pharmaceutical technology. Experimental verification shows that the dietary factor combination can increase the abundance of *Eubacterium rectale*, *Faecalibacterium prausnitzii*, and *Veillonella* species; a combination of galactooligosaccharides, burdock seed, matsutake polysaccharide, and galactooligosaccharides; a combination of burdock seed and matsutake polysaccharide; and a combination of burdock seed and galactooligosaccharides can increase the concentration of fatty acid amides; curcumin and the combination of curcumin, burdock seed, and galactooligosaccharides show the best effect in promoting the content of fecal microbiota after acetic acid fermentation; and a mixed combination of *Bifidobacterium adolescentis* CCFM1066, *Bifidobacterium longum* CCFM1306, curcumin, inulin, burdock seed, matsutake polysaccharide, and galactooligosaccharides shows the most significant effect in increasing the time spent swimming at exhaustion under load in exercised mice.

Description

Technical Field

[0001] This invention relates to a dietary factor and its combination that targets gut microbiota to improve motor performance, belonging to the fields of microbial technology and pharmaceutical technology. Background Technology

[0002] The relationship between gut microbiota and exercise is a highly researched area, with gut microbes potentially linked to athletic performance, recovery, and overall health. Gut microbiota can influence athletic performance by regulating energy production, skeletal muscle turnover, protein metabolism, and nutrient sensing pathways (mTOR, AMPK, etc.). Existing research indicates that some key gut microbiota species and their corresponding metabolites are closely related to athletic performance. Compared to sedentary individuals, active individuals show a significant enrichment of SCFA-producing bacteria (including *Faecalibacterium prausnitzii* and *Eubacterium rectale*). Excess lactic acid produced after exercise can cross the intestinal barrier and be converted into propionic acid by *Veillonella* in the gut, thus improving athletic performance.

[0003] Gut microbiota produce fatty acid amides (FAAs) metabolites that act on cannabinoid 1 receptor (CB1), activating TRPV1 sensory neurons and transmitting signals to the brain, thus downregulating monoamine oxidase (MAO) expression in the striatum. Downregulation of MAO increases post-exercise striatal dopamine levels, thereby enhancing athletic performance and motivation. *Eubacterium rectale* and *Coprococcuseutactus* can mediate the production of FAAs.

[0004] Short-chain fatty acids (SCFAs) are metabolites produced by gut microbes during the digestion of dietary fiber. Exercise can improve gut microbiota structure, promote SCFA production, and the gut microbiota can also have a positive effect on athletic performance. SCFAs produced by gut microbiota may enhance athletic performance by influencing lactate metabolism, providing energy, increasing skeletal muscle glycogen synthesis, and improving the intestinal barrier.

[0005] Polysaccharides and polyphenols play crucial roles in the gut microbiota ecosystem, influencing its composition, function, and overall health. As a primary energy source for gut microbes, polysaccharides are broken down into beneficial SCFAs by certain bacterial enzymes, thus maintaining gut health, immune regulation, and energy balance. Furthermore, the presence of polysaccharides promotes microbial diversity; different types of polysaccharides may selectively promote the proliferation of certain bacterial groups, thereby maintaining gut microbial balance. This further influences the gut environment, intestinal barrier function, the immune system, and overall health. By regulating immune system activity, maintaining mucosal health, and providing energy, polysaccharides play a significant role in promoting gut and overall health.

[0006] With the growing number of fitness enthusiasts, sports nutrition foods are gaining increasing popularity and attention. Current development of sports nutrition foods mainly focuses on energy replenishment, protein intake, electrolyte balance, antioxidants, and immune support to optimize athletic performance and health. A growing body of research indicates a close relationship between gut microbiota and exercise. Rapid screening of potential exercise-promoting dietary factors through in vitro fecal fermentation, targeting gut microbiota and its metabolites, provides a new perspective and approach for developing foods that regulate athletic performance. Summary of the Invention

[0007] This invention utilizes bioinformatics correlation analysis to screen for exercise-promoting bacterial strains and employs an in vitro fecal microbiota fermentation model to screen for dietary factors that specifically regulate exercise capacity. The method, based on publicly available databases such as NCBI, screened 418 metagenomic datasets of gut microbiota containing exercise levels. Species composition annotation was performed on each sample using MetaPhlAn3. Fresh feces from six healthy volunteers were pretreated and homogenized. This homogenate was then inoculated into a culture medium containing dietary polysaccharides and polyphenols at a specific inoculation rate. After 24 hours of anaerobic fermentation, the fermentation broth was collected, centrifuged, and the bacterial sludge and supernatant were separated. The content of fatty acid amides and short-chain fatty acids in the supernatant and the bacterial species composition in the bacterial sludge were measured. The efficacy of the dietary factor combination was verified in animal experiments based on the fermentation results.

[0008] In one embodiment of the invention, the data is based on 418 metagenomic datasets of gut microbiota containing motility levels. Each sample was annotated with species composition using MetaPhlAn3.

[0009] In one embodiment of the present invention, the fecal pretreatment involves mixing fresh feces from six volunteers, mixing them with a sterile buffer solution in a certain proportion, and then removing large particles using a filter screen.

[0010] In one embodiment of the present invention, the buffer solution is sterilized 0.01M PBS at pH 7.3.

[0011] In one embodiment of the present invention, the feces and PBS are mixed in a ratio of 1:10 w / v.

[0012] In one embodiment of the present invention, the fecal homogenate is inoculated into the culture medium at an inoculation rate of 20%.

[0013] In one embodiment of the present invention, the culture medium formulation is as follows: mGAM culture medium formulation: 5g tryptone, 3g soybean peptone. 5g peptone, 10g digested serum powder, 2.5g yeast extract, 2.2g beef extract, 1.2g liver extract, 0.2g L-tryptophan, 1.0g L-arginine, 0.3g L-cysteine, 0.3g sodium thioglycolate, 5mg vitamin K, 10mg heme chloride, 2.5g potassium dihydrogen phosphate, and 3.0g sodium chloride.

[0014] In one embodiment of the present invention, the culture conditions are 37°C, anaerobic (80% N2, 10% CO2, 10% H2) culture.

[0015] In one embodiment of the present invention, the polysaccharides and polyphenols include matsutake polysaccharide, galactooligosaccharide, maitake mushroom polysaccharide, kudzu root polysaccharide, shiitake mushroom polysaccharide, wood ear mushroom polysaccharide, yam polysaccharide, isomaltooligosaccharide, fructooligosaccharide, inulin, wolfberry polysaccharide, mannan, fucoidan, curcumin, burdock fruit, atractylodes lactone, chondroitin sulfate, sesamin, bergamot lactone, perillol, andrographolide, naringin, caffeic acid, phytosterol, leucine, caffeic acid 4-O-glucoside, apigenin, sarcopentol, artemisinin, and limonene.

[0016] In one embodiment of the present invention, high-resolution high-performance liquid chromatography-mass spectrometry is used to detect fatty acid amides in fermentation supernatant.

[0017] In one embodiment of the present invention, the fatty acid amides include oleoylethanolamine and palmitoylethanolamine.

[0018] In one embodiment of the present invention, gas chromatography-mass spectrometry is used to detect the content of short-chain fatty acids in the fermentation supernatant.

[0019] In one embodiment of the present invention, the short-chain fatty acids include acetic acid, propionic acid, and butyric acid.

[0020] In one embodiment of the present invention, the fermented sludge is subjected to metagenomic sequencing using the DNBSEQ-T7 platform PE150.

[0021] This invention provides the application of probiotics, dietary factors, and / or probiotic preparations containing dietary factors in the preparation of products that can improve athletic performance and combat fatigue. The products include food, health products, pharmaceuticals, enteral nutrition products, dietary supplements, and feed additives. The probiotics include *Clostridium plasminogen lysate*, *Eubacterium rectum*, *Bifidobacterium longum* subsp. *longum*, and / or *Bifidobacterium adolescentis*. The dietary factors include one or more of curcumin, matsutake polysaccharides, burdock seeds, and galactooligosaccharides. The probiotic preparations containing dietary factors include *Bifidobacterium longum* subsp. *longum* preparations containing inulin, and probiotic preparations containing *Bifidobacterium adolescentis*, *Bifidobacterium longum* subsp. *longum*, curcumin, inulin, burdock seeds, matsutake polysaccharides, and galactooligosaccharides.

[0022] In one embodiment of the present invention, the food includes, but is not limited to, athlete food, and the medicine includes, but is not limited to, veterinary medicine.

[0023] In one embodiment of the present invention, the Clostridium praosporum is Clostridium praosporum CCFM1203, the Eubacterium rectum is Eubacterium rectum ATCC33656, the Bifidobacterium adolescentis is Bifidobacterium adolescentis CCFM1066, and the Bifidobacterium longum subsp. longum is Bifidobacterium longum subsp. longum CCFM1306.

[0024] In one embodiment of the present invention, the amount of probiotics added to the product is at least 1 × 10⁻⁶. 9 CFU; In the dietary factors, the polysaccharides and polyphenols are added in a mass ratio of 5:1; the curcumin and burdock are added in a ratio of 1:1; and the matsutake polysaccharides and galactooligosaccharides are added in a ratio of 1:1.

[0025] In one embodiment of the present invention, the amount of dietary factor added to the product is at least 0.15 g / kg.

[0026] This invention provides the application of dietary factors in the preparation of products that increase the abundance of Eubacterium rectale in the gut or in the process of increasing the abundance of Eubacterium rectale in the gut microbiota. The product in the application of dietary factors in the preparation of products that increase the abundance of Eubacterium rectale in the gut includes food, pharmaceuticals, or health products.

[0027] In the application of increasing the abundance of Eubacterium rectale in the gut microbiota, the application is to add the gut microbiota to an environment containing dietary factors for cultivation, and the application is for non-disease diagnosis and treatment purposes.

[0028] The dietary factors include Grifola frondosa polysaccharide, galactooligosaccharide, Auricularia auricula-judae polysaccharide, inulin, Lycium barbarum polysaccharide, Lentinan shiitake polysaccharide, paclitaxel, curcumin, bergamot lactone, sesamin, caffeic acid, fructooligosaccharide, andrographolide, mannan, kudzu root polysaccharide, isomaltooligosaccharide, Dioscorea opposita polysaccharide, or Matsutake mushroom polysaccharide;

[0029] Or the dietary factors may include curcumin and galactooligosaccharides, burdock and galactooligosaccharides, curcumin, burdock and galactooligosaccharides, matsutake polysaccharides, burdock and galactooligosaccharides, curcumin, matsutake polysaccharides, burdock and galactooligosaccharides.

[0030] This invention provides the application of dietary factors in the preparation of products that increase the abundance of Faecalibacterium prausnitzii in the gut or in increasing the abundance of Faecalibacterium prausnitzii in the gut microbiota, wherein the product includes food, medicine or health product.

[0031] The application of increasing the abundance of Faecalibacterium prausnitzii in the gut microbiota involves adding the gut microbiota to an environment containing dietary factors for cultivation, and the application is for non-disease diagnosis and treatment purposes.

[0032] The dietary factors include chondroitin sulfate, Grifola frondosa polysaccharide, galactooligosaccharide, galactooligosaccharide group, Auricularia auricula polysaccharide, Lycium barbarum polysaccharide, Lentinan shiitake polysaccharide, paclitaxel, caffeic acid 4-O-glucoside, apigenin, naringin, sesamin, burdock fruit, curcumin, bergamot lactone, fructooligosaccharide, caffeic acid, limonene, andrographolide, phytosterol, mannan, 4,9, kudzu root polysaccharide, inulin, yam polysaccharide, and matsutake mushroom polysaccharide;

[0033] Or the dietary factors may include curcumin and matsutake polysaccharide, curcumin and galactooligosaccharide, burdock and galactooligosaccharide, curcumin, matsutake polysaccharide and burdock, matsutake polysaccharide and burdock, curcumin, matsutake polysaccharide and galactooligosaccharide, curcumin, matsutake polysaccharide, burdock and galactooligosaccharide.

[0034] This invention provides the application of dietary factors in the preparation of products that increase the abundance of Veillonella atypica or Veillonella parvula in the gut, or in the preparation of products that increase the abundance of Veillonella atypica or Veillonella parvula in the gut microbiota. The products include food, pharmaceuticals, or health products.

[0035] The application of increasing the abundance of Veillonella atypica or Veillonella parvula in the gut microbiota involves adding the gut microbiota to an environment containing dietary factors for cultivation, and the application is for non-disease diagnosis and treatment purposes.

[0036] The dietary factors are one or more of curcumin, burdock seed, and matsutake polysaccharide.

[0037] This invention provides the application of dietary factors in the preparation of products that increase the content of fatty acid amides in the intestine or in the preparation of products that increase the content of fatty acid amides after fermentation of intestinal flora. In the application of dietary factors in the preparation of products that increase the content of fatty acid amides in the intestine, the products include food, pharmaceuticals or health products.

[0038] The application of increasing the content of fatty acid amide substances after fermentation of intestinal flora is to add intestinal flora to an environment containing dietary factors for cultivation, and the application is for non-disease diagnosis and treatment purposes.

[0039] When the fatty acid amide is oleoylethanolamine, the dietary factor is one or more of curcumin, matsutake polysaccharide, burdock seed, and galactooligosaccharide;

[0040] When the fatty acid amide is palmitoylethanolamide, the dietary factor is one, three, or four of the following: curcumin, matsutake polysaccharide, burdock seed, and galactooligosaccharide; or the dietary factor is matsutake polysaccharide and burdock seed, or burdock seed and galactooligosaccharide.

[0041] This invention provides the application of dietary factors in the preparation of products that increase the content of short-chain fatty acids in the intestine or in the improvement of the content of short-chain fatty acids after fermentation of intestinal flora. In the application of products that increase the content of short-chain fatty acids in the intestine, the products include food, pharmaceuticals or health products.

[0042] The application of increasing the content of short-chain fatty acids in gut microbiota after fermentation involves adding gut microbiota to an environment containing dietary factors for cultivation. The application is for non-disease diagnosis and treatment purposes.

[0043] When the short-chain fatty acid is acetic acid, the dietary factor is one or more of curcumin, matsutake polysaccharide, burdock seed, and galactooligosaccharide.

[0044] When the short-chain fatty acid is acetic acid, the dietary factors are curcumin, matsutake polysaccharide, burdock seed, galactooligosaccharide, curcumin and matsutake polysaccharide, matsutake polysaccharide and burdock seed.

[0045] In one embodiment of the present invention, the amount of dietary factor added to the product is at least 0.15 (g / kg).

[0046] Beneficial effects

[0047] This invention relates to a method for screening dietary factors that target and regulate exercise capacity based on an in vitro fermentation model and its application. Specifically, this method is used to screen combinations of exercise-promoting dietary factors.

[0048] 1. The overall structure of gut microbiota differs among individuals with different levels of exercise; Faecalibacterium prausnitzii and Eubacterium rectale are enriched in individuals with strong athletic performance and can serve as biomarkers for exercise-promoting gut microbiota.

[0049] 2. This invention has been verified through experiments:

[0050] (1) Galacto-oligosaccharides, Auricularia auricula polysaccharides, inulin, and Pueraria lobata polysaccharides promote the increase of Eubacterium rectale abundance; Galacto-oligosaccharides, curcumin and galacto-oligosaccharide combination, burdock seed, matsutake mushroom polysaccharide and galacto-oligosaccharide combination, and curcumin, burdock seed and galacto-oligosaccharide combination can promote the increase of Eubacterium rectale abundance;

[0051] (2) Matsutake polysaccharide, Grifola frondosa polysaccharide, and Pueraria lobata polysaccharide promote the increase of Faecalibacterium prausnitzii abundance; Matsutake polysaccharide, curcumin, and Matsutake polysaccharide combination, and burdock and Matsutake polysaccharide combination promote the increase of Faecalibacterium prausnitzii abundance;

[0052] (3) Curcumin, burdock seed, and phytosterols promote an increase in the abundance of species related to the genus Veillonella;

[0053] (4) The combination of galactooligosaccharides, burdock seed, matsutake mushroom polysaccharides, galactooligosaccharides, burdock seed, matsutake mushroom polysaccharides, and galactooligosaccharides promotes the concentration of fatty acid amides, among which the combination of burdock seed, matsutake mushroom polysaccharides, and galactooligosaccharides has the best promoting effect.

[0054] (5) Curcumin and curcumin, burdock, and galactooligosaccharide groups had the best promoting effect on the content of acetic acid fecal bacteria after fermentation; matsutake polysaccharide and burdock and matsutake polysaccharide groups had the best promoting effect on the content of butyric acid fecal bacteria after fermentation.

[0055] (6) The combination of Bifidobacterium adolescentis CCFM1066, Bifidobacterium longum subsp. CCFM1306, curcumin, inulin, burdock seed, matsutake polysaccharide, and galactooligosaccharide had the most significant effect on increasing the swimming time of exercise mice under load and exhaustion.

[0056] (7) A mixture of Clostridium praosporum, curcumin, Bifidobacterium adolescentis CCFM1066, Bifidobacterium longum subsp. CCFM1306, curcumin, inulin, burdock seed, matsutake polysaccharide, and galactooligosaccharide significantly increased the rotarod time of exercised mice.

[0057] (8) The combination of burdock seed, matsutake mushroom, and galactooligosaccharide significantly increased the grip strength of mice.

[0058] Therefore, this invention provides an in vitro fecal fermentation model that, based on gut microbiota and its metabolites, rapidly and efficiently screens out combinations of exercise-promoting dietary factors and verifies them in animal experiments. This model has broad application prospects in the development of sports nutrition foods. Attached Figure Description

[0059] Figure 1 α-diversity of gut microbiota in people with different exercise levels.

[0060] Figure 2 β-diversity of gut microbiota in people with different exercise levels.

[0061] Figure 3 Cumulative map of gut microbiota abundance in people with different exercise levels.

[0062] Figure 4 Analysis of Lefse differences in gut microbiota species among individuals with different exercise levels.

[0063] Figure 5 The effects of different dietary factors on the abundance of gut microbiota species.

[0064] Figure 6The effects of different combinations of dietary factors on the abundance of gut microbiota species.

[0065] Figure 7 The effects of different combinations of dietary factors on fatty acid amides.

[0066] Figure 8 The effects of different combinations of dietary factors on short-chain fatty acids.

[0067] Figure 9 Time to exhaustion during weight-bearing swimming in different groups of mice.

[0068] Figure 10 Grasping force of mice in different groups of experiments.

[0069] Figure 11 Rotor fatigue time in mice of different experimental groups. Detailed Implementation

[0070] The chondroitin sulfate, maitake mushroom polysaccharide, galactooligosaccharide, ginseng polysaccharide, wood ear mushroom polysaccharide, wolfberry polysaccharide, shiitake mushroom polysaccharide, lecithin, caffeine-4-O-glucoside, apigenin, naringin, sesamin, burdock fruit, curcumin, bergamot lactone, fructooligosaccharide, caffeic acid, limonene, artemisinin, andrographolide, phytosterol, mannan, kudzu root polysaccharide, inulin, brown algae polysaccharide, isomaltooligosaccharide, yam polysaccharide, and matsutake mushroom polysaccharide involved in the following examples were purchased from Xi'an Shengqing Biotechnology Co., Ltd.

[0071] The culture media involved in the following examples are as follows:

[0072] mGAM medium formulation: 5g tryptone, 3g soybean peptone. 5g peptone, 10g digested serum powder, 2.5g yeast extract, 2.2g beef extract, 1.2g liver extract, 0.2g L-tryptophan, 1.0g L-arginine, 0.3g L-cysteine, 0.3g sodium thioglycolate, 5mg vitamin K, 10mg heme chloride, 2.5g potassium dihydrogen phosphate, 3.0g sodium chloride, add distilled water to 1000ml, adjust pH to 7.2-7.4, sterilize at 115℃ for 20min;

[0073] The following polysaccharides and polyphenols may be added as needed for the experiment:

[0074] Chondroitin sulfate, Grifola frondosa polysaccharide, galactooligosaccharide, ginseng polysaccharide, Auricularia auricula-judae polysaccharide, Lycium barbarum polysaccharide, Lentinus edodes polysaccharide, lecithin, caffeo-4-O-glucoside, apigenin, naringin, sesamin, burdock fruit, curcumin, bergamot lactone, fructooligosaccharide, caffeic acid, limonene, artemisinin, andrographolide, phytosterol, mannan, kudzu root polysaccharide, inulin, brown algae polysaccharide, isomaltooligosaccharide, yam polysaccharide, matsutake mushroom polysaccharide.

[0075] MRS liquid culture medium (g / L): peptone 10g / L, beef extract 10g / L, glucose 20g / L, sodium acetate 2g / L, yeast extract 5g / L, diammonium hydrogen citrate 2g / L, K2PO4·3H2O 2.6g / L, MgSO4·7H2O 0.1g / L, MnSO4 0.05g / L, Tween 80 1mL / L, cysteine ​​0.5g / L.

[0076] M2GSC liquid medium (g / L): yeast extract 5g / L, casein peptone 10g / L, glucose 5g / L, cellobiose 2g / L, fructose 2g / L, sodium bicarbonate 4g / L, sodium chloride 0.9g / L, potassium dihydrogen phosphate 0.45g / L, dipotassium hydrogen phosphate 0.45g / L, magnesium sulfate 0.09g / L, calcium chloride 0.09g / L, cysteine ​​0.5g / L, and clarified rumen fluid 10mL / L.

[0077] The detection methods involved in the following embodiments are as follows:

[0078] Methods for detecting short-chain fatty acids:

[0079] (1) The content of short-chain fatty acids in the culture supernatant was determined by gas chromatography-mass spectrometry (GC-MS).

[0080] (2) Chromatographic detection conditions: Different short-chain fatty acids were separated using an Rtx-Wax column (30m*0.25μm*0.25μm) and a mass spectrometer detector (GC-MS-QP2010Vitta, system); the carrier gas was N2, and the flow rate was 1 mL / min; the injection volume was 1 μL, and the split ratio was 10:1; the initial column temperature was 100℃, increased to 140℃ at a rate of 7.5℃ / min, and then increased to 200℃ at a rate of 60℃ / min. The column was held at 200℃ for 3 min, the injection temperature was 240℃, and the ionization temperature was 220℃.

[0081] (3) Plotting the standard curve: Take 10 μL each of acetic acid, propionic acid, butyric acid, valeric acid, isovaleric acid, and isobutyric acid into a 2 mL centrifuge tube, and dilute to 1 mL with diethyl ether. Mix thoroughly. Take 100 μL of the mixture, dilute it 10 times with diethyl ether, mix well, and then take 200 μL, 100 μL, 50 μL, 25 μL, 15 μL, and 10 μL of the mixture, and dilute to 1 mL with diethyl ether to obtain mixed standards of different concentrations. Filter the mixture through a membrane and then analyze it. Plot the standard curve with the peak area at the corresponding concentration as the x-axis and the corresponding concentration as the y-axis.

[0082] Methods for detecting fatty acid amides in fermentation supernatant:

[0083] (1) The contents of oleoylethanolamine (OEA) and palmitoylethanolamide (PEA) in the fermentation supernatant were detected by high-resolution high-performance liquid chromatography-mass spectrometry (HPLC-MS).

[0084] (2) Detection conditions: The chromatographic column was an ACQUITY UPLC BEH-C18 column (2.1 mm × 100 mm, 1.7 μm). The mobile phase was 0.1% formic acid acetonitrile solution (A) and 0.1% formic acid aqueous solution (A). Elution gradient: 0–2 min, 5% A; 2–14 min, 5–95% A; 14–15 min, 95% A; 15–16 min, 5% A. The injection volume was 5 μl. Detection conditions were positive and negative ion modes, using an electrospray ionization source, with a mass scan range of 100–1200 m / z.

[0085] The *Clostridium pluvialis* CCFM1203, *Bifidobacterium longum* subsp. CCFM1306, and *Bifidobacterium adolescentis* CCFM1066 involved in the following examples are respectively described in Chinese invention patent texts with publication numbers: CN115074277B, CN116590174A, and CN110616167B. The *Eubacterium rectum* ATCC33656 was purchased from Beijing BioBio Biotechnology Co., Ltd.

[0086] The culture method of the strains involved in the following examples is as follows:

[0087] Preparation of *Faecalibacterium prausnitzii* CCFM1203 bacterial suspension: *Faecalibacterium prausnitzii* CCFM1203 was inoculated into M2GSC liquid medium at a 2% inoculum and anaerobically cultured at 37℃ for 24 h. Then, it was transferred to fresh M2GSC liquid medium and cultured under the same conditions for 24 h. The bacterial sludge was obtained by filtration in an anaerobic workstation, followed by washing and resuspending with 0.1M PBS (pH 7.2, containing 0.05% cysteine) to obtain a bacterial concentration of 1×10⁻⁶. 9 CFU / 100ul suspension, use a well-sealed lyophilized bottle with a stopper to preserve the bacterial suspension, and use it on the same day.

[0088] Preparation of Bifidobacterium longum subsp. CCFM1306 bacterial suspension: The culture was activated with MRS-L (MRS + 0.5% cysteine ​​hydrochloride) medium. After three generations of activation with a 4% inoculum, the culture was expanded to collect the Bifidobacterium cells (4th generation). The cells were centrifuged at 8000 rpm for 15 min, and washed three times repeatedly with 0.9% physiological saline (containing 0.5% cysteine ​​hydrochloride) pre-cooled to 4°C. The cells were then collected by centrifugation under the same conditions. The obtained cells were resuspended in pre-cooled 30% sucrose solution, counted, and stored at -80°C for later use. Before use, the cells were diluted with sterile physiological saline to a viable count of 1 × 10⁻⁶.9 CFU / 100ul.

[0089] Preparation of Bifidobacterium tumefaciens CCFM1066 bacterial suspension: The culture was activated with MRS-L (MRS + 0.5% cysteine ​​hydrochloride) medium. After three generations of activation with a 4% inoculum, the culture was expanded to collect the Bifidobacterium cells (4th generation). The cells were centrifuged at 8000 rpm for 15 min, and washed three times repeatedly with 0.9% physiological saline (containing 0.5% cysteine ​​hydrochloride) pre-cooled to 4℃. Finally, the cells were centrifuged under the same conditions to collect the Bifidobacterium cells. The obtained cells were resuspended in pre-cooled 30% sucrose solution, counted, and stored at -80℃ for later use. Before use, the cells were diluted with sterile physiological saline to a viable count of 1×10⁻⁶. 9 CFU / 100ul.

[0090] Example 1: Overall structure of gut microbiota in people with different exercise levels

[0091] Based on publicly available databases such as NCBI, 418 metagenomic datasets of gut microbiota containing exercise levels were selected. Species composition was annotated for each sample using MetaPhlAn3. The 418 metagenomic samples were then categorized according to exercise performance, from highest to lowest, into elite athlete group (Athelete group), moderate athlete group (Moderate athlete group), and sedentary group (Control group).

[0092] The richness and evenness of populations at different activity levels were explored using three alpha diversity indices: Pielou's Evenness, Simpson's, and Shannon's. Pielou's Evenness is a commonly used index for measuring species diversity. Simpson's and Shannon's indices reflect species richness and evenness within the community. The Kruskal-Wallis test (non-parametric) was used to compare alpha diversity between groups. Results are shown below. Figure 1 There were few differences in alpha diversity between groups, but the Shannon index was significantly increased in the Athelete group compared to the moderate athlete group.

[0093] The differences in species distribution among different metagenomic samples were analyzed using statistical distance analysis. PCoA analysis was performed using three distance methods: bray_curtis, WeightedUnifrac, and UnweightedUnifrac. The principal coordinate system with the highest contribution rate was selected for plotting. Results are shown below. Figure 2 PCoA analysis revealed significant differences in the overall bacterial community structure between the Athelete and Moderate Athlete groups and the Control group.

[0094] Example 2: Core species of gut microbiota in people with different exercise levels

[0095] Based on publicly available databases such as NCBI, 418 metagenomic datasets of gut microbiota containing data from different levels of physical activity were selected. Species composition annotation was performed on each sample using MetaPhlAn3. The 418 metagenomic samples were then categorized according to physical activity level, from highest to lowest, into three groups: elite athletes (Athelete group), moderate athletes (moderate athlete group), and sedentary athletes (Control group).

[0096] Species abundance stacking plots were used to analyze the species composition of gut microbiota in individuals with different levels of physical activity. Results are shown below. Figure 3 At the species level, the relative abundance of species such as Prevotella copri, Bacteroides uniformis, Faecalibacterium unknown_species, Faecalibacterium prausnitzii, and Eubacterium rectale was relatively high.

[0097] Linear discriminant analysis (LDA) was used to reduce the dimensionality of the data, identify the most significant influencing factors, and assess the impact of differentially expressed species. A p-value of 0.5 was set for differences between groups, and a threshold of 3.5 was set for LDA; an LDA value greater than 3.5 was considered indicative of differentially expressed species. Results are shown below. Figure 4 .

[0098] The Lefse analysis results showed that there were significant differences in gut microbiota at all species levels among individuals with different exercise levels. Faecalibacterium prausnitzii and Eubacterium rectale were differentially expressed gut microbiota among individuals with different exercise levels, and they were enriched in the Athelete group.

[0099] Example 3: Dietary factors alter the abundance of exercise-related gut microbiota

[0100] The specific steps are as follows:

[0101] (1) Preparation of culture medium containing dietary factors

[0102] Polysaccharide and polyphenol powders were added directly to the mGAM medium at mass fractions of 0.5% and 0.1%, respectively.

[0103] The polysaccharides include: Grifola frondosa polysaccharide, galactooligosaccharide, ginseng polysaccharide, Auricularia auricula polysaccharide, fructooligosaccharide, Lycium barbarum polysaccharide, mannan, kudzu root polysaccharide, inulin, brown algae polysaccharide, isomaltooligosaccharide, yam polysaccharide, and matsutake mushroom polysaccharide.

[0104] The polyphenols include: lentinan, lecithin, caffeo-4-O-glucoside, apigenin, naringin, sesamin, burdock, curcumin, bergapten lactone, caffeic acid, limonene, artemisinin, andrographolide, phytosterol, and chondroitin sulfate.

[0105] The culture medium contained 0.5% dietary polysaccharides and 0.1% dietary polyphenols. Fermentation was performed on individual dietary factors. The group without added carbon source was designated as the negative control group.

[0106] (2) Six healthy volunteers (three males and three females) were selected. They were required to have a normal weight (BMI: 18.5-24), have not received antibiotic treatment in the past 6 months, have no intestinal diseases, not have a habit of consuming probiotics, and not have used probiotic products or consumed alcohol in the past month. Their age was between 20 and 35 years old. Fresh morning stool samples from the six volunteers were collected in 35mL stool sampling tubes. The tube caps were partially tightened and placed in a laboratory-specific self-sealing bag containing an anaerobic gas-generating bag to create an anaerobic environment. The samples were temporarily stored in a 4℃ refrigerator, ensuring processing began within two hours.

[0107] (3) Under anaerobic conditions, equal amounts of fresh feces from six volunteers were weighed and mixed evenly. The mixture was diluted with sterile phosphate buffer (PBS) at a ratio of 1:10 w / v, and then filtered through a sterile filter to remove large particles. The resulting filtrate was inoculated at a volume ratio of 20% into the mGAM medium containing dietary polysaccharides or dietary polyphenols obtained in step (1). The medium was cultured anaerobically at 37°C (80% N2, 10% CO2, 10% H2) for 24 h to obtain a fermentation broth containing intestinal microorganisms.

[0108] (4) Transfer the fermentation broth obtained in step (3) after 24h anaerobic fermentation to a 2ml sterile centrifuge tube and separate the bacterial sludge at a speed of 10000r / min.

[0109] The obtained mycotic sludge was subjected to metagenomic sequencing using the DNBSEQ-T7 platform PE150 from Beijing Novogene Co., Ltd. Quality control of the raw sequencing data was performed as follows: read pairs with adapters were removed; read pairs with a proportion of N (indicating undetermined base information) greater than 10% in single-end sequencing reads were removed; read pairs containing more than 50% low-quality (below 5) bases were removed. Reads were aligned to the human reference genome (Homo sapiens, UCSC hg38) using the BWA tool to remove host-derived genes. Species and functional annotations were performed on the quality-controlled gene sequences using MetaPhlAn3 and HUMAnN3. Species abundance information from various samples was merged using MetaPhlAn3 to obtain a species abundance table for multiple samples. Results are shown below. Figure 5 .

[0110] (5) Experimental results:

[0111] The results show that: Figure 5 It can be seen that after different polysaccharides and polyphenols were fermented by fecal microbiota alone, the relative abundance of Eubacterium rectale, Faecalibacterium prausnitzii and related species of Veillonella changed.

[0112] 1) Effects of gut microbiota fermentation of dietary factors on the abundance of Eubacterium rectale

[0113] After fecal microbiota fermentation, the abundance of *Eubacterium rectale* in the *Yin Shen* group was 0.26, in the chondroitin sulfate group it was 0.19, in the *Grifola frondosa* polysaccharide group it was 0.65, in the galactooligosaccharide group it was 1.3, in the ginseng polysaccharide group it was 0, in the *Auricularia auricula-judae* polysaccharide group it was 0.9, in the *Lycium barbarum* polysaccharide group it was 0.45, in the *Lentinula edodes* polysaccharide group it was 0.59, in the paclitaxel group it was 0.3, in the caffeic acid 4-O-glucoside group it was 0.27, in the apigenin group it was 0.26, in the naringin group it was 0.25, in the sesamin group it was 0.32, in the burdock seed group it was 0.28, and in the turmeric group it was 0. The concentrations of the following polysaccharides were as follows: artemisinin group 0.38, bergamot lactone group 0.32, fructooligosaccharide group 0.69, caffeic acid group 0.31, limonene group 0.24, artemisinin group 0.25, andrographolide group 0.35, phytosterol group 0.28, mannan group 0.76, kudzu root polysaccharide group 0.73, inulin group 0.98, brown algae polysaccharide group 0.2, isomaltooligosaccharide group 0.58, yam polysaccharide group 0.75, and matsutake polysaccharide group 0.56.

[0114] It is evident that the abundance of Eubacterium rectale was increased after fermentation by fecal microbiota, including Grifola frondosa polysaccharides, galactooligosaccharides, Auricularia auricula-judae polysaccharides, inulin, Lycium barbarum polysaccharides, Lentinan, curcumin, mannan, and Pueraria lobata polysaccharides. The relative abundance of the galactooligosaccharide group was 1.3, and that of the inulin group was 0.98, representing increases of 5 times and 3.76 times, respectively, compared to the relative abundance of the *Gynostemma pentaphyllum* group.

[0115] 2) Effects of fermented dietary factors on the abundance of Faecalibacterium prausnitzii in gut microbiota

[0116] After fermentation with fecal microbiota, the abundance of *Faecalibacterium prausnitzii* in the *Yin Shen* group was 3.9, in the chondroitin sulfate group it was 4.7, in the *Grifola frondosa* polysaccharide group it was 6, in the galactooligosaccharide group it was 5.3, in the ginseng polysaccharide group it was 0.0014, in the *Auricularia auricula-judae* polysaccharide group it was 5.5, in the *Lycium barbarum* polysaccharide group it was 4.4, in the *Lentinula edodes* polysaccharide group it was 5.6, in the paclitaxel group it was 4.8, in the caffeic acid 4-O-glucoside group it was 4.4, in the apigenin group it was 4.6, in the naringin group it was 4.5, in the sesamin group it was 4.9, and in the burdock seed group it was 4.9. The group with a concentration of 4.6 was curcumin, bergamot lactone, fructooligosaccharides, caffeic acid, limonene, artemisinin, andrographolide, phytosterols, mannan, kudzu polysaccharides, inulin, brown algae polysaccharides, isomaltooligosaccharides, yam polysaccharides, and matsutake polysaccharides.

[0117] It is evident that the polysaccharides from matsutake mushrooms, galactooligosaccharides, wood ear mushrooms, shiitake mushrooms, yam, maitake mushrooms, and kudzu root mediated an increase in the abundance of *Faecalibacterium prausnitzii* after fermentation by fecal microbiota. Specifically, the relative abundance of the matsutake mushroom polysaccharide group and the maitake mushroom polysaccharide group was 6, representing increases of 1.54 and 1.54 times, respectively, compared to the relative abundance of the *Gynostemma pentaphyllum* group.

[0118] 3) Effects of fermented dietary factors on the abundance of Veillonella spp. in gut microbiota

[0119] Depend on Figure 5The results showed that curcumin, burdock seed, and phytosterol mediated an increase in the abundance of Veillonella-related species after fermentation by fecal microbiota. Specifically, the relative abundance of Veillonella atypica in the *Veillonella stypica* group was 0.082, and the relative abundance in the curcumin group was 0.13, representing a 1.59-fold increase compared to the *Veillonella stypica* group. The relative abundance of Veillonella parvula in the *Veillonella stypica* group was 0.088, the relative abundance in the curcumin group was 0.13, and the relative abundance in the burdock seed group was 0.14, representing increases of 1.48 and 1.59 times, respectively, compared to the *Veillonella stypica* group.

[0120] Example 4: Dietary factor combinations alter the abundance of exercise-promoted gut microbiota

[0121] The specific steps are as follows:

[0122] (1) Preparation of culture medium containing dietary factors

[0123] Polysaccharide and polyphenol powders were added directly to mGAM medium according to the proportions in Table 1; different combinations of dietary factors were fermented; the fermentation groups are shown in Table 1.

[0124] Table 1: Grouping Information of Dietary Polysaccharides and Polyphenols

[0125]

[0126]

[0127] (2) Six healthy volunteers (three males and three females) were selected. They were required to have a normal weight (BMI: 18.5-24), have not received antibiotic treatment in the past 6 months, have no intestinal diseases, not have a habit of consuming probiotics, and not have used probiotic products or consumed alcohol in the past month. Their age was between 20 and 35 years old. Fresh morning stool samples from the six volunteers were collected in 35mL stool sampling tubes. The tube caps were partially tightened and placed in a laboratory-specific self-sealing bag containing an anaerobic gas-generating bag to create an anaerobic environment. The samples were temporarily stored in a 4℃ refrigerator, ensuring processing began within two hours.

[0128] (3) Under anaerobic conditions, equal amounts of fresh feces from six volunteers were weighed and mixed thoroughly. The mixture was then diluted with sterile phosphate buffer (PBS) at a ratio of 1:10 w / v and filtered through a sterile filter to remove large particles. The resulting filtrate was inoculated at a volume ratio of 20% into the mGAM medium containing dietary factors obtained in step (1) and cultured anaerobically at 37°C (80% N2, 10% CO2, 10% H2) for 24 h to obtain a fermentation broth containing intestinal microorganisms.

[0129] (4) Transfer the fermentation broth obtained in step (3) after 24h anaerobic fermentation to a 2ml sterile centrifuge tube and separate the bacterial sludge at a speed of 10000r / min.

[0130] The obtained mycotic sludge was subjected to metagenomic sequencing using the DNBSEQ-T7 platform PE150 from Beijing Novogene Co., Ltd. Quality control of the raw sequencing data was performed as follows: read pairs with adapters were removed; read pairs with a proportion of N (indicating undetermined base information) greater than 10% in single-end sequencing reads were removed; read pairs containing more than 50% low-quality (below 5) bases were removed. Reads were aligned to the human reference genome (Homo sapiens, UCSC hg38) using the BWA tool to remove host-derived genes. Species and functional annotations were performed on the quality-controlled gene sequences using MetaPhlAn3 and HUMAnN3. Species abundance information from various samples was merged using MetaPhlAn3 to obtain a species abundance table for multiple samples. Results are shown below. Figure 6 .

[0131] (5) Experimental results:

[0132] The results show that: Figure 6 It can be seen that the relative abundance of Eubacterium rectale and Faecalibacterium prausnitzii changes after different combinations of polysaccharides and polyphenols are fermented by fecal microbiota.

[0133] 1) Effects of gut microbiota fermentation of dietary factors on the abundance of Eubacterium rectale

[0134] After fermentation, the relative abundance of *Eubacterium rectale* was 2.50475 in the control group, 2.62501 in the J group, 2.28734 in the N group, 2.94651 in the S group, 3.0604 in the D group, 2.77948 in the JS group, 6.61884 in the JD group, 4.40961 in the ND group, 2.53892 in the NS group, 2.22583 in the JNS group, 2.00788 in the JSD group, 9.50193 in the JND group, 10.36435 in the NSD group, and 4.20334 in the JNSD group.

[0135] It can be seen that after fermentation, the relative abundance of Eubacterium rectale in the control group was 2.50475, while the relative abundance in the NSD group was 10.36435, the relative abundance in the JND group was 9.50193, and the abundance in the JD group was 6.61884, which were 4.14, 3.80, and 2.64 times higher than that in the control group, respectively.

[0136] 2) Effects of fermented dietary factors on the abundance of Faecalibacterium prausnitzii in gut microbiota

[0137] After fermentation, the relative abundance of Faecalibacterium prausnitzii was 5.52759 in the control group, 6.23853 in group J, 5.47133 in group N, 10.44086 in group S, 8.57243 in group D, 9.79587 in group JS, 6.47326 in group JD, 6.16557 in group ND, 10.50552 in group NS, 8.42584 in group JNS, 7.34466 in group JSD, 4.57097 in group JND, 5.1589 in group NSD, and 7.93421 in group JNSD.

[0138] It can be seen that after fermentation, the relative abundance of Faecalibacterium prausnitzii in the control group was 5.52759, while the relative abundance in the S group was 10.44086, the relative abundance in the NS group was 10.50552, and the abundance in the JS group was 9.79587, which were 1.89, 1.91, and 1.78 times higher than that in the control group, respectively.

[0139] The above results indicate that the combination of matsutake polysaccharide, curcumin and matsutake polysaccharide, and burdock seed and matsutake polysaccharide promotes the increase of Faecalibacterium prausnitzii abundance; the combination of galactooligosaccharide, curcumin and galactooligosaccharide, burdock seed, matsutake polysaccharide and galactooligosaccharide, and curcumin, burdock seed and galactooligosaccharide can promote the increase of Eubacterium rectale abundance.

[0140] The differences in the abundance results of each strain in Examples 3 and 4 are analyzed as follows:

[0141] Since the samples targeted in Examples 3 and 4 are different, it is equivalent to two fermentations, each using different mixed feces. Due to the large individual differences in the composition of the intestinal flora, the relative abundance of the target bacteria before and after the two fermentations is different, but the fold change of the bacteria before and after the two fermentations is consistent. It can be seen that the overall trend is the same, and the effect of this application is also stable.

[0142] Example 5: Effect of dietary factor combinations on the yield of fatty acid amides.

[0143] The specific steps are as follows:

[0144] (1) Preparation of culture medium containing dietary factors

[0145] Polysaccharide and polyphenol powders were added directly to mGAM medium according to the proportions in Table 1 (Example 4); different combinations of dietary factors were fermented; the grouping of the combined fermentation is shown in Table 1 (Example 4).

[0146] (2) Six healthy volunteers (three males and three females) were selected. They were required to have a normal weight (BMI: 18.5-24), have not received antibiotic treatment in the past 6 months, have no intestinal diseases, not have a habit of consuming probiotics, and not have used probiotic products or consumed alcohol in the past month. Their age was between 20 and 35 years old. Fresh morning stool samples from the six volunteers were collected in 35mL stool sampling tubes. The tube caps were partially tightened and placed in a laboratory-specific self-sealing bag containing an anaerobic gas-generating bag to create an anaerobic environment. The samples were temporarily stored in a 4℃ refrigerator, ensuring processing began within two hours.

[0147] (3) Under anaerobic conditions, equal amounts of fresh feces from six volunteers were weighed and mixed thoroughly. The mixture was then diluted with sterile phosphate buffer (PBS) at a ratio of 1:10 w / v and filtered through a sterile filter to remove large particles. The resulting filtrate was inoculated at a volume ratio of 20% into the mGAM medium containing dietary factors obtained in step (1) and cultured anaerobically at 37°C (80% N2, 10% CO2, 10% H2) for 24 h to obtain a fermentation broth containing intestinal microorganisms.

[0148] (4) Transfer the fermentation broth obtained in step (3) after 24h anaerobic fermentation to a 2ml sterile centrifuge tube and separate the bacterial sludge at a speed of 10000r / min. Determine the content of oleoylethanolamine (OEA) and palmitoylethanolamide (PEA) in the supernatant obtained after centrifugation.

[0149] Take 1 ml of the fermented supernatant into an enzyme-free EP tube, then add 1 ml of chloroform. Vortex the solution for 2 minutes, then concentrate the sample by vacuum evaporation. Before high-resolution high-performance liquid chromatography-mass spectrometry (HPLC-MS) analysis, dry the sample and store it at -20°C. Dissolve the supernatant in 100 μL of methanol and centrifuge. Filter the supernatant through a membrane into a sample vial for injection detection. Results are shown below. Figure 7 .

[0150] Depend on Figure 7 The results showed that, through targeted measurement of the contents of oleoylethanolamine (OEA) and palmitoylethanolamide (PEA) in the fermentation supernatant, the contents of OEA in the blank control group after fermentation were 11.52±0.42 ng / L, in group J it was 15.49±1.80 ng / L, in group N it was 13.20±5.56 ng / L, in group S it was 26.52±7.91 ng / L, in group D it was 49.4±9.80 ng / L, in group JS it was 25.40±3.64 ng / L, in group JD it was 20.60±9.42 ng / L, and in group ND it was 49. The concentrations of OEA in the D, ND, NS, JSD, NSD, and JNSD groups were 49.55±3.80 ng / L, 38.29±2.42 ng / L, 36.28±2.68 ng / L, 70.40±12.60 ng / L, and 36.47±2.49 ng / L, respectively, were significantly higher than those in the blank control group (P<0.05). This indicates that galactooligosaccharides and groups containing galactooligosaccharides can increase the concentration of OEA after fermentation.

[0151] After fermentation, the PEA content was 59.67±3.27 ng / L in the blank control group, 91.80±8.10 ng / L in group J, 70.69±2.64 ng / L in group N, 144.31±3.95 ng / L in group S, 109.64±9.53 ng / L in group D, 38.29±10.80 ng / L in group JS, 50.32±3.21 ng / L in group JD, 105.02±1.81 ng / L in group ND, and 158.27 ng / L in group NS. The PEA concentrations in the JSD group were 106.29±7.89 ng / L, the JND group was 105.06±11.49 ng / L, the NSD group was 185.35±14.13 ng / L, and the JNSD group was 48.22±11.30 ng / L. Compared with the blank control, the PEA concentrations in the J, N, D, ND, JND, JSD, NSD, and JNSD groups were significantly increased (P<0.05). It can be seen that galactooligosaccharides and groups containing galactooligosaccharides can increase the PEA concentration after fermentation.

[0152] The above results indicate that the combination of galactooligosaccharides, burdock seed polysaccharides, matsutake mushroom polysaccharides, galactooligosaccharides, burdock seed polysaccharides, and galactooligosaccharides promotes the concentration of fatty acid amides, with the combination of burdock seed polysaccharides, matsutake mushroom polysaccharides, and galactooligosaccharides showing the best promoting effect.

[0153] Example 6: Effects of dietary factor combinations on SCFA yield

[0154] The specific steps are as follows:

[0155] (1) Preparation of culture medium containing dietary factors

[0156] Polysaccharide and polyphenol powders were added directly to mGAM medium according to the proportions in Table 1 (Example 4); different combinations of dietary factors were fermented; the grouping of the combined fermentation is shown in Table 1 (Example 4).

[0157] (2) Six healthy volunteers (three males and three females) were selected. They were required to have a normal weight (BMI: 18.5-24), have not received antibiotic treatment in the past 6 months, have no intestinal diseases, not have a habit of consuming probiotics, and not have used probiotic products or consumed alcohol in the past month. Their age was between 20 and 35 years old. Fresh morning stool samples from the six volunteers were collected in 35mL stool sampling tubes. The tube caps were partially tightened and placed in a laboratory-specific self-sealing bag containing an anaerobic gas-generating bag to create an anaerobic environment. The samples were temporarily stored in a 4℃ refrigerator, ensuring processing began within two hours.

[0158] (2) Under anaerobic conditions, equal amounts of fresh feces from six volunteers were weighed and mixed thoroughly. The mixture was then diluted with sterile phosphate buffer (PBS) at a ratio of 1:10 w / v and filtered through a sterile filter to remove large particles. The resulting filtrate was inoculated at a volume ratio of 20% into the mGAM medium containing dietary factors obtained in step (1) and cultured anaerobically at 37°C (80% N2, 10% CO2, 10% H2) for 24 h to obtain a fermentation broth containing intestinal microorganisms.

[0159] (4) Transfer the fermentation broth obtained in step (3) after 24h anaerobic fermentation to a 2ml sterile centrifuge tube and separate the bacterial sludge at a speed of 10000r / min. Determine the short-chain fatty acid content of the supernatant obtained after centrifugation.

[0160] Take 500 μL of fermentation supernatant into an enzyme-free EP tube, then add 40 μL of 10% concentrated sulfuric acid for acidification and vortex until homogeneous. Add 1 mL of diethyl ether in a fume hood, vortex for 30 s, and centrifuge at 12000 rpm / min for 15 min at 4 °C. Transfer the ether layer of the supernatant to an EP tube containing 0.25 g of anhydrous sodium sulfate, let stand for 15 min, and then centrifuge at 12000 rpm / min for 15 min at 4 °C. Filter the supernatant through a membrane and add it to a sample vial. Results are shown below. Figure 8 .

[0161] Depend on Figure 8 It was found that after 24 hours of in vitro fermentation, the acetic acid content in the fermentation supernatant was 11.53±0.42 mmol / L in the blank control group, 31.21±4.34 mmol / L in group J, 17.21±2.81 mmol / L in group N, 21.75±1.90 mmol / L in group S, 15.36±2.34 mmol / L in group D, 11.67±2.51 mmol / L in group JS, and 19.47±5.8 mmol / L in group JD. The acetic acid concentrations were 0 mmol / L in the JNS group, 16.52±7.72 mmol / L in the ND group, 16.74±1.65 mmol / L in the JNS group, 19.32±3.30 mmol / L in the JND group, 27.90±3.81 mmol / L in the JSD group, 16.28±1.42 mmol / L in the NSD group, 25.05±4.67 mmol / L in the JNSD group, and 15.27±1.51 mmol / L in the JNSD group. After 24 hours of in vitro fermentation, compared with the blank control group, the J, JND, and NSD groups significantly increased the acetic acid concentration (P<0.05).

[0162] After 24 hours of in vitro fermentation, the butyric acid content in the fermentation supernatant was 9.52±1.33 mmol / L in the blank control group, 27.86±2.50 mmol / L in group J, 20.88±1.96 mmol / L in group N, 26.32±6.39 mmol / L in group S, 18.65±4.38 mmol / L in group D, 21.94±2.33 mmol / L in group JS, and 6.85±1.2 mmol / L in group JD. The butyrate concentrations were 3 mmol / L in the following groups: NS group (23.92±2.46 mmol / L), ND group (4.08±0.95 mmol / L), JNS group (16.67±2.17 mmol / L), JND group (4.45±0.91 mmol / L), JSD group (8.96±0.63 mmol / L), NSD group (2.22±0.70 mmol / L), and JNSD group (8.66±0.78 mmol / L). Compared with the blank control, the butyrate concentrations in the J, N, D, S, JS, NS, and JNS groups were significantly increased (P<0.05).

[0163] The above experimental results show that curcumin, as well as the curcumin, burdock seed, and galactooligosaccharide groups, have the best promoting effect on the content of acetic acid-containing fecal microbiota after fermentation; matsutake polysaccharide, as well as the burdock seed and matsutake polysaccharide groups, have the best promoting effect on the content of butyric acid-containing fecal microbiota after fermentation.

[0164] Example 7: Effects of different combinations of dietary factors on the time to exhaustion during weight-bearing swimming in exercised mice

[0165] The specific steps are as follows:

[0166] One hundred and thirty SPF-grade male ICR mice (6 weeks old, 18-22g) were randomly divided into 13 groups of 10 mice each: a static control group, an exercise control group, *Clostridium plasminoides* group, *E. rectum* group, *Pseudomonas aeruginosa* group, *Matsutake* group, *Pseudomonas aeruginosa* low-potassium group, *Ginger-Pseudomonas aeruginosa* low-potassium ...Ginger-Pseudomonas aeruginosa* low-potassium group, *Ginger-Pseudomonas aeruginosa* low-potassium group, *Inulin-Bifidobacterium longum* group, curcumin group, and a mixed group. The mice were housed at the Experimental Animal Center of Jiangnan University under a constant temperature of 21-26℃, humidity of 40-70%, noise level ≤60dB, and illumination of 15-20LX (all animal experimental procedures were reviewed and approved by the Animal Welfare and Ethics Management Committee of Jiangnan University). The grouping of the animals is shown in Table 2.

[0167] Table 2: Animal Grouping Information

[0168]

[0169]

[0170] The experiment lasted 8 weeks: Mice were initially acclimatized for 7 days. Starting from day 8, different groups were administered different combinations of dietary factors via gavage. After four weeks of gavage, mice underwent 30 minutes of swimming training daily for two weeks. Relevant indicators were measured in the final week.

[0171] Except for the resting control group, all other groups of mice underwent a weight-bearing swimming exhaustion test 30 minutes after the last gavage. A 5% weight was applied to the base of the mouse's tail. Exhaustion was defined as the mouse's hind limb movement becoming sluggish, its swimming range decreasing, and its head sinking to the surface within 3 seconds. Results are shown below. Figure 9 .

[0172] like Figure 9As shown, the time to exhaustion was 12.15±5.02 min in the exercise control group, 27.38±10.34 min in the Clostridium perfringens group, 27.83±8.47 min in the Eubacterium rectum group, 30.63±10.05 min in the inulin-Bifidobacterium longum group, 17.5±8.91 min in the matsutake group, 18.53±7.23 min in the curcumin group, 12.78±4.61 min in the ox pine group, 13.49±5.74 min in the ox pine group, 17.99±5.87 min in the ox pine group, 25.7±5.47 min in the ginger-ox pine group, 25.11±7.32 min in the ginger-ox pine group, and 30.86±6.99 min in the mixed group. Compared with the control group's time to exhaustion of 12.15±5.02 min, the time to exhaustion increased to 27.38±10.37 min in the Clostridium perfringens group, 27.83±8.47 min in the E. rectum group, 25.7±5.47 min in the Ginger-Achyranthes-Polygonatum group, 25.11±7.32 min in the Ginger-Achyranthes-Polygonatum group, 30.63±10.05 min in the Inulin-Bifidobacterium longum group, and 30.86±6.99 min in the mixed group. The time to exhaustion in weighted swimming was significantly increased (P<0.05).

[0173] The above experiments show that the *Clostridium praosporum* group, *E. rectum* group, *Ginger-beta-sinensis* group, *Ginger-beta-sinensis* group, inulin-bifidobacterium longum group, and mixed group can significantly increase the swimming exhaustion time of exercised mice. The mixed group of *Bifidobacterium adolescentis* CCFM1066, *Bifidobacterium longum* subsp. CCFM1306, curcumin, inulin, burdock seed, matsutake polysaccharide, and galactooligosaccharide has the most significant effect.

[0174] Example 8: Effects of different combinations of dietary factors on fatigue rotarod time in exercised mice

[0175] Animal experiments and grouping were conducted as described in Example 7, lasting for 8 weeks. Mice were first acclimatized for 7 days. Starting from day 8, different groups were administered different combinations of dietary factors via gavage. After four weeks of gavage, mice underwent 30 minutes of swimming training daily for two weeks. Relevant indicators were measured in the final week. Except for the resting control group, all other groups of mice underwent fatigue rotarod time measurement 30 minutes after gavage and rest. Before the measurement, mice underwent fatigue rotarod training 3 times. Each group of mice was placed on a fatigue rotarod at a rotation speed of 30 r / min, and the time it took for the mice to fall from the rotarod was recorded. The results are shown below. Figure 10 .

[0176] The results show that: Figure 10As shown, the fatigue rotarod time was 5.46±1.13 min in the exercise control group, 14.89±2.03 min in the Clostridium perfringens group, 11.5±1.38 min in the E. rectum group, 8.83±1.69 min in the inulin-Bifidobacterium longum group, 8.38±1.51 min in the matsutake group, 14.11±1.83 min in the curcumin group, 8.70±2.27 min in the ox pine group, 8.41±2.14 min in the ox pine pine group, 9.4±2.07 min in the ox pine pine pine group, 7.38±1.77 min in the ginger ox pine pine pine pine pine group, 9.13±1.46 min in the ginger ox pine ... Compared with the fatigue time of 5.46±1.13 min in the control group, the fatigue time of the rotarod increased to 14.89±2.03 min in the Clostridium perfringens group, 11.5±1.38 min in the E. rectum group, 14.11±1.83 min in the curcumin group, 8.70±2.2 min in the niu song group, 9.4±2.07 min in the niu song low-dose group, 7.38±1.77 min in the ginger-niu song low-dose group, 9.13±1.46 min in the ginger-niu song low-dose group, 8.83±1.69 min in the inulin-bifidobacterium longum group, and 13.57±1.72 min in the mixed group, with a significant increase in fatigue rotarod time (P<0.05).

[0177] The above experiments show that the *Clostridium praosporum* group, *E. rectum* group, *Bifidobacterium tumefaciens* group, *Bifidobacterium tumefaciens* low-potassium group, ginger-Bifidobacterium tumefaciens low-potassium group, ginger-Bifidobacterium tumefaciens* low-potassium group, inulin-bifidobacterium longum group, and mixed group can significantly improve the fatigue rotarod time of exercised mice. Among them, the *Clostridium praosporum* group, curcumin group, *Bifidobacterium adolescentis* CCFM1066, *Bifidobacterium longum* subsp. CCFM1306, curcumin, inulin, burdock seed, matsutake polysaccharide, and galactooligosaccharide mixed group have the most significant effects.

[0178] Example 9: Effects of different combinations of dietary factors on grip strength in exercising mice

[0179] Animal experiments and grouping were conducted as described in Example 7, lasting for 8 weeks. Mice were first acclimatized for 7 days. Starting from day 8, different groups were administered different combinations of dietary factors via gavage. After four weeks of gavage, mice underwent 30 minutes of swimming training daily for two weeks. Relevant indicators were measured in the final week. Except for the resting control group, all mice in the other groups had their grip strength measured using a grip dynamometer 30 minutes after gavage. The mouse's forelimbs were placed horizontally on a grip bar, the tail was grasped, and the mouse was slowly pulled horizontally, repeated 5 times. Results are shown below. Figure 11 .

[0180] like Figure 11As shown, the fatigue rotarod time was 133.73±10.22g in the exercise control group, 160.07±14.11g in the Clostridium perfringens group, 162.98±10.06g in the E. rectum group, 161.63±13.61g in the inulin-Bifidobacterium longum group, 152.84±22.20g in the matsutake group, 148.11±21.41g in the curcumin group, 145.38±20.43g in the ox pine group, 156±14.27g in the ox pine pine pine group, 175.69±28.36g in the ox pine ... Compared to the control group's grip strength of 133.73±10.22g, the Niu Song low-grip group increased to 175.69±28.36g, and the mixed group increased to 179.17±14.53g.

[0181] The above experiments show that the combination of burdock seed, matsutake mushroom, and galactooligosaccharide, as well as the mixture of Bifidobacterium adolescentis CCFM1066, Bifidobacterium longum subsp. longum CCFM1306, curcumin, inulin, burdock seed, matsutake mushroom polysaccharide, and galactooligosaccharide, has significant effects.

[0182] Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Anyone skilled in the art can make various modifications and alterations without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be determined by the claims.

Claims

1. Dietary factors in the preparation of products that enhance gut microbiota Eubacterium rectale The application of the abundance of the product is characterized by, The dietary factor is a combination of burdock seed, galactooligosaccharide and matsutake polysaccharide. The total mass of galactooligosaccharide and matsutake polysaccharide is added to burdock seed at a mass ratio of 5:1, and the mass ratio of galactooligosaccharide and matsutake polysaccharide is added at a mass ratio of 1:

1. The amount of dietary factor added is at least 0.15 g / kg.

Images