Probiotic composition, fermented yoghurt and application of probiotic composition and fermented yoghurt in improving related indexes of athletes

Abstract

The invention discloses a probiotic composition, fermented yoghurt and application of the probiotic composition and the fermented yoghurt in improving related indexes of athletes. The probiotic composition comprises bacillus subtilis SP-8-5, lactobacillus plantarum YM-4-3, lactobacillus plantarum AY01 and pediococcus acidilactici LH01. The composition of intestinal microbial populations can be adjusted through a specific compounding method, the abundance of harmful bacteria is reduced, the abundance of beneficial bacteria is improved, the immunity of intestinal tracts is enhanced, and the integrity of intestinal barriers of athletes is maintained. Meanwhile, the composition has a positive improvement effect on related exercise indexes such as plasma amino acid, excrement metabolites and exercise performance, and can be used as an effective exercise supplement to be applied to athletes.

Description

Technical Field

[0001] This invention relates to the field of microbial technology, and in particular to a probiotic composition, fermented yogurt, and its application in improving relevant indicators in athletes. Background Technology

[0002] Gut microbiota regulate the body's nervous and immune functions through various metabolic pathways. When the diversity of the gut microbiota decreases, it can cause changes in various systems of the body through the microbiome-gut-brain axis, leading to psychological distress. Microbiome depletion is one of the potential causes of impaired motor performance. Appropriate use of probiotic supplements can improve the structure of the human gut microbiota, allowing the host to spontaneously or reliantly produce beneficial substances and regulate metabolism.

[0003] Athletes have higher physical requirements. Currently, there are relatively few probiotic supplements suitable for athletes, so it is necessary to develop a sports supplement that can improve athletic performance. Summary of the Invention

[0004] The technical problem to be solved by the present invention is to provide a probiotic composition and fermented yogurt suitable for athletes to improve athletic performance.

[0005] This invention provides a probiotic composition comprising Bacillus subtilis SP-8-5, Lactobacillus plantarum YM-4-3, Lactobacillus plantarum AY01, and Pediococcus lactis LH01. Among these, Bacillus subtilis SP-8-5, Lactobacillus plantarum YM-4-3, Lactobacillus plantarum AY01, and Pediococcus lactis LH01 are all high-quality strains screened by the inventors from numerous lactic acid bacteria, and all have been preserved.

[0006] Bacillus subtilis SP-8-5 ( Bacillus subtilis SP-8-5 was deposited at the China Center for Type Culture Collection (CCTCC) on March 6, 2023, with accession number CCTCC NO: M2023254; the deposit address is: Luojia Mountain, Bayi Road, Wuchang District, Wuhan City, Hubei Province; the contact number is 027-68754052.

[0007] Lactobacillus plantarum YM-4-3 ( Lactobacillus plantarum YM-4-3 was deposited at the China Center for Type Culture Collection (CCTCC) on March 6, 2023, with accession number CCTCC NO: M2023253. Address: Luojia Mountain, Bayi Road, Wuchang District, Wuhan City, Hubei Province, China. Tel: 027-68754052.

[0008] Lactobacillus plantarum AY01 ( Lactobacillus plantarumAY01 was deposited at the China Center for Type Culture Collection on September 27, 2022, with accession number CCTCC NO:M 20221517. Address: Luojia Mountain, Bayi Road, Wuchang District, Wuhan City, Hubei Province, China. Tel: 027-68754052.

[0009] Pediococcus acidilactici LH01( Pediococcus acidilactici LH01 was deposited at the Guangdong Provincial Center for Microbial Culture Collection on September 26, 2023, with accession number GDMCCNo: 63840; the deposit address is: 5th Floor, Experimental Building, No. 100 Xianlie Middle Road, Yuexiu District, Guangzhou, Guangdong Province; contact number: 020-87137633.

[0010] Furthermore, the ratio of Bacillus subtilis SP-8-5, Lactobacillus plantarum YM-4-3, Lactobacillus plantarum AY01, and Pediococcus lactis LH01 is 2:1:1:1.

[0011] The present invention also provides a fermented yogurt, which is made by fermenting pure milk using a probiotic composition.

[0012] Furthermore, the preparation method of fermented yogurt is as follows: Measure 1000 mL of commercially available pure milk, pasteurize it, cool it to room temperature, and inoculate it with Bacillus subtilis SP-8-5 bacterial culture to a final concentration of 1.0 × 10⁻⁶. 7 ~1.0×10 8 The sample was incubated at CFU / mL on a shaker at 150 rpm and 37°C for 24 h for initial fermentation. After initial fermentation, *Lactobacillus plantarum* YM-4-3 bacterial culture was inoculated to bring the final concentration to 1.0 × 10⁻⁶. 8 -1.0×10 9 CFU / mL; then inoculate with Lactobacillus plantarum AY01 bacterial culture to a final concentration of 1.0 × 10⁻⁶ CFU / mL; 8 -1.0×10 9 CFU / mL; then inoculate with Pediococcus lactis LH01 bacterial culture to a final concentration of 1.0 × 10⁻⁶ CFU / mL; 8 -1.0×10 9 The concentration of CFU / mL was increased, and the mixture was stirred at 37°C for 48 hours. After fermentation, the mixture was placed in a refrigerator at 4°C for 24 hours to obtain fermented yogurt.

[0013] This invention also relates to the application of probiotic compositions in the preparation of products that regulate plasma metabolites, fecal metabolites, intestinal microbiota, and improve exercise indicators, wherein the probiotic compositions include Bacillus subtilis SP-8-5, Lactobacillus plantarum YM-4-3, Lactobacillus plantarum AY01, and Pediococcus lactis LH01.

[0014] Further, the exercise index includes aerobic / anaerobic training effect, blood lactic acid speed, heart rate variation threshold, internal / external load, and energy supply duration.

[0015] Further, the product is a sports supplement, which is fermented yogurt prepared by using pure milk as a substrate and using the probiotic composition for fermentation.

[0016] During preparation of the fermented yogurt, in addition to the bacterial liquid, a bacterial agent dosage form such as a freeze-dried composite bacterial powder can also be selected according to actual needs. Common adjuvants in the art can also be added.

[0017] The sports supplement can also use other fermentation substrates and use the probiotic composition of the present application for fermentation to prepare other fermented foods.

[0018] The present application also relates to application of the fermented yogurt in regulating plasma metabolites, fecal metabolites, intestinal microorganisms, and improving exercise index products, and the probiotic composition includes Bacillus subtilis SP-8-5, Lactobacillus plantarum YM-4-3, Lactobacillus plantarum AY01, and Pediococcus acidilactici LH01.

[0019] Further, the exercise index includes aerobic / anaerobic training effect, blood lactic acid speed, heart rate variation threshold, internal / external load, and energy supply duration.

[0020] The present application has the following beneficial effects: The fermented yogurt prepared by the present application can regulate intestinal microbial population composition, reduce harmful bacteria abundance, increase beneficial bacteria abundance, enhance intestinal immunity, and maintain intestinal barrier integrity of athletes by a specific compounding method. At the same time, the fermented yogurt has a positive improvement effect on plasma amino acids, fecal metabolites, and exercise performance and other exercise-related indexes, and can be applied to athletes as an effective sports supplement. BRIEF DESCRIPTION OF DRAWINGS

[0021] Figure 1 FIG. 3 is a PCA plot of plasma metabolomics analysis of the probiotic group and the control group in Example 3 3.1; Figure 2 FIG. 4 is a PLS-DA plot of plasma metabolomics analysis of the probiotic group and the control group in Example 3 3.1; Figure 3 FIG. 5 is an OPLS-DA plot of plasma metabolomics analysis of the probiotic group and the control group in Example 3 3.1; Figure 4 FIG. 6 is a heat map of plasma amino acid distribution of the probiotic group and the control group in Example 3 3.1; Figure 5 FIG. 7 is a PCA plot of fecal metabolomics analysis of the probiotic group and the control group in Example 3 3.2; Figure 6PLS-DA plot for fecal metabolomics analysis of probiotic group and control group in Example 3.2; Figure 7 OPLS-DA plot for fecal metabolomics analysis of probiotic group and control group in Example 3.2; Figure 8 Volcano plot for PGW0 vs CGW0 fecal metabolite composition in Example 3.2.2; Figure 9 Volcano plot for PGW2 vs CGW2 fecal metabolite composition in Example 3.2.2; Figure 10 Volcano plot for PGW3 vs CGW3 fecal metabolite composition in Example 3.2.2; Figure 11 Volcano plot for PGW3 vs CGW4 fecal metabolite composition in Example 3.2.2; Figure 12 Volcano plot for PGW2 vs PGW0 fecal metabolite composition in Example 3.2.2; Figure 13 Volcano plot for PGW3 vs PGW0 fecal metabolite composition in Example 3.2.2; Figure 14 Volcano plot for PGW4 vs PGW0 fecal metabolite composition in Example 3.2.2; Figure 15 Plot for gut microbiota composition analysis in Example 3.3; Figure 16 Plot for comparison of aerobic and anaerobic training effects in probiotic group and control group in Example 4 (C group represents control group, T group represents probiotic group); Figure 17 Plot for blood lactate speed in Example 4 (C represents control group, T represents probiotic group); Figure 18 Plot for heart rate variability threshold in Example 4 (C represents control group, T represents probiotic group); Figure 19 Plot for external load vs. internal load in Example 4 (T-E group represents probiotic group external load, C-E group represents control group external load, T-I group represents probiotic group internal load, C-I group represents control group internal load); Figure 20 Plot for energy supply duration in different intensity intervals in Example 4 (C group represents control group, E group represents probiotic group). DETAILED DESCRIPTION

[0022] The specific embodiments of the present application will be further described below with reference to the drawings. It should be noted that the description of these embodiments is intended to help understand the present application, but does not constitute a limitation of the present application. In addition, the technical features involved in the various embodiments of the present application described below can be combined with each other as long as they do not conflict with each other.

[0023] Example 1 A probiotic composition comprising Bacillus subtilis SP-8-5, Lactobacillus plantarum YM-4-3, Lactobacillus plantarum AY01, Pediococcus acidilactici LH01.

[0024] Wherein Bacillus subtilis SP-8-5, Lactobacillus plantarum YM-4-3, Lactobacillus plantarum AY01, Pediococcus acidilactici LH01 have been preserved.

[0025] Bacillus subtilis SP-8-5 Bacillus subtilis SP-8-5) has been preserved in the China Center for Type Culture Collection on March 6, 2023, with a preservation number of CCTCC NO: M2023254, and an address of Luojia Mountain, Bayi Road, Wuchang District, Wuhan City, Hubei Province, with a contact number of 027-68754052.

[0026] Lactobacillus plantarum YM-4-3 Lactobacillus plantarum YM-4-3) has been preserved in the China Center for Type Culture Collection on March 6, 2023, with a preservation number of CCTCC NO: M2023253, and an address of Luojia Mountain, Bayi Road, Wuchang District, Wuhan City, Hubei Province, with a contact number of 027-68754052.

[0027] Lactobacillus plantarum AY01 Lactobacillus plantarum AY01) has been preserved in the China Center for Type Culture Collection on September 27, 2022, with a preservation number of CCTCC NO: M20221517, and an address of Luojia Mountain, Bayi Road, Wuchang District, Wuhan City, Hubei Province, with a contact number of 027-68754052.

[0028] Pediococcus acidilactici LH01 Pediococcus acidilactici LH01) has been preserved in the Guangdong Microbial Culture Collection Center on September 26, 2023, with a preservation number of GDMCCNo: 63840, and an address of Experimental Building 5th Floor, No. 100, Martyrs Road, Yuexiu District, Guangzhou City, Guangdong Province, with a contact number of 020-87137633.

[0029] Specifically, the ratio of Bacillus subtilis SP-8-5, Lactobacillus plantarum YM-4-3, Lactobacillus plantarum AY01, Pediococcus acidilactici LH01 is 2:1:1:1.

[0030] Example 2 A fermented yogurt is prepared as follows: 1000 mL of commercially available pure milk is measured, pasteurized, and cooled to room temperature, and Bacillus subtilis SP-8-5 bacterial solution is added to make the final concentration 1.0 x 10 7 ~1.0×10 8 CFU / mL, and placed in a shaking bed for preliminary fermentation at 150 rpm and 37°C for 24 h; after the preliminary fermentation is completed, Lactobacillus plantarum YM-4-3 bacterial solution is added to make the final concentration 1.0 x 10 8 -1.0×10 9 CFU / mL; then Lactobacillus plantarum AY01 bacterial solution is added to make the final concentration 1.0 x 10 8 -1.0×10 9 CFU / mL; then Pediococcus acidilactici LH01 bacterial solution is added to make the final concentration 1.0 x 10 8 -1.0×10 9 CFU / mL, and then mixed and fermented at 37°C for 48 h; after being taken out, it is placed in a refrigerator at 4°C for fermentation for 24 h, and then the fermented yogurt is obtained.

[0031] The bacterial solution used is prepared as follows: Bacillus subtilis SP-8-5 bacterial solution preparation: Bacillus subtilis SP-8-5 stored at low temperature is taken out, inoculated in NB liquid medium at an inoculation amount of 4‰, and placed in a constant temperature incubator at 37°C for static culture for 24 h and then subcultured once more; then the bacterial solution activated twice and in the logarithmic growth phase is inoculated in 50 mL of NB liquid medium for expansion culture, the concentration of the cultured bacterial solution is calculated by viable bacterial counting method, and the concentration is adjusted to 1 x 10 7 ~1×10 9 CFU / mL for subsequent experiments.

[0032] Lactobacillus plantarum YM-4-3 bacterial solution preparation: Lactobacillus plantarum YM-4-3 stored at low temperature is taken out, inoculated in MRS liquid medium at an inoculation amount of 4‰, and placed in a constant temperature incubator at 37°C for static culture for 24 h and then subcultured once more; then the bacterial solution activated twice and in the logarithmic growth phase is inoculated in 50 mL of MRS liquid medium for expansion culture, the concentration of the cultured bacterial solution is calculated by viable bacterial counting method, and the concentration is adjusted to 1 x 10 9 ~1×10 11 CFU / mL for subsequent experiments.

[0033] Lactobacillus plantarum AY01 bacterial solution preparation: the method is the same as that for YM-4-3 bacterial solution preparation, except that the bacterial strain is replaced by Lactobacillus plantarum AY01.

[0034] Lactococcus lactis LH01 bacterial liquid preparation: the method is the same as that of YM-4-3 bacterial liquid preparation, except that the strain is replaced by Lactococcus lactis LH01.

[0035] MRS liquid medium: 10 g of proteose peptone, 8 g of beef powder, 4 g of yeast powder, 2 g of potassium phosphate dibasic, 5 g of sodium acetate, 2 g of triammonium citrate, 0.2 g of magnesium sulfate, 0.05 g of manganese sulfate, 20 g of glucose, 1 mL of Tween-80, and distilled water are added to make up to 1000 mL, and then mixed, and the pH is adjusted to 6.2±0.2.

[0036] NB liquid medium: 10 g of proteose peptone, 3 g of beef powder, 5 g of sodium chloride, and 1 g of glucose are added to make up to 1000 mL, and then mixed, and the pH is adjusted to 7.4±0.1.

[0037] The application also relates to the application of a probiotic composition in regulating plasma metabolites, fecal metabolites, intestinal microorganisms and improving exercise index products, wherein the probiotic composition comprises Bacillus subtilis SP-8-5, Lactobacillus plantarum YM-4-3, Lactobacillus plantarum AY01 and Lactococcus lactis LH01.

[0038] Specifically, the exercise index includes aerobic / anaerobic training effect, blood lactic acid speed, heart rate variation threshold, internal / external load and energy supply duration.

[0039] Specifically, the product is a sports supplement, which is fermented yogurt prepared by using the probiotic composition to ferment pure milk.

[0040] During the preparation of the fermented yogurt, in addition to the bacterial liquid, a bacterial agent dosage form such as freeze-dried composite bacterial powder can also be selected according to actual needs. Common auxiliary materials in the field can also be added.

[0041] The sports supplement can also select other fermentation substrates, and other fermented foods can be prepared by using the probiotic composition of the application to ferment.

[0042] The application also relates to the application of fermented yogurt in regulating plasma metabolites, fecal metabolites, intestinal microorganisms and improving exercise index products, wherein the probiotic composition comprises Bacillus subtilis SP-8-5, Lactobacillus plantarum YM-4-3, Lactobacillus plantarum AY01 and Lactococcus lactis LH01.

[0043] Specifically, the exercise index includes aerobic / anaerobic training effect, blood lactic acid speed, heart rate variation threshold, internal / external load and energy supply duration.

[0044] In order to verify the application effect of the fermented yogurt, the following test is carried out: Example 3 Plasma amino acid and fecal metabolite detection The participants included 10 triathletes, 6 men and 4 women. One week prior to the start of the experiment, all participants followed a standardized diet and training regimen. All participants were randomly assigned to either the probiotic group (PG group, n = 5) or the control group (CG group, n = 5). The diet and training regimen remained standardized throughout the experiment. Participants consumed 150g of yogurt daily after dinner and before bedtime. The yogurt consumed by the probiotic group was the fermented yogurt prepared in Example 2; the yogurt consumed by the control group was the control yogurt. The control yogurt was a commercially available formula composed of two strains: Lactobacillus bulgaricus and Streptococcus thermophilus. The experiment lasted for 4 weeks.

[0045] Collection of plasma and stool samples Plasma: 5 mL of venous blood was collected using a heparin sodium blood collection tube under fasting conditions, and the plasma was centrifuged at 4°C and 3000 rpm for 10 min.

[0046] Feces: Fecal samples were continuously collected and refrigerated throughout the experiment.

[0047] 3.1 Plasma amino acid detection The test data consisted of six groups: probiotic group week 0 PGW0, probiotic group week 2 PGW2, probiotic group week 3 PGW3, control group week 0 CGW0, control group week 2 CGW2, and control group week 3 CGW3.

[0048] The detection method for each set of data is as follows: Take 50 μl of plasma sample and mix with 200 μL of pre-cooled acetonitrile / methanol (1:1 volume ratio) solution containing 1.25% formic acid. Vortex for 30 s, then sonicate in an ice bath for 30 min. Incubate at -20℃ for 1 h to precipitate proteins. Centrifuge at 14000 rcf / min for 20 min at 4℃, collect the supernatant, add 37 μL of Na2CO3 (200 mg / mL), and vortex for 60 min. Add another 37 μL of Na2CO3 (200 mg / mL) to the supernatant and vortex, then add 50 μL of DSCl solution (10 mg / mL). React on a shaker at 30℃ for 60 min, then add 1 μL of 50% formic acid aqueous solution to adjust the pH to 5. Centrifuge at 4℃ for 5 min, and collect the supernatant for analysis.

[0049] Differential metabolites were screened as potential biomarkers based on p < 0.05, importance of projected variables (VIP) > 1, and |log2FC| > 0. Principal component analysis (PCA), partial least squares discriminant analysis (PLS-DA), and orthogonal partial least squares discriminant analysis (OPLS-DA) were used to analyze plasma amino acids. The PCA plot is shown below. Figure 1 As shown, the PLS-DA diagram is as follows:Figure 2 As shown in Figure 2, OPLS-DA as shown in Figure 3. Figure 3 As shown in Figure 2, OPLS-DA as shown in Figure 3. Figures 1-3 As can be seen from Table 2, there are obvious differences in the plasma metabolite composition between the probiotic group and the control group.

[0050] The detailed information and specific distribution of plasma amino acids in all samples are shown in Table 3. Figure 4 The results show that after two weeks of intervention, the distribution of major amino acids changes significantly, and the content of most amino acids shows a downward trend. The level of tryptophan in the probiotic group decreases and reaches the lowest at two weeks of intervention, and starts to rise at three weeks. As an important amino acid, tryptophan helps to regulate sleep and mood. In the recovery of exercise, tryptophan can help to reduce fatigue and stress after exercise, improve psychological state, and thus promote the recovery of the body. In addition, tryptophan can also increase the level of melatonin in the brain, which helps to regulate the sleep-wake cycle, improve sleep quality, and further promote the recovery of exercise. Although the level of tryptophan in the probiotic group shows a downward trend at the beginning, it shows an upward trend at the third week. It can be speculated that the consumption of the yogurt of the present application may start to work from the third week to the fourth week.

[0051] 3.2 Fecal metabolite detection There are six groups of detection data, which are: probiotic group zero week PGW0, probiotic group second week PGW2, probiotic group third week PGW3, control group zero week CGW0, control group second week CGW2, and control group third week CGW3.

[0052] The detection method of each group of data is as follows: Take 50 μl of fecal sample and 200 μL of pre-cooled acetonitrile / methanol (1:1 by volume) solution containing 1.25% formic acid, vortex for 30 s, then ultrasonic in ice bath for 30 min. Incubate at -20℃ for 1 h to precipitate proteins. After centrifugation at 4℃, 14000rcf / min for 20 min, the supernatant is taken, 37 μL Na2CO3 (200 mg / mL) is added and vortexed for 60 min. Add 37 μL Na2CO3 (200 mg / mL) to the supernatant, vortex, then add 50 μL DSCl solution (10 mg / mL), react for 60 min on a constant temperature shaker at 30℃, then add 1 μL 50% formic acid solution to adjust the pH of the solution to 5. Centrifuge at 4℃ for 5 min, take the supernatant and detect on the machine.

[0053] Fecal metabolite results: According to the metabolomics analysis, there were significant differences in the composition of intestinal metabolites in the fecal samples of athletes in the two groups (probiotic group and control group) within four weeks. Based on p<0.05, projection variable importance (VIP)>1 and |log2FC|>1, the differential metabolites were screened as potential biomarkers. Principal component analysis (PCA), partial least squares discriminant analysis (PLS-DA) and orthogonal partial least squares discriminant analysis (OPLS-DA) were used to analyze the fecal metabolites, as shown in the PCA graph Figure 5 , the PLS-DA graph Figure 6 , and the OPLS-DA Figure 7 .

[0054] Figures 5-7 It is shown that there is a significant difference between the probiotic group and the control group, indicating that consuming the fermented yogurt of the present application can affect the composition of fecal metabolites. The volcano graph Figure 8 is the PGW0 and CGW0 fecal metabolite composition volcano graph, Figure 9 is the PGW2 and CGW2 fecal metabolite composition volcano graph, Figure 10 is the PGW3 and CGW3 fecal metabolite composition volcano graph, Figure 11 is the PGW3 and CGW4 fecal metabolite composition volcano graph, Figure 12 is the PGW2 and PGW0 fecal metabolite composition volcano graph, Figure 13 is the PGW3 and PGW0 fecal metabolite composition volcano graph, Figure 14 is the PGW4 and PGW0 fecal metabolite composition volcano graph. Blue indicates a decrease, and red indicates an increase. Analysis shows that probiotic intervention increases the types of differential metabolites.

[0055] I. Up-regulated metabolites related to exercise after probiotic intervention: 1. δ-valerolactam: may be involved in intestinal flora metabolism-related pathways.

[0056] 2. Docosahexaenoic acid ethyl ester: helps anti-inflammatory and recovery after exercise.

[0057] 3. Palmitoleic acid: involved in energy metabolism, assisting in exercise recovery.

[0058] 4. Lithocholic acid: involved in intestinal digestion and metabolic regulation.

[0059] 5. Nicotinic acid: involved in energy metabolism and maintenance of intestinal health.

[0060] 6. Acetylcholine: a neurotransmitter that may regulate sleep and intestinal peristalsis.

[0061] 7. Leucine proline: involved in intestinal absorption and protein metabolism.

[0062] 8. Imidazoleacetic acid: Histidine metabolite, possibly involved in gut immune regulation.

[0063] 9. Thymine: Involved in nucleic acid synthesis, supports cell repair.

[0064] 10. Thymidine: Involved in DNA synthesis, aids in post-exercise tissue repair.

[0065] 11. Pantothenic acid: Involved in energy metabolism and gut health.

[0066] 12. 12-ketolauric acid: Bile acid derivative, regulates gut microbiota and digestion.

[0067] 13. Threonine: Essential amino acid, involved in protein synthesis, supports exercise recovery.

[0068] 14. 3-(4-Hydroxyphenyl)propionic acid: Polyphenol metabolite, possibly has anti-inflammatory effects, aids in recovery.

[0069] II. Metabolites downregulated after probiotic intervention related to exercise: 1. Palmitic acid: A saturated fatty acid, excess can impact metabolism, downregulation can benefit post-exercise lipid metabolism balance.

[0070] 2. Dimethylglycine-γ-linolenoyl ethanolamide: Fatty acid amide, possibly involved in inflammation regulation, downregulation can be related to recovery phase.

[0071] 3. Arachidonic acid: Polyunsaturated fatty acid, excess can trigger inflammation, downregulation benefits post-exercise anti-inflammatory.

[0072] 4. Adrenic acid: Polyunsaturated fatty acid, downregulation can reduce inflammatory response.

[0073] 5. Benzoic acid: Intestinal microbiota metabolite, downregulation can reflect changes in gut environment.

[0074] 3.3 Fecal microbiome sequencing Genomic DNA of the collected feces during the extraction experiment (DNA extraction used a fecal genomic extraction kit) was used to amplify the V3-V4 region of the 16S rRNA gene using primers 515F and 909R; the reaction system was: DNA template 100 ng, upstream and downstream primers (10 μmol / L) 2 μL each, 2x Taq Master Mix (Novozyme) 25 μL. The reaction program was: 95°C pre-denaturation for 3 min, 95°C denaturation for 15 s, 55°C annealing for 15 s, 72°C extension for 30 s, a total of 30 cycles, and finally 72°C final extension for 10 min. The PCR product was used as a template for a second PCR, and the number of cycles was set to 5 to reduce the influence of non-specific amplification products. The PCR product after the second amplification was sent to Shanghai Yuanxu Biotechnology Co., Ltd. for Illumina second-generation sequencing.

[0075] At the time of intervention for 3 weeks, high-throughput sequencing was performed on the 16S rRNA isolated from the fecal bacterial DNA of the athletes, as shown in Figure 15 The results show that in the first and second weeks after eating the yogurt, although the abundance of the beneficial bacteria phylum Firmicutes decreases, in the fourth week, the abundance of Firmicutes rebounds, and the probiotic group rebounds faster than the control group. Similarly, although the abundance of the harmful bacteria phylum Actinobacteria increases in the first and second weeks, in the fourth week, the abundance of Actinobacteria decreases, and the probiotic group decreases faster than the control group. It is shown that the yogurt eaten starts to work from the third week to the fourth week, and the fermented yogurt of the present application has a positive effect on the intestinal flora, can reduce the abundance of harmful bacteria phylum of the human intestinal flora, and increase the abundance of beneficial bacteria phylum. By increasing the proportion of beneficial bacteria and reducing the proportion of harmful bacteria, the intestinal flora imbalance can be improved, and the intestinal flora healthy balance can be maintained.

[0076] Example 4: Monitoring of sports performance To evaluate sports performance, physiological biomarkers and advanced monitoring techniques were integrated. The FIRSTBEAT heart rate system (sampling rate 1000HZ) was used to collect the raw data of HRV (heart rate variability) of the experimental group and the control group during the training class (1 week before intervention + four weeks after intervention) Figure 18The system acquires complete training duration data via sensors, recording dynamic RR interval data. This data is then scanned using an artifact detection filter to initially correct for false detections, omissions, and premature heartbeats. Linear interpolation is then used to resample the continuous artifact-corrected RR intervals at a rate of 5 Hz to obtain an equidistant time series. From the resampled data, the software uses a polynomial filter and a digital FIR bandpass filter (0.03-1.2Hz) to remove low-frequency trends and variance below and above the band of interest. The system's AI algorithm then derives an aerobic training effectiveness rating score (Aerobic TE) (range 0-5), an anaerobic training effectiveness rating score (Anaerobic TE) (range 0-5), RR interval values, and internal and external load values ​​(Trimp).

[0077] Firstbeat Sports' TRIP calculation system is based on Banister's original TRIP algorithm, with some improvements. Unlike traditional methods, this system no longer uses average heart rate data during training, but instead updates the TRIP score more frequently by monitoring real-time heart rate intervals. This innovative design allows for a more accurate assessment of the intensity of interval training load. Furthermore, Firstbeat has specifically lowered the cumulative intensity threshold for TRIP scores, ensuring that the score value comes solely from performance during actual training. The TRIP calculation formula is: ; In the formula: T represents the duration of the exercise; HRex represents heart rate during exercise; HRrest is the resting heart rate; HRmax is the maximum heart rate.

[0078] From the heart rate variability threshold ( Figure 18 The comparison shows that, starting from the third week of intervention, the difference in heart rate variability between the probiotic group and the control group became more and more obvious. The probiotic group intervened in the control group, indicating that the probiotic group may have a stronger ability to withstand the load, a better recovery level, and be in a more balanced training-recovery state.

[0079] The comparison chart of aerobic and anaerobic training effects between the probiotic group and the control group is shown below. Figure 16 As shown. From Figure 16 As can be seen, the aerobic training effect of the probiotic group was slightly lower than that of the control group, while the anaerobic training effect was higher than that of the control group (Group C represents the control group, and Group T represents the probiotic group).

[0080] The comparison chart of external load and internal load is shown below. Figure 19 As shown. From Figure 19As can be seen, the external load in the probiotic group peaked in the second week after intervention, then gradually decreased over the next two weeks, while the control group reached its lowest value in the second week, followed by an increase. The internal load trends in both the probiotic and control groups were similar to those of the external load, but the magnitude of change was not particularly significant. In the second week after intervention, the internal loads of the probiotic and control groups were relatively similar, but the external load of the probiotic group was significantly higher than that of the control group, indicating that the probiotic group completed higher quality training content. In the third and fourth weeks after intervention, as the training load decreased, the internal load of the probiotic group was lower than that of the control group, making it relatively easier to complete the training content.

[0081] In specialized running training (1km*10), blood lactate levels were collected during the fastest running session, and speeds were recorded under synchronized conditions. A blood lactate-speed scatter plot was then created, as shown below. Figure 17 As shown. All data were statistically analyzed using SPSS Statistics 26, and graphs were generated using Origin software.

[0082] from Figure 17 As can be seen from the data, the speed and blood lactate scattering points of the probiotic group were mostly located in the fourth quadrant, suggesting that athletes in the probiotic group could achieve higher running speeds with relatively lower blood lactate levels, and their aerobic capacity was significantly improved. When achieving higher running speeds, the proportion of anaerobic energy supply may be more involved, thus resulting in a higher anaerobic training effect.

[0083] The duration of energy supply in different intensity zones was tested: 90%-100% HRmax (high-intensity training zone), 80%-90% HRmax (anaerobic training zone), 70-80% HRmax (aerobic training zone 2), 60%-70% HRmax (aerobic training zone 1), and 50-60% HRmax (recovery training zone). A comparison chart of energy supply duration in different intensity zones is shown below. Figure 20 As shown.

[0084] from Figure 20 It can be seen that in the first week after intervention, the duration of >90% intensity and 80%-90% intensity in the probiotic group was shorter than that in the control group, indicating a shorter duration of anaerobic energy supply in the probiotic group. However, from... Figure 16 The evaluation of anaerobic effects showed that the probiotic group was better than the control group, indicating that the probiotic group had better anaerobic energy supply efficiency.

[0085] The embodiments of the present invention have been described in detail above with reference to the accompanying drawings, but the present invention is not limited to the described embodiments. For those skilled in the art, various changes, modifications, substitutions, and variations can be made to these embodiments without departing from the principles and spirit of the present invention, and these variations still fall within the protection scope of the present invention.

Claims

1. Use of a probiotic composition for the preparation of a product for modulating plasma metabolites, faecal metabolites, gut microbiota, and improving exercise markers, characterised in that: The probiotic composition comprises Bacillus subtilis SP-8-5, Lactobacillus plantarum YM-4-3, Lactobacillus plantarum AY01 and Pediococcus acidilactici LH01.

2. The application according to claim 1, wherein the exercise indicators comprise aerobic / anaerobic training effect, blood lactic acid speed, heart rate variation threshold, internal / external load and energy supply duration.

3. Use according to claim 1, characterized in that: The product is a sports supplement.

4. Use according to claim 3, characterized in that: The sports supplement is fermented yogurt fermented by the probiotic composition using pure milk as a substrate.

5. A probiotic composition, characterized in that: The probiotic composition comprises Bacillus subtilis SP-8-5, Lactobacillus plantarum YM-4-3, Lactobacillus plantarum AY01 and Pediococcus acidilactici LH01.

6. The probiotic composition according to claim 5, characterized in that: The ratio of Bacillus subtilis SP-8-5, Lactobacillus plantarum YM-4-3, Lactobacillus plantarum AY01 and Pediococcus acidilactici LH01 is 2:1:1:

1.

7. A fermented yoghurt, characterized in that, The product is fermented by the probiotic composition of claim 4 or 5 using pure milk as a substrate.

8. The fermented yoghurt according to claim 7, characterized in that: The preparation method is as follows: The pure milk is sterilized and cooled to room temperature, then Bacillus subtilis SP-8-5 bacterial solution is inoculated and placed on a shaking table for preliminary fermentation; after the preliminary fermentation is completed, Lactobacillus plantarum YM-4-3 bacterial solution, Lactobacillus plantarum AY01 bacterial solution and Pediococcus acidilactici LH01 bacterial solution are inoculated in sequence, then mixed and continue to ferment; after being taken out, it is placed in a refrigerator to obtain fermented yogurt.

9. Use of the fermented yoghurt according to claim 7 or 8 for modulating plasma metabolites, faecal metabolites, gut microbiota and for improving exercise markers, characterized in that: The probiotic composition comprises Bacillus subtilis SP-8-5, Lactobacillus plantarum YM-4-3, Lactobacillus plantarum AY01 and Pediococcus acidilactici LH01.

10. The application according to claim 9, wherein the exercise indicators comprise aerobic / anaerobic training effect, blood lactic acid speed, heart rate variation threshold, internal / external load and energy supply duration.

Images