Complex probiotics capable of increasing exercise tolerance and resisting exercise fatigue and application of complex probiotics

Abstract

The invention discloses a compound probiotic capable of increasing exercise tolerance and resisting exercise fatigue and application of the compound probiotic. The compound probiotic comprises lactobacillus plantarum SHY21-2 and lactobacillus fermentum SHY10, the preservation number of the lactobacillus plantarum is CCTCC NO: M 2020043, and the preservation number of the lactobacillus fermentum is CCTCC NO: M 2020042. The invention further discloses a preparation method of the compound probiotic capable of increasing exercise tolerance and resisting exercise fatigue. The invention discloses the compound probiotics capable of accurately regulating and controlling the mitochondrial homeostasis of the gastrocnemius muscle for the first time, and the compound probiotics can remarkably improve exercise tolerance and relieve exercise fatigue.

Description

Technical Field

[0001] The present invention relates to the technical field of biomedicine, and in particular to a composite probiotic for increasing sports endurance and resisting sports fatigue and an application thereof. Background Art

[0002] Sports fatigue refers to a physiological phenomenon in which "the body's physiological processes cannot maintain normal functional levels or predetermined exercise intensity." Sports fatigue can easily lead to sports injuries, directly affect normal exercise performance, and reduce the body's athletic ability. At present, research on the mechanism of sports fatigue mainly involves energy depletion theory, metabolite accumulation theory, and free radical theory, among which the theory of reactive oxygen free radicals is the most widely studied. Studies have shown that under high-intensity exercise, mitochondria are in a hyperoxic state, and the oxidoreductases in the electron transfer chain are in an abnormally reduced state, resulting in a significant increase in reactive oxygen generation and a decrease in energy generation. At the same time, calcium ion overload in mitochondria changes the permeability and fluidity of the mitochondrial membrane, ultimately leading to cell death by apoptosis, which in turn causes sports fatigue and tissue damage. Therefore, exploring safe and effective strategies to improve sports fatigue is of great significance for improving human athletic ability and promoting the development of competitive sports.

[0003] The intestinal flora refers to all microorganisms present in the gastrointestinal tract, including bacteria, viruses, fungi and protozoa. The intestinal flora in the human body consists of about 10 13 to 10 14 The intestinal flora is composed of cells, and each individual has as many as 100 to 1,000 species of bacteria. Studies have shown that compared with normal mice, the "pseudo-germ-free mice" treated with antibiotics have significantly decreased exercise capacity and obvious muscle damage. Further studies have found that high-intensity exercise significantly reduced the expression level of intestinal tight junction proteins, reduced the diversity and richness of intestinal flora, and changed the composition of the flora. Although the relationship between intestinal flora and exercise fatigue has not been fully elucidated, many studies have shown that the metabolites of intestinal flora can affect the function and biosynthesis of mitochondria. Therefore, targeting mitochondria may be an effective strategy to improve exercise performance and improve exercise fatigue.

[0004] Probiotics are a type of active microorganisms that can have beneficial effects on the host when ingested in sufficient quantities. They are currently an effective way to regulate intestinal flora and intestinal metabolism. Exploring probiotic strains that can target mitochondria and improve sports fatigue is of great significance to the development of functional nutritional supplements, and has broad market prospects. Summary of the invention

[0005] The purpose of the present invention is to provide a composite probiotic for increasing sports endurance and resisting sports fatigue and its application in view of the above problems.

[0006] In order to achieve its purpose, the present invention adopts the following technical solution:

[0007] The first aspect of the present invention provides a composite probiotic for increasing sports endurance and resisting sports fatigue, comprising Lactobacillus plantarum SHY21-2 and Lactobacillus fermentum SHY10, wherein the Lactobacillus plantarum has a preservation number of CCTCCNO: M 2020043, and the Lactobacillus fermentum has a preservation number of CCTCCNO: M 2020042.

[0008] In the composite probiotics, the ratio of the live bacteria count of Lactobacillus plantarum SHY21-2 to that of Lactobacillus fermentum SHY10 is 1-5:1-5 (preferably 1-3:1-3 or 1-2:1-2).

[0009] Preferably, the composite probiotic is a mixture of freeze-dried powder of Lactobacillus plantarum SHY21-2 and freeze-dried powder of Lactobacillus fermentum SHY10 in a mass ratio of 1-5:1-5 (preferably 1-3:1-3 or 1-2:1-2).

[0010] The second aspect of the present invention provides a microbial agent comprising the above-mentioned composite probiotics.

[0011] The third aspect of the present invention provides the use of the above-mentioned composite probiotics or the above-mentioned microbial agent in the preparation of a product having the effects of increasing sports endurance and resisting sports fatigue.

[0012] Preferably, the product is food, food additive, health product, feed, feed additive, reagent or medicine.

[0013] The fourth aspect of the present invention provides a product having the effects of increasing sports endurance and resisting sports fatigue, wherein the active ingredients thereof include the above-mentioned composite probiotics or the above-mentioned microbial agent.

[0014] Preferably, the product is food, food additive, health product, feed, feed additive, reagent or medicine.

[0015] Preferably, the product further includes excipients; the excipients are selected from any one or a combination of at least two of fillers, binders, wetting agents, disintegrants, emulsifiers, cosolvents, solubilizers, osmotic pressure regulators, colorants, pH regulators, antioxidants, antibacterial agents or buffers.

[0016] The beneficial effects of the present invention are:

[0017] After being ingested by a subject, the composite probiotic of the present invention can significantly upregulate the gene expression of the key genes PGC-1α, Mfn2, Fis1, PINK1 and Parkin in the gastrocnemius mitochondrial homeostasis, and significantly prolong the exhaustive running time and the weighted swimming time. The composite probiotic can be used to prepare products (such as health products, medicines, etc.) that increase sports endurance and relieve sports fatigue.

[0018] Compared with the prior art, the present invention discloses for the first time a compound probiotic that can precisely regulate the homeostasis of gastrocnemius mitochondria. After intervention with the compound probiotic, the exhaustion running time and weighted swimming time of mice increased by at least 20% compared with the probiotics reported in existing patent applications, significantly improving exercise endurance and relieving exercise fatigue. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 Effects of probiotics on running endurance in mice.

[0020] Figure 2 The effect of probiotics on the weighted swimming time of mice.

[0021] Figure 3 To investigate the effects of probiotics on the expression of key genes in mitochondrial biogenesis of gastrocnemius muscle.

[0022] Figure 4 To investigate the effects of probiotics on the expression of key genes in gastrocnemius muscle mitochondrial dynamics.

[0023] Figure 5 To investigate the effect of probiotics on the expression of key genes of mitochondrial autophagy in gastrocnemius muscle.

[0024] Above Figures 1 to 5 There are no significant differences between the groups marked with the same lowercase letters (p>0.05); there are significant differences between the groups marked with different lowercase letters (p<0.05). DETAILED DESCRIPTION

[0025] The present invention will be further described below in conjunction with embodiments, but the present invention is not limited thereto.

[0026] The experimental methods in the following examples are conventional methods unless otherwise specified.

[0027] The deposit information of Lactobacillus plantarum SHY21-2 and Lactobacillus fermentum SHY 10 in the embodiments of the present invention is as follows:

[0028] The Lactobacillus plantarum strain SHY21-2 was sent to the China Center for Type Culture Collection (CCTCC) for preservation in January 2020, with the address at Luojia Mountain, Bayi Road, Wuchang District, Wuhan City, Hubei Province. The preservation date is January 13, 2020, the preservation number is CCTCCNO: M 2020043, and the classification name is: Lactobacillus plantarum SHY21-2 Lactobacillus plantarum SHY21-2. The Lactobacillus plantarum strain SHY21-2 has been disclosed in Chinese patent ZL202110069093.3 (authorization announcement number CN112760253 B).

[0029] The fermented Lactobacillus strain SHY10 was sent to the China Center for Type Culture Collection (CCTCC) for preservation in January 2020. The address is Wuhan University, No. 299 Bayi Road, Wuchang District, Wuhan City, Hubei Province. The preservation date is January 13, 2020. The preservation number is CCTCCNO: M 2020042. The classification name is: fermented Lactobacillus SHY10

[0030] Lactobacillusfermentum SHY10. Lactobacillus fermentum SHY10 has been disclosed in Chinese patent ZL202110069095.2 (authorization announcement number CN 113025518 B).

[0031] Example 1 Study on the effect and mechanism of compound probiotics in increasing exercise endurance and relieving exercise fatigue

[0032] 1 Experimental Materials

[0033] The experimental strains are Lactobacillus plantarum SHY21-2, with a preservation number of CCTCC NO: M 2020043, and Lactobacillus fermentum SHY10, with a preservation number of CCTCC NO: M 2020042. Freeze-dried bacterial powders of SHY21-2 and SHY10 are mixed in a mass ratio of 1:1 to obtain a composite probiotic.

[0034] The experimental animals were 7-week-old male ICR mice purchased from Hunan Slake Jingda Experimental Animal Co., Ltd. They were kept in a standardized laboratory with a room temperature of 25±2°C, a relative humidity of 50±5%, and 12h light / 12h dark. The experiment began after one week of adaptive feeding.

[0035] 2 Experimental methods

[0036] 2.1 Experimental animal grouping and treatment

[0037] Eighty ICR mice were randomly divided into a quiet group (SED), a quiet group + compound probiotics group (SED + PRO, the dose of each probiotic was 1×10 9cfu / kg·bw), quiet group + SHY 21-2 group (SED + SHY 21-2, probiotic dose 1×10 9 cfu / kg·bw), quiet group + SHY10 group (SED + SHY10, probiotic dose 1×10 9 cfu / kg·bw), running training group (RUNNING), running training + compound probiotics group (RUNNING+PRO, each probiotic dose was 1×10 9 cfu / kg·bw), RUNNING+SHY 21-2 group (RUNNING+SHY 21-2, probiotic dose 1×10 9 cfu / kg·bw), and the RUNNING+SHY10 group (RUNNING+SHY10, probiotic dose 1×10 9 cfu / kg·bw), 10 mice in each group. For the probiotic intervention group, probiotics were gavaged once a day according to the above corresponding doses for 7 consecutive weeks. The running training program for mice was as follows: in the first week, 12m / min, 35min, treadmill inclination 0 O , once a day; second week, 14m / min, 35min, treadmill inclination 0 O , once a day; third week, 16m / min, 35min, treadmill inclination 5 O , once a day; fourth week, 18m / min, 35min, treadmill inclination 0 O , once a day; Week 5, 20m / min, 35min, treadmill inclination 10 O , once a day; Week 6, 22m / min, 35min, treadmill inclination 10 O , once a day; Week 7, 24m / min, 35min, treadmill inclination 10 O , once a day. In the 8th week, the mice were tested for exercise endurance (running test and weight-bearing swimming test).

[0038] 2.2 Mouse running test

[0039] Before the running test, the SED group, SED+PRO, SED+SHY21-2 and SED+SHY10 groups were subjected to 2 days of adaptive training on the treadmill (speed of 10m / min). After the training, the test officially began. One hour after intragastric administration of bacterial solution, the mice were subjected to a running endurance test. One mouse was placed in each grid, and the mice started running at a speed of 14m / min, increasing by 2m / min until 30m / min, and maintained this speed until exhaustion, that is, unable to run for 10s, and the exhaustion time was recorded.

[0040] 2.3 Weighted swimming test in mice

[0041] After the running test, the mice rested for two days and then underwent a weight-bearing swimming test. All groups of mice were trained in a constant temperature water pool without weights for 2 days. After the training, the test officially began. The mice were subjected to a swimming test 1 hour after intragastric administration of the bacterial solution. The tail of the mouse in the constant temperature water pool was loaded with 5% of its own weight. The mouse was placed in the pool and the timing was started (the mouse was kept swimming all the time). The time when the mouse sank under the water for 8 seconds and could not float to the surface was taken as the exhaustion swimming time.

[0042] 2.4 RT-PCR determination of the expression of key genes in gastrocnemius mitochondrial homeostasis

[0043] After the experiment, the gastrocnemius muscle tissue of the mouse calf was collected, washed with PBS, and stored at -80℃ for future use. 100mg gastrocnemius muscle tissue was homogenized with 1mL Trizol. The tissue homogenate was transferred to a 1.5mL centrifuge tube, 200μL chloroform was added in a fume hood, and the mixture was centrifuged at 14000r / min and 4℃ for 15min. The supernatant was collected, and an equal volume of isopropanol was added, and the mixture was placed at 4℃ for 15min after mixing, and then centrifuged at 14000r / min and 4℃ for 20min. After the centrifugation, the supernatant was discarded to obtain RNA precipitation. 75% ethanol solution was added to the RNA precipitation for washing, and then centrifuged at 14000r / min and 4℃ for 15min. After the centrifugation, the supernatant was discarded, the centrifuge tube cap was opened, and it was left to air for 3-5min, and 20μLRNase-Free water was added to dissolve the RNA. According to the instructions of the Thermo Fisher RevertAid First Strand cDNA Synthesis Kit, RNA was reverse transcribed into cDNA. After determining the concentration and purity of cDNA, it was diluted to 1 μg / μL. After mixing 1 μL cDNA, 10 μL Master Mix, 1 μL upstream primer, 1 μL downstream primer and 7 μL ddH2O in an eight-tube strip, cover the eight-tube strip with a cap, centrifuge briefly and place on a StepOnePlus Real-Time PCR instrument. After pre-denaturation at 95°C for 15 minutes, perform 40 cycles at 95°C, 15s, 60°C, 1min, and 72°C, 30s. Using GAPDH as the internal reference, according to formula 2 -ΔΔCT Calculate the relative mRNA expression of each target gene. The primer sequence information is shown in Table 1.

[0044] Table 1 Primer sequences used in the experiment

[0045]

[0046] 3 Experimental results and analysis

[0047] 3.1 Effects of compound probiotics on running endurance of mice

[0048] Exhaustive running time is an important indicator for evaluating the exercise endurance of mice. Figure 1 As shown in the results, running training significantly prolonged the exhaustive running time of mice compared with the quiet control group (SED) mice. After the intervention of compound probiotics, the exhaustive running time of mice in the SED group was further significantly prolonged (52.00±2.22min). In addition, the administration of compound probiotics on the basis of running training also significantly enhanced the exhaustive running time (65.00±2.89min). Moreover, whether it was the quiet group or the running training group, the exhaustive running time of mice after the intervention of the compound probiotics SHY 21-2+SHY10 was significantly higher than the effect of SHY 21-2 or SHY10 alone. These results show that compound probiotics can not only improve the exercise endurance of SED mice, but also show a more significant promoting effect on mice that have undergone running training.

[0049] 3.2 Effect of compound probiotics on weight-bearing swimming time of mice

[0050] The weighted swimming test in mice is a model commonly used to evaluate the anti-fatigue effects of drugs or foods. Figure 2 As shown in the results, compared with the quiet control group (SED), exercise training significantly prolonged the weighted swimming time of mice. After intervention with the compound probiotics of SHY 21-2+SHY10, the weighted swimming time of mice in the SED group was further significantly prolonged (33.20±6.11min). In addition, the administration of compound probiotics on the basis of running training also significantly increased the weighted swimming time of mice (36.50±3.86min). Moreover, whether it was the quiet group or the running training group, the weighted swimming time of mice after intervention with the compound probiotics of SHY21-2+SHY10 was significantly higher than the effect of SHY 21-2 or SHY10 alone. This shows that compound probiotics can effectively improve exercise fatigue in SED mice and exercise-trained mice.

[0051] 3.3 Effects of compound probiotics on mitochondrial synthesis, dynamics and autophagy-related gene expression in gastrocnemius muscle of mice

[0052] Skeletal muscle mitochondria provide the energy required for physical activities through biogenesis, and peroxisome proliferator-activated receptor-γ coactivator-1α (PGC-1α) plays a key role in the activation of genes related to skeletal muscle mitochondrial biogenesis. Figure 3The results showed that the compound probiotics significantly increased the gene expression of gastrocnemius PGC-1α in SED mice and exercise-trained mice after intervention, and the regulatory effect on exercise-trained mice was more significant. Moreover, the effect of compound probiotics SHY21-2+SHY10 after intervention was significantly higher than that of SHY 21-2 or SHY10 alone, regardless of whether it was the quiet group or the running training group.

[0053] Mitochondrial fusion and fission are a dynamic balance that participates in the rapid response of cells to energy demands. Mitochondrial fusion protein 2 (Mfn2) and fission protein 1 (Fis1) are key regulatory proteins for mitochondrial fusion and fission, respectively. Figure 4 The results showed that the compound probiotics intervention significantly increased the expression of Mfn2 and Fis1 genes in the gastrocnemius of SED mice and exercise-trained mice, and the regulatory effect on exercise-trained mice was more prominent. Moreover, the effect of the compound probiotics SHY 21-2+SHY10 after intervention was significantly higher than that of SHY 21-2 or SHY10 alone, whether in the quiet group or the running training group.

[0054] The PINK1 / Parkin signaling pathway is an important pathway for mitophagy. PINK1 accumulates on the outer membrane of damaged mitochondria and initiates mitophagy by recruiting Parkin protein, thereby removing excess or damaged mitochondria and maintaining cell homeostasis. Figure 5 The results showed that the compound probiotic intervention significantly increased the expression of PINK1 and Parkin genes in the gastrocnemius of SED mice and exercise-trained mice. Moreover, the effect of the compound probiotic intervention of SHY 21-2+SHY10 was significantly higher than that of SHY21-2 or SHY10 alone, regardless of whether it was the quiet group or the running training group.

[0055] These results indicate that the compound probiotics can significantly improve the exercise endurance and exercise fatigue of mice by regulating mitochondrial biogenesis, fusion fission and autophagy processes in the gastrocnemius muscle.

[0056] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention rather than to limit it. Although the present invention has been described with reference to the preferred embodiments of the present invention, it should be understood by those skilled in the art that various changes may be made in form and details without departing from the spirit and scope of the present invention as defined in the appended claims.

Claims

1. A composite probiotic for increasing sports endurance and resisting sports fatigue, characterized in that: The invention comprises Lactobacillus plantarum SHY 21-2 and Lactobacillus fermentum SHY10. The preservation number of the Lactobacillus plantarum is CCTCC NO: M 2020043, and the preservation number of the Lactobacillus fermentum is CCTCC NO: M 2020042.

2. The composite probiotic according to claim 1, characterized in that: The ratio of the number of live bacteria of Lactobacillus plantarum SHY21-2 and Lactobacillus fermentum SHY10 is 1~5:1~5.

3. The composite probiotic according to claim 1, characterized in that: The composite probiotics are a mixture of freeze-dried powder of Lactobacillus plantarum SHY21-2 and freeze-dried powder of Lactobacillus fermentum SHY10 in a mass ratio of 1-5:1-5.

4. A microbial agent, characterized in that: The invention comprises the composite probiotics as claimed in claim 1.

5. Use of the composite probiotics according to claim 1 or the microbial agent according to claim 4 in preparing a product having the effects of increasing sports endurance and resisting sports fatigue.

6. The use according to claim 5, characterized in that: The product is food, food additive, health product, feed, feed additive, reagent or medicine.

7. A product that has the effect of increasing sports endurance and resisting sports fatigue, characterized in that: The active ingredients thereof include the composite probiotics described in claim 1 or the microbial agent described in claim 4.

8. The product according to claim 7, characterized in that: The product is food, food additive, health product, feed, feed additive, reagent or medicine.

9. The product according to claim 8, characterized in that: The product also includes auxiliary materials; The auxiliary material is selected from any one or a combination of at least two of fillers, binders, wetting agents, disintegrants, emulsifiers, cosolvents, solubilizers, osmotic pressure regulators, colorants, pH regulators, antioxidants, antibacterial agents or buffers.

Images