Lactobacillus fermentum TKSN042 as well as application and probiotic preparation thereof

Abstract

The invention relates to the technical field of microorganisms, in particular to lactobacillus fermentum TKSN042 as well as application and a probiotic preparation thereof. The lactobacillus fermentum TKSN042 is preserved in the China General Microbiological Culture Collection Center on July 15, 2019, and the preservation number is CGMCC No.18223. The lactobacillus fermentum TKSN042 is preserved in the China General Microbiological Culture Collection Center on July 15, 2019. According to the invention, the intervention effect of the LFTKSN042 capable of regulating the balance of the intestinal flora on the reduction of the running capacity of mice is researched, and in combination with the regulation effect of the LFTKSN042 on the intestinal flora, the action mechanism of the LFTKSN042 on the regulation of the intestinal flora and the improvement of the motor function is preliminarily clarified, and a theoretical basis is accumulated for the practical application of the strain.

Description

Technical Field

[0001] The present invention relates to the technical field of microorganisms, and particularly to a Lactobacillus fermentum TKSN042, its application and a probiotic preparation. Background Art

[0002] The causes of exercise-induced fatigue can be mainly summarized into four theories: energy depletion theory, metabolite accumulation theory, central protective inhibition theory, and free radical theory. In recent years, the research on the association between oxidative stress damage caused by free radicals and exercise-induced fatigue has received increasing attention, and oxidative stress plays an important role in the occurrence mechanism of fatigue. The changes in oxidative stress and cellular energy metabolism can activate AMP-activated protein kinase (AMPK), thereby regulating energy metabolism. In addition, peroxisome proliferator-activated receptor γ coactivator 1α (PGC-1α) enhances the function of energy metabolism enzymes and improves skeletal muscle resistance to fatigue by promoting the activation of factors related to mitochondrial gene transcription. When the AMPK / PGC-1α signaling pathway is activated and strengthened, the oxidative phosphorylation process in mitochondria in muscle tissue is accelerated, metabolic by-products are reduced, and the energy generation efficiency in muscle cells is improved.

[0003] In recent years, the research on the application of probiotics in sports and their use as nutritional supplements has been continuously advancing. According to existing research, several specific types of probiotics, such as Lactiplantibacillus plantarum PS128, have an improving effect on the post-race endurance decline and fatigue state of triathlon athletes; Lactobacillus casei Shirota can reduce the incidence of upper respiratory tract infections in long-distance runners; Lactobacillus acidophilus SPP can increase the maximal oxygen uptake of swimmers; Bifidobacterium SPP 07 / 3 can enhance energy supply and improve athletes' endurance; at the same time, multiple Streptococcus salivarius subsp. thermophilus are added to sports drinks and yogurts for use as drinks for athletes. The results of animal experiments have clarified the principle of how probiotics improve the running endurance of mice from the mechanism, further corroborating the manifestations obtained from clinical research, which provides a new direction for exploring how to use probiotics to improve the physical limit of humans. Existing research has shown that lactic acid bacteria in Sichuan pickles have good intestinal colonization effects and can regulate intestinal function and promote the health of the body. Some research has found through DNA fingerprint maps of the structural characteristics of the intestinal microbiota that the stability of the intestinal microbiota is one of the factors for athletes to maintain a good sports state, indicating that there is a close relationship between the intestinal microbiota and sports performance.

[0004] Regular and scientific physical exercise is an effective means to prevent and treat various chronic diseases and their complications. Aerobic exercise, in particular, can significantly improve physiological adaptability by enhancing cardiovascular function and energy metabolism. However, the fast-paced life and work often put people in a state of high intensity and unhealthy living habits, leading to oxidative stress and a sense of fatigue. This continuous fatigue and high-intensity work will hinder normal exercise, and then lead to a decline in physical fitness and a continuous sub-healthy state. Therefore, it is very important to find effective intervention measures to improve physical fitness, enhance exercise performance and improve the body's oxidative stress state. Summary of the Invention

[0005] To solve the above problems, the present invention provides a strain of Lactobacillus fermentum TKSN042, its application and probiotic preparation. The present invention studied the intervention effect of LFTKSN042, which has the function of regulating the balance of intestinal flora, on the decline of running ability in mice. Combining with the regulatory effect of LFTKSN042 on intestinal flora, the mechanism of LFTKSN042 in regulating intestinal flora and improving exercise function was preliminarily clarified, so as to accumulate theoretical basis for the practical application of this strain.

[0006] To achieve the above object, the present invention provides the following technical solutions:

[0007] The present invention provides a strain of Lactobacillus fermentum TKSN042, which was deposited in the China General Microbiological Culture Collection Center on July 15, 2019, and the deposit number is CGMCC No. 18223.

[0008] The present invention also provides the application of the Lactobacillus fermentum TKSN042 described in the above technical solution in the preparation of products for promoting exercise endurance.

[0009] The present invention also provides the application of the Lactobacillus acidophilus XY27 described in the above technical solution in the preparation of products for preventing lactate accumulation under exhaustive exercise.

[0010] The present invention also provides the application of the Lactobacillus fermentum TKSN042 described in the above technical solution in the preparation of products for relieving oxidative stress and enhancing exercise durability.

[0011] Preferably, the indicators of oxidative stress include muscle glycogen, liver glycogen, creatine kinase and urea nitrogen.

[0012] The present invention also provides the application of the Lactobacillus fermentum TKSN042 described in the above technical solution in the preparation of products for enhancing the activity of antioxidant enzymes under exhaustive exercise.

[0013] The present invention also provides the use of Lactobacillus fermentum TKSN042 as described in the above technical solution in the preparation of a product for regulating the intestinal flora under exhaustive exercise.

[0014] Preferably, the method for regulating the intestinal flora under exhaustive exercise includes: increasing the numbers of Firmicutes microorganisms, lactic acid bacteria, and Bifidobacterium, and decreasing the number of Bacteroidetes microorganisms.

[0015] The present invention also provides the use of Lactobacillus fermentum TKSN042 as described in the above technical solution in the preparation of a product for alleviating liver injury under exhaustive exercise.

[0016] The present invention also provides a probiotic preparation, which contains Lactobacillus fermentum TKSN042 as described in the above technical solution; the bacterial content of Lactobacillus fermentum TKSN042 in the probiotic preparation is 1.875×10 8 CFU / mL.

[0017] Advantages of the present invention:

[0018] The present invention constructs a mouse oxidative stress state model to evaluate the antioxidant performance of LFTKSN042 and its effect on the exercise function of mice. Experimental data show that LFTKSN042 can significantly reduce the oxidative stress in mice, promote the energy metabolism of the gastrocnemius muscle, and thus improve the ability of the test animals to resist fatigue and perform physical activities. Further analysis reveals that, on the premise of following the daily recommended intake for humans, LFTKSN042 exhibits a better effect than vitamin C. In summary, the present invention explores the mechanism of action of LFTKSN042 in enhancing the exercise ability of oxidative stress mice, laying a foundation for the subsequent development of food-grade antioxidant components that can alleviate oxidative damage and exercise ability decline caused by high-intensity work or natural aging, and facilitating the research and development process of probiotic products with independent intellectual property rights. Nevertheless, the above conclusions still need to be verified by more clinical trials for their general applicability, which will be a key point for future scientific research on LFTKSN042. Description of the Drawings

[0019] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required in the embodiments.

[0020] Figure 1 is the running time of oxidative stress mice in the exhausted state. Note: The same lowercase letters in the bar graph indicate no significant difference between the two groups, while different lowercase letters indicate a significant difference between the two groups (P<0.05);

[0021] Figure 2For the changes in serum lactate concentration in mice with oxidative stress after excessive exercise. Note: The data before exhaustive running were the first measurement data of mice numbered 1-10, the data 5 minutes after exhaustive running were the second measurement data of mice numbered 1-10, and the data 30 minutes after exhaustive running were the measurement data of mice numbered 11-20. At the same time condition, the same lowercase English letters indicate no significant difference between the two groups, while different lowercase letters indicate a significant difference between the two groups (P<0.05);

[0022] Figure 3 For the MG level in the gastrocnemius muscle of mice with oxidative stress. Note: In the bar chart, the same lowercase letters indicate no significant difference between the two groups, while different lowercase letters indicate a significant difference between the two groups (P<0.05);

[0023] Figure 4 For the HG level in the gastrocnemius muscle of mice with oxidative stress. Note: In the bar chart, the same lowercase letters indicate no significant difference between the two groups, while different lowercase letters indicate a significant difference between the two groups (P<0.05);

[0024] Figure 5 For the serum CK level in mice with oxidative stress. Note: In the bar chart, the same lowercase letters indicate no significant difference between the two groups, while different lowercase letters indicate a significant difference between the two groups (P<0.05);

[0025] Figure 6 For the serum BUN level in mice with oxidative stress. Note: In the bar chart, the same lowercase letters indicate no significant difference between the two groups, while different lowercase letters indicate a significant difference between the two groups (P<0.05);

[0026] Figure 7 For the pathological observation of the liver tissue of mice with oxidative stress;

[0027] Figure 8 For the mRNA expression level of AMPK in the gastrocnemius muscle of mice with oxidative stress. Note: In the bar chart, the same lowercase letters indicate no significant difference between the two groups, while different lowercase letters indicate a significant difference between the two groups (P<0.05);

[0028] Figure 9 For the mRNA expression level of PGC1-α in the gastrocnemius muscle of mice with oxidative stress. Note: In the bar chart, the same lowercase letters indicate no significant difference between the two groups, while different lowercase letters indicate a significant difference between the two groups (P<0.05);

[0029] Figure 10The mRNA expression level of SOD2 in the gastrocnemius muscle of oxidative stress mice. Note: The same lowercase letters in the bar graph indicate no significant difference between the two groups, while different lowercase letters indicate a significant difference between the two groups (P<0.05);

[0030] Figure 11 The mRNA expression level of GPx1 in the gastrocnemius muscle of oxidative stress mice. Note: The same lowercase letters in the bar graph indicate no significant difference between the two groups, while different lowercase letters indicate a significant difference between the two groups (P<0.05);

[0031] Figure 12 The mRNA expression level of AMPK in the liver of oxidative stress mice. Note: The same lowercase letters in the bar graph indicate no significant difference between the two groups, while different lowercase letters indicate a significant difference between the two groups (P<0.05);

[0032] Figure 13 The mRNA expression level of PGC1-α in the liver of oxidative stress mice. Note: The same lowercase letters in the bar graph indicate no significant difference between the two groups, while different lowercase letters indicate a significant difference between the two groups (P<0.05);

[0033] Figure 14 The mRNA expression level of SOD2 in the liver of oxidative stress mice. Note: The same lowercase letters in the bar graph indicate no significant difference between the two groups, while different lowercase letters indicate a significant difference between the two groups (P<0.05);

[0034] Figure 15 The mRNA expression level of GPx1 in the liver of oxidative stress mice. Note: The same lowercase letters in the bar graph indicate no significant difference between the two groups, while different lowercase letters indicate a significant difference between the two groups (P<0.05);

[0035] Figure 16 The mRNA expression level of Firmicutes in the intestinal contents of oxidative stress mice. Note: The same lowercase letters in the bar graph indicate no significant difference between the two groups, while different lowercase letters indicate a significant difference between the two groups (P<0.05);

[0036] Figure 17 The mRNA expression level of Bacteroidetes in the intestinal contents of oxidative stress mice. Note: The same lowercase letters in the bar graph indicate no significant difference between the two groups, while different lowercase letters indicate a significant difference between the two groups (P<0.05);

[0037] Figure 18The mRNA expression levels of Lactobacillus in the intestinal contents of oxidative stress mice. Note: The same lowercase letters in the bar graph indicate no significant difference between the two groups, while different lowercase letters indicate a significant difference between the two groups (P < 0.05).

[0038] Figure 19 The mRNA expression levels of Bifidobacterium in the intestinal contents of oxidative stress mice. Note: The same lowercase letters in the bar graph indicate no significant difference between the two groups, while different lowercase letters indicate a significant difference between the two groups (P < 0.05).

[0039] Biological deposit description

[0040] Lactobacillus fermentum TKSN042, with the Latin name Lactobacillus fermentum, was deposited at the General Microbiological Center of the China Committee for Culture Collection of Microorganisms on July 15, 2019, with the deposit number CGMCC No. 18223. The address is: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing 100101. Detailed implementation manners

[0041] The present invention provides a strain of Lactobacillus fermentum TKSN042, which was deposited at the General Microbiological Center of the China Committee for Culture Collection of Microorganisms on July 15, 2019, with the deposit number CGMCC No. 18223.

[0042] The present invention also provides the application of Lactobacillus fermentum TKSN042 described in the above technical solution in the preparation of products for promoting exercise endurance. The present invention has no special limitation on the products, such as drugs, health products, etc.

[0043] The present invention also provides the application of Lactobacillus fermentum TKSN042 described in the above technical solution in the preparation of products for preventing lactic acid accumulation under exhaustive exercise. The present invention has no special limitation on the products, such as drugs, health products, etc.

[0044] The present invention also provides the application of Lactobacillus fermentum TKSN042 described in the above technical solution in the preparation of products for relieving oxidative stress and enhancing exercise durability. In the present invention, the indexes of oxidative stress preferably include muscle glycogen, liver glycogen, creatine kinase, and urea nitrogen. The present invention has no special limitation on the products, such as drugs, health products, etc.

[0045] The present invention also provides the application of Lactobacillus fermentum TKSN042 described in the above technical solution in the preparation of products for enhancing the antioxidant enzyme activity under exhaustive exercise. The present invention has no special limitation on the products, such as drugs, health products, etc.

[0046] The present invention also provides the use of Lactobacillus fermentum TKSN042 as described in the above technical solution in the preparation of a product for regulating the intestinal flora under exhaustive exercise. In the present invention, the method for regulating the intestinal flora under exhaustive exercise includes: increasing the numbers of Firmicutes microorganisms, lactic acid bacteria and Bifidobacterium, and decreasing the number of Bacteroidetes microorganisms. The present invention has no special limitation on the product, such as drugs, health products, etc.

[0047] The present invention also provides the use of Lactobacillus fermentum TKSN042 as described in the above technical solution in the preparation of a product for relieving liver injury under exhaustive exercise. The present invention has no special limitation on the product, such as drugs, health products, etc.

[0048] The present invention also provides a probiotic preparation, which contains Lactobacillus fermentum TKSN042 as described in the above technical solution; the bacterial content of Lactobacillus fermentum TKSN042 in the probiotic preparation is 1.875×10 8 CFU / mL. The present invention has no special limitation on the preparation method of the probiotic preparation, and those skilled in the art can prepare it by using the preparation method of probiotic preparations of Lactobacillus acidophilus.

[0049] In order to further illustrate the present invention, the following examples are used to describe the present invention in detail, but they should not be construed as limiting the protection scope of the present invention.

[0050] Example 1

[0051] 1 Materials and Methods

[0052] 1.1 Materials and Instruments

[0053] Lactobacillus fermentum TKSN042 was isolated and identified from traditional natural fermented yogurt from the household of a herdsman in Mogutai Village, Kuoketireke Township, Tekes County, Ili Kazakh Autonomous Prefecture, Xinjiang Uygur Autonomous Region by the Collaborative Innovation Center for Child Nutrition and Health Development of Chongqing University of Education. It has been registered and preserved at the General Microbiology Center of the China Committee for Culture Collection of Microorganisms (CGMCC No. 18223); DeMan, Rogosa and Sharpe (MRS) medium, Beijing LandBridge Technology Co., Ltd.; 4% paraformaldehyde universal tissue fixative, Biosharp; Mouse blood urea nitrogen (BUN) kit, Beijing Solarbio Science & Technology Co., Ltd.; Mouse muscle glycogen (MG), liver glycogen (HG), creatine kinase (CK), ELISA kit, Shanghai Enzyme-linked Biotechnology Co., Ltd.; Mouse blood lactic acid determination ELISA kit, Shanghai Enzyme Australia Biotechnology Co., Ltd.; TRlzol reagent, Thermo Fisher Scientific; RNase-Free water, SYBR Green PCR Master Mix, Yeasen Biotechnology (Shanghai) Co., Ltd.; D-galactose, Sinopharm Chemical Reagent Co., Ltd.; All organic solvents used for separation are domestic analytical pure grade.

[0054] Nano-300 micro-spectrophotometer, AMR-100 automatic microplate reader, Hangzhou Allsheng Instruments Co., Ltd.; BI-I50A low-temperature biochemical incubator, Shiduokai Instrument Equipment (Shanghai) Co., Ltd.; ZH-PT eight-channel experimental animal treadmill, Anhui Zhenghua Biological Instrument Equipment Co., Ltd.; A200 gene amplifier, Hangzhou Langji Scientific Instruments Co., Ltd.; StepOnePlus real-time fluorescence quantitative PCR instrument, Thermo Fisher Scientific; OLYMPUS-BX43 upright microscope, Olympus Instruments Co., Ltd.

[0055] The Kunming mice used in the experiments were all SPF grade, 6 weeks old, with half males and half females, and were purchased from the Experimental Animal Center of Chongqing Medical University (License No.: SCXK (Yu) 2022-0010).

[0056] The identification and isolation of Lactobacillus fermentum TKSN042 are as follows:

[0057] 1.1.1 Experimental materials

[0058] Traditional natural fermented yogurt collected from the household of a herdsman in Mogutai Village, Kuoketireke Township, Tekes County, Ili Kazakh Autonomous Region, Xinjiang Uygur Autonomous Region. After thoroughly stirring the yogurt with a sterile spoon, 50 mL was aspirated with a sterile syringe into a sterilized centrifuge tube, placed in a low-temperature food sampling box and taken back to the laboratory for freezing preservation in an ultra-low temperature refrigerator at -80 °C for standby.

[0059] 1.1.2 Experimental methods

[0060] 1.1.2.1 Isolation and Identification of Lactic Acid Bacteria

[0061] 1.1.2.1.1 Isolation and Purification of Lactic Acid Bacteria

[0062] Take 1 mL of yogurt sample respectively, and dilute it 10-fold serially with sterile normal saline to 10 -6 , then take 10 -4 , 10 -5 , 10 -6 100 μL of the bacterial suspensions at 3 dilution gradients are spread on plates and cultured at 37 °C for 24 - 48 h, and the colony morphology is observed and recorded. Colonies with different morphologies on the plates are picked for streak isolation. After culturing at 37 °C for 48 h, single colonies with different morphologies on the plates are picked again for streak isolation. Repeat this process multiple times until pure single colonies with consistent morphology are obtained.

[0063] 1.1.2.1.2 DNA Extraction of Lactic Acid Bacteria

[0064] Inoculate the purified suspected target strain into MRS broth, and after culturing at 37 °C for 18 - 24 h, extract DNA using a bacterial genomic DNA extraction kit. Number the extracted DNA and store it at -20 °C in a freezer for future use.

[0065] 1.1.2.1.3 PCR Amplification of Genomic DNA

[0066] Perform PCR amplification on the extracted DNA. Among them, 1 μL of upstream primer 27F (SEQ ID No.1 5'-AGA GTT TGATCC TGGCTC AG-3'), 1 μL of downstream primer 1495R (SEQ ID No.2 5'-CTACGG CTACCTTGT TAC GA-3'), 12.5 μL of 2×Taq plus Buffer, 1 μL of template DNA, and make up the system to 25 μL with sterile dd H2O. Use sterile ultrapure water to replace the template DNA as a negative control. The amplification conditions are: 94 °C for 5 min; 94 °C for 30 s, 55 °C for 30 s, 72 °C for 1 min, for a total of 29 cycles, and finally extend at 72 °C for 5 min. Then send the PCR product to Beijing Tsingke Biotechnology Co., Ltd. for sequencing, and use the BLAST (Basic Local Alignment Search Tool) program in NCBI to perform comparative analysis on the successfully sequenced sequences.

[0067] 1.1.2.1.4 In Vitro Resistance Screening of Lactic Acid Bacteria

[0068] 1.1.2.1.4.1 Ability of Lactic Acid Bacteria to Tolerate 0.3% Bile Salt

[0069] Add porcine bile salt to MRS-THIO medium (MRS broth containing 0.2% sodium thioglycollate) to make its concentration 0.3%, sterilize at 121 °C for 15 min, and inoculate 5 mL of the activated strain into MRS-THIO medium without bile salt (0.0%) and MRS-THIO medium containing 0.3% bile salt at an inoculation amount of 2% (v / v). Using the blank medium (MRS-THIO medium without inoculation) as a control, after culturing at 37 °C for 24 h, measure the OD 600nm value of the above-mentioned media with different concentrations, and calculate the tolerance of the strain to bile salt according to formula (1):

[0070]

[0071] 1.1.2.1.4.2 Artificial gastric juice tolerance test

[0072] Preparation of artificial gastric juice: Artificial gastric juice consists of 0.2% NaCl and 0.35% pepsin. Weigh the required NaCl and pepsin for the test according to the corresponding mass-to-volume ratio for preparation. Adjust the pH of the prepared artificial gastric juice to 3.0 with 1 mol / L HCl, and then filter and sterilize it with a 0.22 μm filter membrane for standby.

[0073] In a laminar flow hood, pipette 5 mL of the cultured bacterial culture into a 10 mL sterile centrifuge tube, centrifuge at 4000 r / min for 10 min, discard the upper layer of the medium and collect the bacterial cells, add an equal volume (5 mL) of sterile normal saline and mix well to make a bacterial suspension. Then take 1 mL of the bacterial suspension and mix it with 9 mL of artificial gastric juice with pH 3.0. At this time, take 1 mL of the above-mentioned mixture as the sample treated with artificial gastric juice for 0 h, and place the remaining 9 mL of the mixture in a constant temperature water bath shaker (37 °C, 150 r / min) for 3 h. The samples at 0 h and 3 h are respectively diluted by 10-fold gradient, and the appropriate gradient is selected to measure the viable cell count by the method of plate coating, and culture at 37 °C for 48 h on MRS solid medium, and calculate the survival rate (%) according to formula 2.

[0074]

[0075] 1.1.2.2 Results

[0076] 1.1.2.2.1 Sequencing results of PCR amplification products of lactic acid bacteria

[0077] The 16s rDNA sequence of Lactobacillus fermentum TKSN042 is as follows (SEQ ID No.3):

[0078]

[0079] 1.1.2.2.2 In vitro resistance results of Lactobacillus

[0080] As can be seen from Table 1, the survival rate of TKSN042 in artificial gastric juice at pH 3.0 exceeded 80%; LFHFY13 had a relatively high efficiency in 0.3% bile salts, reaching 58.33%, indicating that this strain of bacteria has a strong ability to tolerate bile salts.

[0081] Table 1 Survival rates of lactic acid bacteria in artificial gastric juice at pH 3.0 and 0.3% bile salts

[0082]

[0083] 1.2 Experimental methods

[0084] 1.2.1 Preparation of experimental bacterial suspension

[0085] Add the freeze-dried bacterial powder of LFTKSN042 based on skim milk powder into sterilized MRS liquid medium (1:20, w / w), then place it in a low-temperature biochemical incubator at 37°C for 48 h, then centrifuge at 4000 r / min for 10 min, and finally collect the precipitated bacteria. Then add normal saline at 5 times the mass of the bacteria, and directly count the number of bacteria in the bacterial liquid using a hemocytometer under a microscope to calculate the bacterial liquid concentration. Finally, adjust the bacterial liquid concentration to 3×10 8 CFU / mL for standby, and dilute the bacterial liquid according to the mouse body weight before animal experiments for gavage.

[0086] 1.2.2 Animal experiments

[0087] Adaptively raise 100 mice in an environment with a temperature maintained at 20±1°C and a humidity of 30% - 40% for 7 h. Subsequently, randomly divide the mice into 5 groups, namely the normal group, the model group, the vitamin C gavage group (Vc group), and two groups given LFTKSN042 gavage at two different dose levels (LFTKSN042-L group and LFTKSN042-H group), with 20 mice in each group. Each group of mice is labeled from 1 to 20, among which numbers 1 - 5 and 11 - 15 are female, and numbers 6 - 10 and 16 - 20 are male. Referring to the recommended daily safe intake of vitamin C for humans of 1000 mg / d in the "Scientific Research Report on Chinese Residents' Dietary Guidelines (2021)", in this study, the amount of vitamin C required to be supplemented per kilogram of body weight of experimental animals per day was set at 150 mg. In addition, according to the requirements of the GB / T21732-2008 "Milk-containing Beverages" standard, that is, when the product leaves the factory, live bacteria-containing beverages need to ensure that each milliliter contains at least 10 7active microorganisms, and combined with the recommended daily dosage of probiotic drinks on the market, it is estimated that the daily probiotic intake suitable for experimental mice should be adjusted to 1.5×10 9 CFU / kg body weight. During the experiment, all other groups of mice except the normal group will receive a 5% (w / v) D-galactose solution by intraperitoneal injection every day for 6 consecutive weeks, with a dosage of 100 mg / kg BW; while the normal group will be replaced with an equal volume of physiological saline. After the induction of the D-galactose solution ends, starting from the 7th week, the Vc group will be gavaged with a vitamin C solution at 150 mg / kg BW daily; the model group and the normal group will be gavaged with 2 mL of distilled water (placebo) daily; the LFTKSN042-L group and the LFTKSN042-H group will be gavaged with 0.75×10 9 CFU / kg and 1.50×10 9 CFU / kg of the LFTKSN042 bacterial suspension respectively according to the body weight of the mice, and the gavage of samples for each group will last for 4 weeks. On the 2nd day after the gavage of samples, all the mice participating in the experiment will undergo a physical strength test, a exhaustive running experiment, and finally be euthanized by cervical dislocation, and the liver tissue will be collected for further analysis.

[0088] 1.2.3 Exhaustive running exercise experiment

[0089] An 8-channel mouse treadmill is used for the exhaustive running test, the slope is set at 10°, and the speed of the treadmill is gradually increased. The specific steps are as follows: run at a speed of 10 m / min for 5 min, and then run at speeds of 16 m / min, 21 m / min, 26 m / min, 31 m / min, 36 m / min, 41 m / min, and 46 m / min for 10 min each until the mice show exhaustion. The criterion for exhaustion is that the number of electric shocks reaches 10 times, or the mice cannot continue to run continuously for more than 10 s. Record the time taken for the mice to run to exhaustion.

[0090] 1.2.4 Determination of CK, BUN, and lactic acid levels in mouse serum

[0091] After 2 h of the end of gavage, blood samples are drawn from the orbits of mice No. 1-20 by capillary blood sampling; 5 min after the end of the exhaustive running test on the 2nd day, blood samples are drawn from the orbits of mice No. 1-10 by capillary blood sampling; 30 min after the end of the exhaustive running test, blood samples are drawn from the orbits of mice No. 11-20 by capillary blood sampling. The collected mouse blood is centrifuged at 4°C (1500 r / min, for 10 min), the upper layer of serum is separated, and according to the instructions provided by the detection kit, the lactic acid levels in the serum of the 2 blood samples of mice No. 1-10 are measured and the CK, BUN, and lactic acid levels in the serum of mice No. 11-20 are measured.

[0092] 1.2.5 Determination of MG levels in mouse gastrocnemius muscle tissue and HG levels in liver tissue

[0093] Take an appropriate amount of mouse tissue, add normal saline at a ratio of 1:9, and homogenize. According to the instructions provided by the detection kit, determine the MG levels in the gastrocnemius muscle tissue and HG levels in the liver tissue of mice numbered 11 - 20.

[0094] 1.2.6 H&E staining and pathological sections of mouse liver

[0095] After dissecting the liver tissue of mice numbered 11 - 20, first rinse it 3 times with normal saline, and then immediately fix it in 10% (v / v) formalin solution. After 48 h of dehydration at 4°C, embed these samples in paraffin. Then, cut the embedded tissue into sections 5 - 10 μm thick, stain them using the H&E staining method, and finally examine the changes in their pathological characteristics under an optical microscope.

[0096] 1.2.7 Determination of mRNA expression in mouse tissues

[0097] To detect the mRNA expression of AMPK, PGC1-α, SOD2, and GPx1 in mouse liver and gastrocnemius muscle, first accurately weigh 0.2 g of tissue samples from mice numbered 11 - 20 and wash them with normal saline. Then, cut these tissues into small pieces and add 1.0 mL of TRlzol reagent to them. Next, determine its purity by measuring the absorbance values of the obtained RNA solution at wavelengths of 260 nm and 280 nm, and adjust the RNA concentration to 1 μg / μL accordingly. After completing the above steps, perform the reverse transcription process to generate cDNA. On this basis, construct a reaction system for fluorescence quantitative PCR analysis, which consists of 1 μL of cDNA, 10 μL of SYBR Green PCR Master Mix, 1 μL each of self-designed specific primers (see Table 2), and 7 μL of sterile distilled water. Place the prepared reaction solution in a real-time fluorescence quantitative PCR instrument and perform the amplification cycle according to the preset conditions: initially heat at 95°C for 60 s; then there are 40 cycles of denaturation stage (95°C, 15 s), annealing extension stage (55°C, 30 s), and final synthesis stage (72°C, 35 s). In addition, set the melting curve analysis step (95°C, 30 s; 55°C, 35 s) to verify the product specificity. Throughout the experiment, the GAPDH gene was selected as the internal reference standard, and the relative expression level of the target gene was calculated according to the 2 -ΔΔCt method.

[0098] Table 2 Sequences of primers used for tissue determination in animal experiments

[0099]

[0100] 1.2.8 Determination of the relative content of microorganisms in intestinal contents

[0101] After dissecting mice numbered 11 - 20, 0.2 g samples of intestinal contents were collected from their large intestines. Subsequently, according to the method described in Section 1.2.8, the mRNA levels of total bacteria, Firmicutes, Bacteroidetes, Lactobacillus, and Bifidobacterium in these intestinal samples were measured (see Table 3) to explore the specific composition of the intestinal microbial community of the mice.

[0102] Table 3 Sequences of primers used for the determination of microorganisms in the large intestinal contents in the animal experiment

[0103]

[0104] 1.3 Data processing

[0105] The mice participating in the experiment were measured in detail, and the obtained data were presented in the form of mean ± standard deviation. In addition, one-way analysis of variance was performed using SPSS 22.0 software to evaluate whether there were statistically significant differences among groups at the significance level of P < 0.05.

[0106] 2 Results and analysis

[0107] 2.1 Effect of LFTKSN042 on the running state of oxidative stress mice

[0108] In the running experiment under exhaustion state, the performance time of the mice in the normal group was the longest. In contrast, the exercise duration of the mice in the model group was the shortest ( Figure 1)。Research findings indicate that compared with the model group, both LFTKSN042 and Vc can significantly increase the performance duration of oxidative stress mice in this running test (P<0.05). Among them, the effect of LFTKSN042-H is the most significant, being significantly better than LFTKSN042-L and Vc. The accumulation of excessive free radicals in the body may lead to organ damage and reduced function, especially in tissues and systems closely related to exercise, thereby causing a decrease in exercise performance and frequent fatigue. Appropriate physical activity helps to reduce the damage caused by free radicals to cell membranes, support the effective operation of the oxidative respiratory chain, and maintain the stability of mitochondrial structure and function; in addition, good physical condition is also crucial for maintaining a high level of physical strength. In scientific experiments, the maximum endurance running distance of animals is often measured to evaluate their exercise ability; improving such ability not only directly reflects the enhanced ability of an individual to resist fatigue but is also an important indicator for measuring the antioxidant stress response. Existing research has pointed out that certain lactic acid bacteria species with probiotic characteristics exhibit excellent antioxidant properties, which may help improve body vitality and enhance exercise ability. This study established an oxidative stress model in mice to observe the effect of lactic acid bacteria LFTKSN042 on the exhaustion of endurance running in mice, and further explored whether this strain can enhance the exercise potential of the host in the face of oxidative stress. Similar to previous studies, the time to exhaustion running has increased after the improvement of exercise ability by intervention. The results of this study also show that the persistence of mice treated with LFTKSN042 has increased in both tests compared with the control group, indicating that this bacterium may have the effect of promoting running endurance.

[0109] 2.2 Effect of LFTKSN042 on blood lactic acid concentration in oxidative stress mice after excessive running exercise

[0110] As Figure 2As shown, there was no significant difference in blood lactic acid concentration among groups of mice at the quiet moment before exhaustive running (P<0.05), and it was generally at a low level. However, 5 minutes after exhaustive exercise, the blood lactic acid content in each group of mice increased, and there were significant differences among groups (P<0.05). Among them, the blood lactic acid concentration of the model group mice was the highest among all groups, followed by the Vc group, the LFTKSN042-L group, the LFTKSN042-H group, and the normal group in turn. 30 minutes after exhaustive exercise, the blood lactic acid concentration in each group of mice decreased, and there were significant differences among groups (P<0.05). Except for the normal group, the LFTKSN042-H group was the lowest, followed by the LFTKSN042-L group and the Vc group, and the model group had the highest blood lactic acid content. Blood lactic acid is a metabolite produced by muscles during anaerobic metabolism, and its concentration is closely related to the intensity and duration of muscle activity. When the exercise intensity increases, muscle activity enhances, leading to accelerated anaerobic metabolism and increased production of lactic acid, thus causing the blood lactic acid concentration to rise; exercise causes muscle fatigue, affects the uptake and utilization of oxygen by muscles, promotes the synthesis of lactic acid, and results in lactic acid accumulation. A high level of blood lactic acid usually indicates that the body consumes more energy during exercise and causes fatigue of the body. The animal experiment in this study also supported the above conclusion. The blood lactic acid content increased significantly after exhaustive exercise, and gradually decreased with the extension of time after exercise. During this process, LFTKSN042 could better prevent lactic acid accumulation and protect the body under exhaustive exercise.

[0111] 2.3 Effects of LFTKSN042 on MG in gastrocnemius muscle tissue, HG in liver tissue, CK and BUN in serum of oxidative stress mice

[0112] As Figures 3 - 6As shown, the normal group showed the highest levels of MG and HG, while the model group had the lowest levels of MG and HG. Vc and LFTKSN042 could increase the levels of MG and HG in mice with oxidative stress. The results showed that the levels of MG and HG in the LFTKSN042-H group were higher than those in the LFTKSN042-L group and the Vc group. The levels of BUN and CK in the model group showed the opposite trend, being higher than those in the other groups. The other groups, from high to low, were the Vc group, the LFTKSN042-L group, the LFTKSN042-H group, and the normal group. After experiencing oxidative stress, long-term physical activity may lead to abnormalities in carbohydrate metabolism and lipid metabolism, and a large consumption of proteins and amino acids. During high-intensity or continuous exercise, a large number of metabolites are produced in the body, such as urea nitrogen (BUN) and various oxygen free radicals, which are the main factors causing exercise fatigue. The large accumulation of metabolites in the body not only causes metabolic imbalance in the internal environment but may also damage organs and tissues. Creatine kinase (CK) is an important enzyme involved in the body's energy metabolism and is mainly present in skeletal muscle. When high-intensity exercise causes damage to skeletal muscle, the permeability of the muscle cell membrane changes, and CK is released into the blood in large amounts, resulting in an increase in the concentration of CK in the serum. Glycogen is mainly stored in the liver and muscle. Liver glycogen maintains blood glucose stability, and muscle glycogen directly provides energy for muscles. During long-term exercise, glycogen is gradually decomposed to maintain energy supply. Depletion of glycogen leads to fatigue and affects exercise performance. The regulation of glycogen is of great significance for the body's exercise metabolism. It has been confirmed that the intervention of beneficial microorganisms in MG, HG, CK, and BUN can improve exercise performance. Similar results were also observed in this study. LFTKSN042 could significantly regulate the indexes of MG, HG, CK, and BUN in the body of mice affected by oxidative stress and the lactic acid content in the blood, effectively alleviating the oxidative stress condition and enhancing its exercise durability and efficacy.

[0113] 2.4 Effects of LFTKSN042 on the pathological changes of the liver tissue of mice with oxidative stress

[0114] Through microscopic observation of the liver tissue of mice ( Figure 7) It can be seen that in the normal group, the lobular structure of the liver remained intact and the boundaries were clear, and the hepatocytes were arranged radially around the central vein in an orderly manner. In contrast, within the model group, the lobular structure of the mouse liver was significantly damaged, manifested as the hepatocytes no longer arranged in the normal radial pattern, accompanied by partial rupture of the cell membrane and nucleus, and the presence of apoptotic bodies was observed. Through treatment, LFTKSN042 and Vc were able to reduce the hepatocyte damage in mice (model group) caused by oxidative stress. After treatment with the LFTKSN042-H group, the lobular structure of the mouse liver almost returned to normal, while in the LFTKSN042-L group and the Vc group, some hepatocytes were still damaged and there were obvious disruptions in the cell structure. During strenuous exercise, the metabolic demands of the body increase significantly, the blood circulation speed accelerates, and more metabolites are produced in muscle tissues. Exhaustive exercise will increase the burden on the liver, thus causing liver damage. The pathological sections of this study also showed the liver damage caused by exhaustive exercise, and LFTKSN042 could effectively relieve liver damage, protect the liver, and may play an effect of assisting the liver in metabolism, thereby regulating the body to maintain a good exercise state and reducing fatigue.

[0115] 2.5 Effects of LFTKSN042 on the mRNA expression levels of factors related to the AMPK / PGC1-α pathway in mouse tissues

[0116] The mRNA expression levels of AMPK, PGC1-α, SOD2, and GPx1 in gastrocnemius muscle and liver tissues were detected, and the results are as Figures 8 - 11 and Figures 12 - 15As shown. Compared with the model group mice, the mRNA expression levels of AMPK, PGC1-α, SOD2 and GPx1 in the Vc, LFTKSN042-L and LFTKSN042-H groups were all significantly increased (P<0.05), and the increase degree in the LFTKSN042-H group was significantly higher than that in the Vc group and the LFTKSN042-L group. At the same time, the mRNA expression levels of AMPK, PGC1-α, SOD2 and GPx1 in the normal group mice were the highest. AMPK plays a central role in energy metabolism regulation. When cells face stress conditions such as hypoxia, ischemia or physical exercise, this kinase system will be activated, thereby promoting the acceleration of glucose transport and fatty acid oxidation processes, and at the same time inhibiting activities such as gluconeogenesis, protein synthesis and lipid metabolism. In addition, PGC-1α is also one of the key factors regulating physiological functions such as fatty acid oxidation, glucose utilization and mitochondrial biogenesis. Therefore, regulating the AMPK / PGC-1α pathway can promote energy metabolism during exercise, help delay the occurrence of fatigue and improve exercise endurance. The results of this experiment showed that LFTKSN042 could significantly increase the mRNA expression levels of AMPK and PGC-1α in the tissues of oxidative stress mice. Excessive exercise can lead to in vivo peroxidation reactions and cause oxidative stress damage. Preclinical studies have shown that endurance training can enhance the activities of key antioxidant enzymes in skeletal muscle, such as superoxide dismutase (SOD) and glutathione peroxidase (GPX). Consistent with this result, this study also found that the mice receiving LFTKSN042 gavage showed higher levels of SOD2 and GPx1 compared with the model group, indicating that LFTKSN042 has a significant positive effect on enhancing antioxidant enzymes in mice, thus reflecting the beneficial effects related to liver protection and enhanced exercise ability of the body.

[0117] 2.6 Effects of LFTKSN042 on the expression of microorganisms in the intestinal contents of oxidative stress mice

[0118] Firmicutes and Bacteroidetes bacteria are the two largest types of microorganisms at the phylum level in the human intestine, and these two phylum microorganisms can be used as preliminary indicators for flora evaluation. Bifidobacterium is one of the most important beneficial microorganisms in the intestine. The sample gavaged in this study is lactic acid bacteria. Therefore, by measuring the expressions of each Firmicutes bacterium, Bacteroidetes bacterium, Lactobacillus and Bifidobacterium in the intestinal contents, and using the total bacteria (all microorganisms in the contents) as a reference, the relative intensities of the expressions of various bacteria are detected to judge the relative contents of various bacteria. According to Figures 16 - 19For the data shown, the expression level of Bacteroidetes mRNA in the intestines of normal group mice was the lowest (P<0.05), while the expressions of Firmicutes and Bifidobacterium were relatively higher. This result indicates that in the normal group, the proportion of Bacteroidetes microorganisms is the smallest, while the proportion of Firmicutes microorganisms is the largest. After treatment with LFTKSN042, the proportions of Firmicutes and Bifidobacterium in the intestines of model group mice increased significantly. At the same time, the proportion of Bacteroidetes decreased. In addition, after the intake of LFTKSN042, the proportion of Lactobacillus in the LFTKSN042-L group and LFTKSN042-H group mice increased significantly, even exceeding the level of the normal group, and this change was statistically significant compared with the Vc group and the untreated model group (P<0.05). Research shows that there is a close connection between the health status of human internal organs and redox balance and the gut microbiota. In addition, gut microbiota also has an important impact on organ damage and inflammatory responses. Relevant research indicates that healthy gut flora can convert specific foods into nutrients beneficial to the body, thereby promoting the generation of metabolic regulators, enhancing muscle strength, and improving sports performance. Clinical observations have found that certain specific types of gut bacteria can consume the lactic acid produced during exercise, reducing its adverse effects on muscles, thus helping to improve the endurance level of athletes. When the body faces oxidative stress, the microbial composition in the intestines will directly affect the functionality of the intestines. Imbalanced gut flora may interfere with normal intestinal peristalsis, mucus production, and barrier function, leading to toxins being more likely to enter the bloodstream, further exacerbating the degree of oxidative stress. Under oxidative stress conditions, the proportions of Firmicutes and Bacteroidetes bacteria in the human intestine change significantly, with the proportion of Bacteroidetes bacteria increasing and Bifidobacterium decreasing relatively. It is worth noting that Lactobacillus, which belongs to Firmicutes, has the effect of increasing the activity of antioxidant enzymes. The results of this example also confirm this view, that LFTKSN042 can enhance the activity of antioxidant enzymes in the gastrocnemius and liver tissues of mice. In addition, under the intervention of LFTKSN042, the proportions of Firmicutes microorganisms and Bifidobacterium in oxidative stress mice both increased, while the proportion of Bacteroidetes decreased. In addition, since LFTKSN042 is a kind of Lactobacillus, the proportion of Lactobacillus in the intestines of oxidative stress mice under its intervention increased significantly, reaching a state higher than that of normal group mice. The experimental results show that LFTKSN042 can effectively regulate the gut microbiota of mice, which is consistent with the results related to the gut microbiota and sports status of athletes in clinical experiments, suggesting that the role of LFTKSN042 in improving gut microbiota is also one of the important factors for its improvement of body function and enhancement of sports ability.

[0119] 3 Conclusions

[0120] In this embodiment, a mouse oxidative stress state model was constructed to evaluate the antioxidant performance of LFTKSN042 and its effect on the motor function of mice. Experimental data showed that LFTKSN042 could significantly reduce the oxidative stress in mice, promote the energy metabolism of the gastrocnemius muscle, and thus improve the ability of the test animals to resist fatigue and perform physical activities. Further analysis found that, on the premise of following the daily recommended intake for humans, LFTKSN042 showed a better effect than vitamin C. In summary, this embodiment explored the mechanism of action of LFTKSN042 in enhancing the motor ability of oxidative stress mice, laid a foundation for the subsequent development of food-grade antioxidant components that can alleviate oxidative damage and motor ability decline caused by high-intensity work or natural aging, and was conducive to promoting the research and development process of probiotic products with independent intellectual property rights. Nevertheless, the above conclusions still need to be verified by more clinical trials for their general applicability, which will be a key point for future scientific research on LFTKSN042.

[0121] Although the above embodiments have described the present invention in detail, they are only a part of the embodiments of the present invention, not all of them. People can also obtain other embodiments according to this embodiment without creative efforts, and these embodiments all belong to the protection scope of the present invention.

Claims

1. A Lactobacillus fermentum TKSN042, characterized in that, The Lactobacillus fermentum TKSN042 was deposited at the China General Microbiological Culture Collection Center on July 15, 2019, with the deposit number of CGMCC No. 18223.

2. Use of the Lactobacillus fermentum TKSN042 according to claim 1 in the preparation of a product for promoting exercise endurance.

3. Use of the Lactobacillus fermentum TKSN042 according to claim 1 in the preparation of a product for preventing lactic acid accumulation under exhaustive exercise.

4. Use of the Lactobacillus fermentum TKSN042 according to claim 1 in the preparation of a product for relieving oxidative stress and enhancing exercise durability.

5. The application according to claim 4, wherein The indicators of the oxidative stress include muscle glycogen, liver glycogen, creatine kinase and blood urea nitrogen.

6. Use of the Lactobacillus fermentum TKSN042 according to claim 1 in the preparation of a product for enhancing the activity of antioxidant enzymes under exhaustive exercise.

7. Use of the Lactobacillus fermentum TKSN042 according to claim 1 in the preparation of a product for regulating the intestinal flora under exhaustive exercise.

8. The application according to claim 7, characterized in that, The method for regulating the intestinal flora under exhaustive exercise includes: increasing the numbers of Firmicutes microorganisms, lactic acid bacteria and Bifidobacterium, and decreasing the numbers of Bacteroidetes microorganisms.

9. Use of the Lactobacillus fermentum TKSN042 according to claim 1 in the preparation of a product for relieving liver injury under exhaustive exercise.

10. A probiotic preparation, characterized in that, The probiotic preparation contains Lactobacillus fermentum TKSN042 as described in claim 1; the bacterial content of Lactobacillus fermentum TKSN042 in the probiotic preparation is 1.875×10 8 CFU / mL.

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