Lactobacillus fermentum HFY13 and application thereof

Abstract

The invention relates to the technical field of microorganisms, in particular to lactobacillus fermentum HFY13 and application thereof. The lactobacillus fermentum HFY13 is preserved in the China General Microbiological Culture Collection Center (CGMCC) on October 29, 2018, and the preservation number is CGMCC No.16646. The lactobacillus fermentum HFY13 is preserved in the China General Microbiological Culture Collection Center. The action mechanism of the LFHFY13 in the aspect of improving the exercise ability of oxidative stress mice is discussed, a foundation is laid for subsequent development of food-grade antioxidant components capable of relieving oxidative damage and exercise ability decline caused by high-intensity work or natural aging, and the research and development process of probiotic products with proprietary intellectual property rights is promoted.

Description

Technical Field

[0001] The present invention relates to the technical field of microorganisms, and particularly to a strain of Lactobacillus fermentum HFY13 and its applications. Background Art

[0002] The causes of exercise-induced fatigue can be mainly summarized into four theories: the energy depletion theory, the metabolite accumulation theory, the central protective inhibition theory, and the 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 improvement effect on the post-race endurance decline and fatigue state of triathletes; Lactobacillus casei Shirota can reduce the incidence of upper respiratory tract infections in long-distance runners; Lactobacillus acidophilus SPP can increase the maximum oxygen uptake of swimmers; Bifidobacterium SPP 07 / 3 can enhance energy supply and improve the endurance of athletes; at the same time, multiple Streptococcus salivarius subsp. thermophilus are added to sports drinks and yogurt 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.

[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 lifestyle habits, leading to oxidative stress and feelings of fatigue. This continuous fatigue and high-intensity work can hinder normal exercise, thereby causing 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 HFY13 and its applications. The present invention studied the intervention effect of LFHFY13 (Lactobacillus fermentum HFY13 is hereinafter referred to as LFHFY13) on oxidative stress-induced liver injury and the decline of exercise ability in mice, and preliminarily clarified the mechanism of action of LFHFY13 in protecting the liver and improving exercise performance, so as to accumulate a 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 HFY13, which was deposited at the General Microbiological Center of the China Committee for Culture Collection of Microorganisms on October 29, 2018, with the deposit number CGMCC No. 16646.

[0008] The present invention also provides the application of the Lactobacillus fermentum HFY13 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 fermentum HFY13 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 HFY13 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 HFY13 described in the above technical solution in the preparation of products for relieving liver injury under exhaustive exercise.

[0013] The present invention also provides the application of the Lactobacillus fermentum HFY13 described in the above technical solution in the preparation of products for enhancing the activity of antioxidant enzymes under exhaustive exercise.

[0014] The present invention also provides the use of Lactobacillus fermentum HFY13 as described in the above technical solution in the preparation of a product for increasing the expression levels of AMPK and PGC1-α in the gastrocnemius muscle under exhaustive exercise.

[0015] The present invention also provides the use of Lactobacillus fermentum HFY13 as described in the above technical solution in the preparation of a product for increasing the expression levels of SOD2 and GPx1 in liver tissue under exhaustive exercise.

[0016] The present invention also provides a probiotic preparation, which is characterized in that the probiotic preparation contains Lactobacillus fermentum HFY13 as described in the above technical solution; the bacterial content of Lactobacillus fermentum HFY13 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 LFHFY13 and its effect on the exercise function of mice. Experimental data show that LFHFY13 can significantly reduce the oxidative stress condition in mice and promote the energy metabolism of the gastrocnemius muscle, thereby improving 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, LFHFY13 exhibits a better effect than vitamin C. In summary, the present invention explores the mechanism of action of LFHFY13 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 is conducive to promoting the research and development process of probiotic products with independent intellectual property rights. Brief 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 for use in the embodiments.

[0020] Figure 1 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 2 Swimming 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);

[0022] Figure 3Changes in serum lactate concentration in mice with oxidative stress after excessive exercise. Note: The data before exhaustive exercise (swimming) were the first measurement data of mice numbered 1-10, the data 5 minutes after exhaustive exercise (swimming) were the second measurement data of mice numbered 1-10, and the data 30 minutes after exhaustive exercise (swimming) 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);

[0023] Figure 4 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);

[0024] Figure 5 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);

[0025] Figure 6 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);

[0026] Figure 7 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);

[0027] Figure 8 Pathological observation of the liver tissue of mice with oxidative stress;

[0028] Figure 9 For the mRNA expression level of AMPK in the gastrocnemius muscle of mice with oxidative stress;

[0029] Figure 10 For the mRNA expression level of PGC1-α in the gastrocnemius muscle of mice with oxidative stress;

[0030] Figure 11 For the mRNA expression level of SOD2 in the gastrocnemius muscle of mice with oxidative stress;

[0031] Figure 12 For the mRNA expression level of GPx1 in the gastrocnemius muscle of mice with oxidative stress;

[0032] Figure 13The mRNA expression level of AMPK in the liver of oxidative stress mice. Note: The same lowercase letters in the column chart 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 PGC1-α in the liver of oxidative stress mice. Note: The same lowercase letters in the column chart 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 SOD2 in the liver of oxidative stress mice. Note: The same lowercase letters in the column chart 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 GPx1 in the liver of oxidative stress mice. Note: The same lowercase letters in the column chart indicate no significant difference between the two groups, while different lowercase letters indicate a significant difference between the two groups (P<0.05).

[0036] Biological deposit description

[0037] Lactobacillus fermentum HFY13, with the Latin name Lactobacillus fermentum, was deposited in the China General Microbiological Culture Collection Center on October 29, 2018, with the deposit number CGMCC No. 16646. The address is: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing 100101. Detailed implementation manners

[0038] The present invention provides a strain of Lactobacillus fermentum HFY13, which was deposited in the China General Microbiological Culture Collection Center on October 29, 2018, with the deposit number CGMCC No. 16646.

[0039] The present invention also provides the application of Lactobacillus fermentum HFY13 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.

[0040] The present invention also provides the application of Lactobacillus fermentum HFY13 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.

[0041] The present invention also provides the use of Lactobacillus fermentum HFY13 as described in the above technical solution in the preparation of a product for alleviating oxidative stress and enhancing exercise durability. In the present invention, the indicators of oxidative stress preferably include muscle glycogen, liver glycogen, creatine kinase, and urea nitrogen. The present invention places no special limitation on the product, such as drugs, health products, etc.

[0042] The present invention also provides the use of Lactobacillus fermentum HFY13 as described in the above technical solution in the preparation of a product for alleviating liver injury under exhaustive exercise. The present invention places no special limitation on the product, such as drugs, health products, etc.

[0043] The present invention also provides the use of Lactobacillus fermentum HFY13 as described in the above technical solution in the preparation of a product for enhancing the activity of antioxidant enzymes under exhaustive exercise. The present invention places no special limitation on the product, such as drugs, health products, etc.

[0044] The present invention also provides the use of Lactobacillus fermentum HFY13 as described in the above technical solution in the preparation of a product for increasing the expression levels of AMPK and PGC1-α in the gastrocnemius muscle under exhaustive exercise. The present invention places no special limitation on the product, such as drugs, health products, etc.

[0045] The present invention also provides the use of Lactobacillus fermentum HFY13 as described in the above technical solution in the preparation of a product for increasing the expression levels of SOD2 and GPx1 in liver tissue under exhaustive exercise. The present invention places no special limitation on the product, such as drugs, health products, etc.

[0046] The present invention also provides a probiotic preparation, which contains Lactobacillus fermentum HFY13 as described in the above technical solution; the bacterial content of Lactobacillus fermentum HFY13 in the probiotic preparation is 1.875×10 8 CFU / mL. The present invention places 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.

[0047] 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.

[0048] Example 1

[0049] 1 Materials and Methods

[0050] 1.1 Materials and Instruments

[0051] Lactobacillus fermentum HFY13 was isolated and identified from traditional naturally fermented yak yogurt in the herdsmen's homes in Hongyuan County, Aba Tibetan and Qiang Autonomous Prefecture, Sichuan Province. This strain has been registered and preserved at the General Microbiology Center of the China Committee for Culture Collection of Microorganisms (CGMCC No. 16646); DeMan, Rogosa and Sharpe (MRS) medium, Beijing Land Bridge Technology Co., Ltd.; 4% paraformaldehyde general 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 lactate 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.

[0052] Nano-300 Micro-spectrophotometer, AMR-100 Automatic Microplate Analyzer, 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 Fluorescent Quantitative PCR Instrument, Thermo Fisher Scientific; OLYMPUS-BX43 Upright Microscope, Olympus Instruments Co., Ltd.

[0053] 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).

[0054] The isolation and identification of Lactobacillus fermentum HFY13 are as follows:

[0055] 1.1.1 Experimental materials

[0056] Traditional naturally fermented yak yogurt from the herdsmen's homes in Hongyuan County, Aba Tibetan and Qiang Autonomous Prefecture, Sichuan Province. 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 and preservation in an ultra-low temperature refrigerator at -80 °C for standby.

[0057] 1.1.2 Experimental methods

[0058] 1.1.2.1 Isolation and identification of lactic acid bacteria

[0059] 1.1.2.1.1 Isolation and purification of lactic acid bacteria

[0060] Take 1 mL of yogurt sample respectively, and perform 10-fold serial dilution with sterile normal saline to 10 -6 , and then take 10 -4 , 10 -5 , 10 -6 100 μL of bacterial suspensions at 3 gradients are spread on plates and cultured at 37 °C for 24 - 48 h, and the colony morphology is observed and recorded. Pick colonies with different morphologies on the plate for streak isolation. After culturing at 37 °C for 48 h, pick different single colonies on the plate for streak isolation again. Repeat this process multiple times until pure single colonies with consistent morphology are obtained.

[0061] 1.1.2.1.2 DNA extraction of lactic acid bacteria

[0062] Inoculate the purified suspected target strain into MRS broth. 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.

[0063] 1.1.2.1.3 PCR amplification of genomic DNA

[0064] 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'-CTA CGG CTA CCTTGT TACGA-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.

[0065] 1.1.2.1.4 In vitro resistance screening of lactic acid bacteria

[0066] 1.1.2.1.4.1 Ability of lactic acid bacteria to tolerate 0.3% bile salt

[0067] Add sodium cholate to MRS-THIO medium (MRS broth containing 0.2% sodium thioglycolate) to make its concentration 0.3%, sterilize at 121 °C for 15 min. Inoculate 5 mL of the activated strain into MRS-THIO medium without cholate (0.0%) and MRS-THIO medium containing 0.3% cholate at an inoculation amount of 2% (v / v) respectively. Using the blank medium (uninoculated MRS-THIO medium) 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 cholate according to formula (1):

[0068]

[0069] 1.1.2.1.4.12 Artificial gastric juice tolerance test

[0070] 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 experiment according to the corresponding mass-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.

[0071] 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 at pH 3.0. At this time, take 1 mL of the above mixture as the sample of artificial gastric juice treatment 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 diluted by 10-fold gradient respectively, and the appropriate gradient is selected to measure the viable bacteria 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.

[0072]

[0073] 1.1.3 Sequencing results of PCR amplification products of lactic acid bacteria

[0074] The 16s rDNA sequence of HFY13 is as follows (SEQ ID No.3):

[0075]

[0076] 1.1.4 In vitro resistance results of Lactobacillus

[0077] As can be seen from Table 1, the survival rate of LFHFY13 in artificial gastric juice at pH 3.0 was close to 80%; LFHFY13 had a high efficiency in 0.3% bile salt, reaching 54.38%, indicating that this strain had a strong ability to tolerate bile salt.

[0078] Table 1 Survival rates of lactic acid bacteria in artificial gastric juice at pH 3.0 and 0.3% bile salt

[0079]

[0080] 1.2 Experimental methods

[0081] 1.2.1 Preparation of experimental bacterial suspension

[0082] The lyophilized bacterial powder of LFHFY13 based on skim milk powder was added to sterilized MRS liquid medium (1:20, w / w), then placed in a low-temperature biochemical incubator at 37 °C for 48 h, and then centrifuged at 4000 r / min for 10 min. Finally, the precipitated bacteria were collected. Then, physiological saline was added at 5 times the mass of the bacteria, and the number of bacteria in the bacterial liquid was directly counted under a microscope using a hemocytometer to calculate the concentration of the bacterial liquid. Finally, the concentration of the bacterial liquid was adjusted to 3×10 8 CFU / mL for standby, and the bacterial liquid was diluted according to the mouse body weight before animal experiments for gavage.

[0083] 1.2.2 Animal experiments

[0084] 100 mice were adaptively fed in an environment with a temperature maintained at 20 ± 1 °C and a humidity of 30% - 40% for 7 h. Subsequently, the mice were randomly divided into 5 groups, namely the normal group, the model group, the vitamin C gavage group (Vc group), and two groups given LFHFY13 gavage at two different dose levels (LFHFY13-L group and LFHFY13-H group), with 20 mice in each group. Each group of mice was labeled from 1 to 20, among which numbers 1 - 5 and 11 - 15 were female, and numbers 6 - 10 and 16 - 20 were male. Referring to the standard of the recommended daily safe intake of vitamin C for humans of 1000 mg / d in the "Scientific Research Report on Dietary Guidelines for Chinese Residents (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 standard requirements of GB / T 21732-2008 "Milk-containing beverages", that is, live bacteria-containing beverages need to ensure at least 10 7 active microorganisms per milliliter at the time of product factory, and combined with the recommended daily intake of probiotic beverages on the market, it was deduced that the average daily probiotic intake suitable for experimental mice should be adjusted to 1.5×109 CFU / kg body weight. During the experiment, all groups of mice except the normal group will receive a 5% (w / v) D-galactose solution by intraperitoneal injection at a dose of 100 mg / kg BW daily for 6 consecutive weeks; while the normal group will be replaced with an equal volume of normal saline. Starting from the 7th week after the induction of the D-galactose solution, the Vc group will be gavaged with 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 LFHFY13-L group and the LFHFY13-H group will be gavaged with 0.75×10 9 CFU / kg and 1.50×10 9 CFU / kg of LFHFY13 bacterial suspension respectively according to the body weight of the mice, and the samples of each group will be gavaged for 4 weeks

[16] . On the 2nd day after the gavage of the samples, all the mice participating in the experiment will undergo a physical strength test. First, a exhaustion running experiment will be carried out, and after a 6-hour interval, an exhaustion swimming test will be carried out. Finally, euthanasia will be performed by cervical dislocation, and the liver tissue will be collected for further analysis.

[0085] 1.2.3 Exhaustion running experiment

[0086] An 8-channel mouse treadmill will be used for the exhaustion running test. The slope is set at 10°, and the speed of the treadmill will be gradually increased. The specific steps are as follows: running at a speed of 10 m / min for 5 min, and then running 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 judgment 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.

[0087] 1.2.4 Exhaustion swimming experiment

[0088] Before the exhaustion swimming test of the mice, a 10-min load-bearing swimming adaptation training was first carried out (the water temperature was maintained at 28±1°C and the water depth was about 40 cm). During the training, the mice's tails were loaded with lead wires about 5% of their body weight. After the training, the mice's fur was immediately dried with a towel and then dried with a hair dryer until completely dry. The formal exhaustion swimming experiment was carried out on the second day after the end of the adaptation training. The experimental conditions were the same as those of the adaptation training to ensure that the mice's limbs continued to move. The judgment criterion for exhaustion was that the mice's nostrils were completely immersed in the water for 7 s and they could not surface, and record the time required for the mice to swim to exhaustion.

[0089] 1.2.5 Determination of CK, BUN and lactic acid levels in mouse serum

[0090] After 2 h of intragastric administration, blood samples were collected from the orbits of mice numbered 1 - 20 by capillary blood sampling; 5 min after the exhaustion swimming test on the second day, blood samples were collected from the orbits of mice numbered 1 - 10 by capillary blood sampling; 30 min after the exhaustion swimming test, blood samples were collected from the orbits of mice numbered 11 - 20 by capillary blood sampling. The collected mouse blood was centrifuged at 4°C (1500 r / min for 10 min), and the upper serum was separated. According to the instructions provided by the detection kit, the serum lactate levels of the two blood samples from mice numbered 1 - 10 were measured, and the CK, BUN, and lactate levels in the serum of mice numbered 11 - 20 were measured.

[0091] 1.2.6 Determination of MG levels in gastrocnemius muscle tissue and HG levels in liver tissue of mice

[0092] An appropriate amount of mouse tissue was homogenized after adding normal saline at a ratio of 1:9. According to the instructions provided by the detection kit, the MG levels in gastrocnemius muscle tissue and HG levels in liver tissue of mice numbered 11 - 20 were measured.

[0093] 1.2.7 H&E staining and pathological sections of mouse liver

[0094] After dissecting the liver tissue of mice numbered 11 - 20, it was first rinsed 3 times with normal saline and then immediately fixed in 10% (v / v) formalin solution. After 48 h of dehydration at 4°C, these samples were embedded in paraffin. Then, the embedded tissue was cut into sections 5 - 10 μm thick and stained with H&E staining method, and finally the pathological characteristic changes were examined under an optical microscope.

[0095] 1.2.8 Determination of mRNA expression in mouse tissues

[0096] To detect the mRNA expressions of AMPK, PGC1-α, SOD2, and GPx1 in the livers and gastrocnemius muscles of mice, first, accurately weigh 0.2 g of tissue samples from mice numbered 11 - 20, and wash them with normal saline. Subsequently, cut these tissues into small pieces and add 1.0 mL of TRIzol reagent to them. Then, determine the purity of the obtained RNA solution by measuring the absorbance values at wavelengths of 260 nm and 280 nm, and adjust the RNA concentration to 1 μg / μL accordingly. After completing the above steps, a reverse transcription process was carried out to generate cDNA. On this basis, a reaction system for fluorescence quantitative PCR analysis was constructed, which consisted 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 amplification cycles 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 and extension stage (55 °C, 30 s), and final synthesis stage (72 °C, 35 s). In addition, a melting curve analysis step (95 °C, 30 s; 55 °C, 35 s) was set to verify the product specificity. Throughout the experiment, the GAPDH gene was selected as the internal reference standard, and the relative expression levels of the target genes were calculated according to 2 -ΔΔCt the method.

[0097] Table 2 Sequences of primers used for tissue determination in animal experiments

[0098]

[0099] 1.3 Data processing

[0100] 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.

[0101] 2 Results and analysis

[0102] 2.1 Effects of LFHFY13 on the exercise status of mice with oxidative stress

[0103] In the running and swimming experiments under exhaustion conditions, the normal group of mice had the longest performance time. In contrast, the model group of mice had the shortest exercise duration ( Figure 1-2)。Research found that compared with the model group, both LFHFY13 and Vc could significantly increase the performance duration of oxidative stress mice in these two tests (P<0.05). Among them, the effect of LFHFY13-H was the most significant, significantly better than that of LFHFY13-L and Vc. The accumulation of excessive free radicals in the body may lead to organ damage and functional decline, especially those tissues and systems closely related to exercise, thus 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 exercise ability of animals is often evaluated by measuring the maximum endurance running or swimming distance; improving such ability not only directly reflects the enhanced ability of individuals to resist fatigue, but also is an important indicator for measuring the antioxidant stress response. Existing research has pointed out that some lactic acid bacteria species with probiotic characteristics exhibit excellent antioxidant properties, which may help improve body vitality and enhance exercise ability. In this study, an oxidative stress model of mice was established to observe the effects of lactic acid bacteria LFHFY13 on the endurance running and swimming exhaustion of mice, and then to explore whether this strain can enhance the exercise potential of the host in the face of oxidative stress. Similar to previous studies, the time of exhaustion running and swimming increased after the improvement of exercise ability by intervention. The results of this study also showed that the persistence of mice treated with LFHFY13 increased in both tests compared with the control group, indicating that this bacterium may have the effect of promoting exercise endurance.

[0104] 2.2 Effects of LFHFY13 on blood lactic acid concentration in oxidative stress mice after excessive exercise (swimming)

[0105] As Figure 3As shown, before the exhaustive exercise (swimming) at rest, there was no significant difference in blood lactic acid concentration among the groups of mice (P < 0.05), and it was generally at a low level. After 5 minutes of exhaustive exercise, the blood lactic acid content in each group of mice increased, and there were significant differences among the groups (P < 0.05). Among them, the blood lactic acid concentration of the model group mice was the highest among the groups, followed by the Vc group, the LFHFY13-L group, the LFHFY13-H group, and the normal group in turn. After 30 minutes of exhaustive exercise, the blood lactic acid concentration in each group of mice decreased, and there were significant differences among the groups (P < 0.05). Among them, except for the normal group, the LFHFY13-H group was the lowest, followed by the LFHFY13-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 an accelerated anaerobic metabolism, and the production of lactic acid also increases accordingly, 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 experiments in this study also supported the above conclusions. The blood lactic acid content increased significantly after exhaustive exercise, and gradually decreased with the extension of time after exercise. During this process, LFHFY13 can better prevent lactic acid accumulation and protect the body under exhaustive exercise.

[0106] 2.3 Effects of LFHFY13 on MG in gastrocnemius muscle tissue, HG in liver tissue, CK and BUN in serum of oxidative stress mice

[0107] As Figure 4-7As 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 LFHFY13 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 LFHFY13-H group were higher than those in the LFHFY13-L group and the Vc group. The BUN and CK levels 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 LFHFY13-L group, the LFHFY13-H group, and the normal group. After experiencing oxidative stress, long-term physical activity may lead to abnormalities in carbohydrate and lipid metabolism and consume large amounts of protein and amino acids. During high-intensity or continuous exercise, a large number of metabolites are produced in the body, such as blood urea nitrogen (BUN) and various oxygen free radicals. These metabolites are the main factors causing exercise fatigue. The massive 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 CK concentration in the serum. Glycogen is mainly stored in the liver and muscles. Liver glycogen maintains blood glucose stability, and muscle glycogen directly supplies energy to the 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 beneficial microorganisms can intervene in MG, HG, CK, and BUN to improve exercise performance, and similar results were also observed in this study

[22] , LFHFY13 could significantly regulate the MG, HG, CK, BUN indexes in mice affected by oxidative stress and the lactic acid content in the blood, effectively alleviating the oxidative stress condition and enhancing their exercise durability and efficacy.

[0108] 2.4 Effects of LFHFY13 on the pathological changes of the liver tissue of mice with oxidative stress

[0109] Through microscopic observation of the liver tissue of mice ( Figure 8) 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 orderly in a radial pattern around the central vein. In contrast, in 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 the rupture of some cell membranes and cell nuclei, and the presence of apoptotic bodies was observed. Through treatment, LFHFY13 and Vc could reduce the hepatocyte damage in mice (model group) caused by oxidative stress. After treatment with the LFHFY13-H group, the lobular structure of the mouse liver almost returned to normal, while in the LFHFY13-L group and the Vc group, some hepatocytes were still damaged and the cell structure was significantly damaged. During strenuous exercise, the metabolic demand of the body increases 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 LFHFY13 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.

[0110] 2.5 Effects of LFHFY13 on the mRNA expression levels of factors related to the AMPK / PGC1-α pathway in mouse tissues

[0111] The mRNA expression levels of AMPK, PGC1-α, SOD2, and GPx1 in gastrocnemius muscle and liver tissues were detected, and the results are as Figure 9-12 and Figure 13-16As shown. Compared with the model group of mice, the mRNA expression levels of AMPK, PGC1-α, SOD2, and GPx1 in the Vc, LFHFY13-L, and LFHFY13-H groups were all significantly increased (P<0.05), and the increase in the LFHFY13-H group was significantly higher than that in the Vc group and the LFHFY13-L group. At the same time, the mRNA expression levels of AMPK, PGC1-α, SOD2, and GPx1 in the normal group of mice were the highest. AMPK plays a central role in energy metabolism regulation. When cells are faced with stress conditions such as hypoxia, ischemia, or physical exercise, this kinase system is activated, which then promotes the acceleration of glucose transport and fatty acid oxidation processes, while 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 LFHFY13 could significantly increase the mRNA expression levels of AMPK and PGC-1α in the tissues of oxidative stress mice. Excessive exercise can lead to peroxidation reactions in the body 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 mice receiving LFHFY13 gavage showed higher levels of SOD2 and GPx1 compared with the model group, indicating that LFHFY13 has a significant positive effect on enhancing antioxidant enzymes in mice, thus reflecting beneficial effects related to liver protection and enhanced exercise ability of the body.

[0112] 3 Conclusions

[0113] This invention constructed a mouse oxidative stress state model to evaluate the antioxidant performance of LFHFY13 and its effects on the exercise function of mice. The experimental data showed that LFHFY13 could significantly reduce the oxidative stress status 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, LFHFY13 showed better effects than vitamin C. In summary, this invention explored the mechanism of action of LFHFY13 in improving the exercise ability of oxidative stress mice, laid a foundation for the subsequent development of food-grade antioxidant components that can relieve oxidative damage and exercise ability decline caused by high-intensity work or natural aging, and was conducive to promoting the R & D process of probiotic products with independent intellectual property rights.

[0114] Although the above embodiments have described the present invention in detail, they are only a part of the embodiments of the present invention, rather than all of them. People can also obtain other embodiments based on these embodiments without creative efforts, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A Lactobacillus fermentum HFY13, characterized in that, The Lactobacillus fermentum HFY13 was preserved in the China General Microbiological Culture Collection Center on October 29, 2018, with the preservation number of CGMCC No. 16646.

2. Use of the Lactobacillus fermentum HFY13 according to claim 1 in the preparation of a product for promoting exercise endurance.

3. Use of the Lactobacillus fermentum HFY13 according to claim 1 in the preparation of a product for preventing lactic acid accumulation under exhaustive exercise.

4. Use of the Lactobacillus fermentum HFY13 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 urea nitrogen.

6. Use of the Lactobacillus fermentum HFY13 according to claim 1 in the preparation of a product for relieving liver injury under exhaustive exercise.

7. Use of the Lactobacillus fermentum HFY13 according to claim 1 in the preparation of a product for enhancing the antioxidant enzyme activity under exhaustive exercise.

8. Use of the Lactobacillus fermentum HFY13 according to claim 1 in the preparation of a product for increasing the expression levels of AMPK and PGC1-α in gastrocnemius under exhaustive exercise.

9. Use of the Lactobacillus fermentum HFY13 according to claim 1 in the preparation of a product for increasing the expression levels of SOD2 and GPx1 in liver tissue under exhaustive exercise.

10. A probiotic preparation, characterized in that, The probiotic preparation contains Lactobacillus fermentum HFY13 as described in claim 1; the bacterial content of Lactobacillus fermentum HFY13 in the probiotic preparation is 1.875×10 8 CFU / mL.

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