Lactobacillus acidophilus XY27 as well as application and probiotic preparation thereof

Abstract

The invention relates to the technical field of microorganisms, in particular to lactobacillus acidophilus XY27 as well as application and a probiotic preparation thereof. The lactobacillus acidophilus XY27 is preserved in the China General Microbiological Culture Collection Center (CGMCC) on July 15, 2019, and the preservation number is CGMCC No.18227. The lactobacillus acidophilus XY27 can be used for preparing the lactobacillus acidophilus XY27. According to the present invention, the intervention effect of the LAXY27 capable of regulating the balance of the intestinal flora on the decline of the swimming ability of the mouse is researched, the regulation effect of the LAXY27 on the intestinal flora is combined, the action mechanism of the LAXY27 on the regulation of the intestinal flora and the improvement of the motor function is preliminarily clarified, and the theoretical basis is accumulated for the practical application of the strain.

Description

Technical Field

[0001] The invention relates to the technical field of microorganisms, in particular to a strain of Lactobacillus acidophilus XY27 and an application thereof and a probiotic preparation. Background Art

[0002] The causes of sports fatigue can be summarized into four theories: energy depletion theory, metabolite accumulation theory, central nervous system protection inhibition theory and free radical theory. In recent years, the relationship between oxidative stress damage caused by free radicals and sports fatigue has received increasing attention, and oxidative stress plays an important role in the occurrence of fatigue. Oxidative stress and changes in cellular energy metabolism can activate adenylate-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 byproducts are reduced, and the energy generation efficiency in muscle cells is improved.

[0003] In recent years, the application of probiotics in sports and their use as nutritional supplements has been continuously promoted. According to existing studies, several specific types of probiotics, such as Lactobacillus plantarum PS128, have an effect on improving the endurance decline and fatigue state of triathletes after the game; Lactobacillus casei Shirota can reduce the incidence of upper respiratory tract infections in long-distance runners; Lactobacillus acidophilus SPP can improve the maximum oxygen uptake of swimmers; Bifidobacterium SP 07 / 3 can enhance the energy supply and improve the endurance of athletes; at the same time, a variety of thermophilic subspecies of Streptococcus salivarius are added to sports drinks and yogurt as drinks for athletes. The results of animal experiments explain the mechanism of probiotics improving the running endurance of mice, further confirming the expression of clinical research, which provides a new direction for exploring how to use probiotics to improve the physical limit of humans. Studies have shown that lactic acid bacteria in Sichuan pickles have a good intestinal colonization effect, which can regulate intestinal function and promote the health of the body. A study found that the stability of the intestinal flora is one of the factors for athletes to maintain a good athletic state through the DNA fingerprint of the structural characteristics of the intestinal microbial flora. This shows that the intestinal flora is closely related to athletic function.

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

[0005] In order to solve the above problems, the present invention provides a strain of Lactobacillus acidophilus XY27 and its application and probiotic preparation. The present invention studies the intervention effect of LAXY27, which has the function of regulating the balance of intestinal flora, on the decreased swimming ability of mice. Combined with the regulatory effect of LAXY27 on intestinal flora, the mechanism of action of LAXY27 on intestinal flora regulation and motor function improvement is preliminarily clarified, accumulating theoretical basis for the practical application of the strain. The "LAXY27" mentioned in the present invention is Lactobacillus acidophilus XY27.

[0006] In order to achieve the above object, the present invention provides the following technical solutions: The present invention provides a strain of Lactobacillus acidophilus ( Lactobacillus acidophilus ) XY27, the Lactobacillus acidophilus XY27 was deposited in the General Microbiology Center of China National Microbiological Culture Collection Administration on July 15, 2019, with the deposit number CGMCC No.18227.

[0007] The present invention also provides the use of Lactobacillus acidophilus XY27 described in the above technical solution in the preparation of a product promoting sports endurance.

[0008] The present invention also provides the use of Lactobacillus acidophilus XY27 described in the above technical solution in the preparation of a product for preventing lactic acid accumulation during exhaustive exercise.

[0009] The present invention also provides the use of Lactobacillus acidophilus XY27 described in the above technical solution in the preparation of a product for alleviating oxidative stress and enhancing sports durability.

[0010] Preferably, the indicators of oxidative stress include muscle glycogen, liver glycogen, creatine kinase and urea nitrogen.

[0011] The present invention also provides the use of Lactobacillus acidophilus XY27 described in the above technical solution in the preparation of a product for enhancing the activity of antioxidant enzymes under exhaustive exercise.

[0012] The present invention also provides the use of Lactobacillus acidophilus XY27 described in the above technical solution in the preparation of a product for regulating intestinal flora under exhaustive exercise.

[0013] Preferably, the method of regulating the intestinal flora under exhaustive exercise includes: increasing the number of Firmicutes microorganisms, lactic acid bacteria and Bifidobacterium, and reducing the number of Bacteroidetes microorganisms.

[0014] The present invention also provides the use of Lactobacillus acidophilus XY27 described in the above technical solution in the preparation of a product for alleviating liver damage caused by exhaustive exercise.

[0015] The present invention also provides a probiotic preparation, which contains the Lactobacillus acidophilus XY27 described in the above technical solution; the bacterial content of Lactobacillus acidophilus XY27 in the probiotic preparation is 1.875×10 8 CFU / mL.

[0016] Beneficial effects of the present invention: The present invention constructs a mouse oxidative stress state model, aiming to evaluate the antioxidant properties of LAXY27 and its effect on the motor function of mice. Experimental data show that LAXY27 can significantly reduce the oxidative stress in mice and promote the energy metabolism of gastrocnemius muscle, thereby improving the ability of the test animals to resist fatigue and perform physical activities. Further analysis found that, under the premise of following the daily recommended intake of humans, LAXY27 showed an effect superior to vitamin C. In summary, the present invention explores the mechanism of action of LAXY27 in improving the motor ability of oxidative stress mice, laying the foundation for the subsequent development of food-grade antioxidant ingredients that can alleviate oxidative damage and motor decline caused by high-intensity work or natural aging, and is conducive to promoting the research and development of probiotic products with independent intellectual property rights. Nevertheless, the above conclusions still need to be verified through more clinical trials to verify their general applicability, which will be a key point for future scientific research around LAXY27. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required to be used in the embodiments are briefly introduced below.

[0018] Figure 1 is the swimming time of mice with oxidative stress under exhaustion. Note: The same lowercase letters in the bar graph indicate that there is no significant difference between the two groups, while different lowercase letters indicate that there is a significant difference between the two groups (P<0.05); Figure 2This is the change of serum lactate concentration in mice with oxidative stress after excessive exercise. Note: The data before exhaustive exercise (swimming) are the first measurement data of mice No. 1-10, the data 5 minutes after exhaustive exercise (swimming) are the second measurement data of mice No. 1-10, and the data 30 minutes after exhaustive exercise (swimming) are the measurement data of mice No. 11-20. The same lowercase English letters under the same time conditions indicate that there is no significant difference between the two groups, while different lowercase letters indicate that there is a significant difference between the two groups (P<0.05); Figure 3 is the HG level of gastrocnemius muscle in mice with oxidative stress. Note: The same lowercase letters in the bar graph indicate no significant difference between the two groups, while different lowercase letters indicate significant difference between the two groups (P<0.05); Figure 4 is the MG level of gastrocnemius muscle in mice with oxidative stress. Note: The same lowercase letters in the bar graph indicate no significant difference between the two groups, while different lowercase letters indicate significant difference between the two groups (P<0.05); Figure 5 is the serum CK level of mice with oxidative stress. Note: The same lowercase letters in the bar graph indicate no significant difference between the two groups, while different lowercase letters indicate significant difference between the two groups (P<0.05); Figure 6 is the serum BUN level of mice with oxidative stress. Note: The same lowercase letters in the bar graph indicate no significant difference between the two groups, while different lowercase letters indicate significant difference between the two groups (P<0.05); Figure 7 Pathological observation of liver tissues in mice with oxidative stress; Figure 8 is the mRNA expression level of AMPK in gastrocnemius muscle of mice with oxidative stress. Note: The same lowercase letters in the bar graph indicate no significant difference between the two groups, while different lowercase letters indicate significant difference between the two groups (P<0.05); Fig. 9 is the mRNA expression level of PGC1-α in gastrocnemius muscle of mice with oxidative stress. Note: The same lowercase letters in the bar graph indicate no significant difference between the two groups, while different lowercase letters indicate significant difference between the two groups (P<0.05); Fig.10 is the mRNA expression level of SOD2 in gastrocnemius muscle of mice with oxidative stress. Note: The same lowercase letters in the bar graph indicate no significant difference between the two groups, while different lowercase letters indicate significant difference between the two groups (P<0.05); Fig.11is the mRNA expression level of GPx1 in gastrocnemius muscle of mice with oxidative stress. Note: The same lowercase letters in the bar graph indicate no significant difference between the two groups, while different lowercase letters indicate significant difference between the two groups (P<0.05); Fig.12 is the mRNA expression level of AMPK in the liver of mice with oxidative stress. Note: The same lowercase letters in the bar graph indicate that there is no significant difference between the two groups, while different lowercase letters indicate that there is a significant difference between the two groups (P<0.05); Fig.13 is the mRNA expression level of PGC1-α in the liver of mice with oxidative stress. Note: The same lowercase letters in the bar graph indicate that there is no significant difference between the two groups, while different lowercase letters indicate that there is a significant difference between the two groups (P<0.05); Fig.14 is the mRNA expression level of SOD2 in the liver of mice with oxidative stress. Note: The same lowercase letters in the bar graph indicate that there is no significant difference between the two groups, while different lowercase letters indicate that there is a significant difference between the two groups (P<0.05); Fig.15 is the mRNA expression level of GPx1 in the liver of mice with oxidative stress. Note: The same lowercase letters in the bar graph indicate that there is no significant difference between the two groups, while different lowercase letters indicate that there is a significant difference between the two groups (P<0.05); Fig.16 is the mRNA expression level of Firmicutes in the intestinal contents of mice with oxidative stress. Note: The same lowercase letters in the bar graph indicate that there is no significant difference between the two groups, while different lowercase letters indicate that there is a significant difference between the two groups (P<0.05); Fig.17 is the mRNA expression level of Bacteroidetes in the intestinal contents of oxidative stress mice. Note: The same lowercase letters in the bar graph indicate that there is no significant difference between the two groups, while different lowercase letters indicate that there is a significant difference between the two groups (P<0.05); Fig.18 is the mRNA expression level of Lactobacillus in the intestinal contents of mice with oxidative stress. Note: The same lowercase letters in the bar graph indicate that there is no significant difference between the two groups, while different lowercase letters indicate that there is a significant difference between the two groups (P<0.05); Fig.19 is the mRNA expression level of Bifidobacterium in the intestinal contents of mice with oxidative stress. Note: The same lowercase letters in the bar graph indicate that there is no significant difference between the two groups, while different lowercase letters indicate that there is a significant difference between the two groups (P<0.05).

[0019] Biological Deposit Description Lactobacillus acidophilus XY27, Latin for Lactobacillus acidophilus , deposited at the General Microbiology Center of China Microorganism Culture Collection on July 15, 2019, with the deposit number CGMCC No.18227. The address is: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Postal Code 100101. DETAILED DESCRIPTION

[0020] The present invention provides a strain of Lactobacillus acidophilus ( Lactobacillus acidophilus ) XY27, the Lactobacillus acidophilus XY27 was deposited in the General Microbiology Center of China National Microbiological Culture Collection Administration on July 15, 2019, with the deposit number CGMCC No.18227.

[0021] The present invention also provides the use of Lactobacillus acidophilus XY27 described in the above technical solution in the preparation of a product for promoting sports endurance. The present invention has no special limitation on the product, such as medicine, health care product, etc.

[0022] The present invention also provides the use of Lactobacillus acidophilus XY27 described in the above technical solution in the preparation of a product for preventing lactic acid accumulation during exhaustive exercise. The present invention has no special limitation on the product, such as medicine, health care product, etc.

[0023] The present invention also provides the use of Lactobacillus acidophilus XY27 described in the above technical solution in the preparation of a product for alleviating oxidative stress and enhancing sports endurance. In the present invention, the index of oxidative stress preferably includes muscle glycogen, liver glycogen, creatine kinase and urea nitrogen. The present invention has no special limitation on the product, such as medicine, health care product, etc.

[0024] The present invention also provides the use of Lactobacillus acidophilus XY27 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 has no special limitation on the product, such as medicine, health care product, etc.

[0025] The present invention also provides the use of Lactobacillus acidophilus XY27 described in the above technical solution in the preparation of a product for regulating intestinal flora under exhaustive exercise. In the present invention, the method for regulating intestinal flora under exhaustive exercise includes: increasing the number of Firmicutes microorganisms, lactic acid bacteria and Bifidobacterium, and reducing the number of Bacteroidetes microorganisms. The present invention has no special limitation on the product, such as medicine, health care product, etc.

[0026] The present invention also provides the use of Lactobacillus acidophilus XY27 described in the above technical solution in preparing a product for relieving liver damage caused by exhaustive exercise. The present invention has no special limitation on the product, such as medicine, health care product, etc.

[0027] The present invention also provides a probiotic preparation, which contains Lactobacillus acidophilus XY27 described in the above technical solution; the bacterial content of Lactobacillus acidophilus XY27 in the probiotic preparation is 1.875×10 8 CFU / mL. The present invention does not have any 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.

[0028] To further illustrate the present invention, the present invention will be described in detail below with reference to examples, but they should not be construed as limiting the protection scope of the present invention.

[0029] Example 1 1 Materials and Methods 1.1 Materials and Instruments Lactobacillus acidophilus XY27 was isolated and identified from traditional natural fermented yogurt in the herdsman's home in Xinyuan County, Xinjiang Uygur Autonomous Region by the Collaborative Innovation Center for Child Nutrition and Health Development of Chongqing University of Education. This strain has been registered and preserved in the China General Microbiological Culture Collection Center (CGMCC No. 18227); DeMan, Rogosa and Sharpe (MRS) medium, Beijing Land Bridge 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 for separation are domestic analytical pure.

[0030] 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.; A200 gene amplifier, Hangzhou Longgene Scientific Instruments Co., Ltd.; StepOnePlus real-time fluorescence quantitative PCR instrument, Thermo Fisher Scientific; OLYMPUS-BX43 upright microscope, Olympus Instruments Co., Ltd.

[0031] The Kunming mice used in the experiment 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 number: SCXK (Yu) 2022-0010).

[0032] The isolation and identification of Lactobacillus acidophilus XY27 are as follows: 1.1.1 Experimental Materials The yogurt was collected from the traditional naturally fermented yogurt of herders in Xinyuan County, Xinjiang Uygur Autonomous Region. After the yogurt was fully stirred with a sterile spoon, 50 mL was drawn into a sterilized centrifuge tube with a sterile syringe, which was then placed in a low-temperature food sampling box and brought back to the laboratory for freezing and storage at -80 o C ultra-low temperature refrigerator for later use.

[0033] 1.1.2 Experimental methods 1.1.2.1 Isolation and identification of lactic acid bacteria 1.1.2.1.1 Isolation and purification of lactic acid bacteria Take 1 mL of yogurt sample and dilute it 10 times to 10 with sterile saline. -6 , then take 10 -4 , 10 -5 , 10 -6 100 μL of the three gradient bacterial solutions were spread on the plates and cultured at 37°C for 24-48 h. The colony morphology was observed and recorded. The colonies of different morphologies on the plates were picked for streaking and separated. o After culturing at C for 48 h, single colonies with different morphologies on the plate were picked up again for streaking separation, and this process was repeated several times until pure single colonies with consistent morphology were obtained.

[0034] 1.1.2.1.2 Lactic acid bacteria DNA extraction The purified suspected target strain was inoculated into MRS broth. o After culturing at C for 18-24 h, DNA was extracted using a bacterial genomic DNA extraction kit. The extracted DNA was numbered and stored in a -20°C freezer for later use.

[0035] 1.1.2.1.3 PCR amplification of genomic DNA The extracted DNA was subjected to PCR amplification, including 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, and 1 μL of template DNA. Sterile dd H2O was used to make up the system to 25 μL. Sterile ultrapure water was used to replace the template DNA as a negative control. The amplification conditions were: 94 o C 5 min; 94 o C 30s, 55 oC 30 s, 72 o C for 1 min, 29 cycles in total, and 72 o C extension for 5 min. The PCR products were then sent to Beijing Qingke Biotechnology Co., Ltd. for sequencing, and the successfully sequenced sequences were compared and analyzed using the BLAST (Basic Local Alignment Search Tool) program in NCBI.

[0036] 1.1.2.1.4 In vitro resistance screening of lactic acid bacteria 1.1.2.1.4.1 Ability of lactic acid bacteria to tolerate 0.3% bile salts Porcine bile salt was added to MRS-THIO medium (MRS broth containing 0.2% sodium thioglycolate) to make the concentration 0.3%, and sterilized at 121℃ for 15 min. 5 mL of activated bacteria were inoculated at a 2% (v / v) inoculum into MRS-THIO medium without bile salt (0.0%) and MRS-THIO medium containing 0.3% bile salt, respectively. Blank medium (MRS-THIO medium without bacteria) was used as control. o After culturing for 24 h, the OD values ​​of the above culture media with different concentrations were measured. 600nm The value was used to calculate the tolerance of the strain to bile salts according to formula (1): 1.1.2.1.4.2 Artificial gastric juice tolerance test Preparation of artificial gastric juice: Artificial gastric juice is composed of 0.2% NaCl and 0.35% pepsin. The NaCl and pepsin required for the test are weighed according to the corresponding mass-to-volume ratio for preparation. The pH of the prepared artificial gastric juice is adjusted to 3.0 with 1 mol / L HCl, and then filtered with a 0.22 μm filter membrane for sterilization and set aside.

[0037] In the clean bench, 5 mL of the culture medium containing bacteria was taken into a 10 mL sterile centrifuge tube and centrifuged at 4000 r / min for 10 min. The upper culture medium was discarded and the bacteria were collected. An equal volume (5 mL) of sterile saline was added and mixed to make a bacterial suspension. Then 1 mL of the bacterial suspension was mixed with 9 mL of artificial gastric juice at pH 3.0. At this time, 1 mL of the above mixture was taken as the sample treated with artificial gastric juice for 0 h, and the remaining 9 mL of the mixture was placed in a constant temperature water bath shaker (37 o C, 150 r / min) for 3 h. The samples at 0 h and 3 h were diluted 10 times, and the number of viable bacteria was determined by plate coating method at appropriate gradients. o C for 48 h, and the survival rate (%) was calculated according to Formula 2.

[0038] 1.1.3 Experimental Results 1.1.3.1 Sequencing results of lactic acid bacteria PCR amplification products The sequence of 16s rDNA of Lactobacillus acidophilus XY27 is as follows (SEQ ID No. 3):

[0039] 1.1.3.2 Results of in vitro resistance of lactobacilli As shown in Table 1, the survival rate of XY27 in pH 3.0 artificial gastric juice exceeded 80%; LFHFY13 was more efficient in 0.3% bile salt, reaching 62.71%, indicating that this strain has a strong ability to tolerate bile salt.

[0040] Table 1 Survival rate of lactic acid bacteria in pH 3.0 artificial gastric juice and 0.3% bile salt 1.2 Experimental methods 1.2.1 Preparation of experimental bacterial suspension The LAXY27 freeze-dried powder based on skim milk powder was added to the sterilized MRS liquid medium (1:20, w / w), and then placed in a low-temperature biochemical incubator at 37°C for 48 h, and then centrifuged at 4000 r / min for 10 min, and finally the precipitated bacteria were collected. Then physiological saline was added according to 5 times the mass of the bacteria, and the number of bacteria in the bacterial solution was directly counted under a microscope using a hemocytometer to calculate the concentration of the bacterial solution. Finally, the concentration of the bacterial solution was adjusted to 3×10 8 CFU / mL was reserved for future use. Before animal experiments, the bacterial solution was diluted according to the weight of mice for intragastric administration.

[0041] 1.2.2 Animal experiments 100 mice were adaptively raised for 7 hours in an environment maintained at a temperature of 20 ± 1℃ and a humidity of 30%~40%, and then randomly divided into 5 groups, namely a normal group, a model group, a vitamin C gavage group (Vc group), and two groups given LAXY27 gavage at different dose levels (LAXY27-L group and LAXY27-H group), with 20 mice in each group, and each group of mice was marked as 1-20, of which 1-5 and 11-15 were female, and 6-10 and 16-20 were male. Referring to the vitamin C standard of 1000 mg / d for human daily safe intake recommended in the "Scientific Research Report on the Dietary Guidelines for Chinese Residents (2021)", this study set the amount of vitamin C supplemented per kilogram of body weight per day for experimental animals to be 150 mg. In addition, according to the GB / T 21732-2008 "Milk Beverages" standard requirements, that is, the live bacteria beverage must ensure that it contains at least 10 per milliliter when the product leaves the factory. 7 active microorganisms, and combined with the recommended daily intake of probiotic drinks on the market, it was calculated that the average daily probiotic intake suitable for experimental mice should be adjusted to 1.5×10 9CFU / kg body weight. During the experiment, mice in all groups except the normal group received 5% (w / v) D-galactose solution at a dose of 100 mg / kg BW by intraperitoneal injection every day for 6 consecutive weeks; the normal group received an equal volume of saline instead. Starting from the 7th week after the end of the D-galactose solution induction, the Vc group was gavaged with vitamin C solution at 150 mg / kg BW daily; the model group and the normal group were gavaged with 2 mL of distilled water (placebo) daily; the LAXY27-L group and the LAXY27-H group were gavaged with 0.75×10 9 CFU / kg and 1.50×10 9 CFU / kg of LAXY27 bacterial suspension, and each group was gavaged for 4 weeks. On the second day after the gavage, all mice participating in the experiment were tested for physical strength and exhaustive swimming test, and finally euthanized by cervical dislocation, and liver tissue was collected for further analysis.

[0042] 1.2.3 Exhaustive swimming test Before the exhaustive swimming test of mice, a 10-min weighted swimming adaptation training was first performed (the water temperature was maintained at 28±1℃ and the water depth was about 40 cm). During the training, the tail of the mouse was loaded with a lead wire weighing about 5% of its body weight. After the training, the fur of the mouse was immediately dried with a towel and blown dry with hot air until completely dry. The formal exhaustive swimming experiment was performed the day after the adaptive training. The experimental conditions were the same as those of the adaptive training, ensuring that the limbs of the mouse continued to move. The criterion for exhaustion was that the mouse's nostrils were completely immersed in water for 7 s and could not float to the surface, and the time required for the mouse to swim to exhaustion was recorded.

[0043] 1.2.4 Determination of CK, BUN and lactate levels in mouse serum Blood samples were drawn from the eye sockets of mice No. 1-20 by capillary blood sampling 2 hours after the end of oral gavage; blood samples were drawn from the eye sockets of mice No. 1-10 by capillary blood sampling 5 minutes after the end of the exhaustive swimming test on the second day; blood samples were drawn from the eye sockets of mice No. 11-20 by capillary blood sampling 30 minutes after the end of the exhaustive swimming test. The collected mouse blood was centrifuged at 4°C (1500 r / min, for 10 minutes) to separate the upper serum. According to the instructions provided by the detection kit, the serum lactate levels of the two blood samples from mice No. 1-10 and the CK, BUN and lactate levels in the serum of mice No. 11-20 were determined.

[0044] 1.2.5 Determination of MG levels in gastrocnemius muscle and HG levels in liver tissue of mice Take an appropriate amount of mouse tissue and add physiological saline at a ratio of 1:9 and homogenize. According to the instructions provided by the detection kit, measure the MG level of gastrocnemius muscle tissue and HG level of liver tissue of mice No. 11-20.

[0045] 1.2.6 H&E staining and pathological sections of mouse liver After dissecting the liver tissues of mice No. 11-20, they were rinsed three times with saline and then immediately fixed in 10% (v / v) formalin solution. After 48 hours of low-temperature dehydration at 4°C, the samples were embedded in paraffin. After that, the embedded tissues were cut into 5-10 µm thick sections and stained with H&E, and finally the pathological changes were examined under an optical microscope.

[0046] 1.2.7 Determination of mRNA expression in mouse tissues To detect the mRNA expression of AMPK, PGC1-α, SOD2, and GPx1 in mouse liver and gastrocnemius muscle, 0.2 g of mouse tissue samples No. 11-20 were first accurately weighed and washed with physiological saline. Subsequently, the tissues were minced and 1.0 mL of TRlzol reagent was added to them. Next, the purity of the obtained RNA solution was determined by measuring the absorbance values ​​at wavelengths of 260 nm and 280 nm, and the RNA concentration was adjusted to 1 μg / μL accordingly. After completing the above steps, the reverse transcription process was performed to generate cDNA. On this basis, a reaction system for fluorescence quantitative PCR analysis was constructed, which consisted of 1 μL cDNA, 10 μL SYBR Green PCR Master Mix, 1 μL of each self-designed specific primer (see Table 2), and 7 μL sterile distilled water. The prepared reaction solution was placed in a real-time fluorescence quantitative PCR instrument, and the amplification cycle was performed according to the preset conditions: initial heating at 95°C for 60 s; followed by 40 cycles of denaturation phase (95°C, 15 s), annealing extension phase (55°C, 30 s) and final synthesis phase (72°C, 35 s). In addition, a melting curve analysis step (95°C, 30 s; 55°C, 35 s) was set to verify 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 based on 2 −ΔΔCt Calculated by method.

[0047] Table 2 Sequences of primers used for tissue determination in animal experiments 1.2.8 Determination of relative microbial content in intestinal contents After the autopsy of mice No. 11-20, 0.2 g samples of the 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 were measured for these intestinal samples (see Table 3) to explore the specific composition of the intestinal microbial community of the mice.

[0048] Table 3 Sequences of primers used for determination of microorganisms in large intestine contents in animal experiments 1.3 Data processing The mice involved in the experiment were measured in detail, and the obtained data are presented as mean ± SD. In addition, one-way ANOVA was performed using SPSS 22.0 software to evaluate the P Whether there is a statistically significant difference between the groups at a significance level of <0.05.

[0049] 2 Results and analysis 2.1 Effect of LAXY27 on the swimming state of mice with oxidative stress In the swimming test under exhaustion, the mice in the normal group performed the longest, while the mice in the model group had the shortest swimming duration ( Figure 1 ). The study found that compared with the model group, both LAXY27 and Vc could significantly improve the performance time of oxidative stress mice in this swimming test ( P<0.05). Among them, LAXY27-H had the most significant effect, which was significantly better than LAXY27-L and Vc. Excessive free radical accumulation in the body may cause organ damage and decreased function, especially those tissues and systems closely related to exercise, resulting in weakened athletic 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 essential for maintaining a high level of physical strength. In scientific experiments, the maximum endurance swimming distance of animals is often measured to evaluate their athletic ability; improving such ability not only directly reflects the individual's enhanced ability to resist fatigue, but is also an important indicator for measuring anti-oxidative stress response. Existing studies have shown that some lactic acid bacteria with probiotic properties have excellent antioxidant properties, which may help improve body vitality and enhance athletic ability. In this study, an oxidative stress model was established in mice to observe the effect of lactic acid bacteria LAXY27 on mouse swimming exhaustion, and then explore whether this strain can enhance the host's athletic potential in the face of oxidative stress. Similar to previous studies, the duration of exhaustive swimming was improved after the intervention. The results of this study also showed that the mice treated with LAXY27 had increased endurance in the swimming test compared with the control group, indicating that this bacterium may have the effect of promoting exercise endurance.

[0050] 2.2 Effect of LAXY27 on blood lactate concentration in mice with oxidative stress after excessive exercise (swimming) like Figure 2 As shown, at rest time before exhaustive exercise (swimming), there was no significant difference in blood lactate concentration among mice in each group ( P <0.05), and the overall level was low. After 5 min of exhaustive exercise, the blood lactate content of mice in each group increased, and the differences among the groups were significant (P<0.05). Among them, the blood lactate concentration of mice in the model group was the highest among all groups, followed by the Vc group, LAXY27-L, LAXY27-H group and the normal group. After 30 min of exhaustive exercise, the blood lactate concentration of mice in each group decreased, and there were significant differences among the groups ( P<0.05). Among them, except for the normal group, the LAXY27-H group had the lowest blood lactate content, followed by the LAXY27-L group and the Vc group, and the model group had the highest blood lactate content. Blood lactate 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 intensity of exercise increases, muscle activity increases, resulting in accelerated anaerobic metabolism, and the production of lactate also increases, thereby increasing the blood lactate concentration; exercise causes muscle fatigue, affects the intake and utilization of oxygen by muscles, promotes the synthesis of lactate, and causes lactate accumulation. High levels of blood lactate usually indicate that the body consumes more energy during exercise and causes fatigue. Animal experiments in this study also support the above conclusions. The blood lactate content increased significantly after exhaustive exercise, and the blood lactate content gradually decreased as the time after exercise increased. In this process, LAXY27 can better prevent lactic acid accumulation and protect the body under exhaustive exercise.

[0051] 2.3 Effects of LAXY27 on MG in gastrocnemius muscle, HG in liver tissue, and serum CK and BUN in mice with oxidative stress like Figure 3-6As shown, the normal group showed the highest MG and HG levels, while the model group had the lowest MG and HG levels. Vc and LAXY27 can increase the MG and HG levels of mice with oxidative stress. The results showed that the MG and HG levels of the LAXY27-H group were higher than those of the LAXY27-L group and the Vc group. The BUN and CK levels of the model group showed an opposite trend, which were higher than those of the other groups. The other groups were Vc group, LAXY27-L group, LAXY27-H group and normal group from high to low. After experiencing oxidative stress, long-term physical activity may cause abnormal carbohydrate metabolism and lipid metabolism, and consume a large amount of protein and amino acids. During high-intensity or continuous exercise, a large amount of metabolites will be produced in the body, such as urea nitrogen (BUN) and various oxygen free radicals. These metabolites are the main factors leading to exercise fatigue. The large accumulation of metabolites in the body will not only cause metabolic imbalance in the internal environment, but also damage organs and tissues. Creatine kinase (CK) is an important enzyme involved in the body's energy metabolism, mainly present in skeletal muscle. When high-intensity exercise causes skeletal muscle damage, the permeability of the muscle cell membrane will change, and CK will be released into the blood in large quantities, resulting in an increase in serum CK concentration. Glycogen is mainly stored in the liver and muscles. Liver glycogen maintains blood sugar stability, and muscle glycogen directly supplies energy to the muscles. During long-term exercise, glycogen is gradually broken down to maintain energy supply. Glycogen depletion can cause fatigue and affect athletic performance. Glycogen regulation is of great significance to the body's exercise metabolism. It has been confirmed that beneficial microorganisms can intervene in MG, HG, CK, and BUN to improve exercise function. Similar results were also observed in this study. LAXY27 can significantly regulate MG, HG, CK, BUN indicators and lactic acid content in the blood of mice affected by oxidative stress, effectively alleviating oxidative stress and enhancing their exercise endurance and performance.

[0052] 2.4 Effect of LAXY27 on pathological changes in liver tissue of mice with oxidative stress Microscopic observation of mouse liver tissue ( Figure 7), it can be seen that in the normal group, the lobular structure of the liver remained intact and had clear boundaries, and the hepatocytes were arranged in an orderly manner around the central vein in a radial pattern. In contrast, in the model group, the lobular structure of the mouse liver was significantly damaged, as shown by the fact that the hepatocytes were no longer arranged in a normal radial pattern, accompanied by the rupture of some cell membranes and nuclei, and the presence of apoptotic bodies was observed. Through treatment, LAXY27 and Vc can alleviate the hepatocyte damage caused by oxidative stress in mice (model group). After treatment with the LAXY27-H group, the hepatic lobular structure of the mice almost returned to normal, while some hepatocytes in the LAXY27-L group and the Vc group were still damaged, and the cell structure was significantly damaged. During strenuous exercise, the body's metabolic needs increase significantly, the blood circulation speed increases, and muscle tissue produces more metabolites. Exhaustive exercise will increase the burden on the liver, thereby causing liver damage. The pathological sections of this study also showed liver damage caused by exhaustive exercise, and LAXY27 can effectively alleviate liver damage, protect the liver, and may have the effect of assisting the liver in metabolism, thereby regulating the body to maintain a good state of exercise and reducing fatigue.

[0053] 2.5 Effect of LAXY27 on mRNA expression levels of AMPK / PGC1-α pathway-related factors in mouse tissues The mRNA expression levels of AMPK, PGC1-α, SOD2, and GPx1 in gastrocnemius muscle and liver tissues were detected. Figure 8-11 and Figure 12-15 Compared with the model group, the mRNA expression levels of AMPK, PGC1-α, SOD2 and GPx1 in the Vc, LAXY27-L and LAXY27-H groups were significantly increased ( P<0.05), among which the increase in the LAXY27-H group was significantly higher than that in the Vc group and the LAXY27-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 the regulation of energy metabolism. When cells face stress conditions such as hypoxia, ischemia or physical exercise, the kinase system will be activated, which will promote the acceleration of glucose transport and fatty acid oxidation, while inhibiting activities such as gluconeogenesis, protein synthesis and lipid metabolism. In addition, PGC-1α is also one of the key factors in regulating physiological functions such as fatty acid oxidation, glucose utilization and mitochondrial generation. 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 LAXY27 can significantly increase the mRNA expression levels of AMPK and PGC-1α in the tissues of mice with oxidative stress. Excessive exercise can lead to peroxidation in the body and induce oxidative stress damage. Preclinical studies have shown that endurance training can enhance the activity 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 that received LAXY27 gavage showed higher levels of SOD2 and GPx1 compared with the model group, indicating that LAXY27 has a significant positive effect on enhancing antioxidant enzymes in mice, thereby reflecting the beneficial effects related to liver protection and enhanced exercise capacity.

[0054] Effect of LAXY27 on microbial expression in the intestinal contents of oxidative stress mice Firmicutes and Bacteroidetes are the two largest types of microorganisms in the human intestine, and these two types of microorganisms can be used as preliminary indicators for microbial flora assessment. Bifidobacterium is one of the most important beneficial microorganisms in the intestine. The sample administered by gavage in this study was lactic acid bacteria. Therefore, the expression of each Firmicutes, Bacteroidetes, Lactobacillus and Bifidobacterium in the intestinal contents was measured, and the total bacteria (all microorganisms in the contents) was used as a reference to detect the relative intensity of the expression of each type of bacteria in order to determine the relative content of each type of bacteria. Figure 16-19 The data shown show that the expression level of Bacteroidetes mRNA in the intestine of mice in the normal group is the lowest ( P<0.05), while the expression of Firmicutes and Bifidobacterium was relatively high. This result shows that in the normal group, the proportion of Bacteroidetes microorganisms was the smallest, while the proportion of Firmicutes microorganisms was the largest. After LAXY27 treatment, the proportion of Firmicutes and Bifidobacterium in the intestines of the model group mice increased significantly, while the proportion of Bacteroidetes decreased. In addition, after LAXY27 intake, the proportion of Lactobacillus in mice in the LAXY27-L group and LAXY27-H group 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). Studies have shown that there is a close connection between the health status and redox balance of the human internal organs and the intestinal microbial community. In addition, intestinal microorganisms also have an important impact on organ damage and inflammatory responses. Related studies have shown that healthy intestinal flora can convert specific foods into nutrients that are beneficial to the body, thereby promoting the production of metabolic regulatory factors, enhancing muscle strength, and improving athletic performance. Clinical observations have found that certain types of intestinal bacteria can consume lactic acid produced during exercise, reduce its adverse effects on muscles, and thus help improve athletes' endurance levels. When the body faces oxidative stress, the composition of microorganisms in the intestine will directly affect the functionality of the intestine. Unbalanced intestinal flora may interfere with normal intestinal peristalsis, mucus production, and barrier function, making it easier for toxins to enter the blood circulation system, further aggravating the degree of oxidative stress. Under oxidative stress conditions, the ratio of Firmicutes and Bacteroidetes bacteria in the human intestine will change significantly, among which the proportion of Bacteroidetes bacteria increases and Bifidobacterium decreases relatively. It is worth noting that Lactobacillus, one of the Firmicutes, has the effect of increasing the activity of antioxidant enzymes. The results of this embodiment also confirm this viewpoint, LAXY27 can enhance the antioxidant enzyme activity in gastrocnemius and liver tissue of mice. In addition, under the intervention of LAXY27, the proportion of Firmicutes microorganisms and Bifidobacterium in oxidative stress mice increased, while the proportion of Bacteroidetes decreased. In addition, since LAXY27 is a lactobacillus, the proportion of lactobacillus in the intestine of oxidative stress mice increased significantly under its intervention, reaching a state higher than that of normal group mice. The experimental results show that LAXY27 can effectively regulate the intestinal flora of mice, which is consistent with the results of the intestinal flora of athletes related to exercise status in clinical experiments, suggesting that the effect of LAXY27 on improving intestinal flora is also one of the important factors for improving body function and enhancing exercise ability.

[0055] 3 Conclusion The present invention constructs a mouse oxidative stress state model, aiming to evaluate the antioxidant properties of LAXY27 and its effect on the motor function of mice. Experimental data show that LAXY27 can significantly reduce the oxidative stress in mice and promote the energy metabolism of gastrocnemius muscle, thereby improving the ability of the test animals to resist fatigue and perform physical activities. Further analysis found that, under the premise of following the daily recommended intake of humans, LAXY27 showed an effect superior to vitamin C. In summary, this study explored the mechanism of action of LAXY27 in improving the motor ability of oxidative stress mice, laying the foundation for the subsequent development of food-grade antioxidant ingredients that can alleviate oxidative damage and motor decline caused by high-intensity work or natural aging, and is conducive to promoting the research and development of probiotic products with independent intellectual property rights. Nevertheless, the above conclusions still need to be verified through more clinical trials to verify their general applicability, which will be a key point for future scientific research around LAXY27.

[0056] Although the above embodiment describes the present invention in detail, it is only a part of the embodiments of the present invention, not all of the embodiments. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A strain of Lactobacillus acidophilus ( Lactobacillus acidophilus ) XY27, characterized in that, The Lactobacillus acidophilus XY27 was deposited in the General Microbiology Center of the China Microorganism Culture Collection Administration on July 15, 2019, with the deposit number CGMCC No.18227.

2. Use of the Lactobacillus acidophilus XY27 according to claim 1 in the preparation of a product for promoting sports endurance.

3. Use of the Lactobacillus acidophilus XY27 described in claim 1 in preparing a product for preventing lactic acid accumulation during exhaustive exercise.

4. Use of the Lactobacillus acidophilus XY27 according to claim 1 in preparing a product for alleviating oxidative stress and enhancing sports endurance.

5. The use according to claim 4, characterized in that: The indicators of oxidative stress include muscle glycogen, liver glycogen, creatine kinase and urea nitrogen.

6. Use of the Lactobacillus acidophilus XY27 described in claim 1 in preparing a product for enhancing the activity of antioxidant enzymes under exhaustive exercise.

7. Use of the Lactobacillus acidophilus XY27 described in claim 1 in preparing a product for regulating intestinal flora under exhaustive exercise.

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

9. Use of the Lactobacillus acidophilus XY27 described in claim 1 in preparing a product for alleviating liver damage caused by exhaustive exercise.

10. A probiotic preparation, characterized in that: The probiotic preparation contains the Lactobacillus acidophilus XY27 according to claim 1; the bacterial content of Lactobacillus acidophilus XY27 in the probiotic preparation is 1.875×10 8 CFU / mL.

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