Bifidobacterium longum subsp. Longum capable of improving exercise fatigue and application thereof

Abstract

The invention provides a bifidobacterium longum subsp. Longum HX-BL11 capable of improving exercise fatigue, which is preserved in the China General Microbiological Culture Collection Center (CGMCC), the preservation number is CGMCC No.32960, and the preservation date is December 6, 2024. Meanwhile, the invention further provides a fermentation composition obtained by fermenting spina date seeds, hawthorn fruits, perilla seeds and dried tangerine or orange peel with the bifidobacterium longum subsp. Longum HX-BL11, and a fermentation product of the fermentation composition is rich in ferulic acid, phlorizin and hesperidin. The probiotic preparation and the fermentation product can improve the swimming exercise time of mice, improve indexes of fatigue damage degrees such as BUN, LD, LDH and CK-MB in the exercise mice, improve related indexes of energy metabolism such as GLU, MG and ATP, improve the exercise tolerance, improve antioxidant indexes such as T-SOD level and MDA content, improve the antioxidant capacity, play a synergistic effect and improve the health care effect of the mice. Therefore, the effect of relieving exercise fatigue is achieved. And meanwhile, the probiotic preparation of the bifidobacterium longum subsp. Longum HX-BL11 and the fermented product are simple in preparation method, can be produced in a large scale, and have a good application prospect.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of microbial engineering, and relates to a Bifidobacterium longum subsp. longum capable of improving exercise fatigue and an application thereof. BACKGROUND

[0002] Exercise is an indispensable part of human life and has a very important influence on physical and mental health. With the progress of society and the improvement of living standards, people have gradually paid more attention to exercise, and the research on exercise function has always been concerned. Physical exercise is generally considered to be an effective way to maintain physical health, and is beneficial to strong physique, improved immunity and enhanced cardiopulmonary function. However, a large amount of energy supply is required during exercise, and at the same time, the body produces complex physiological changes. When the body cannot maintain its specific physiological level or exercise intensity, it is defined as exercise fatigue. Exercise fatigue can cause muscle soreness, mental fatigue and loss of appetite, and even lead to abnormalities in metabolism, endocrine and immune systems. Therefore, it is necessary to effectively delay the occurrence of exercise fatigue or promote the recovery of exercise fatigue in time. It is one of the effective methods to relieve exercise fatigue to pay attention to a balanced diet, ensure sufficient intake of carbohydrates, proteins, appropriate fats, water and vitamins and minerals to meet the body's energy needs and help the body recover, and ensure sufficient intake of carbohydrates, proteins, appropriate fats, water and vitamins and minerals to meet the body's energy needs and help the body recover. At present, the components with anti-fatigue activity reported mainly include branched-chain amino acids, caffeine, electrolytes, trace nutrients, caffeine proteins and protease hydrolysates / peptides. Although most of the supplements are safe for the human body, some exercise supplements have side effects on the human body, especially some chemical drugs and biological products, which can cause allergic reactions and toxicity problems. Therefore, it has become a hot spot in the field of sports to find more safe and effective exercise supplements.

[0003] As a natural, safe and convenient nutritional supplement, probiotics produce health benefits to the host by regulating the intestinal flora homeostasis. Studies have shown that probiotics have the effects of improving lactose intolerance, preventing intestinal infection, regulating immune function, reducing serum cholesterol, anti-tumor and regulating bone mass. Supplementing probiotics in the exercise body can relieve oxidative stress, inflammatory response and material and energy metabolism imbalance caused by high-intensity exercise and other stress responses. Therefore, appropriate supplementation of probiotics can help to reduce exercise-induced fatigue and improve exercise performance.

[0004] As a natural, safe and convenient nutritional supplement, probiotics produce health benefits to the host by regulating the intestinal flora homeostasis. Studies have shown that probiotics have the effects of improving lactose intolerance, preventing intestinal infection, regulating immune function, reducing serum cholesterol, anti-tumor and regulating bone mass. Supplementing probiotics in the exercise body can relieve oxidative stress, inflammatory response and material and energy metabolism imbalance caused by high-intensity exercise and other stress responses. Therefore, appropriate supplementation of probiotics can help to reduce exercise-induced fatigue and improve exercise performance.

[0005] ​The medicinal and edible material is a special material with dual attributes of food and medicine, which is stronger in food than in medicine, mild and safe in action, and more suitable for athletes or sports enthusiasts to improve and adjust their physical condition for a long time. In recent years, research has found that due to the rich bioactive compounds such as polyphenols, terpenes and flavonoids, the medicinal and edible material has the effects of slowing down exercise fatigue and improving exercise capacity. How to excavate the active ingredients and their contents that can improve exercise capacity from numerous medicinal and edible materials has important research significance and market prospects for developing medicinal and edible materials with safe and effective effects of improving exercise capacity and / or slowing down exercise fatigue. SUMMARY

[0006] In view of the deficiencies of the prior art, the purpose of the present application is to provide a Bifidobacterium longum subsp. Longum and its application for improving exercise fatigue.

[0007] The present application is realized by the following technical solutions: A Bifidobacterium longum subsp. Longum HX-BL11, which is preserved in the China General Microbiological Culture Collection Center, with a preservation number of CGMCC No. 32960 and a preservation date of December 6, 2024.

[0008] Further, a probiotic preparation is provided, which comprises the Bifidobacterium longum subsp. Longum HX-BL11 and a freeze-drying protective agent.

[0009] Further, the freeze-drying protective agent comprises the following components: 8-10% of skimmed milk powder, 10-12% of trehalose, 8-10% of sucrose, 1-2% of sodium glutamate, 0.5-1.5% of ascorbic acid, 0.5-1.5% of glycerol, 0.3-0.5% of hydroxypropyl-β-cyclodextrin, 0.01-0.08% of vitamin E, and the balance of distilled water.

[0010] Further, a preparation method of the probiotic preparation is provided, which comprises the following steps: Step 1: after streak culture of the Bifidobacterium longum subsp. Longum HX-BL11 for 48 h, a single colony is picked and inoculated into a liquid culture medium for activation culture for 24 h, Step 2: after activation, the strain is inoculated into a liquid culture medium at an inoculation amount of 1%, and cultured at 37°C for 24 h. The fermentation is completed when the strain reaches the stable phase according to the growth curve, and a probiotic slurry is obtained, Step 3: the probiotic slurry is mixed with the freeze-drying protective agent at a ratio of 1:1, and then freeze-dried.

[0011] Further, the liquid culture medium ingredient composition is: galactooligosaccharide 5-10 g / L, lactose 2-3 g / L, yeast extract 3-5 g / L, casein hydrolysate 1-2 g / L, inulin 1-1.5 g / L, glutamine 0.8-1 g / L, folic acid 0.2-0.5 mg / L, vitamin B2 0.06-0.2 mg / L, vitamin B6 0.06-0.10 mg / L.

[0012] In another aspect, a fermentation composition is provided, which is obtained by fermentation of Z. longum subsp. longum HX-BL11 on Zizyphus jujuba 5-20 parts by weight, Crataegus pinnatifida 7-12 parts, Perilla frutescens 5-15 parts, and Citrus reticulata 1-10 parts.

[0013] Further, a preparation method of the fermentation composition is provided, which comprises the following steps: Step S1: mixing and sterilization: Preparation of composition 1: weigh the raw materials Zizyphus jujuba and Perilla frutescens, mix them uniformly after being crushed, add deionized water, and stir to form a uniform suspension; Preparation of composition 2: weigh the raw materials Crataegus pinnatifida and Citrus reticulata, mix them uniformly after being crushed, add deionized water, and stir to form a uniform suspension; Sterilization: sterilize the prepared compositions 1 and 2 respectively, and cool them to room temperature for standby; Step S2: first-stage fermentation: inoculate the composition 1 prepared in step S1 with Z. longum subsp. longum HX-BL11 bacterial liquid, and ferment at 37°C for 8 hours; Step S3: feeding and second-stage fermentation: when the fermentation in step S2 reaches 8 hours, feed the composition 2 prepared in step S1, and continue to ferment at 37°C for 8 hours; Step S4: post-treatment: centrifuge the fermentation liquid prepared in step S3, and take the supernatant to obtain the desired substance.

[0014] In step S1, the ratio of fermentation raw material to water is 1 g:(5-10 mL); the sterilization temperature is 105°C, and the sterilization time is 5-10 min.

[0015] In step S2, the viable bacterial count of the bacterial liquid Z. longum subsp. longum HX-BL11 is 10 7 CFU / mL; In step S4, the centrifugation speed is 3000-5000 r / min, and the centrifugation time is 5-10 min.

[0016] The centrifugation speed is 3000-5000 r / min, and the centrifugation time is 5-10 min.

[0017] Further, the application provides the use of the probiotic preparation or the fermentation product, including the use in the preparation of food or medicine for improving exercise fatigue.

[0018] Compared with the prior art, the application has the following advantages: The longissimus bifidobacterium longum HX-BL11 provided by the application has strong probiotic functions, including gastric acid resistance, bile salt resistance, self-aggregation and bacteriostatic ability, and the probiotic preparation of the longissimus bifidobacterium longum HX-BL11 provided by the application helps to improve exercise fatigue.

[0019] Meanwhile, the application provides a fermentation composition obtained by fermenting a combination of jujube kernel, hawthorn, perilla seed and dried tangerine or orange peel by the longissimus bifidobacterium longum HX-BL11, and through the close combination of the specific strain (HX-BL11), the specific raw material combination (jujube kernel, hawthorn, perilla seed and dried tangerine or orange peel) and the specific fermentation method, the directional enrichment of ferulic acid, phlorizin and hesperidin is realized, and the “golden triangle” of anti-fatigue is formed. They have synergistic effects on energy metabolism, antioxidant and function improvement.

[0020] The probiotic preparation and the fermentation product provided by the application can significantly improve the swimming time of mice, reduce the BUN, LD and LDH levels in the serum of mice after exercise and the CK level in the skeletal muscle of mice, improve the skeletal muscle damage caused by exercise, improve the GLU level in the serum of mice, improve the MG and ATP levels in the skeletal muscle of mice, improve energy metabolism, improve endurance, improve the T-SOD and GSH-Px activities and CAT level in the skeletal muscle tissue of mice, inhibit the MDA level in the skeletal muscle tissue of mice, improve the antioxidant capacity, and overall realize the multi-target intervention on the three core links of energy, damage and oxidation, and comprehensively realize the beneficial effects of relieving exercise fatigue and accelerating physical recovery.

[0021] The preparation method of the probiotic preparation and the fermentation product provided by the application is simple and can be mass-produced. BRIEF DESCRIPTION OF DRAWINGS

[0022] Figure 1 Fig. 1 is a microscope picture of the longissimus bifidobacterium longum HX-BL11 according to an embodiment of the application; Figure 2 Fig. 2 is a growth curve of the longissimus bifidobacterium longum HX-BL11 according to an embodiment of the application. DETAILED DESCRIPTION

[0023] The application will be further described in conjunction with specific embodiments. The following examples are not intended to limit the application, but merely to illustrate the application. Unless otherwise specified, the experimental methods used in the following examples were carried out according to conventional conditions. Unless otherwise specified, the materials and reagents used in the following examples were obtained from commercial sources.

[0024] The Bifidobacterium longum subsp. Longum HX-BL11 used in the following examples has the Latin name Bifidobacterium longum subsp. Longum, and the strain number is CGMCC NO. 32960. The strain was deposited at the China General Microbiological Culture Collection Center on December 6, 2024, and the deposit unit code is CGMCC. The deposit unit address is No. 1, Beichen West Road, Yard 3, Beijing Chaoyang District, Institute of Microbiology, Chinese Academy of Sciences, Beijing 100101. The strain was alive at the time of deposit.

[0025] Example 1: Isolation and identification of Bifidobacterium longum subsp. Longum HX-BL11 0.1 g of the collected fresh infant feces was weighed, 0.9 mL of 60% glycerol was added, and vortexed for 3-5 min. After centrifugation at 1000 rpm for 5-7 min, the supernatant was transferred to a sterile centrifuge tube. After centrifugation of the supernatant at 5000 rpm for 10-15 min, the supernatant was discarded, and the fecal suspension was resuspended with physiological saline to obtain the fecal suspension. The fecal suspension was inoculated into TPY liquid medium at a proportion of 2%, and cultured anaerobically at 37°C for 36 h. The fecal suspension after enrichment culture was sequentially diluted to 10 -1 , 10 -2 , 10 -3 , 10 -4 , 10 -5 , 10 -6 , 10 -7 After mixing, 100 uL of the diluted solution was taken and spread on the surface of modified TPY solid medium, and cultured anaerobically at 37°C for 48 h. A single colony with a regular white edge was picked and inoculated on the surface of TPY solid medium for repeated streaking and purification, and cultured at 37°C for 24 h to obtain pure bacteria.

[0026] One strain of Bifidobacterium was obtained in the preliminary screening, which was identified as Bifidobacterium longum subsp. Longum. The probiotic functions of the strain were compared, and the results are shown in Table 1.

[0027] Table 1: Determination of probiotic function of the strain

[0028] As shown in Table 1, the strain HX-BL11 has good bacteriostatic ability, and has strong bacteriostatic ability on common pathogenic bacteria such as Escherichia coli, Staphylococcus aureus and Salmonella, indicating that the HX-BL11 can regulate the balance of intestinal flora and inhibit pathogen infection, and is an optimal strain.

[0029] The strain HX-BL11 is sequenced by using 16S rDNA universal primers, and the gene sequence of Bifidobacterium longum subsp. longum HX-BL11 is shown in SEQ ID No: 1.

[0030] SEQ ID No: 1: Example 2: Preparation of Bifidobacterium longum subsp. longum HX-BL11 probiotic preparation (1) Strain activation Bifidobacterium longum subsp. longum HX-BL11 was streaked and cultured for 48 h, and single colonies on the plate were picked for microscopic examination to observe the strain morphology, as shown in FIG. 1. Single colonies were then inoculated into liquid medium, respectively, and cultured at 37°C for 24 h for standby use. Figure 1

[0031] (2) Determination of probiotic growth curve Bifidobacterium longum subsp. longum HX-BL11 was inoculated into liquid medium at an inoculation amount of 1%, and cultured for 24 h. The OD value of the bacterial solution was determined every 2 h to draw a growth curve, as shown in FIG. 2. Figure 2

[0032] The liquid medium comprises 5-10 g / L of galactooligosaccharide, 2-3 g / L of lactose, 3-5 g / L of yeast extract, 1-2 g / L of casein hydrolysate, 1-1.5 g / L of inulin, 0.8-1 g / L of glutamine, 0.2-0.5 mg / L of folic acid, 0.06-0.2 mg / L of vitamin B2, and 0.06-0.10 mg / L of vitamin B6.

[0033] (3) Fermentation culture of probiotics Bifidobacterium longum subsp. longum HX-BL11 was inoculated into liquid medium at an inoculation amount of 1%, and cultured at 37°C. After fermentation to the stable phase according to the strain growth curve, centrifugal freeze-drying was performed.

[0034] (4) Centrifugal freeze-drying of probiotics The fermentation broth after fermentation was centrifuged at 6000 rpm and 4°C for 10 min to obtain probiotic slurry. The freeze-drying protectant was added at a ratio of 1:1, and the mixture was thoroughly mixed, transferred to -80°C for 2 h of pre-freezing, and then vacuum freeze-dried to calculate the freeze-drying survival rate.

[0035] The freeze-drying protectant comprises 8-10% of skim milk powder, 10-12% of trehalose, 8-10% of sucrose, 1-2% of sodium glutamate, 0.5-1.5% of ascorbic acid, 0.5-1.5% of glycerol, 0.3-0.5% of hydroxypropyl-β-cyclodextrin, 0.01-0.08% of vitamin E, and the balance of distilled water. The specific results are shown in Table 2.

[0036] Table 2: Test results of bacterial powder

[0037] Example 3: Screening of synergistically enhanced fermented medicinal and edible substances ​​To screen the medicinal and edible materials that can improve the fermentation synergistic effect of the probiotics for improving exercise fatigue, the medicinal and edible materials that have been proved to have anti-fatigue activity or antioxidant activity are initially screened, including: red dates, jujube kernel, codonopsis, wolfberry, perilla seed, eucommia leaf, hawthorn, sea buckthorn, cistanche, turmeric, orange peel, yam, angelica, ginger, longan, licorice, ginseng.

[0038] To evaluate the synergistic growth effect of B. longum subsp. longum HX-BL11 and medicinal and edible materials, 2% (w / v) of the materials are added to the MRS liquid medium, sterilized at 121°C for 15 min, and B. longum subsp. longum HX-BL11 is inoculated into the medium containing the materials. The medium containing the materials without inoculation is set as the blank control group. The OD difference (ΔOD600nm) of the medium cultured for 24 hours is calculated based on the blank medium, for comparing the synergistic growth effect of different materials and B. longum subsp. longum HX-BL11.

[0039] The specific results are shown in Table 3: Table 3: Synergistic effect of different materials and HX-BL11 growth (ΔOD600nm)

[0040] As shown in Table 3, jujube kernel, perilla seed, orange peel, and hawthorn can promote the growth of B. longum subsp. longum HX-BL11. The results provide a key basis for subsequent compounding or fermentation. To improve the bioavailability, the four medicinal and edible materials such as jujube kernel are combined, and fermentation technology is used for fermentation, which not only helps to improve the growth of probiotics, but also may enhance the synergistic potential of the final product in improving exercise fatigue function through metabolic conversion of active ingredients in jujube kernel and other medicinal and edible materials.

[0041] Example 4: Preparation of fermented composition The composition provided in this example includes, by weight, 5-20 parts of jujube kernel, 7-12 parts of hawthorn, 5-15 parts of perilla seed, and 1-10 parts of orange peel. The above-mentioned materials are fermented by B. longum subsp. HX-BL11 to obtain the fermented composition.

[0042] The specific fermentation material properties are as follows: The jujube kernel is dried jujube kernel, which is roasted at a low temperature of 40-50 degrees for about 5-8 minutes, ground and crushed, and then filtered through a 60-80 mesh sieve for standby; The hawthorn is dried hawthorn, which is ground and crushed after removing the core, and then filtered through a 60-80 mesh sieve for standby; The perilla seed is directly ground and crushed to a 60-80 mesh sieve for standby; The dried tangerine peel is directly ground and crushed, and then passed through a 60-80 mesh filter for standby; Fermentation strain: Bifidobacterium longum subsp. longum HX-BL11, with a viable bacterial count of 10 billion CFU / g.

[0043] The specific preparation method of the fermentation composition is as follows: S1: mixing and sterilization Preparation of composition 1: weigh the pretreated raw materials of Zizyphus jujuba Mill. var. spinosa (Bunge) Hu ex H. F. Chou 5-20 parts and Perilla frutescens (L.) Britt. 5-15 parts, and mix uniformly; add deionized water, with a material-to-liquid ratio of 1: (5-10) (g:mL), and stir to form a uniform suspension; Preparation of composition 2: weigh the pretreated raw materials of Crataegus pinnatifida Bunge 7-12 parts and dried tangerine peel 1-10 parts, and mix uniformly; add deionized water, with a material-to-liquid ratio of 1: (5-10) (g:mL), and stir to form a uniform suspension; Sterilization: high-pressure sterilization at 105°C for 5-10 min, and then cool to room temperature for standby.

[0044] S2: first-stage fermentation (0-8 hours) Inoculate the composition 1 prepared in step S1 into the Bifidobacterium longum subsp. longum HX-BL11 bacterial solution, with a viable bacterial count of 10 7 CFU / mL, and set the fermentation parameters to a temperature of 37°C and a fermentation time of 8 h.

[0045] S3: feeding and second-stage fermentation (9-16 hours) When the fermentation is carried out for 8 h, add the composition 2 prepared in step S1 to the above-mentioned HX-BL11 primary fermentation liquid, and continue to ferment at 37°C for 8 h, so that the total fermentation time reaches 16 h.

[0046] S4: post-treatment Centrifuge the fermentation liquid prepared in step S3 at 3000-5000 r / min for 5-10 min to obtain the supernatant, which is the desired substance.

[0047] Zizyphus jujuba Mill. var. spinosa (Bunge) Hu ex H. F. Chou has the effects of nourishing the spleen and stomach, and tonifying and benefiting qi, and contains abundant ferulic acid in the cell wall, which usually exists in the form of combination with polysaccharides, and is rich in proteins, so as to quickly supplement energy and relieve fatigue; Crataegus pinnatifida Bunge is rich in organic acids, vitamin C and other components, and can promote digestion and relieve fatigue; Perilla frutescens (L.) Britt. is rich in proteins and has a comprehensive amino acid composition, and is rich in α-linolenic acid, and has good health care effects; dried tangerine peel contains abundant volatile oils, flavonoids, vitamins and other components, and has multiple biological activities such as antioxidant, anti-inflammatory and antibacterial. In order to verify whether it has a synergistic effect, the activity components of the fermentation liquid are determined.

[0048] Example 5: activity component detection 1. Activity component content detection The contents of ferulic acid, phloridzin, hesperidin, hesperetin and phloretin were determined by HPLC method.

[0049] Comparative Example 1: The difference from Example 4 is that the fermentation is carried out with commercially available equal concentration of commercial strain Bifidobacterium longum subsp. longum BB536, and the rest of the operating conditions remain unchanged. Comparative Example 2: The difference from Example 4 is that the fermentation is carried out with commercially available equal concentration of commercial strain Lactobacillus plantarum LPL28, and the rest of the operating conditions remain unchanged. Comparative Example 3: The difference from Example 4 is that the Suanzaoren is removed, and the rest of the operating conditions remain unchanged. Comparative Example 4: The difference from Example 4 is that the Shanzha is removed, and the rest of the operating conditions remain unchanged. Comparative Example 5: The difference from Example 4 is that the Zisugeng is removed, and the rest of the operating conditions remain unchanged. Comparative Example 6: The difference from Example 4 is that the Chenpi is removed, and the rest of the operating conditions remain unchanged. Comparative Example 7: The difference from Example 4 is that the Shanzha and Chenpi are fermented first in the first stage, and then the Suanzaoren and Zisugeng are fermented in the second stage. Comparative Example 8: The difference from Example 4 is that the Shanzha and Chenpi are fed after 4 hours of fermentation in the first stage, and then the second stage of fermentation is continued until 16 hours of fermentation. Comparative Example 9: The difference from Example 4 is that the Shanzha and Chenpi are fed after 12 hours of fermentation in the first stage, and then the second stage of fermentation is continued until 16 hours of fermentation. Comparative Example 10: The difference from Example 4 is that all 4 plant raw materials are added at once at 0 hours, and HX-BL11 is inoculated at the same time, and co-fermented for 16 hours.

[0050] Table 4 Determination of active ingredient content before and after fermentation

[0051] Ferulic acid is a plant aromatic acid, which has been proven to activate AMPK (adenosine acid activated protein kinase), promote glucose uptake and fatty acid oxidation, provide more energy for muscles during exercise, promote mitochondrial biosynthesis, enhance the aerobic metabolism capacity of cells, thereby reducing the accumulation of lactic acid, improving the LD (lactic acid) index, thereby reducing the accumulation of lactic acid, and helping to reduce the BUN (blood urea nitrogen) level.

[0052] Phlorizin, as a natural SGLT (glucose transporter) inhibitor, can mildly regulate glucose reabsorption in the intestine and kidneys, helping to maintain stable blood glucose levels during exercise and promoting fat oxidation for energy, thereby conserving limited muscle glycogen reserves and prolonging exercise duration. Furthermore, some literature reports that phlorizin has an anti-fatigue effect in exhaustive exercise model mice, inhibiting oxidative stress damage caused by exercise fatigue.

[0053] Hesperidin, as the aglycone form of hesperidin, exhibits significantly enhanced bioavailability. It can effectively scavenge free radicals, inhibit inflammatory signaling pathways, reduce exercise-induced acute oxidative damage and muscle inflammation, and accelerate functional recovery.

[0054] Table 4 shows that, compared with before fermentation, the ferulic acid content significantly increased from 0.42 mg / mL to 1.85 mg / mL. Furthermore, the phlorizin content did not decrease but instead increased dramatically from 58.75 μg / mL to 156.32 μg / mL. Simultaneously, the hesperidin content decreased from 480.51 μg / mL to 185.60 μg / mL, and 115.06 μg / mL of hesperidin was generated. In contrast, the comparative experiments failed to achieve optimal results for all three indicators simultaneously.

[0055] The results of the examples and comparative examples demonstrate that the technical solution of the present invention is an indivisible organic whole. Comparative examples 3-6 (raw material deficiency) provide direct evidence: the lack of any raw material affects the content of ferulic acid, phlorizin, or hesperidin, especially when tangerine peel is lacking, the content of phlorizin is much lower than in the examples. Comparative example 7 (reversed fermentation order) demonstrates the importance of timing control from the opposite perspective. Although hesperidin is efficiently converted, phlorizin is retained but fails, and the production of ferulic acid is also reduced. In addition, the results of strain specificity (comparative examples 1-2) and fermentation time allocation (comparative examples 8-10) indicate that the fermentation characteristics of the strain and appropriate segmentation time are necessary conditions to ensure the efficient operation of the fermentation system. Only through the close combination of the specific strain (HX-BL11), the specific raw material combination (jujube seed, hawthorn, perilla seed, tangerine peel), and the specific fermentation method provided by the present invention can excellent synergistic effects be achieved, realizing the targeted enrichment of the target active ingredients.

[0056] 2. Enzyme activity expression detection at key fermentation time points The activity of β-glucosidase was measured using the pNPG colorimetric method with pNPG (p-nitrobenzene-β-D-glucopyranoside) as a substrate, in units of U / mL. The activity of ferulic acid esterase was also measured using the colorimetric method with methyl ferulic acid ester as a substrate, in units of U / mL.

[0057] The enzyme activity results after fermentation for each group are detailed in Tables 5-6.

[0058] Table 5 Enzyme activity expression of ferulic acid esterase (U / ml)

[0059] Table 6 Enzyme activity expression of β-glucosidase (U / ml)

[0060] According to the results of Tables 4-6, it can be seen that in the present application, the strain HX-BL11 responds extremely well to the Zizyphus jujuba seed / Perilla seed substrate, and ferulic acid esterase is rapidly induced to 1.18 U / mL in 8H, laying a foundation for ferulic acid enrichment. At the same time, it precisely suppresses the β-glucosidase activity at a low level (0.11 U / mL) in the first stage, avoiding energy waste. After the β-glucosidase is induced by dried tangerine or orange peel in the second stage, it shows higher substrate preference for hesperidin, realizing the competitive protection of phlorizin. Secondly, the 8H segmented fermentation in the first stage is exactly the "plateau period" when the strain completes adaptation, the enzyme activity of ferulic acid esterase reaches the peak, and the environment tends to be stable. At this time, the introduction of dried tangerine or orange peel and hawthorn is the best opportunity to induce β-glucosidase with substrate selectivity; the enzyme activity of β-glucosidase is directed to hesperidin, and then the 8H fermentation in the second stage provides sufficient time for the complex "induction-competition-protection" dynamic process between hesperidin and phlorizin, so that both conversion and protection can tend to be balanced. Secondly, the four raw materials synergize with each other, Zizyphus jujuba and Perilla seed are highly utilized by HX-BL11, inducing the enzyme activity of ferulic acid esterase while partially inhibiting the activity of most β-glucosidases, dried tangerine or orange peel and hawthorn complement each other, dried tangerine or orange peel is the key competitive substrate for protecting phlorizin, and the hesperidin element converted from it can also form an anti-fatigue "golden triangle" with the simultaneously enriched ferulic acid and phlorizin. They synergize in energy metabolism, antioxidant and function improvement, and the improved fermentation composition of the present application can significantly improve exercise fatigue.

[0061] Example 6: Experimental verification of improving exercise fatigue 1. Experimental grouping and design Male BALB / c mice aged 4-5 weeks and weighing 20±2g were selected as experimental subjects. All mice were housed in separate cages of 3 and subjected to a one-week acclimatization period at room temperature (22±2)℃ and relative humidity of 50%-60%. After the one-week acclimatization period, 30 mice were randomly divided into five different groups: blank group (NC group), model group (MC group), probiotic preparation group (Example 2 group), fermentation product group (Example 4 group), and positive control group, with 6 mice in each group. The NC and MC groups were administered distilled water by gavage daily; the Example 2 group was administered 75 mg / kg / day of the probiotic powder prepared in Example 2 by gavage; the Example 4 group was administered 75 mg / kg / day of the fermented composition prepared in Example 4 by gavage; the positive control group was administered 200 mg / kg / day of Rhodiola rosea by gavage, wherein Rhodiola rosea soft capsules were dissolved in double-distilled water (purchased from Gansu Alcon Biotechnology Co., Ltd.), and double-distilled water was purchased from Nanjing Yixun Biotechnology Co., Ltd. The intervention lasted for 4 weeks, and body weight was monitored weekly. Except for the NC group, which did not exercise, the other groups of mice underwent non-weight-bearing adaptive swimming training twice a week (water temperature 25±1℃, water depth >30cm).

[0062] Thirty minutes after the last administration on day 35, mice were weighed down with a lead weight (5% of their body weight) and forced to swim to exhaustion (the mice's noses sank to the surface for 5 seconds, their bodies remained still, they could not maintain a floating state, and their escape response was weakened when they were pulled out of the water). The time to exhaustion was recorded. During the swimming process, if any mice were suspended and resting, the water surface was continuously stirred with a glass rod to force them to continue swimming.

[0063] Mice were swam to exhaustion under load. Afterward, they were removed from the water, dried, and their eyes were enucleated. Approximately 1 mL of blood was collected. 0.05 mL of whole blood was added to 0.3 mL of protein precipitant, and the mixture was centrifuged at 4000 rpm for 10 min to prepare the LD supernatant. The whole blood LD content was measured on the same day. The remaining blood sample was centrifuged at 3000 rpm for 15 min at 4°C, and the serum was collected, transferred to EP tubes, and stored at -80°C for subsequent detection of BUN, LDH, and GLU levels.

[0064] After blood collection from each group of mice, they were euthanized by cervical dislocation, and skeletal muscle from both legs was harvested and washed with pre-cooled PBS solution (0.1 mol / L, pH 7.4). Approximately one-third of the skeletal muscle from each side was added to 4% paraformaldehyde general-purpose tissue fixative and then transferred to a -80°C freezer for subsequent biochemical analysis.

[0065] 2. Analysis of the effect on the exhaustive swimming time of mice The exercise time is an important indicator for evaluating muscle fatigue, and the length of the exercise time of the body directly reflects the strength of the exercise capacity. Among them, the exhaustive swimming test as a classic behavior experiment for reflecting the exercise capacity of the body is widely used to evaluate the exercise endurance level of mice. As shown in Table 7, the exhaustive swimming time of the mice in the MC group was 10.23±1.21 min, and the exhaustive swimming time of the mice in the example 2, 4 groups and the positive control group was 14.28±1.12 min, 17.85±1.42 min and 15.85±1.26 min, respectively. Compared with the MC group, the exercise capacity of the mice in the three intervention groups was significantly improved. Compared with the example 2 group and the positive control group, the exhaustive swimming time of the mice in the example 4 group was prolonged by 25.0% and 12.62%, respectively. It is proved that the example 2 group, the example 4 group and the positive control group can all prolong the exhaustive swimming time of the mice, and the effect of the example 4 group is better than that of the positive control group.

[0066] Table 7: Exhaustive swimming time of mice

[0067] 3. BUN, LD, LDH and CK and other related indicators for detecting the degree of fatigue injury The contents of BUN, LD and LDH in the serum of mice were detected according to the test kit instruction provided by Nanjing Jiancheng Biological Engineering Institute. The 5% tissue homogenate concentration was used to detect the absorbance value of CK at 660 nm wavelength according to the operation steps of the corresponding instruction of the reagent kit.

[0068] The relevant detection results are shown in Table 8 below: Table 8: BUN, LD, LDH and CK and other related indicators for detecting the degree of fatigue injury

[0069] The serum BUN (blood urea nitrogen) is the metabolic product of protein and amino acid decomposition in vivo, and the BUN concentration reflects the degree of protein decomposition and affects the muscle strength during exercise, which is a commonly used indicator for evaluating the degree of fatigue after exercise. As shown in Table 8, compared with the NC group, the BUN content in the serum of the mice in the MC group was significantly increased. Compared with the MC group, the BUN content in the serum of the mice in the example 4 group was significantly decreased and tended to be close to the NC group. Compared with the example 2 group and the positive control group, the BUN content in the serum of the mice in the example 4 group was significantly reduced. It is proved that the example 2 group, the example 4 group and the positive control group can all reduce the BUN content in the serum of the mice, and the example 4 group is more effective in inhibiting the accumulation of BUN in vivo.

[0070] Intense exercise destroys the metabolic balance in skeletal muscle cells, the body is in a state of relative hypoxia, glycolysis is accelerated, a large amount of LD (lactic acid) is produced and accumulated in the body, reducing the pH value of the body, causing fatigue and reduced exercise capacity, which is a sensitive indicator for evaluating the degree of fatigue. As can be seen from Table 8, compared with the NC group, the LD level in the serum of the mice in the MC group significantly increased after exhaustive swimming. Compared with the MC group, the LD content in the serum of the mice in the example 2 group, the example 4 group and the positive control group was significantly reduced, and the LD content in the example 4 group was the least. Therefore, it can be concluded that the example 2 group, the example 4 group and the positive control group all reduce the accumulation of LD in the body, and the example 4 group has the best effect.

[0071] LDH (lactate dehydrogenase) is an oxidoreductase in the glycolysis process, which catalyzes the reversible conversion of lactic acid to GLU (glucose), and the activity of LDH reflects the degree of LD metabolism. As can be seen from Table 8, compared with the NC group, the LDH activity in the serum of the mice in the MC group significantly increased. Compared with the MC group, the LDH content in the serum of the mice in the example 2 group, the example 4 group and the positive control group was reduced. And the LDH content in the example 4 group was the least.

[0072] CK (creatine kinase) is an enzyme that catalyzes the transphosphorylation reaction between ATP and creatine, which is directly related to the energy operation in cells, muscle contraction and ATP regeneration, and reflects the degree of skeletal muscle damage. As can be seen from Table 8, compared with the NC group, the skeletal muscle CK level of the MC group significantly increased; compared with the MC group, the CK level of the example 2 group, the example 4 group and the positive control group was inhibited to different extents; and the example 4 group tended to be close to the NC group. It is proved that the example 2 group, the example 4 group and the positive control group can all improve the skeletal muscle damage caused by exercise and relieve fatigue, but the example 4 group has the best effect.

[0073] 4. Detection of energy metabolism GLU, MG and ATP related indicators According to the test kit instruction provided by Nanjing Jiancheng Bioengineering Institute, the GLU content in the serum of mice was detected, and the enzyme label instrument was used to measure the GLU absorbance value at a wavelength of 505 nm.

[0074] Take 1% tissue homogenate concentration, according to the operation steps of the corresponding instruction, use the enzyme label instrument to detect the MG absorbance value at 620 nm wavelength.

[0075] Take 5% tissue homogenate concentration, according to the operation steps of the corresponding instruction, use the enzyme label instrument to detect the ATP absorbance value at 636 nm wavelength.

[0076] The relevant detection results are shown in the following table 9: Table 9: Energy metabolism GLU, MG and ATP related indicators

[0077] GLU is the main energy substance of body movement, and its level directly affects the performance of movement and the feeling of fatigue. As shown in Table 9, compared with the NC group, the GLU content in the serum of the mice in the MC group decreased significantly, indicating that exhaustive swimming consumed a large amount of energy in the body and significantly reduced the GLU level in the body. Compared with the MC group, the GLU level in the serum of the mice in the Example 2 group, the Example 4 group and the positive control group was improved. It is indicated that exhaustive swimming consumed a large amount of energy in the body, and the intervention of the Example 2 group, the Example 4 group and the positive control group can improve the GLU level in the serum of the mice, relieve exercise fatigue, and the effect of the Example 4 group is the best.

[0078] In the early stage of movement, the body uses MG (muscle glycogen) for energy supply. With the extension of movement time and the increase of movement intensity, MG is continuously consumed, the GLU concentration is reduced, and the movement ability is decreased. As shown in Table 9, compared with the NC group, the MG level in the skeletal muscle tissue of the mice in the MC group decreased significantly. Compared with the MC group, the MG level in the skeletal muscle tissue of the mice in the Example 4 group and the positive control group was significantly improved. The MG content in the Example 4 group was higher than that in the Example 2 group. It is indicated that the improvement of the MG level in the skeletal muscle tissue of the movement mice in the Example 2 group needs to be strengthened, and the effect of the Example 4 group on relieving exercise fatigue by improving the MG level in the skeletal muscle of the mice is better than that of the positive control group.

[0079] In intense exercise, ATP (adenosine triphosphate) is the direct energy substance consumed for muscle contraction and relaxation, which ensures normal metabolism of the body. As shown in Table 9, compared with the NC group, the ATP content in the MC group decreased significantly. Compared with the MC group, the ATP content in the skeletal muscle tissue of the mice in the Example 2 group, the Example 4 group and the positive control group increased to different degrees. Compared with the Example 2 group, the ATP content in the skeletal muscle tissue of the mice in the Example 4 group and the positive control group increased significantly. It can be seen that the positive control group and the Example 4 group can regulate the ATP content in the skeletal muscle tissue of the movement mice to relieve fatigue, and the effect of the Example 4 group is better.

[0080] 5. Detection of T-SOD, MDA, GSH-Px and CAT and other antioxidant activity related indexes in the skeletal muscle tissue of the mice The 1% tissue homogenate concentration was taken, and the absorbance values of CAT, GSH-Px and T-SOD indexes were detected at 405 nm, 412 nm and 550 nm wavelengths respectively according to the operation steps of the corresponding instruction manual of the test kit provided by Nanjing Jiancheng Biological Engineering Institute.

[0081] The 5% tissue homogenate concentration was taken, and the absorbance value of MDA was detected at 532 nm wavelength using an enzyme marker according to the operation steps of the corresponding instruction manual of the test kit provided by Nanjing Jiancheng Biological Engineering Institute.

[0082] The related detection results are shown in Table 10 below: Table 10: T-SOD, MDA, GSH-Px and CAT and other antioxidant activity related indexes in mouse skeletal muscle tissue

[0083] T-SOD (total superoxide dismutase) can alleviate oxidative stress damage caused by exercise by removing free radicals, and its activity may directly affect the speed of recovery after exercise and the perception of exercise fatigue. As shown in Table 10, compared with the NC group, the T-SOD activity of the skeletal muscle tissue of the MC group was significantly reduced; compared with the MC group, the T-SOD activity of the Example 2 group, the Example 4 group and the positive control group was significantly increased; the T-SOD activity of the skeletal muscle tissue of the Example 4 group was significantly higher than that of the Example 2 group and the positive control group. It shows that the Example 2 group, the Example 4 group and the positive control group can all play a role in relieving exercise fatigue by increasing the T-SOD activity of the skeletal muscle tissue of the mouse, and the Example 4 group is the best.

[0084] MDA (malondialdehyde) is a product of lipid peroxidation, and during exercise, free radicals cause damage to the body, with lipid peroxidation being a major damage mechanism. As shown in Table 10, compared with the MC group, the MDA content of the skeletal muscle tissue of the Example 2 group, the Example 4 group and the positive control group was significantly relieved to varying degrees, and the Example 4 group had the best inhibitory effect on MDA, even lower than the NC group. It shows that the Example 2 group, the Example 4 group and the positive control group can all inhibit the production of peroxide (MDA) to relieve exercise fatigue, and the Example 4 group is the best.

[0085] During exercise, a large number of free radicals and peroxides are produced in the muscle, leading to muscle damage and fatigue. Free radical attack caused by exercise can cause GSH-Px (glutathione peroxidase) to be inactivated, further exacerbating exercise fatigue. As shown in Table 10, compared with the NC group, the GSH-Px content in the skeletal muscle tissue of the MC group mice after exhaustive swimming decreased significantly; compared with the MC group, the antioxidant capacity of the Example 2 group was improved, and the antioxidant capacity of the Example 4 group and the positive control group was significantly enhanced. It shows that the Example 4 group and the positive control group can both regulate the GSH-Px activity of the skeletal muscle tissue of the mouse, and the Example 4 group is better.

[0086] CAT (catalase) can alleviate oxidative stress produced during exercise to some extent by removing hydrogen peroxide, thereby slowing down the occurrence of exercise fatigue. As shown in Table 10, compared with the MC group, the CAT content in the skeletal muscle tissue of the mice in the Example 2 group, the Example 4 group and the positive control group was significantly increased. It shows that the Example 4 group can increase the CAT content in the skeletal muscle tissue of the mouse, thereby relieving exercise fatigue, but the Example 2 group needs to be improved in improving the CAT capacity.

[0087] From the above experiments, the groups of examples 2 and 4 can improve the fatigue damage by improving the indicators such as BUN, LD, LDH and CK-MB, improve the exercise endurance by improving the related indicators such as GLU, MG and ATP, improve the T-SOD, GSH-Px and CAT levels and reduce the MDA content, play a synergistic effect, improve the exercise endurance, improve the energy metabolism, fatigue damage, improve the antioxidant capacity and play a role in relieving exercise fatigue, but the effect of the group of example 4 is the best, and the effect of the group of example 2 is worse than that of the group of example 4 and the positive control group.

[0088] The specific embodiments of the present application are described in detail above, but they are only examples, and the present application is not limited to the specific embodiments described above. Any equivalent modifications and substitutions made to the present application by those skilled in the art are also within the scope of the present application. Therefore, equivalent transformations and modifications made without departing from the spirit and scope of the present application should be included in the scope of the present application.

Claims

1. A subsp. longum of Bifidobacterium longum HX-BL11, deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 32960 and deposit date of December 6, 2024.

2. A probiotic preparation, characterized in that, Includes Bifidobacterium longum subsp. HX-BL11 as described in claim 1 and a freeze-drying protectant.

3. The method for preparing the probiotic preparation according to claim 2, characterized in that, The lyophilization protectant composition, by weight percentage, is as follows: Skim milk powder 8-10%, trehalose 10-12%, sucrose 8-10%, monosodium glutamate 1-2%, ascorbic acid 0.5-1.5%, glycerol 0.5-1.5%, hydroxypropyl-β-cyclodextrin 0.3-0.5%, vitamin E 0.01%-0.08%, water balance.

4. The method for preparing the probiotic preparation according to claim 2, characterized in that, Includes the following steps: Step 1: After streaking the *Bifidobacterium longum* subsp. *HX-BL11* as described in claim 1 for 48 hours, pick a single colony and inoculate it into liquid culture medium for activation and culture for 24 hours. Step 2: After activation, inoculate 1% of the culture medium into liquid culture medium and incubate at 37℃ for 24 hours. Fermentation is completed after reaching the stationary phase according to the strain's growth curve, yielding probiotic sludge. Step 3: Add freeze-drying protectant to the probiotic slurry at a 1:1 ratio, mix thoroughly, and then freeze-dry.

5. The method for preparing the probiotic preparation according to claim 4, characterized in that, The liquid culture medium is composed of: galactooligosaccharides 5-10 g / L, lactose 2-3 g / L, yeast extract 3-5 g / L, casein hydrolysate 1-2 g / L, inulin 1-1.5 g / L, glutamine 0.8-1 g / L, folic acid 0.2-0.5 mg / L, vitamin B2 0.06-0.2 mg / L, and vitamin B6 0.06-0.10 mg / L.

6. A fermentation composition, characterized in that, The product is obtained by fermenting 5-20 parts by weight of Ziziphus jujuba seed, 7-12 parts of hawthorn, 5-15 parts of perilla seed, and 1-10 parts of dried tangerine peel using Bifidobacterium longum subsp. HX-BL11 as described in claim 1.

7. The method for preparing the fermentation composition according to claim 6, characterized in that: Includes the following steps: Step S1: Mixing and Sterilization Preparation of Composition 1: Weigh the raw materials jujube kernel and perilla seed, crush them and mix them evenly; add deionized water and stir to form a homogeneous suspension; Preparation of Composition 2: Weigh the raw materials hawthorn and dried tangerine peel, pulverize them and mix them evenly; add water and stir to form a homogeneous suspension; Sterilization: Sterilize the prepared compositions 1 and 2 separately and then cool them to room temperature for later use; Step S2 First stage fermentation: Inoculate the composition 1 prepared in step S1 with a culture of Bifidobacterium longum subsp. HX-BL11, ferment at 37°C for 8 hours after fermentation; Step S3 Feeding and Second Stage Fermentation: When the fermentation in step S2 reaches 8 hours, the composition 2 prepared in step S1 is fed in, the fermentation temperature is 37℃, and fermentation continues for another 8 hours; Post-processing in step S4: After centrifuging the fermentation broth obtained in step S3, the supernatant is collected to obtain the desired substance.

8. The method for preparing the fermentation composition according to claim 7, characterized in that: In step S1, the ratio of fermentation raw material to water is 1g:(5-10mL); the sterilization temperature is 105℃ and the sterilization time is 5-10min.

9. The method for preparing the fermentation composition according to claim 8, characterized in that: In step S2, the viable count of *Bifidobacterium longum* subsp. HX-BL11 in the bacterial culture is 10. 7 CFU / mL; In step S4, the centrifugation speed is 3000-5000 r / min; the centrifugation time is 5-10 min.

10. The application of the probiotic preparation according to claim 2 or the fermentation product according to claim 6, characterized in that, This includes applications in the preparation of foods or medicines that improve exercise fatigue.

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