Bacillus coagulans NSL1204 capable of improving exercise tolerance and application thereof

Abstract

The invention provides bacillus coagulans NSL1204 capable of improving exercise tolerance and application of the bacillus coagulans NSL1204, and belongs to the technical field of microorganisms. The classification name of the bacillus coagulans NSL1204 disclosed by the invention is bacillus coagulans, and the preservation number of the bacillus coagulans NSL1204 is CCTCC (China Center for Type Culture Collection) NO: M 2025206. The bacillus coagulans NSL1204 can improve the levels of hepatic glycogen and muscle glycogen and enhance continuous supply of energy in the exercise process so as to improve exercise tolerance, has the functions of maintaining the blood sugar level and reducing the content of blood lactic acid after exercise, can be used for preparing sports drinks or granules, and has a probiotic function; the bacillus coagulans strain NSL1204 can also be applied to preparation of food resistant to gastric acid, bile salt and heat; the bacillus coagulans NSL1204 is wide in application range and high in biological safety performance in the application of preparing food.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of microbial technology, and particularly relates to a Bacillus coagulans NSL1204 capable of improving exercise endurance and application thereof. BACKGROUND

[0002] Exercise endurance is an important indicator for measuring the body's anti-fatigue ability. If fatigue is not eliminated in time, it will further develop into "overwork", causing endocrine disorders, decreased immunity, and even organic lesions. Fatigue has become a major factor threatening human health in today's society. Existing research shows that probiotics can affect exercise performance by regulating intestinal flora, improving metabolism and anti-inflammatory effects. They can replace exercise nutritional supplements (such as caffeine, branched-chain amino acids) with limited side effects or limited effects. However, traditional probiotics (such as lactobacillus and bifidobacterium) generally have poor acid tolerance, short shelf life and other problems, making it difficult to maintain activity during food processing and storage, and the effect of improving exercise endurance is limited. Therefore, it is of great significance to develop a probiotic strain with high stability and significant exercise enhancement function.

[0003] Bacillus coagulans is a facultative anaerobic gram-positive probiotic bacteria that ferments to produce L-lactic acid. It has good tolerance to harsh environments and has become a research hotspot and gradually been applied due to its significant probiotic properties. SUMMARY

[0004] The present application provides a Bacillus coagulans NSL1204 capable of improving exercise endurance and application thereof. The Bacillus coagulans NSL1204 can resist gastric acid and bile salts, and is also heat-resistant. The present application also verifies the significant property of the Bacillus coagulans NSL1204 in improving exercise endurance, which widens the application of the strain in the preparation of food or drugs, and has promotional significance.

[0005] To achieve the above-mentioned application purposes, the present application adopts the following technical solutions:

[0006] The present application provides a Bacillus coagulans NSL1204, which is classified as Bacillus coagulans, and has a preservation number of CCTCC NO: M 2025206.

[0007] Further, the Bacillus coagulans NSL1204 is a gram-positive bacillus that forms a round, neat-edged, opaque, milky-white colony on an agar plate, with a slightly rough surface. It forms an oval spore inside the cell, with the spore position biased towards one end, and the spore cyst is slightly swollen.

[0008] Further, the 16S rDNA sequence of Bacillus coagulans NSL1204 is shown as SEQ ID No. 1.

[0009] The application further provides the use of the Bacillus coagulans NSL1204 in the preparation of food and / or medicine resistant to gastric acid and / or bile salt.

[0010] Further, the resistant concentration of the bile salt is 0.1%-0.5% by mass ratio; and the resistant pH value of the gastric acid is 1.5-4.

[0011] Further, the pH value of the gastric acid is 1.5-2.5, and the spore rate of the Bacillus coagulans NSL1204 is >75%; the pH value of the gastric acid is 2.5-4, and the spore rate of the Bacillus coagulans NSL1204 is >90%; and the pH value of the gastric acid is 3-4, and the spore rate of the Bacillus coagulans NSL1204 is >92%.

[0012] Further, the concentration of the bile salt is 0.1%-0.5%, and the spore rate of the Bacillus coagulans NSL1204 is >94%; the concentration of the bile salt is 0.1%-0.3%, and the spore rate of the Bacillus coagulans NSL1204 is >96%; and the concentration of the bile salt is ≤0.1%, and the spore rate of the Bacillus coagulans NSL1204 is >98%.

[0013] The application further provides the use of the Bacillus coagulans NSL1204 in the preparation of food and / or medicine resistant to heat.

[0014] Further, the resistant temperature of the heat resistance is 80℃-100℃.

[0015] Further, in the use of the preparation of the heat-resistant food, when the treatment temperature is ≥80℃ and the treatment time is ≤2h, the spore rate of the Bacillus coagulans NSL1204 is ≥80%; and when the treatment temperature is ≥80℃ and the treatment time is ≤5h, the spore rate of the Bacillus coagulans NSL1204 is ≥78%.

[0016] The application further provides the use of the Bacillus coagulans NSL1204 in the preparation of medicine and / or food for improving exercise endurance.

[0017] Further, the Bacillus coagulans NSL1204 can improve the content of muscle glycogen and liver glycogen in the body, and can improve the blood glucose level and reduce the serum lactic acid level in the body.

[0018] Compared with the prior art, the application has the following advantages and beneficial effects:

[0019] 1. The Bacillus coagulans NSL1204 provided by the application has excellent heat resistance, and the spore survival rate is 96% after 2h of water bath treatment at 80℃; the spore survival rate is 92% after 10min of high-temperature treatment at 100℃. The bacterial powder can be used in food or beverage, especially hot beverage, so that the food or beverage has a probiotic function.

[0020] 2. The Bacillus coagulans NSL1204 provided by the application has excellent gastric acid resistance and bile salt resistance, and the spore survival rate is 76.75% after 2h of pH 2.0 gastric acid treatment; the spore survival rate is 93.63% after 2h of pH 4.0 gastric acid treatment; and the spore survival rate is 94.08% after 2h of 0.5% bile salt treatment.

[0021] 3. The Bacillus coagulans NSL1204 provided by the application can improve the levels of liver glycogen and muscle glycogen, enhance the continuous supply of energy during exercise to improve exercise endurance, and has the functions of maintaining blood glucose level after exercise and reducing blood lactic acid content, and can be used for preparing sports drinks or granules, and has a probiotic function.

[0022] 4. The Bacillus coagulans NSL1204 provided by the application has fast growth rate and high cell density, and is widely applicable in the preparation of food, and has high biological safety, and also provides a reference for the later research of related food. BRIEF DESCRIPTION OF DRAWINGS

[0023] Figure 1 is the growth curve of the Bacillus coagulans NSL1204;

[0024] Figure 2 is the image graph of the Bacillus coagulans NSL1204 under an optical microscope;

[0025] Figure 3 is the effect of the Bacillus coagulans NSL1204 on the exhaustive swimming time of mice under load;

[0026] Figure 4 is the effect of the Bacillus coagulans NSL1204 on the blood glucose in serum of mice after non-load swimming exercise;

[0027] Figure 5 is the effect of the Bacillus coagulans NSL1204 on the lactic acid in serum of mice after non-load swimming exercise;

[0028] Figure 6 is the effect of the Bacillus coagulans NSL1204 on the liver glycogen and muscle glycogen content of mice after non-load swimming exercise. DETAILED DESCRIPTION

[0029] The application will be further described in detail in combination with examples, but the embodiments of the application are not limited thereto.

[0030] Example 1: Screening, identification and preservation of strains

[0031] 1. Screening of Bacillus coagulans strains

[0032] The Bacillus coagulans NSL1204 provided by the present application is isolated from the feces of healthy adults, and the isolation method is as follows: 1 g of feces sample that has been refrigerated for no more than 24 h is added to 9 mL of sterile normal saline, vortexed and mixed, allowed to stand for 5 minutes to precipitate large particles, and the supernatant is heated at 70°C for 30 minutes to kill non-sporulating bacteria. After cooling, plate separation is performed. Incubate at 37°C for 24 h on nutrient agar (NA) medium, and pick colonies that are milky white, round, with neat edges, and about 1-3 mm in diameter for Gram staining. Pick Gram-positive bacilli, spore-forming, and non-swollen or slightly swollen spore cysts for isolation and purification. The purified strain is subjected to catalase test, and the strain that shows a positive reaction is preliminarily identified as Bacillus. Then, the strain is identified at the species level using sugar fermentation test and 16S rDNA method.

[0033] 2. Strain identification

[0034] Molecular identification: After isolation and screening, the Bacillus coagulans strain is identified by PCR amplification of the corresponding 16S rDNA gene sequence (the sequence is shown as SEQ ID No. 1), and the strain is preserved in a cryogenic glycerol tube in the laboratory of Qingdao Noson Biotech Co., Ltd. The Bacillus coagulans uses PCR amplification of the corresponding 16S rDNA gene sequence, and analysis with the gene sequences registered in the NCBI database Genbank shows that the strain has 99% similarity with the 16S rDNA sequences of different Bacillus coagulans strains in the database.

[0035] Morphological identification: Gram-positive bacteria, rod-shaped under a microscope, blunt at both ends, straight rod-shaped when present alone, and slightly curved when arranged in pairs or short chains. Form round, neat-edged, opaque, milky white colonies on agar plates, with a slightly rough surface. Oval spores are formed inside the cells, with the spore position mostly biased towards one end, and the spore cyst is slightly swollen. The affinity of the vegetative cells to basic dyes is strong, and they appear dark purple after Gram staining.

[0036] 3. Preservation of strains

[0037] The screened Bacillus coagulans NSL1204 is preserved, and the preservation unit of the Bacillus coagulans NSL1204 is China Center for Type Culture Collection (CCTCC); the address is Wuhan, Wuhan University, China; the preservation number of the Bacillus coagulans NSL1204 is CCTCC NO: M 2025206, and the preservation date is February 13, 2025.

[0038] Example 2: Analysis of growth characteristics of Bacillus coagulans NSL1204

[0039] (1) From the glycerol tube, inoculate a ring on the PCA test tube slant medium, and culture at 42°C for 48-72 h, and then add 3-5 mL of sterile water to elute the test tube slant mycelium to prepare a bacterial suspension.

[0040] (2) Take the bacterial suspension and inoculate into a seed culture medium in a shake flask at a 10-20% inoculation amount, 6 mL of liquid is filled in a 50 mL shake flask, and culture at 40°C, 200 r / min for 4-6 h to obtain a first-stage seed liquid.

[0041] (3) The first-stage seed liquid is inoculated into a seed culture medium in a shake flask at a 10-20% inoculation amount, 30 mL of liquid is filled in a 250 mL shake flask, and culture at 40°C, 200 r / min for 2-4 h to obtain a second-stage seed liquid.

[0042] (4) The second-stage seed liquid is inoculated into a fermentation culture medium in a shake flask at a 5% inoculation amount, 30 mL of liquid is filled in a 250 mL shake flask, and culture at 40°C, 200 r / min for growth curve determination.

[0043] The growth of Bacillus coagulans is shown in Figure 1 , and the image of Bacillus coagulans under an optical microscope is shown in Figure 2 .

[0044] The growth curve of Bacillus coagulans in Figure 1 indicates that Bacillus coagulans is in a growth lag phase for 0-6 h, in a logarithmic growth phase for 6-16 h, in a relatively stable phase for 16-26 h, reaches a first peak at 24 h, and then the OD 600 absorbance value increases again due to spore formation under the microscope, and reaches a highest peak at 30 h, and then starts to decrease, and enters a decline phase. The fermentation culture medium of Bacillus coagulans is cultured for 20 h, and the culture for 30 h is suitable for biomass and spore plating count.

[0045] The seed culture medium formula: 2.0% glucose, 1.0% yeast extract powder, 1.0% peptone, 0.03% MgSO4·7H2O, 0.01% MnSO4, 0.05% NaCl, 0.1% K2HPO4, 0.001% FeSO4·7H2O, 0.05% CaCO3, the balance is water, adjust the initial pH value to 7.0±0.1, sterilize at 118°C for 20 min for standby.

[0046] The fermentation medium formula: 3.0% glucose, 3.5% yeast extract powder, 4.0% peptone, 0.06% MgSO4·7H2O, 0.01% MnSO4, 0.1% NaCl, 0.2% K2HPO4, 0.002% FeSO4·7H2O, 0.1% CaCO3, the balance is water, adjust the initial pH value to 7.0±0.1, sterilize at 118°C for 20 min for standby.

[0047] Example 3: Effect of Bacillus coagulans NSL1204 on the exhaustive swimming time of mice under load

[0048] Preparation of Bacillus coagulans strain bacterial suspension of different dose concentrations: centrifuge the activated culture solution of Bacillus coagulans NSL1204 to collect the bacterial cells, and dilute with 0.85% sterile normal saline to prepare a bacterial suspension of low dose 1×10 7 cfu / mL, medium dose 1×10 8 cfu / mL and high dose 1×10 9 cfu / mL.

[0049] Mouse caging and exercise intervention: After 7-week-old healthy male Kunming mice (a total of 30) were adaptively fed for one week, they were randomly grouped according to body weight and labeled, a total of 5 cages, 6 mice per cage, respectively, blank control group, positive control group, low-dose test group, medium-dose test group and high-dose test group. In the 30 days of continuous feeding, the blank control group did not undergo any exercise training, and the rest of the mice in each group underwent 15 minutes of weightless swimming training every day at a fixed time, and the mice were free to eat and drink during the test period. The control group was given 0.85% sterile normal saline, and the test group was given the corresponding bacterial suspension of different doses, and the continuous gavage was performed for 30 days (100 μL / day).

[0050] Daily weightless swimming training of mice: the rest of the groups except the blank control group were subjected to weightless swimming test at 2 pm every day, the water temperature of the pool was 25±5°C, the water depth was 25 cm (more than 1.5 times the body length of the mouse, so that the tail could not touch the bottom support), the swimming time was 15 minutes, and the training was continued for 30 days.

[0051] Weight-bearing exhaustion swimming test: After 30 days of continuous weightless swimming training, the weight of mice was measured. On the 31st day, after 30 minutes of gavage, lead sheet equivalent to 5% of the mouse's body weight was attached to the base of the mouse's tail and the mouse was placed alone in a swimming pool. The time from entering the pool to exhaustion was recorded. The time when the mouse could not float to the surface for 8 seconds after sinking was taken as the weight-bearing exhaustion swimming time.

[0052] like Figure 3 As shown, the effect of Bacillus coagulans on the time to exhaustion in weight-bearing swimming in mice revealed that, compared with the blank control group, the time to exhaustion in weight-bearing swimming in the positive control group was significantly increased after weightless swimming training, indicating that the exercise model was successfully established. Compared with the positive control group, the time to exhaustion in weight-bearing swimming in the experimental group was significantly increased, and the time to exhaustion in weight-bearing swimming in the high-dose experimental group reached as high as 78.59±2.07 min, which was nearly 3 times higher than the weight-bearing swimming endurance of mice that did not receive Bacillus coagulans NSL1204 bacterial suspension. These results indicate that Bacillus coagulans NSL1204 has the characteristic of significantly improving exercise endurance.

[0053] Example 4: Effects of Bacillus coagulans NSL1204 administration on serum biochemical parameters in mice after swimming exercise

[0054] Mouse ocular blood collection test: After the weighted swimming exhaustion test, mice rested for 24 hours, during which time they were fed and hydrated normally. After fasting for 12 hours, they swam for 15 minutes without weight, rested for 30 minutes, and were anesthetized with ether. Immediately afterward, the eyeballs were removed with forceps to collect blood. The collected blood samples were placed in 2 mL centrifuge tubes and incubated at 4°C for 1 hour to allow clotting. The samples were then centrifuged at 3500 rpm for 10 minutes at 4°C. The separated serum was transferred and stored at 4°C for later use.

[0055] Determination of serum lactate content: Add 450 μL of 10% TCA to 50 μL of serum, vortex to mix, incubate on ice for 10 min, centrifuge at 10000 rpm for 5 min, and collect the supernatant for analysis. (Based on Solarbio...) ® Follow the instructions for the micro-method of the lactate (LA) assay kit to prepare the sample and perform the colorimetric reaction (purple). Measure the absorbance at 570 nm using an ELISA reader, plot a standard curve, and calculate the lactate concentration in the supernatant.

[0056] Determination of serum glucose levels: Serum was diluted 1:10 with physiological saline (ensuring glucose concentration <20 mmol / L). (Based on LEAGENE...) ® Follow the instructions for the Glucose Assay Kit (GOD-POD Microplate Method) to perform sample preparation and colorimetric reaction (red), measure the absorbance at 505 nm using an ELISA reader, plot a standard curve, and calculate the serum glucose concentration.

[0057] Conclusions Figure 4 The results showed that the blood glucose levels of mice in each group were between 4 and 5.5 mmol / L. Compared with the positive control group, the blood glucose levels of mice after taking Bacillus coagulans NSL1204 were slightly elevated, but still maintained a relatively stable state. The results showed that the serum lactate levels of mice after taking Bacillus coagulans NSL1204 were significantly reduced, and compared with the positive control group, the serum lactate levels of mice in the high-dose test group were reduced by 43.46%. Figure 5

[0058] These results showed that taking Bacillus coagulans NSL1204 could reduce the serum lactate levels after exercise, reduce fatigue after exercise, and maintain blood glucose levels to some extent.

[0059] Example 5: Effect of taking Bacillus coagulans NSL1204 on muscle glycogen and liver glycogen content of mice after swimming exercise

[0060] Mouse dissection test: After taking blood from the eyeball, the mice were killed by cervical dislocation method, and the liver and gastrocnemius muscle tissues were stripped, washed with 0.85% pre-cooled physiological saline to remove blood, and dried with filter paper. Liquid nitrogen was used for preservation.

[0061] Liver glycogen extraction test: 100 mg of liver tissue was weighed, cut into pieces and added with 1 mL of 5% TCA, and homogenized at 10000 rpm in ice bath for 30 s. The homogenate was centrifuged at 4°C and 3000 rpm for 10 min, and the supernatant was taken, 2 volumes of absolute ethanol were added, mixed well, and then placed at 4°C for 1 h (glycogen precipitation), then centrifuged at 10000 rpm for 15 min, the supernatant was discarded, and the precipitate was dissolved with 1 mL of distilled water, which was the liver glycogen extract.

[0062] Muscle glycogen extraction test: 100 mg of gastrocnemius muscle tissue was weighed, cut into pieces and added with 1 mL of 30% KOH, and boiled in water bath for 20 min to completely dissolve the tissue. After cooling, 1.25 mL of absolute ethanol was added, vortexed, and mixed well, and then placed for 10 min (glycogen precipitation), then centrifuged at 12000 rpm for 15 min, the supernatant was discarded, and the precipitate was dissolved with 1 mL of distilled water, which was the muscle glycogen extract.

[0063] Glycogen content determination: According to the Solarbio ® Glycogen content detection kit (anthrone method) instruction, sample preparation and color reaction (blue-green) were performed, and the absorbance was determined at 620 nm by enzyme label instrument, the standard curve was drawn, and the glycogen content of unit tissue (mg / g) was calculated.

[0064] Conclusions​Figure 6 The results showed that the contents of liver glycogen and muscle glycogen of the mice in the test groups were significantly higher than those in the positive control group after exercise. Among them, the liver glycogen content of the mice in the high-dose test group was as high as 14.43±0.20 mg / g, which was about 1.9 times the liver glycogen level of the positive control group, and the muscle glycogen content was as high as 1.71±0.26 mg / g, which was about 2.48 times the muscle glycogen level of the positive control group.

[0065] These results show that taking Bacillus coagulans NSL1204 can enable the body to have higher levels of muscle glycogen and liver glycogen to sustain energy for the body during exercise, thereby improving exercise endurance.

[0066] Example 6: Bacillus coagulans NSL1204 tolerance test to gastric acid and bile salt

[0067] Artificial gastric juice with pH values of 2.0, 3.0, and 4.0 was prepared by adjusting with 1 mol / L hydrochloric acid. The bacterial cells of Bacillus coagulans NSL1204 activated culture were collected by centrifugation, and diluted with sterile normal saline to about 10 9 CFU / mL, and inoculated into artificial gastric juice with different pH values at a 1% inoculation amount. After 2 h of constant temperature treatment at 37°C in a water bath, viable cell counting was performed, and the survival rate of the bacteria was calculated after 24 h of culture at 40°C. Similarly, 1% inoculation was added to pig bile salt solutions with mass concentrations of 0.1%, 0.3%, and 0.5%, respectively. After 2 h of constant temperature treatment at 37°C in a water bath, viable cell counting was performed, and the survival rate of the bacteria was calculated after 24 h of culture at 40°C. The results are shown in Table 1.

[0068] Table 1. Survival rate of Bacillus coagulans NSL1204 treated with different gastric acid / bile salt conditions for 2 h

[0069] The results of the gastric acid and bile salt tolerance experiments of Bacillus coagulans NSL1204 showed that the survival rate of the spores was 93.63% after 2 h of pH 4.0 gastric acid treatment, 92.08% after 2 h of pH 3.0 gastric acid treatment, and still 76.75% after 2 h of pH 2.0 gastric acid treatment. The survival rate of the spores was 98.13% after 2 h of 0.1% bile salt treatment, 96.36% after 2 h of 0.3% bile salt treatment, and still 94.08% after 2 h of 0.5% bile salt treatment.

[0070] Example 7: Bacillus coagulans NSL1204 heat resistance performance test

[0071] The activated culture solution of Bacillus coagulans NSL1204 was centrifuged to collect the bacterial cells, and the bacterial cells were diluted with 0.85% sterile normal saline to prepare 10 9 A bacterial suspension of Bacillus coagulans with 10 cfu / mL was prepared for determining the heat resistance of Bacillus coagulans.

[0072] Each 1 mL of the bacterial suspension of Bacillus coagulans NSL1204 was subjected to 80°C water bath treatment and 100°C high-temperature treatment, respectively, and the bacterial suspension was treated at 80°C for 2h and 5h and at 100°C for 10min and 30min, respectively. The treated solution was appropriately diluted, and the viable cell count was determined. The Bacillus coagulans solution without treatment was used as a blank control group, and the spore survival rate of Bacillus coagulans was calculated. Each experiment was repeated three times, and the results are shown in Table 2.

[0073] Table 2. Evaluation of heat resistance of Bacillus coagulans NSL1204

[0074] The results of the heat resistance experiment of Bacillus coagulans showed that the spore survival rate was 96% after 2h of 80°C water bath treatment, and 78% of the spores were still alive after 5h of treatment. The spore survival rate was 92% after 10min of 100°C high-temperature treatment, and 80% of the spores were still alive after 30min of treatment.

[0075] The above examples only illustrate the technical solutions of the present application, but do not limit the same; although the present application has been described in detail with reference to the foregoing examples, the technical solutions recorded in the foregoing examples can still be modified by those of ordinary skill in the art, or some technical features can be replaced by equivalents; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the spirit and scope of the technical solutions claimed by the present application.

Claims

1. Bacillus coagulans NSL1204, which is capable of enhancing exercise performance, characterized in that, The classification name is Bacillus coagulans Bacillus coagulans and the accession number is CCTCC NO: M 2025206.

2. Bacillus coagulans NSL1204 according to claim 1, characterized in that, The Bacillus coagulans NSL1204 is a gram-positive bacillus, which forms round, neat-edged, opaque, milky-white and slightly rough-surfaced colonies on agar plates, oval spores in the cell interior, the spore position is biased to one end, and the spore capsule is slightly swollen.

3. Use of the Bacillus coagulans NSL1204 according to claim 1 or 2 for the preparation of a food and / or a pharmaceutical product resistant to gastric acid and / or resistant to bile salt.

4. Use according to claim 3, characterized in that, The resistant concentration of the bile salt resistance is 0.1%-0.5%.

5. Use according to claim 3, characterized in that, The resistant pH value of the gastric acid resistance is 1.5-4.

6. Use of the Bacillus coagulans NSL1204 according to claim 1 or 2 for the preparation of a heat-resistant food and pharmaceutical product.

7. Use according to claim 6, characterized in that, The resistant temperature of the heat resistance is 80℃-100℃.

8. Use of the Bacillus coagulans NSL1204 according to claim 1 or 2 for the preparation of a food and pharmaceutical product for improving exercise endurance.

9. Use according to claim 8, characterized in that, The food is a probiotic beverage, and the bacterial content is 1 x 10 7 cfu / mL - 1 x 10 9 cfu / mL.

10. Use according to claim 9, characterized in that, The Bacillus coagulans NSL1204 can increase the content of muscle glycogen and liver glycogen in the body, increase the blood glucose level in the body, and reduce the lactic acid level in the serum.

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