Bifidobacterium lactis BL-PG-09 strain capable of relieving diarrhea and preparation method thereof

Abstract

The invention belongs to the technical field of microorganisms, and relates to a bifidobacterium lactis BL-PG-09 strain capable of relieving diarrhea and a preparation method of the bifidobacterium lactis BL-PG-09 strain, the strain is bifidobacterium lactis BL-PG-09, the preservation number is CGMCC No.30076, the preservation time is March 21, 2024, the preservation unit is China General Microbiological Culture Collection Center, and the preservation number is CGMCC No.30076. The address is No.3, Yard 1, Beichen West Road, Chaoyang District, Beijing. The bifidobacterium lactis BL-PG-09 disclosed by the invention is separated from faeces of healthy infants, has the function of relieving mouse diarrhea caused by antibiotics, has a certain antibacterial effect on pathogenic bacteria such as staphylococcus aureus, bacillus subtilis and escherichia coli, and has relatively strong acid resistance, salt resistance, cholate resistance and artificial simulated gastrointestinal fluid tolerance.

Description

Technical Field

[0001] This invention relates to the field of microbial technology, and in particular to a strain of Bifidobacterium lactis BL-PG-09 that can relieve diarrhea and its preparation method. Background Technology

[0002] Antibiotics are an effective treatment for diseases and are widely used to treat respiratory, digestive, and urinary system disorders. However, while antibiotics kill pathogenic bacteria, they can also indiscriminately attack the normal flora in the human gut, disrupting the gut microbiota balance and thus inducing antibiotic-associated diarrhea (AAD). Various antibiotics can cause AAD, with cephalosporins, clindamycin, and aminopenicillins having a particularly high incidence, affecting approximately 5% to 30% of patients during or after antibiotic treatment.

[0003] When antibiotics disrupt the balance of gut microbiota, the number of beneficial bacteria in the gut decreases significantly, while harmful bacteria proliferate excessively, affecting intestinal digestion and absorption, triggering intestinal inflammation, and causing diarrhea. Studies have shown that probiotics can alleviate acute inflammatory bowel disease (AAD). Lactic acid bacteria exert their health benefits by regulating the host's gut microbiota composition, increasing beneficial metabolites such as short-chain fatty acids (SCFAs), inhibiting the growth of pathogenic bacteria, strengthening the intestinal mucosal barrier function, regulating the intestinal immune response, and reducing inflammation.

[0004] Among probiotic strains used to intervene in antibiotic-associated diarrhea (AAD), Bifidobacterium animalis stands out as a highly representative strain in clinical applications due to its strong intestinal colonization ability, acid and bile salt resistance, and well-defined probiotic effects. It can be used concurrently with antibiotic treatment to prevent AAD, or after the onset of AAD symptoms to accelerate symptom relief. For patients experiencing slow recovery of gut microbiota and recurrent diarrhea after antibiotic discontinuation, long-term supplementation with this strain can promote long-term stability of the gut microbiota and reduce the risk of AAD recurrence. Summary of the Invention

[0005] The purpose of this invention is to provide a Bifidobacterium lactis strain BL-PG-09 that can relieve diarrhea, in order to solve the above-mentioned problems.

[0006] This invention discloses a strain of Bifidobacterium lactis BL-PG-09 (newly named Bifidobacterium animalis subsp. Lactis, but the old name is used for preservation) that has the effect of relieving diarrhea. The strain is classified as Bifidobacterium lactis, with accession number CGMCC No. 30076, deposited on March 21, 2024, at the China General Microbiological Culture Collection Center, located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing.

[0007] Furthermore, the present invention proposes a Bifidobacterium lactis strain BL-PG-09 that relieves diarrhea, which includes the following preparation steps:

[0008] Fecal samples were diluted with physiological saline and spread onto modified MRS solid medium containing 0.05 g / L cysteine ​​hydrochloride and 0.05 g / L mupirocin lithium. The samples were then anaerobically cultured at 37°C for 48 h. Single colonies of different morphologies and sizes were picked from the culture dishes and purified by streaking multiple times on the modified MRS solid medium until they were identified as single colonies. Single colonies were then examined under a microscope, and physiological and biochemical tests and 16S rDNA homology comparisons were performed. The isolated strains were then cryopreserved in glycerol.

[0009] Furthermore, the fecal sample was from a healthy infant, and the Bifidobacterium lactis strain BL-PG-09 was isolated from the fecal sample of a healthy infant.

[0010] The present invention also protects the use of the above-mentioned Bifidobacterium lactis strain BL-PG-09, which has the effect of relieving diarrhea, in the preparation of a drug for treating antibiotic-induced diarrhea in mice.

[0011] The present invention has the following technical effects:

[0012] 1. The Bifidobacterium lactis BL-PG-09 prepared in this invention was isolated from the feces of healthy infants. This strain has the function of relieving diarrhea in mice, which proves that the Bifidobacterium lactis BL-PG-09 prepared in this invention has broad application prospects in relieving diarrhea in mice and provides high-quality strain resources for the probiotic industry.

[0013] 2. The Bifidobacterium lactis BL-PG-09 prepared by this invention has good resistance to acid, salt, bile salts and simulated gastrointestinal fluids. When it enters the human body, it can reach the intestine with a high survival rate.

[0014] 3. The Bifidobacterium lactis BL-PG-09 prepared by this invention has the function of inhibiting Staphylococcus aureus, Bacillus subtilis, Escherichia coli, Pseudomonas aeruginosa, Listeria monocytogenes and Salmonella. The diameters of the inhibition zones are 17.13±0.76, 20.00±0.20, 19.53±0.50, 18.73±0.64, 19.07±0.50 and 18.33±0.31, respectively, showing strong antibacterial ability.

[0015] 4. The Bifidobacterium lactis BL-PG-09 prepared in this invention can significantly (P<0.05) increase the body weight of mice with diarrhea. Compared with the model group, the prevention group and the treatment group significantly (P<0.05) increased the body weight of mice with diarrhea, and the effect of the prevention group was significantly better than that of the treatment group (P<0.05), indicating that Bifidobacterium lactis BL-PG-09 has the effect of increasing the body weight of mice with antibiotic-induced diarrhea; and the effect is better when Bifidobacterium lactis BL-PG-09 is used at the same time as antibiotics.

[0016] 5. The Bifidobacterium lactis BL-PG-09 prepared in this invention has the function of relieving diarrhea in mice. After mice were given antibiotics by gavage, the diarrhea score of the prevention group was significantly lower (P<0.05) than that of the model group and the positive group, indicating that Bifidobacterium lactis BL-PG-09 can be used during antibiotic treatment to prevent the occurrence of diarrhea in mice. Attached Figure Description

[0017] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0018] Figure 1 Image showing the colony morphology and Gram staining results of Bifidobacterium lactis BL-PG-09 in a petri dish;

[0019] Figure 2 The image shows the results of the hemolysis test for Bifidobacterium lactis BL-PG-09.

[0020] Figure 3 A graph showing the acid resistance of Bifidobacterium lactis BL-PG-09;

[0021] Figure 4 A graph showing the salt tolerance of Bifidobacterium lactis BL-PG-09;

[0022] Figure 5 A graph showing the bile salt tolerance of Bifidobacterium lactis BL-PG-09;

[0023] Figure 6A graph showing the tolerance of Bifidobacterium lactis BL-PG-09 to simulated gastrointestinal fluid.

[0024] Figure 7 Figure 1 shows the results of the test on the inhibition of pathogenic bacteria by the supernatant of Bifidobacterium lactis BL-PG-09 fermentation broth.

[0025] Figure 8 The graph shows the rate of increase in body weight of mice in the blank group, model group, prevention group, treatment group, and positive control group.

[0026] Figure 9 The graph shows the water consumption of mice in the blank group, model group, prevention group, treatment group, and positive control group.

[0027] Figure 10 The graph shows the food intake of mice in the blank group, model group, prevention group, treatment group, and positive control group.

[0028] Figure 11 The graph shows the fecal water content of mice in the blank group, model group, prevention group, treatment group, and positive control group.

[0029] Figure 12 Diarrhea score charts for mice in the blank group, model group, prevention group, treatment group, and positive control group.

[0030] in, Figure 8 , 11 The different lowercase letters in 12 represent significant differences between different groups (P<0.05). Detailed Implementation

[0031] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0032] To make the above-mentioned objects, features and advantages of the present invention more apparent and understandable, the present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments.

[0033] Example 1: Screening and identification of Bifidobacterium lactis BL-PG-09

[0034] 1. Strains Isolation and Purification

[0035] This strain was isolated and purified from the feces of healthy infants. First, 1g of fecal sample was diluted with 9mL of sterile physiological saline. After shaking and mixing, it was further diluted 10-fold with sterile physiological saline. Different dilutions were spread on modified MRS solid medium containing 0.05g / L cysteine ​​hydrochloride and 0.05g / L mupirocin lithium salt and cultured anaerobically at 37℃ for 48h.

[0036] Further, single colonies of varying morphology and size were picked from the culture dishes and repeatedly streaked on modified MRS solid medium for purification until a pure strain was confirmed. Single colonies were then examined under a microscope, and physiological and biochemical analyses and 16S rDNA homology comparisons were performed. The isolated strains were then cryopreserved in glycerol. The purified strains were milky white, convex in the center, round in shape, with a smooth and moist surface, and relatively small in diameter. Figure 1 Left).

[0037] 2. Microscopic examination

[0038] Single colonies were picked and inoculated into MRS broth containing 0.5 g / L L-cysteine. After culturing for 24 hours, Gram staining was performed. The bacterial suspension was smeared, and cell morphology and arrangement were observed under a light microscope after Gram staining. Strains conforming to the typical morphology of Bifidobacterium were selected through microscopic observation, and colony morphology was photographed and recorded. The strains were Gram-positive, and the cells were short rod-shaped (…). Figure 1 right).

[0039] Physiological and biochemical tests

[0040] Physiological and biochemical tests were conducted using hydrogen peroxide, indole, and carbohydrate utilization tests to identify and screen strains. The strains exhibited the following physicochemical characteristics: negative hydrogen peroxide test, negative indole reaction; positive aescin hydrolysis test; and the ability to ferment cellobiose, maltose, mannitol, salicin, sorbitol, sucrose, raffinose, inulin, and lactose.

[0041] Molecular biological identification

[0042] For 16S rDNA homology alignment, the bacterial strain was expanded and cultured, the bacterial culture was centrifuged to obtain bacterial cells, and sequencing was performed. The sequencing was completed by Sangon Biotech (Shanghai) Co., Ltd. The rDNA sequence of Bifidobacterium lactis BL-PG-09 is as follows:

[0043]

[0044] Example 2: Activation and culture of Bifidobacterium lactis strain BL-PG-09

[0045] The following steps were taken to fully activate and expand the cryopreserved Bifidobacterium lactis BL-PG-09:

[0046] When activating Bifidobacterium lactis BL-PG-09, it was inoculated into sterile MRS liquid medium containing 0.5 g / L L-cysteine. It was first cultured anaerobically at 37°C for 24 h, and then cultured anaerobically at 37°C for another 24 h to fully activate Bifidobacterium lactis BL-PG-09.

[0047] Example 3 Hemolysis test of Bifidobacterium lactis BL-PG-09

[0048] The hemolysis test for Bifidobacterium lactis strain BL-PG-09 includes the following steps:

[0049] When fully activated and expanded Bifidobacterium lactis BL-PG-09 culture is inoculated onto Columbia blood agar medium, it is incubated upside down in a 37°C incubator for 24 hours. Then, the colonies are observed for hemolysis. A translucent hemolysis zone on Columbia blood agar indicates α-hemolysis, a clearly defined, completely transparent hemolysis zone indicates β-hemolysis, and the absence of a hemolysis zone indicates γ-hemolysis (no hemolysis).

[0050] The results of the hemolysis test for Bifidobacterium lactis BL-PG-09 are as follows: Figure 2 As shown in the image, using Staphylococcus aureus (β-hemolytic) as the control strain (left image), a well-defined, completely transparent hemolytic zone of 2-4 mm can be observed around Staphylococcus aureus colonies on Columbia blood agar medium. Bifidobacterium lactis BL-PG-09 (right image) did not produce a hemolytic zone and was not hemolytic, thus it is a safe strain.

[0051] Example 4: Acid resistance test of Bifidobacterium lactis strain BL-PG-09

[0052] When performing an acid resistance test on Bifidobacterium lactis strain BL-PG-09, the following steps are included:

[0053] In the acid tolerance test of the strain, the pH of the MRS medium was adjusted with 4.0 mol / L HCl, and MRS media with different pH values ​​were prepared: 1.0, 2.0, 3.0, 4.0, 5.0, and 6.0. The fully activated and expanded Bifidobacterium lactis strain BL-PG-09 was inoculated into the media with different pH values. After anaerobic culture at 37°C for 24 h, the OD of the bacterial suspension was measured. 600The nm value was set up in 3 replicates, with the OD value of MRS medium (pH 6.0) as a control. The survival rate was calculated to determine the acid tolerance of Bifidobacterium lactis BL-PG-09.

[0054] That is, survival rate = N t / N0;

[0055] Where: N t The values ​​are OD values ​​of MRS media with pH values ​​ranging from 1.0 to 5.0.

[0056] N0 is the OD value of the MRS medium at pH 6.0.

[0057] The acid resistance test results of Bifidobacterium lactis strain BL-PG-09 are as follows: Figure 3 As shown. From Figure 3 It can be seen that Bifidobacterium lactis BL-PG-09 exhibits poor tolerance at pH 1.0, with a survival rate of only 7.18% after 24 hours of treatment. However, it demonstrates strong tolerance at pH 4.0, with a survival rate as high as 62.59% after 24 hours of treatment. This indicates that Bifidobacterium lactis BL-PG-09 has a strong ability to tolerate acidic conditions.

[0058] Example 5 Salt tolerance test of Bifidobacterium lactis strain BL-PG-09

[0059] When performing a salt tolerance test on Bifidobacterium lactis strain BL-PG-09, the following steps are included:

[0060] In the salt tolerance test, fully activated and expanded Bifidobacterium lactis BL-PG-09 bacterial suspension was inoculated at a 3% inoculum into MRS medium containing 1.0, 2.0, 3.0, 4.0, and 5.0 g / 100 mL NaCl concentrations, respectively. After anaerobic culture at 37°C for 24 h, the OD of the bacterial suspension was measured. 600 The nm value was used as a control, with the OD value of the strain in MRS medium without NaCl added. Three replicates were set up to calculate the survival rate and determine the effect of different NaCl concentrations on the activity of Bifidobacterium lactis BL-PG-09.

[0061] That is, survival rate = N t / N0;

[0062] Where: N t The values ​​represent the OD values ​​of MRS media with NaCl concentrations ranging from 1.0 to 5.0 g / mL.

[0063] N0 is the OD value of the MRS medium when the NaCl concentration is 0 g / mL.

[0064] The results of the salt tolerance test for Bifidobacterium lactis strain BL-PG-09 are as follows: Figure 4As shown. The tolerance of the strain to NaCl was tested by selecting NaCl concentrations ranging from 1.0 to 5.0 g / mL. Figure 4 It can be seen that the survival rate of Bifidobacterium lactis BL-PG-09 decreased slightly with the increase of NaCl concentration, but after culturing for 24 h under NaCl concentration of 5.0 g / mL, its survival rate was still 59.19%, indicating that the bacterium has a strong ability to tolerate salt conditions.

[0065] Example 6: Bile salt tolerance test of Bifidobacterium lactis strain BL-PG-09

[0066] When performing a bile salt tolerance test on Bifidobacterium lactis strain BL-PG-09, the following steps are included:

[0067] In the bile salt tolerance test, fully activated and expanded Bifidobacterium lactis strain BL-PG-09 was inoculated at a 3% inoculum into MRS medium containing different concentrations of bile salt No. 3 (0.1%, 0.3%, 0.5%, 0.7%, and 0.9%, respectively). After anaerobic culture at 37°C for 24 hours, the OD of the bacterial suspension was measured. 600 The nm value was used as a control, with the OD value of the strain in MRS medium without bile salt No. 3 as a control. Three replicates were set up, the survival rate was calculated, and the effect of bile salt concentration on the activity of Bifidobacterium lactis BL-PG-09 was determined.

[0068] Survival rate = N t / N0;

[0069] Where: N t The viable bacterial count in MRS culture media containing different concentrations of bile salt No. 3;

[0070] N0 represents the number of viable bacteria in the MRS medium without the addition of bile salt No. 3.

[0071] The results of the bile salt tolerance test for Bifidobacterium lactis strain BL-PG-09 are as follows: Figure 5 As shown. In this study, five different mass fractions of bile salt No. 3 (0.1%, 0.3%, 0.5%, 0.7%, and 0.9%) were used to treat Bifidobacterium lactis strain BL-PG-09 for 24 hours (with the strain cultured without bile salts as a control). Figure 5 It can be seen that as the concentration of bile salt No. 3 increases, the survival rate of the strain gradually decreases. After 24 hours of treatment with 0.9% bile salt No. 3, the survival rate is still 33.43%, which shows that Bifidobacterium lactis BL-PG-09 has good bile salt tolerance.

[0072] Example 7: Simulated gastrointestinal test of Bifidobacterium lactis strain BL-PG-09

[0073] A naked microbial gastrointestinal assay was performed on the Bifidobacterium lactis strain BL-PG-09, including the following steps:

[0074] In the simulated gastrointestinal tract test using naked bacteria, the strain was activated and passaged twice in MRS medium at 37°C. Under aseptic conditions, 1.0 g of bacterial sludge was centrifuged and added to 5.0 mL of simulated gastric fluid. After incubating in a 37°C water bath for 3 hours, the mixture was centrifuged at 8000 rpm for 10 minutes. The precipitate was collected, and sterile physiological saline was added at a ratio of 1.0 g bacterial sludge to 5.0 mL physiological saline. The mixture was diluted, spread, and colony counts were performed. Then, 1.0 g of the bacterial sludge treated with simulated gastric fluid was added to 5.0 mL of simulated bile. After treatment at 37°C for 20 minutes, the mixture was centrifuged at 8000 rpm for 10 minutes. For each time, take bacterial sludge, add sterile physiological saline at a ratio of 1.0g bacterial sludge: 5.0mL physiological saline, mix well, dilute and spread, and count colonies. Then take 1.0g of bacterial sludge treated with artificial bile and add it to 5.0mL of artificial intestinal fluid. Treat at 37℃ for 4 or 8 hours respectively, centrifuge at 8000r / min for 10 minutes, take bacterial sludge, add sterile physiological saline at a ratio of 1.0g bacterial sludge: 5.0mL physiological saline, mix well, dilute and spread, and count colonies. Use the colony count in MRS medium that has not undergone simulated gastrointestinal treatment as a control to calculate the survival rate of the strain.

[0075] Survival rate of artificial gastrointestinal fluid = N t / N0;

[0076] Where: N t To simulate the number of viable bacteria after treatment with gastric juice, bile, and intestinal fluid;

[0077] N0 represents the number of viable bacteria before treatment.

[0078] Results of the simulated gastrointestinal test of Bifidobacterium lactis BL-PG-09 are as follows Figure 6 As shown. By Figure 6 It can be seen that after 3 hours of treatment with gastric juice, the survival rate of Bifidobacterium lactis BL-PG-09 was 76.24%, with a viable count of 9.80±0.07lg(cfu / g). After 20 minutes of treatment with bile, the survival rate was 74.68%, with a viable count of 9.60±0.06lg(cfu / g). After 4 hours of treatment with intestinal juice, the survival rate was still 59.93%, with a viable count of 7.70±0.02lg(cfu / g). This shows that Bifidobacterium lactis BL-PG-09 has a strong ability to tolerate artificial simulated gastrointestinal juice.

[0079] Example 8: Antibacterial test of Bifidobacterium lactis strain BL-PG-09

[0080] An antibacterial test was performed on the Bifidobacterium lactis strain BL-PG-09, including the following steps:

[0081] The indicator bacteria were activated and expanded in LB liquid medium and cultured at 37°C for 24 hours; Bifidobacterium lactis BL-PG-09 was activated and expanded in MRS liquid medium and cultured anaerobically at 37°C for 24 hours, and antibacterial tests were conducted.

[0082] When conducting the antibacterial test of Bifidobacterium lactis BL-PG-09, the Oxford cup agar diffusion method was used. Staphylococcus aureus, Bacillus subtilis, Escherichia coli, Pseudomonas aeruginosa, Listeria monocytogenes, and Salmonella typhimurium were used as indicator bacteria. The bacteria were plated on LB agar medium, with physiological saline as a blank control. Four Oxford cups were evenly placed in each petri dish. 200 μL of Bifidobacterium lactis BL-PG-09 bacterial suspension was added to three Oxford cups, and physiological saline was added to one Oxford cup. After anaerobic incubation at 37°C for 24 h, the presence of inhibition zones was observed, and the size of the inhibition zones was measured with calipers.

[0083] The antibacterial results of Bifidobacterium lactis BL-PG-09 are as follows: Figure 7 As shown in Table 1. From Figure 7 As shown in Table 1, inhibition zones appeared in all the petri dishes, and the diameter of the inhibition zones was greater than 17.00 mm. This indicates that Bifidobacterium lactis BL-PG-09 has an inhibitory effect on the growth of Staphylococcus aureus, Bacillus subtilis, Escherichia coli, Pseudomonas aeruginosa, Listeria monocytogenes, and Salmonella typhimurium. The diameters (mm) of the inhibition zones were 17.13±0.76, 20.00±0.20, 19.53±0.50, 18.73±0.64, 19.07±0.50, and 18.33±0.31, respectively, indicating that Bifidobacterium lactis BL-PG-09 has a significant antibacterial effect and strong antibacterial ability.

[0084] Table 1. Experimental data on the antibacterial activity of Bifidobacterium lactis BL-PG-09

[0085] indicator bacteria Diameter of the inhibition zone (mm) Staphylococcus aureus 17.13±0.76 Bacillus subtilis 20.00±0.20 E. coli 19.53±0.50 Pseudomonas aeruginosa 18.73±0.64 Listeria monocytogenes 19.07±0.50 Salmonella muscularis 18.33±0.31

[0086] Note: The diameter of the Oxford cup is 7.83±0.17mm.

[0087] Example 9: Baseline Indicators of Experimental Mice

[0088] The basic indicators of the experimental mice were measured, including the following steps:

[0089] Forty SPF-grade 6-8 week old BALB / c mice were acclimatized for 7 days and then randomly divided into five groups: blank control group, model group, prevention group, treatment group, and positive control group, with eight mice in each group. The specific experimental treatments are as follows.

[0090] Control group: Each mouse was administered 0.2 mL of physiological saline by gavage twice a day for a total of 14 days;

[0091] Model group: From day 8 to 14, each mouse was administered 0.2 mL of 150 mg / mL lincomycin hydrochloride by gavage (the dosage was calculated based on 3 g / kg BW). From day 15 to 21, each mouse was administered 0.2 mL of physiological saline by gavage, twice a day.

[0092] Prevention group: From day 8 to 14, each mouse was administered 0.2 mL of 150 mg / mL lincomycin hydrochloride (based on a dosage of 3 g / kg BW) + 0.2 mL of 1×10 9 CFU / mL Bifidobacterium lactis BL-PG-09 live bacterial dilution was administered to each mouse via gavage from days 15 to 21, at a concentration of 0.2 mL per 10⁻⁶ cfu / mL. 9 CFU / mL Bifidobacterium lactis BL-PG-09 live bacteria dilution, twice daily;

[0093] Treatment group: From days 8 to 14, each mouse was administered 0.2 mL of 150 mg / mL lincomycin hydrochloride by gavage (dosage calculated at 3 g / kg BW); from days 15 to 21, each mouse was administered 0.2 mL of 1×10⁻⁶ lincomycin hydrochloride by gavage. 9 CFU / mL Bifidobacterium lactis BL-PG-09 live bacteria dilution, twice daily;

[0094] Positive group: From day 8 to 14, each mouse was administered 0.2 mL of 150 mg / mL lincomycin hydrochloride by gavage (dosage calculated at 3 g / kg BW); from day 15 to 21, each mouse was administered 0.2 mL of 1×10⁻⁶ lincomycin hydrochloride by gavage. 9 CFU / mL commercially available Bifidobacterium triple live bacteria capsules (Shanghai Shangyao Xinyi Pharmaceutical Co., Ltd.), twice daily.

[0095] During the experiment, the weight gain rate of the mice was calculated using the following formula.

[0096] Weight gain rate (%) = (Final weight - Initial weight) / Final weight × 100

[0097] Weight gain results as follows Figure 8 As shown. From Figure 8 It can be seen that the weight gain rate of mice in both the prevention and treatment groups was significantly greater than that in the model group (P<0.05), and the weight gain rate of mice in the prevention group was significantly higher than that in the treatment group (P<0.05). This indicates that the administration of Bifidobacterium lactis BL-PG-09 has a significant effect on both preventing and treating weight loss in mice caused by diarrhea, with the prevention group showing a better effect. However, the improvement effect of both groups on weight loss in diarrheal mice was lower than that in the positive control group.

[0098] from Figure 9 and Figure 10 It can be seen that from day 7 to day 18 of the experiment, the food intake and water intake of mice in each group were within the normal range, and there was no significant difference in the mental state of the mice.

[0099] Example 10: Procedures and Results for Determining Diarrhea Indicators in Experimental Mice

[0100] The following steps were taken to determine diarrhea markers in experimental mice:

[0101] On the last day of the experiment, feces were collected, and the weight of feces output over 120 minutes was observed. The wet weight of feces in each group of mice was recorded. After the feces were dried and lost water, the water content of the feces was calculated to assess the diarrhea status of the mice. Based on the changes in mouse weight and feces, the severity of diarrhea in each group was calculated, and the diarrhea status of each group was statistically analyzed and scored.

[0102] Scoring criteria: 0 points, no diarrhea; 1 point, stool is formed but lighter in color and sticky, mental state is average; 2 points, stool is unformed, stool is stuck to the anus and tail, mental state is lethargic, appetite is reduced, and weight is lost.

[0103] Fecal moisture content (%) = (wet weight - dry weight) / wet weight × 100

[0104] Diarrhea status score results are shown in Figure 11 .from Figure 11 It can be seen that from day 8 to day 14 of the experiment, after antibiotic treatment, the diarrhea scores of all groups were greater than or equal to 1, indicating that diarrhea occurred, and except for the prevention group, all groups had obvious watery stools; the diarrhea score of the prevention group was relatively low, indicating that the diarrhea symptoms were milder.

[0105] During days 15–21 of the experiment, the diarrhea symptoms in the prevention and treatment groups, who were administered Bifidobacterium lactis BL-PG-09 via gavage, significantly decreased, indicating that Bifidobacterium lactis BL-PG-09 alleviated antibiotic-induced diarrhea. Furthermore, the diarrhea recovery in the prevention and treatment groups was significantly better than that in the positive control group (commercially available Bifidobacterium triple live bacteria capsules), suggesting that Bifidobacterium lactis BL-PG-09 is superior to commercially available Bifidobacterium triple live bacteria capsules in alleviating antibiotic-related diarrhea in mice, and that the effect of a single strain is superior to that of commercially available Bifidobacterium triple live bacteria capsules. It is predicted that the production cost can be reduced when single strains are used as drugs in the future.

[0106] Fecal water content results as follows Figure 12 As shown, the fecal water content of mice in the treatment group treated with Bifidobacterium lactis BL-PG-09 of this invention decreased to 61.89%, while the fecal water content of mice in the prevention group was 57.87%, both significantly (P<0.05) lower than the fecal water content of 66.43% in the model group. The fecal water content of mice in both the prevention and treatment groups was significantly (P<0.05) lower than that of the positive group (commercially available Bifidobacterium triple live bacteria capsules). Therefore, it can be concluded that the single strain of Bifidobacterium lactis BL-PG-09 of this invention is more effective than commercially available Bifidobacterium triple live bacteria in alleviating diarrhea in mice.

[0107] In conclusion, the results show that the Bifidobacterium lactis strain BL-PG-09 can alleviate diarrhea in mice and has broad application prospects, providing high-quality strain resources for the probiotic industry.

[0108] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A strain of Bifidobacterium lactis BL-PG-09 that relieves diarrhea, characterized in that, The strain is classified and named Bifidobacterium lactis, with accession number CGMCC No. 30076, deposited on March 21, 2024, at the China General Microbiological Culture Collection Center, located at No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing.

2. The Bifidobacterium lactis strain BL-PG-09 with diarrhea-relieving properties according to claim 1, characterized in that, The preparation steps include the following: Fecal samples were diluted with physiological saline and spread onto modified MRS solid medium containing 0.05 g / L cysteine ​​hydrochloride and 0.05 g / L mupirocin lithium. The samples were then anaerobically cultured at 37°C for 48 h. Single colonies of different morphologies and sizes were picked from the culture dishes and purified by streaking multiple times on the modified MRS solid medium until they were identified as single colonies. Single colonies were then examined under a microscope, and physiological and biochemical tests and 16S rDNA homology comparisons were performed. The isolated strains were then cryopreserved in glycerol.

3. The Bifidobacterium lactis strain BL-PG-09 with diarrhea-relieving properties according to claim 2, characterized in that, The stool sample was from a healthy infant, and the Bifidobacterium lactis strain BL-PG-09 was isolated from the stool of the healthy infant.

4. The application of the Bifidobacterium lactis strain BL-PG-09 with diarrhea-relieving properties according to claim 1 in the preparation of a drug, characterized in that, The drug is used to treat antibiotic-induced diarrhea in mice.

Images