Bifidobacterium breve strain with constipation relieving effect

Abstract

The invention belongs to the technical field of microorganisms, and relates to a bifidobacterium breve strain capable of relieving constipation, the strain is bifidobacterium breve Bb-PG-14, the classification name of the strain is Bifidobacterium breve, the preservation number is CGMCC No.31861, the preservation time is September 4, 2024, the preservation unit is China General Microbiological Culture Collection Center, the preservation number is CGMCC No.31861, the preservation number is CGMCC No.31861, the preservation number is CGMCC No.31861, the preservation number is CGMCC No.31861, the preservation number is CGMCC No.31861, and the preservation number is CGMCC No.31861. The address is No.3, Yard 1, Beichen West Road, Chaoyang District, Beijing. The strain is separated and purified from excrement of healthy babies, has relatively strong acid resistance, alkali resistance, cholate resistance and artificial simulated gastrointestinal fluid tolerance, and has the characteristics of probiotics.

Description

Technical Field

[0001] This invention relates to the field of microbial technology, and in particular to a strain of Bifidobacterium breve that has the effect of relieving constipation. Background Technology

[0002] Constipation is a common gastrointestinal disorder caused by various factors, which can further lead to intestinal dysfunction. The main clinical manifestations include reduced bowel movement frequency (less than 3 times per week), hard stools, straining during defecation, and a feeling of incomplete evacuation. Constipation not only reduces quality of life but can also lead to hemorrhoids, anal fissures, and subsequently infections, intestinal inflammation, and even increase the risk of colon cancer. Clinical relief methods for constipation mainly include dietary adjustments and medication. Dietary adjustments that increase dietary fiber and water intake are the most common method, but their effectiveness is not ideal due to individual differences. Stimulant laxatives (such as senna) have a rapid short-term effect, but long-term use can easily lead to intestinal mucosal damage, decreased intestinal motility, and drug dependence. Osmotic laxatives (such as lactulose) may cause bloating, diarrhea, and other gastrointestinal discomfort. Therefore, all of the above relief or treatment methods have certain limitations.

[0003] In recent years, in-depth research on gut microbiota has revealed a close relationship between gut microbiota and the occurrence and development of constipation. Under normal circumstances, probiotics in the gut maintain normal intestinal physiological functions through various pathways, including participating in food digestion, promoting intestinal peristalsis, and maintaining an acidic intestinal environment. When the gut microbiota is imbalanced, the number of probiotics decreases, while harmful bacteria proliferate excessively, which may lead to slowed intestinal peristalsis and prolonged stool retention time in the intestine, thus inducing constipation. Probiotics can colonize the gut, adjusting the microbiota structure by inhibiting the proliferation of harmful bacteria and increasing the proportion of probiotics; they also indirectly improve the gut microenvironment by producing lactic acid to maintain an acidic environment. This synergistic effect of "directly regulating the microbiota + indirectly improving the environment" ultimately effectively relieves constipation. Because probiotics are safe for humans, they can also avoid the side effects of using medication to treat constipation.

[0004] Bifidobacterium breve, as a native intestinal bacterium of the human gut, is characterized by its resistance to gastric acid and bile, and its strong colonization ability. Bifidobacterium breve can improve the intestinal environment and enhance intestinal motility by regulating the balance of intestinal flora, promoting mucus secretion from goblet cells in the intestinal mucosa, and improving gastrointestinal regulatory mediators, thus effectively relieving constipation. However, most commercially available bifidobacteria drugs currently use imported strains, which are costly and not entirely suitable for the "Chinese gut." Therefore, screening for "native" bifidobacteria suitable for the Chinese gut is of great significance. Summary of the Invention

[0005] The purpose of this invention is to provide a strain of Bifidobacterium breve that can relieve constipation, in order to solve the above-mentioned problems. The present invention provides a strain of Bifidobacterium breve that has the effect of relieving constipation, namely Bifidobacterium breve Bb-PG-14, which was deposited on September 4, 2024 at the China General Microbiological Culture Collection Center, with accession number CGMCCNo.31861, at No.3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing. Furthermore, the present invention also protects the use of this strain in the preparation of products for relieving constipation. Furthermore, its applications include: 1) Increase the water content of feces; 2) Shorten the time it takes to pass the first black stool; 3) Improve intestinal propulsion rate; 4) Improves colon tissue structure; 5) Regulates the increase of serum motilin; 6) Regulates the reduction of vasoactive intestinal peptide. The present invention has the following technical effects: 1. In existing technologies, most studies on probiotics relieving constipation use common commercial strains, which may have generalized effects but lack specificity. This invention screens out Bifidobacterium breve strains through in vitro experiments (such as the survival ability of strains in a simulated intestinal environment), and combines this with hemolysis tests to eliminate potential pathogenic risks, ensuring the safety of the strains. It proves that the strains have a higher survival rate in gastric acid and bile environments, and can more efficiently colonize the intestines and exert their effects. 2. This invention combines in vitro tests (such as acid resistance, bile salt resistance, and antibacterial tests) and in vivo tests (such as measuring the intestinal propulsion rate of mice and the levels of related hormones (such as motilin and vasoactive intestinal peptide) in serum) to clarify that Bifidobacterium breve exerts its effects through multiple pathways, with a clearer mechanism and more sufficient scientific evidence. In summary, this invention, through a systematic study of "in vitro screening-in vivo verification," has advantages in strain specificity, mechanism of action, safety, and application prospects. It has both practical application value and scientific theoretical significance, providing a new solution for the development of probiotics in the field of constipation intervention. Attached Figure Description

[0019] 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.

[0020] Figure 1 The colony morphology and Gram staining results of Bifidobacterium breve Bb-PG-14 plate;

[0021] Figure 2 The results of the acid resistance test for Bifidobacterium breve Bb-PG-14;

[0022] Figure 3 The results of the bile salt tolerance test for Bifidobacterium breve Bb-PG-14;

[0023] Figure 4 The results of the test on the ability of Bifidobacterium breve Bb-PG-14 to tolerate artificial simulated gastrointestinal fluid;

[0024] Figure 5 The results of the test on the inhibition of pathogenic bacteria by the supernatant of Bifidobacterium breve Bb-PG-14 fermentation broth;

[0025] Figure 6 The results are from the hemolytic activity test of Bifidobacterium breve Bb-PG-14.

[0026] Figure 7 The results of an experiment on fecal water content in mice to alleviate constipation with Bifidobacterium breve Bb-PG-14;

[0027] Figure 8 The results of an experiment on the time to first black stool in mice with constipation relieved by Bifidobacterium breve Bb-PG-14;

[0028] Figure 9 The results of an intestinal ink propulsion rate test for Bifidobacterium breve Bb-PG-14 relieving constipation in mice;

[0029] Figure 10 The results of intestinal histopathological experiments on Bifidobacterium breve Bb-PG-14 relieving constipation in mice;

[0030] Figure 11 Results of a study on the effects of Bifidobacterium breve Bb-PG-14 on constipation in mice;

[0031] Figure 12 The results of the vasoactive intestinal peptide assay for Bifidobacterium breve Bb-PG-14 in relieving constipation in mice. Detailed Implementation

[0032] 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.

[0033] 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.

[0034] Example 1: Screening and identification of Bifidobacterium breve Bb-PG-14

[0035] 1. Strains Isolation and Purification

[0036] Take 1.0 g of infant stool, dilute it with physiological saline, and then take 100 μL of the diluted solution at different dilution gradients of 10 μg / mL. -2 -10 -7 The fermentation broth was evenly spread onto MRS medium and incubated at 37°C for 48 hours. Single colonies of different morphologies and sizes were picked from the culture dishes and streaked multiple times on MRS agar medium for purification until a pure strain was confirmed.

[0037] 2. Microscopic examination

[0038] Single colonies were picked and inoculated into MRS broth medium. After culturing for 24 hours, Gram staining was performed. The bacterial culture was smeared, Gram-stained, and the cell morphology and arrangement were observed under a light microscope. The observations were recorded and photographed for preservation. Finally, Gram-positive cultures were identified as suspected lactic acid bacteria, and 1.0 mL of pure culture was transferred to a storage tube containing 50% glycerol for later use.

[0039] 3. Physiological and biochemical tests for identification

[0040] Physiological and biochemical tests, including hydrogen peroxide test, indole test, and carbohydrate utilization test, were used to identify and screen bacterial strains. The isolated and purified strain was milky white, with a raised center, a smooth and moist surface, and a diameter of 1.07 mm; the strain was Gram-positive, and the cells were short rod-shaped, occurring singly, in pairs, or arranged in a V-shape. Figure 1 Its physicochemical characteristics include a negative hydrogen peroxide test; a negative indole reaction; a positive aescin hydrolysis test; and the ability to ferment cellobiose, maltose, mannitol, salicin, sorbitol, sucrose, raffinose, inulin, and lactose.

[0041] 4. Molecular biological identification

[0042]

[0043] Example 2: Tolerance test of Bifidobacterium breve Bb-PG-14

[0044] 1. Results of acid resistance test

[0045] After two generations of anaerobic activation in MRS medium at 37℃, bacterial suspensions were prepared and resuspended in MRS medium with pH values ​​adjusted to 5.0, 4.0, 3.0, 2.5, 2.0, and 1.5 using 4.0 mol / L HCl. After anaerobic culture at 37℃ for 24 h, the bacterial suspensions were collected. The absorbance of the bacterial suspensions was measured at 600 nm with three replicates, using the absorbance of MRS medium (pH 6.0) at 600 nm as a control. The survival rate was calculated.

[0046] Survival rate = N t / N0

[0047] Where: N t The absorbance values ​​are for MRS media with pH values ​​of 1.5-5.0.

[0048] N0 represents the absorbance of the MRS medium at pH 6.0.

[0049] In healthy individuals, the pH of gastric juice on an empty stomach is between 0.9 and 1.8. After ingestion, the pH of gastric juice changes to approximately 1.8 to 5.0. Therefore, this experiment selected pH values ​​of 1.5, 2.0, 2.5, 3.0, 4.0, and 5.0 to test the acid tolerance of the bacterial strain. The results are as follows: Figure 2 As shown, by Figure 2 It can be seen that *Bifidobacterium breve* Bb-PG-14 exhibits poor tolerance at pH 1.5, with a survival rate of only 8.77% after 24 hours of treatment; however, it demonstrates strong tolerance at pH 4.0, with a survival rate of 53.92% after 24 hours of treatment. This indicates that the bacterium has a strong ability to tolerate acidic conditions.

[0050] 2. Results of bile salt tolerance test

[0051] After two generations of anaerobic activation, *Bifidobacterium breve* strain Bb-PG-14 was inoculated at a 3% inoculum into MRS medium containing 0.1%, 0.3%, 0.5%, 0.7%, and 0.9% bile salts (Bifidobacterium breve No. 3), respectively. After anaerobic incubation at 37°C for 24 h, the bacterial suspension was collected, and the absorbance of the bacterial suspension was measured at a wavelength of 600 nm. Three replicates were set up, with the absorbance of MRS medium without bile salts at a wavelength of 600 nm as a control. The survival rate of the strain was calculated.

[0052] Survival rate = N t / N0

[0053] Where: Nt The number of viable bacteria in MRS culture media containing different concentrations of bile salts;

[0054] N0 represents the number of viable bacteria in MRS medium without bile salts.

[0055] The concentration of bile salts in the small intestine of most humans is around 0.3% (w / v). Probiotics need to tolerate the bile salts in the intestine to exert their beneficial effects. In this study, *Bifidobacterium breve* strain Bb-PG-14 was treated with five different mass fractions of bile salts (0.1%, 0.3%, 0.5%, 0.7%, and 0.9%) for 24 hours (with the strain cultured without bile salts serving as a control). The results are as follows: Figure 3 As shown. From Figure 3 It can be seen that the survival rate of the strain gradually decreases with the increase of bile salt concentration. After 24 hours of treatment with 0.3% bile salt, the survival rate was still 64.22%, indicating that Bifidobacterium breve Bb-PG-14 has good bile salt tolerance.

[0056] 3. Results of simulated gastrointestinal fluid tolerance test

[0057] Bifidobacterium breve Bb-PG-14 strain was anaerobically activated and passaged twice in MRS medium at 37°C. A bacterial suspension was prepared, resuspended in simulated gastric fluid, and treated at 37°C for 3 hours. After centrifugation and discarding the supernatant, the bacterial sludge was collected and washed three times with sterile physiological saline. The sludge precipitate was aseptically collected and mixed with physiological saline at a ratio of 1:5. The mixture was then serially diluted before colony counting.

[0058] Take 1.0g of bacterial sludge after 3 hours of treatment with the gastric fluid, add it to simulated bile, and treat at 37℃ for 20 minutes. Centrifuge and discard the supernatant, collect the bacterial sludge, and wash it three times with sterile physiological saline. Collect the precipitate under aseptic conditions, add physiological saline at a ratio of 1:5 to bacterial sludge, mix thoroughly, perform serial dilution, and count the colonies.

[0059] Take 1.0g of bacterial sludge after treating the bile for 20 minutes, add it to simulated intestinal fluid, and treat at 37℃ for 4 hours. Centrifuge and discard the supernatant, collect the bacterial sludge, and wash it three times with sterile physiological saline. Collect the precipitate under aseptic conditions, add physiological saline at a ratio of bacterial sludge to physiological saline of 1:5, mix well, perform serial dilution, and count the colonies.

[0060] Survival rate was calculated using the number of colonies in MRS medium that had not undergone simulated gastrointestinal treatment as a control.

[0061] Survival rate of artificial gastrointestinal fluid and bile = N t / N0

[0062] Where: N tThe number of viable bacteria after artificially simulating gastric juice, bile, and intestinal juice treatment; N0 is the number of viable bacteria before treatment.

[0063] Gastric juice contains mucus, gastric acid, pepsin, etc. For probiotics to exert their beneficial effects, they must survive in the stomach, meaning they need to be able to tolerate the acidic environment and resist pepsin. Food typically stays in the stomach for 3 hours; therefore, this study used simulated gastric juice to treat Bifidobacterium breve Bb-PG-14 bacterial culture for 3 hours. After digestion in the stomach, food enters the intestines. The small intestine has a weakly alkaline environment and contains trypsin and bile. The viable count of probiotics in the intestines reaches 10-10. 6 Probiotics need a concentration of at least CFU / mL to exert their probiotic function; therefore, they must be able to tolerate the weakly alkaline environment of the intestine and resist trypsin and bile. Food typically stays in the small intestine for 3-8 hours. Therefore, this experiment used simulated intestinal fluid to treat Bifidobacterium breve Bb-PG-14 bacterial culture for 4 hours. The results are as follows... Figure 4 As shown. By Figure 4 It can be seen that after 3 hours of treatment with gastric juice, the survival rate of Bifidobacterium breve Bb-PG-14 was 69.11%, with a viable count of 12.44 ± 0.22 lg (cfu / g). After 20 minutes of treatment with bile, the survival rate was 58.22%, with a viable count of 10.48 ± 0.29 lg (cfu / g). After 4 hours of treatment with intestinal juice, the survival rate was still 55.20%, with a viable count of 9.94 ± 0.22 lg (cfu / g), indicating that Bifidobacterium breve Bb-PG-14 has a strong ability to tolerate simulated gastric and intestinal juices and bile.

[0064] Example 4: Antibacterial activity test of Bifidobacterium breve Bb-PG-14

[0065] Antibacterial test results

[0066] Indicator bacteria *Escherichia coli*, *Listeria monocytogenes*, *Staphylococcus aureus*, *Bacillus subtilis*, *Pseudomonas aeruginosa*, and *Salmonella typhimurium* were activated separately and inoculated into LB liquid medium, incubated at 37°C for 24 h, and the resulting bacterial suspensions were prepared for later use. *Bifidobacterium breve* Bb-PG-14 was activated and inoculated into liquid MRS medium, anaerobically incubated at 37°C for 24 h, centrifuged at 8000 rpm for 5 min, and the supernatant was collected. Using the Oxford cup agar diffusion method, *Escherichia coli*, *Listeria monocytogenes*, *Staphylococcus aureus*, *Bacillus subtilis*, *Pseudomonas aeruginosa*, and *Salmonella typhimurium* were used for plate spreading, with sterile physiological saline as a blank control. Four Oxford cups were evenly placed in each culture dish; 200 μL of fermentation supernatant was added to three Oxford cups, and 200 μL of sterile physiological saline was added to one Oxford cup. The plates were anaerobically incubated at 37°C for 24 h. Observe whether there is an inhibition zone around the bottom of the Oxford cup, measure the diameter of the inhibition zone, and take the average value.

[0067] From Table 1 and Figure 5 As can be seen, inhibition zones appeared in all the petri dishes, and the diameter of the inhibition zones was all above 16.0 mm. This indicates that Bifidobacterium breve Bb-PG-14 has an inhibitory effect on the growth of Escherichia coli, Listeria monocytogenes, Staphylococcus aureus, Bacillus subtilis, Pseudomonas aeruginosa, and Salmonella typhimurium. The diameters of the inhibition zones were 20.87±0.09 mm, 18.53±0.04 mm, 21.93±0.09 mm, 17.73±0.03 mm, 16.40±0.05 mm, and 22.47±0.06 mm, respectively, showing that Bifidobacterium breve Bb-PG-14 has a significant antibacterial effect and strong antibacterial ability.

[0068] Table 1. Test data on the antibacterial activity of Bifidobacterium breve Bb-PG-14

[0069] indicator bacteria Diameter of the inhibition zone (mm) E. coli 20.87±0.09 Listeria monocytogenes 18.53±0.04 Staphylococcus aureus 21.93±0.09 Bacillus subtilis 17.73±0.03 Pseudomonas aeruginosa 16.40±0.05 Salmonella typhimurium 22.47±0.06

[0070] Note: The diameter of the Oxford cup is 7.833±0.172mm.

[0071] Example 5: Hemolysis test of Bifidobacterium breve Bb-PG-14 strain

[0072] Hemolysis test results

[0073] Bifidobacterium breve Bb-PG-14 bacterial suspension was inoculated onto Columbia blood agar medium and incubated in an anaerobic incubator at 37°C for 24 hours. Hemolysis was then observed around the colonies. The appearance of a translucent hemolytic zone on the plate indicated α-hemolysis, while a clearly defined, completely transparent hemolytic zone indicated β-hemolysis. The absence of a hemolytic zone indicated γ-hemolysis, i.e., no hemolysis.

[0074] The test results for Bifidobacterium breve Bb-PG-14 are as follows: Figure 6 As shown, with Staphylococcus aureus as the control strain (left image), Bifidobacterium breve Bb-PG-14 (right image) is non-hemolytic and is a safe strain.

[0075] Example 6: The relieving effect of Bifidobacterium breve Bb-PG-14 on constipation in mice.

[0076] The method for evaluating the effects of Bifidobacterium breve Bb-PG-14 on fecal water content, time to first black stool, intestinal propulsion rate, histopathological analysis, and serum levels of peptides affecting gastrointestinal regulation in mice with loperamide hydrochloride-induced constipation includes the following steps:

[0077] 1. Animal handling and experimental grouping

[0078] Forty mice (BALB / c mice, 6-8 weeks old) were acclimatized for 7 days and then randomly divided into four groups: blank group (NC), model group (MC), positive control group (PC), and Bifidobacterium breve Bb-PG-14 intervention test group (BLD), with 10 mice in each group.

[0079] A constipation model was established by continuous gavage for 14 days, and the specific treatment with drugs and experimental bacterial strains is as follows:

[0080] The blank control group was administered 0.2 mL of normal saline by gavage; the model group was administered 0.2 mL (1 mg / mL) of loperamide hydrochloride (based on a modeling standard of 10 mg / kg b.w) by gavage + 0.2 mL of normal saline; the positive control group was administered 0.2 mL of loperamide hydrochloride + 0.2 mL of 1.0 × 10⁻⁶ mcg of 1000 mg / kg b.w by gavage. 9 CFU / mL Bifidobacterium triple live bacteria capsules (Shanghai Shangyao Xinyi Pharmaceutical Co., Ltd.); the intervention group was administered 0.2 mL loperamide hydrochloride + 0.2 mL 1.0 × 10⁻⁶ CFU / mL via gavage. 9 cfu / mL Bifidobacterium breve Bb-PG-14 bacterial suspension.

[0081] The experimental mice were housed at the SPF-grade experimental animal center of Harbin University of Commerce. Environmental control parameters were strictly set at a temperature of 20±2℃ and a relative humidity of 50±5%, and an automatic light control system was installed to achieve a 12-hour day / night cycle. Strict animal welfare standards were followed during the experiment, with cage cleaning and bedding changes performed twice weekly. All animal experimental procedures involved in this study strictly followed the relevant requirements of the Experimental Animal Ethics Committee of Heilongjiang University (Experimental Animal Ethics Review Acceptance Number: 20220523006). After a 2-week intervention, the mice were fasted for 12 hours. All mice were then administered activated charcoal ink by gavage for 30 minutes, anesthetized, and blood samples were collected by enucleation for subsequent experimental measurements.

[0082] 2. Determination of fecal moisture content

[0083] Fecal moisture content results

[0084] Accurately weigh the mass of fresh mouse feces (m1), place it in an oven at 105℃ and dry it to a constant mass (m2), and calculate the water content of the feces according to the formula.

[0085] The formula is as follows: Fecal water content % = (m1g − m2(g) / m1(g)) × 100

[0086] Depend on Figure 7It was found that the water content of feces in the model group mice was significantly lower than that in the control group (P < 0.05), indicating that the modeling with loperamide hydrochloride was successful. The influence of bacteria showed that both the positive control group (Bifidobacterium triple live bacteria) and the intervention group (Bifidobacterium breve Bb-PG-14) effectively increased the water content of feces, achieving a laxative effect. This indicates that a single Bifidobacterium breve Bb-PG-14 can achieve the same level of fecal water content regulation as commercially available Bifidobacterium triple live bacteria capsules.

[0087] 3. Time of passing the first black stool

[0088] Results of the first black stool expulsion time

[0089] On the day before the end of the experiment, the blank control group was administered 0.2 mL of physiological saline by gavage, while the model group, positive control group, and intervention group were administered 0.2 mL of loperamide hydrochloride by gavage to induce the model. One hour later, the blank control group and model group were administered 0.2 mL of physiological saline and activated charcoal ink by gavage, the positive control group was administered 0.2 mL of Bifidobacterium triple live bacteria capsules and activated charcoal ink by gavage, and the intervention group was administered 0.2 mL of Bifidobacterium breve Bb-PG-14 bacterial solution and activated charcoal ink by gavage. Each mouse was placed individually in a clean cage lined with filter paper, and the start time of administration of activated charcoal ink was recorded. The time after the mouse excreted its first black stool was recorded as the end time, and the interval between the two times was the time of the first black stool.

[0090] The time it takes for a mouse to pass its first black stool can reflect the transit time of chyme throughout the intestine. Figure 8 It was found that there was a significant difference in the time to first black stool between the blank group and the model group (P < 0.05), indicating that the loperamide hydrochloride model was successfully established. Both the positive control group (Bifidobacterium triple live bacteria) and the intervention group (Bifidobacterium breve Bb-PG-14) significantly shortened the time to first black stool in mice. This indicates that single-strain Bifidobacterium breve Bb-PG-14 can achieve the same time to first black stool as commercially available Bifidobacterium triple live bacteria capsules.

[0091] 4. Intestinal propulsion rate measurement

[0092] Intestinal propulsion rate results

[0093] On the last day of the animal experiment, after fasting for 12 hours but not water, mice in the blank group, model group, positive control group, and intervention group were administered 0.2 mL of activated charcoal ink by gavage. After 30 minutes, the mice were euthanized, and the intestinal segments from the pylorus to the ileocecal region were dissected and placed on a white board. Sterile water was sprayed on the segments to prevent small intestinal adhesion. The small intestine was gently pulled into a straight line, and the total length of the intestinal segment (L1) was measured. The length from the pylorus to the leading edge of the ink was measured as the ink propulsion length (L2). The intestinal propulsion rate was calculated according to the formula.

[0094] The formula is as follows: Intestinal propulsion rate % = (L2 (cm) L1 (cm)) × 100

[0095] Intestinal propulsion rate directly reflects the motility of the small intestine. Loperamide hydrochloride acts on intestinal smooth muscle, inhibiting intestinal smooth muscle contraction and peristalsis, thereby prolonging the residence time of food in the small intestine. Figure 9 The intestinal propulsion rate in the model group was significantly different from that in the control group (P < 0.05), indicating that the modeling with loperamide hydrochloride was successful. Both the positive control group (Bifidobacterium triple live bacteria) and the intervention group (Bifidobacterium breve Bb-PG-14) effectively alleviated the slowed small intestinal motility caused by loperamide hydrochloride. This demonstrates that a single Bifidobacterium breve Bb-PG-14 can achieve the same intestinal propulsion rate in constipated mice as commercially available Bifidobacterium triple live bacteria capsules.

[0096] 5. Intestinal histopathological examination

[0097] Intestinal histopathological results

[0098] All surviving mice were euthanized, and their colons were harvested, fixed overnight in 4% paraformaldehyde solution, and then dehydrated in a gradient manner, embedded in paraffin, and prepared into 5μm serial sections. Hematoxylin-eosin (H&E) staining was used for histopathological examination of the intestinal tissue under a microscope. The pathological examination of these sections was commissioned to Wuhan Sewell Biotechnology Co., Ltd.

[0099] like Figure 10 In the control group, the colonic mucosal epithelial cells were intact, without atrophy, erosion, or necrosis; goblet cells were abundant and regularly arranged; and the colonic tissue layer was relatively thick. Compared to the control group, the model group showed incomplete and irregularly arranged goblet cells, focal aggregation of inflammatory cells with intestinal wall thickening, and a few cell ruptures. Compared to the model group, the positive control group (Bifidobacterium triple live bacteria) and the intervention group (Bifidobacterium breve Bb-PG-14) showed an increased number of goblet cells, more regular arrangement, improved villus morphology, and a thickened colonic tissue layer, similar to the colonic characteristics of the control group. This indicates that Bifidobacterium breve Bb-PG-14 alone can achieve the remission level of commercially available Bifidobacterium triple live bacteria capsules.

[0100] 6. Measurement of serum gastrointestinal regulatory peptide levels in mice

[0101] Results of serum gastrointestinal regulatory peptide levels in mice

[0102] After anesthetizing mice, blood was collected by enucleation. Serum was collected by centrifugation at 3500 rpm and 4℃ for 10 min. The levels of motilin (MTL) and vasoactive intestinal peptide (VIP) in mouse serum were measured using an ELISA kit. Each sample was measured three times, and the average value was taken.

[0103] Motilin (MTL) is an excitatory neurotransmitter in the gastrointestinal tract that regulates gastrointestinal motility and stimulates intestinal peristalsis. MTL can promote segmental movement of the small intestine, thereby shortening the transit time of chyme in the small intestine. Figure 11 It was found that the serum MTL levels in constipated mice modeled by loperamide hydrochloride gavage were significantly lower than those in the control group (P<0.05). Both the positive control group (Bifidobacterium triple live bacteria) and the intervention group (Bifidobacterium breve Bb-PG-14) significantly increased the serum MTL concentration in mice (P<0.05). This indicates that a single Bifidobacterium breve Bb-PG-14 can achieve the same MTL-regulating effect as commercially available Bifidobacterium triple live bacteria capsules.

[0104] Vasoactive intestinal peptide (VIP) is an inhibitory neurotransmitter in the gastrointestinal tract that regulates gastrointestinal motility and can relax the gastrointestinal tract. Figure 12 It was found that the serum VIP concentration in constipated mice induced by loperamide hydrochloride gavage was significantly higher than that in the control group (P<0.05). Both the positive control group (Bifidobacterium triple live bacteria) and the intervention group (Bifidobacterium breve Bb-PG-14) significantly reduced the serum VIP concentration in mice (P<0.05). This indicates that a single Bifidobacterium breve Bb-PG-14 can achieve the same level of VIP regulation as commercially available Bifidobacterium triple live bacteria capsules.

[0105] In conclusion, the results show that Bifidobacterium breve Bb-PG-14 has a good ability to relieve constipation in mice, providing a new solution for the development of probiotics in the field of constipation intervention.

[0106] 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 breve having a constipation-relieving effect, characterized in that, Bifidobacterium breve Bb-PG-14, which was preserved in China General Microbiological Culture Collection Center on September 4, 2024, with a preservation number of CGMCC No. 31861 and a preservation address of No. 3, Xibahe West Road, Yard 1, Chaoyang District, Beijing.

2. The use of the Bifidobacterium breve strain with the effect of relieving constipation in claim 1 in the preparation of a product for relieving constipation.

3. Use according to claim 2, wherein the compound is ###0002### The effects include: 1) increasing the water content of feces; 2) shortening the time of the first black stool; 3) improving the intestinal propulsion rate; 4) improving the structure of the colon tissue; 5) regulating the increase of serum motilin; 6) regulating the decrease of vasoactive intestinal peptide.

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