Application of bifidobacterium breve and exopolysaccharide produced by the same in prevention and treatment of hyperlipidemia acute pancreatitis

Abstract

The application discloses Bifidobacterium breve and application of exopolysaccharide produced by the Bifidobacterium breve in prevention and treatment of hyperlipidemia acute pancreatitis, and belongs to the technical field of biological medicine. The application screens a Bifidobacterium breve with high yield of glucuronan exopolysaccharide, and the strain or the exopolysaccharide produced by the strain can help to reduce hyperlipidemia acute pancreatitis and reduce pancreatic lipid metabolism and inflammatory injury caused by HTG-AP.

Description

Technical Field

[0001] This invention relates to the application of Bifidobacterium breve and its produced extracellular polysaccharides in the prevention and treatment of acute pancreatitis with hyperlipidemia, and belongs to the field of biomedical technology. Background Technology

[0002] Acute pancreatitis (AP) is characterized by an acute inflammatory process of the pancreas, primarily manifested by severe abdominal pain and elevated levels of pancreatic lipases. With the continued rise in the global prevalence of hyperlipidemia and changes in dietary patterns, the incidence of hyperlipidemic acute pancreatitis (HLAP) is increasing year by year. In China, hypertriglyceridemia (HTG) accounts for 10% to 20% of the causes of acute pancreatitis. Patients with HLAP have significantly elevated blood triglyceride levels, which, after being hydrolyzed by pancreatic enzymes, release large amounts of free fatty acids (FFA), which can directly damage pancreatic acinar cells and disrupt cell membrane structure.

[0003] Bifidobacterium ( Bifidobacterium breve Extracellular polysaccharides (EPS) are large glycomolecules secreted by bacteria outside the cell wall or attached to the cell wall surface. Increasing evidence suggests that Bifidobacterial EPS play a multifaceted role in maintaining intestinal homeostasis. They can act as prebiotics, selectively promoting the growth of beneficial bacteria; as immunomodulators, inducing the host immune response towards an anti-inflammatory phenotype; and as physical barriers, protecting the producing bacteria and host epithelial cells from environmental stresses and pathogen invasion. Notably, the anti-inflammatory properties of certain Bifidobacterial EPS have attracted widespread research interest, particularly in inflammatory bowel disease (IBD), metabolic syndrome, and other inflammation-related diseases.

[0004] Existing technologies disclose various uses of Bifidobacterium extracellular polysaccharides, such as CN202211584760.2 Bifidobacterium longum YS108R extracellular polysaccharide for alleviating ulcerative colitis, CN 117487693 for alleviating colitis, and CN115261426B Bifidobacterium bifidum E3 for antioxidant effects. Although existing technologies disclose that the EPS produced by Bifidobacterium can alleviate colitis in mice, enhance the survival rate and colonization ability of the strain in the intestine, and strengthen the intestinal barrier, the above uses are significantly different from the treatment mechanism of pancreatitis and cannot meet the clinical treatment needs of pancreatitis. Summary of the Invention

[0005] [Technical Issues] To address the aforementioned technical problems, the purpose of this invention is to provide a novel application of extracellular polysaccharides derived from Bifidobacterium breve in the prevention and treatment of acute pancreatitis with hyperlipidemia.

[0006] [Technical Solution] To achieve the above objectives, the present invention provides a strain of Bifidobacterium breve (Bifidobacterium breve). Bifidobacterium breve MYQQ-7 was deposited at the Guangdong Provincial Center for Microbial Culture Collection on March 4, 2026, with accession number GDMCC No: 67879; this strain can produce high yields of extracellular polysaccharides with glucuronic acid as the main monosaccharide component.

[0007] In one embodiment, the Bifidobacterium breve MYQQ-7 is isolated from human breast milk.

[0008] In one embodiment, the growth characteristics of the Bifidobacterium MYQQ-7 are as follows: the strain is a strict anaerobic bacterium, inoculated into a culture medium, and cultured at a temperature of 37°C for 48 hours.

[0009] In one embodiment, the colony characteristics of the Bifidobacterium MYQQ-7 are as follows: it is milky white, round and raised, with neat and smooth edges on MRS solid medium.

[0010] The present invention also provides an extracellular polysaccharide prepared using the aforementioned Bifidobacterium MYQQ-7.

[0011] In one embodiment, the method for preparing the extracellular polysaccharide includes: culturing the Bifidobacterium breve MYQQ-7 and collecting the extracellular polysaccharide from the fermentation broth.

[0012] In one embodiment, the culture medium used for culturing includes, but is not limited to, MRS medium.

[0013] In one embodiment, the method involves inoculating the Bifidobacterium breve into MRS medium, performing anaerobic fermentation, heat inactivating it, and then collecting the supernatant.

[0014] In one embodiment, the heat inactivation condition is boiling for 10-15 minutes.

[0015] In one embodiment, the inoculation amount of the Bifidobacterium breve MYQQ-7 is 2%-8% (v / v), preferably 5% (v / v).

[0016] In one embodiment, the heat-inactivated fermentation broth is centrifuged at 9000 × g for 20-30 min.

[0017] In one embodiment, the method further includes separating extracellular polysaccharides from the supernatant, specifically comprising the following steps: (1) Centrifuge to remove proteins from the supernatant; (2) Precipitation of polysaccharides in the supernatant obtained in step (1); (3) The polysaccharide solution obtained from dialysis step (2); (4) Freeze-dry the polysaccharide solution obtained in step (3) to obtain freeze-dried extracellular polysaccharide.

[0018] In one embodiment, step (1) uses trichloroacetic acid to remove proteins.

[0019] In one embodiment, anhydrous ethanol is used to precipitate polysaccharides in step (2).

[0020] In one embodiment, step (3) involves dialysis using a dialysis bag with a capacity of 8000-12000 Da.

[0021] In one embodiment, step (4) involves freeze-drying at 0-4 °C for at least 48 h.

[0022] The present invention also provides the use of the aforementioned Bifidobacterium MYQQ-7 and / or the aforementioned extracellular polysaccharide in the preparation of a drug for the prevention and treatment of acute pancreatitis with hyperlipidemia.

[0023] In one embodiment, the monosaccharide composition of the extracellular polysaccharide of the *Bifidobacterium breve* is: rhamnose, arabinose, glucosamine, galactose, glucose, mannose, and glucuronic acid. The molar ratio is 1:0.03:1.59:0.19:1.17:0.55:4.40.

[0024] The present invention also provides compositions containing the aforementioned Bifidobacterium MYQQ-7 and / or the aforementioned extracellular polysaccharide.

[0025] In one embodiment, the composition is a drug.

[0026] In one embodiment, the drug further comprises a pharmaceutically acceptable carrier and / or excipients.

[0027] In one embodiment, the prevention and / or treatment of hyperlipidemic acute pancreatitis includes at least one of the following (1) to (2): (1) Relieves pancreatic tissue damage in acute pancreatitis caused by hyperlipidemia; (2) Reduce the serum biochemical levels of patients with hyperlipidemia and acute pancreatitis; the serum biochemical levels include at least one of cholesterol, triglycerides, amylase or lipase.

[0028] Beneficial effects: (1) This invention isolates a new strain of Bifidobacterium breve from fresh breast milk. Bifidobacterium breve The strain, named MYQQ-7, has a clear origin and can produce functional extracellular polysaccharides. These extracellular polysaccharides are mainly composed of glucuronic acid and contain monosaccharides such as rhamnose, arabinose, glucosamine, galactose, glucose, and mannose.

[0029] (2) The present invention also provides the application of extracellular polysaccharides in the treatment of pancreatitis, especially hyperlipidemic acute pancreatitis. The extracellular polysaccharides as a whole have significant effects on improving pancreatitis-related indicators and are significantly better than single glucuronic acid components, indicating that its multiple monosaccharide components have played a synergistic role and achieved unexpected technical effects.

[0030] The live and heat-inactivated Bifidobacterium MYQQ-7 preparations provided by this invention can significantly improve pancreatic tissue edema and inflammatory damage in mice with acute pancreatitis. They can be used as active ingredients in drugs for the prevention and treatment of acute pancreatitis and have broad industrialization prospects.

[0031] Preservation of biological materials Bifidobacterium breve ( Bifidobacterium breve MYQQ-7, categorized as Bifidobacterium breve It was deposited on March 4, 2026 at the Guangdong Provincial Center for Microbial Culture Collection, with accession number GDMCC No: 67879, located at 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou. Attached Figure Description

[0032] Figure 1 This image shows stained sections of pancreatic tissue cells from mice treated with Bifidobacterium and antibiotics.

[0033] Figure 2 The levels of serum cholesterol, triglycerides, amylase, and lipase in mice treated with Bifidobacterium and antibiotics were measured.

[0034] Figure 3 Results of high-pressure ion chromatography analysis of EPS monosaccharide composition in Bifidobacterium breve.

[0035] Figure 4 This is a stained image of pancreatic tissue cell sections from mice treated with extracellular polysaccharides and glucuronic acid.

[0036] Figure 5 The levels of serum cholesterol, triglycerides, amylase, and lipase in mice treated with extracellular polysaccharides and glucuronic acid were measured. In the above figures, * represents P < 0.05, ** represents P < 0.01, *** represents P < 0.001, and ns represents P > 0.05. Detailed Implementation

[0037] MRS medium: peptone 10.0 g / L, beef extract 8.0 g / L, yeast extract 4.0 g / L, glucose 20.0 g / L, dipotassium hydrogen phosphate 2.0 g / L, diammonium hydrogen citrate 2.0 g / L, sodium acetate 5.0 g / L, magnesium sulfate 0.2 g / L, manganese sulfate 0.04 g / L, Tween 80 1.0 mL / L.

[0038] Example 1: Screening of Bifidobacterium breve MYQQ-7 In this embodiment, *Bifidobacterium breve* was derived from fresh breast milk. Specifically, fresh breast milk was cultured in MRS broth supplemented with 0.05% L-cysteine ​​hydrochloride. The bacterial culture was then plated onto MRS solid agar plates supplemented with 1% mupirocin (final concentration 100 μg / mL) and 0.5% nystatin (final concentration 25 U / mL). After PCR identification, the bacteria were purified and isolated. The isolated *Bifidobacterium breve* was named MYQQ-7 and cultured in an anaerobic environment at 37°C, subculturing every two days.

[0039] Example 2 Preparation of Bifidobacterium breve inoculum (1) Live bacterial preparation: The *Bifidobacterium breve* MYQQ-7 selected in Example 1 was inoculated into MRS liquid medium at an inoculum of 5% (v / v) and cultured anaerobically at 37°C for two days. After the second generation, the bacterial cells were collected by centrifugation and resuspended in PBS to obtain a bacterial concentration of 1×10⁻⁶. 9 Live bacteria preparation at CFU / mL. (2) Preparation of sterilizing agent: The *Bifidobacterium breve* MYQQ-7 strain screened in Example 1 was inoculated into MRS liquid culture medium and cultured anaerobically at 37°C for 48 h. Then, 5% (v / v) of the inoculum was transferred to a fresh MRS liquid culture medium and cultured under the same conditions for 48 h. After the culture was completed, the culture was sterilized at 4°C with 2500 × 10⁻⁶ ppm. g Centrifuge for 5 min, then resuspend in PBS to a final concentration of 1×10⁻⁶. 9 The concentration of CFU / mL was then inactivated by heat at 95°C for 10-15 min, followed by centrifugation and resuspending to obtain the heat-inactivated bacterial preparation.

[0040] Example 3: Pharmacodynamic Study of Heat-Inactivated Bacteria in the HTG-AP Process The animal experiment procedure is as follows: 6-8 week old SPF-grade C57BL / 6J male mice were divided into 5 groups of 8 mice each, and the following treatments were performed: Control group: 200 μL sterile PBS was administered intragastrically daily, 200 μL sterile PBS was administered intraperitoneally from day 1 to day 28, and 200 μL sterile PBS was administered intraperitoneally 8 times on day 29.

[0041] Model group: 200 μL of sterile PBS was administered intragastric daily, and 200 μL of poloxamer (50 mg / mL) was administered intraperitoneally from day 1 to day 28, with continuous administration from day 1 to day 7 and administration every other day from day 8 to day 28. On day 29, 200 μL of thymol (5 μg / mL) was administered intraperitoneally eight times every hour.

[0042] Antibiotic control group: 200 μL metronidazole solution (10 mg / mL) was administered intragastric daily, followed by 200 μL poloxamer intraperitoneally from day 1 to 28, with continuous administration from day 1 to 7 and every other day from day 8 to 28. On day 29, 200 μL of hygroscin (concentration 5 μg / mL) was administered intraperitoneally eight times every hour.

[0043] Live bacteria preparation group: Example 2 prepared a live bacteria preparation, and administered 200 μL (1×10⁻⁶) orally daily. 9 Administer 200 μL poloxamer (50 mg / mL) intraperitoneally from day 1 to 28, with continuous administration from day 1 to 7 and every other day from day 8 to 28. On day 29, administer 200 μL of crotonin (5 μg / mL) intraperitoneally eight times every hour.

[0044] Heat-inactivated bacterial preparation group: Example 2 prepared a heat-inactivated bacterial preparation, and administered 200 μL (1×10⁻⁶) orally daily. 9 Administer 200 μL poloxamer (50 mg / mL) intraperitoneally from day 1 to 28, with continuous administration from day 1 to 7 and every other day from day 8 to 28. On day 29, administer 200 μL of crotonin (5 μg / mL) intraperitoneally eight times every hour.

[0045] Pancreatic tissue was collected from mice in each group, dehydrated, embedded, sectioned, stained, and the condition of mouse tissue cells was observed. Compared with the model group and the antibiotic control group, heat-inactivated Bifidobacterium breve significantly improved pancreatic tissue edema and inflammatory damage (e.g., ...). Figure 1 (As shown).

[0046] Fresh blood was collected from mice in each group and centrifuged at 3000 rpm for 20 minutes at room temperature. The supernatant was collected, and serum cholesterol, triglyceride, and lipase levels were measured using a biochemical analyzer. Compared with the model group, the live bacteria group did not improve serum biochemical indicators, and the antibiotic group did not improve serum amylase, triglyceride, and cholesterol levels; these differences were not statistically significant. Compared to the model group, *Bifidobacterium shortis* heat-inactivated bacteria significantly improved serum cholesterol levels (from 19.84±2.165 mmol / L to 13.22±4.4 mmol / L), serum amylase levels (from 5920±604.6 U / L to 4949±572.4 U / L), serum lipase levels (from 433.1±84.09 U / L to 202.4±42.78 U / L), and serum triglyceride levels (from 19.68±0.5506 mmol / L to 18.19±0.7051 mmol / L), all of which were statistically significant (e.g., ...). Figure 2 (as shown in the figure), and the effect is better than that of the antibiotic group to some extent.

[0047] Example 4: Extracellular polysaccharide monosaccharide composition of Bifidobacterium breve MYQQ-7 The MYQQ-7 strain screened in Example 1 was anaerobically cultured in MRS medium at 37 °C for 48 h. Fermentation was stopped when OD600 = 1 to obtain the MYQQ-7 fermentation broth. The obtained fermentation broth was centrifuged (9000 × 10⁻⁶) after being in a 100 °C water bath for 10 min. g The supernatant was treated with 80% trichloroacetic acid to a final concentration of 4% overnight at 4°C for 20 min (4°C) to precipitate excess protein. Subsequently, the mixed supernatant was subjected to a 9000 × 10⁻⁶ rpm high-temperature treatment. g Centrifuge for 20 min to remove excess protein. Add triploid volume of anhydrous ethanol to the centrifuged supernatant to precipitate the polysaccharide. Dissolve the precipitated polysaccharide in distilled water. Subsequently, dialyze the solution in a dialysis bag with double-distilled water for 72 hours (molecular weight cutoff 8000-12000 Da), changing the water every 8 hours. Finally, freeze-dry to obtain the extracellular polysaccharide of Bifidobacterium breve MYQQ-7.

[0048] High-pressure ion chromatography was used to analyze the extracellular polysaccharides of MYQQ-7, such as... Figure 3 The components represent rhamnose, arabinose, glucosamine, galactose, glucose, mannose, and glucuronic acid, respectively. As shown in Table 1, their molar ratios are 1:0.03:1.59:0.19:1.17:0.55:4.40. These results indicate that the extracellular polysaccharide of *Bifidobacterium breve* contains a high molar amount of glucuronic acid.

[0049] Table 1. Molar ratio of monosaccharide components in extracellular polysaccharides

[0050] Example 5: Effect of Bifidobacterium breve MYQQ-7 extracellular polysaccharide on acute pancreatitis with hyperlipidemia. Six- to eight-week-old SPF-grade male C57BL / 6J mice were divided into four groups of eight mice each: control group, model group, extracellular polysaccharide group, and glucuronic acid group.

[0051] Control group: 200 μL sterile PBS was administered intragastrically daily, 200 μL sterile PBS was administered intraperitoneally from day 1 to day 28, and 200 μL sterile PBS was administered intraperitoneally 8 times on day 29.

[0052] Model group: 200 μL of sterile PBS was administered intragastric daily, and 200 μL of poloxamer (50 mg / mL) was administered intraperitoneally from day 1 to day 28, with continuous administration from day 1 to day 7 and administration every other day from day 8 to day 28. On day 29, 200 μL of thymol (5 μg / mL) was administered intraperitoneally eight times every hour.

[0053] Extracellular polysaccharide group: Extracellular polysaccharides were prepared according to the method in Example 4; 200 μL of extracellular polysaccharide solution (concentration 200 μg / mL) was administered intragastric daily, and 200 μL of poloxamer (concentration 50 mg / mL) was administered intraperitoneally from day 1 to day 28, with continuous administration from day 1 to day 7 and administration every other day from day 8 to day 28. On day 29, 200 μL of crotonin (concentration 5 μg / mL) was administered intraperitoneally eight times every hour.

[0054] Glucuronic acid group: 200 μL of glucuronic acid solution (concentration 200 μg / mL) was administered intragastric daily. From days 1 to 28, 200 μL of poloxamer (concentration 50 mg / mL) was administered intraperitoneally, with continuous administration from days 1 to 7 and every other day from days 8 to 28. On day 29, 200 μL of thymol (concentration 5 μg / mL) was administered intraperitoneally eight times every hour.

[0055] After the experiment, pancreatic tissue was collected from mice in each group, dehydrated, embedded, sectioned, stained, and the condition of mouse tissue cells was observed. Compared with the model group and the glucuronic acid group, heat-inactivated Bifidobacterium breve significantly improved pancreatic tissue edema and inflammatory damage (e.g., Figure 4 (As shown).

[0056] Fresh blood was collected from mice in each group and centrifuged at 3000 rpm for 20 minutes at room temperature. The supernatant was collected, and serum cholesterol, triglyceride, lipase, and amylase levels were measured using a biochemical analyzer. Compared with the model group, the extracellular polysaccharide with glucuronic acid as the main monosaccharide significantly improved serum cholesterol, decreasing from 24.56±1.321 mmol / L in the model group to 15.55±1.642 mmol / L; triglyceride level decreased from 24.29±0.0498 mmol / L to 23.58±0.6372 mmol / L; serum amylase level decreased from 6465±252.8 U / L to 5294±550.2 U / L; and serum cholesterol level decreased from 328.8±145.3 U / L to 134.2±27.75 U / L, all of which were statistically significant.

[0057] Although the glucuronic acid group improved serum lipase (167.4±40.73 U / L) and serum cholesterol (18.63±3.72 mmol / L), the effect was not as good as that of the extracellular polysaccharide group. Furthermore, the glucuronic acid group showed poor improvement in serum amylase (5953±871.1 U / L) and serum triglycerides (23.69±0.3362 mmol / L), and could not effectively improve the inflammatory infiltration state of pancreatic tissue (e.g., Figure 5 (As shown).

[0058] The above data demonstrate that there is a synergistic promoting effect among the various monosaccharide components, mainly glucuronic acid, in extracellular polysaccharides, which may be an important mechanism for their therapeutic effects. Comparative example: The specific implementation method is the same as in Example 3, using the commonly used nonsteroidal anti-inflammatory drug metronidazole (at an effective dose of 10 mg / mL) as a positive control. The serum biochemical indicators in the metronidazole group were: lipase 98.14±25.43 U / L, triglycerides 22.91±0.3955 mmol / L, total cholesterol 18.02±0.8375 mmol / L, and amylase 5790±698.5 U / L. Compared with the extracellular polysaccharide group, the extracellular polysaccharide showed better improvement in serum biochemical indicators, except for serum lipase. Furthermore, heat-inactivated Bifidobacterium breve improved pancreatic tissue edema and inflammatory invasion compared to metronidazole.

[0059] Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Anyone skilled in the art can make various modifications and alterations without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be determined by the claims.

Claims

1. Bifidobacterium breve ( Bifidobacterium breve MYQQ-7 was deposited at the Guangdong Provincial Center for Microbial Culture Collection on March 4, 2026, with accession number GDMCC No: 67879.

2. The extracellular polysaccharide prepared using the Bifidobacterium MYQQ-7 as described in claim 1.

3. The extracellular polysaccharide according to claim 2, characterized in that, The monosaccharide composition of the extracellular polysaccharide includes: rhamnose, arabinose, glucosamine, galactose, glucose, mannose, and glucuronic acid.

4. A method for preparing the extracellular polysaccharide according to claim 2 or 3: characterized in that, Culture the Bifidobacterium shortis MYQQ-7 as described in claim 1 and collect the extracellular polysaccharides from the fermentation broth.

5. The method according to claim 4, characterized in that, It also includes the separation of extracellular polysaccharides from the supernatant, with specific steps including: (1) Centrifuge to remove proteins from the supernatant; (2) Precipitation of polysaccharides in the supernatant obtained in step (1); (3) The polysaccharide solution obtained from dialysis step (2); (4) Freeze-dry the polysaccharide solution obtained in step (3) to obtain freeze-dried extracellular polysaccharide.

6. A composition containing the Bifidobacterium MYQQ-7 of claim 1 and / or the extracellular polysaccharide of any one of claims 2 to 3.

7. The composition according to claim 6, characterized in that, The composition is a drug.

8. The composition according to claim 7, characterized in that, The drug also contains pharmaceutically acceptable carriers and / or excipients.

9. The use of the Bifidobacterium MYQQ-7 of claim 1 and / or the extracellular polysaccharide of any one of claims 2 to 3 in the preparation of a drug for the prevention and treatment of acute pancreatitis with hyperlipidemia.

10. The application according to claim 9, characterized in that, The prevention and / or treatment of hyperlipidemic acute pancreatitis includes at least one of the following (1) to (2): (1) Relieves pancreatic tissue damage in acute pancreatitis caused by hyperlipidemia; (2) Reduce the serum biochemical levels of patients with hyperlipidemia and acute pancreatitis; the serum biochemical levels include at least one of cholesterol, triglycerides, amylase or lipase.

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