New use of bifidobacterium breve ccfm1078 for the relief of colic

Abstract

This invention discloses a novel application of Bifidobacterium breve CCFM1078 in relieving intestinal colic, belonging to the fields of microbial technology and pharmaceutical technology. The Bifidobacterium breve CCFM1078 provided by this invention can relieve intestinal colic in infants and young children, specifically in the following ways: (1) increasing the visceral pain threshold and reducing the frequency of defecation in rats to a certain extent; (2) reducing the number of chromaffin cells and 5-HT content in the colon of rats with intestinal colic; (3) reducing the serum TNF-α and IL-1β content in rats with intestinal colic; (4) reducing the hippocampal TNF-α and IL-6 content in rats with intestinal colic; and (5) increasing the propionic acid content in the cecal contents of rats with intestinal colic.

Description

Technical Field

[0001] This invention relates to a novel application of Bifidobacterium breve CCFM1078 in relieving intestinal colic, belonging to the fields of microbial technology and pharmaceutical technology. Background Technology

[0002] Infant colic (IC) is a common condition in infants under 5 months old. Its clinical features include recurrent, inconsolable crying. Its core characteristics are recurrent, intense crying without organic lesions, with crying lasting more than 3 hours a day, occurring at least 3 days a week, and lasting for more than 1 week. It seriously affects the sleep quality and growth and development of infants and young children, and brings heavy care pressure to families.

[0003] The exact cause of infantile colic is not fully understood. Current research suggests possible causes include dysbiosis / inflammation, visceral hypersensitivity, gastrointestinal factors, hormones, neurodevelopment, and psychosocial factors. Among these, visceral hypersensitivity is a core characteristic of functional gastrointestinal disorders, referring to increased pain sensitivity in the abdominal region, and is closely related to early life stress.

[0004] Existing interventions include improving feeding methods, abdominal massage, and the use of simethicone, but these methods suffer from unstable efficacy, limited applicability, and potential side effects with long-term use of chemical drugs. Probiotics, due to their high safety and significant efficacy, have become a research hotspot for colic intervention. However, currently only some strains have been shown to be effective in breastfed infants, and there are issues such as high strain specificity and unclear efficacy in formula-fed infants. The allergic effects of most probiotic strains lack sufficient clinical evidence. Therefore, further screening and validation of superior strains with potential functions in relieving infant colic is of great significance. Summary of the Invention

[0005] [Technical Issues] The technical problem to be solved by this invention is to provide a strain of Bifidobacterium shortissis that has the effect of relieving colic in infants and young children. Bifidobacterium breve ).

[0006] [Technical Solution] To solve the technical problem of this invention, this invention provides a strain of Bifidobacterium breve (Bifidobacterium breve). Bifidobacterium breve The use of CCFM1078 in the preparation of medicines for the prevention and / or treatment of infantile colic.

[0007] In one embodiment of the present invention, the Bifidobacterium breve CCFM1078 was isolated from fecal samples of infants in Wuxi. After isolation and screening, the strain was identified as Bifidobacterium breve by steps including bacterial genomic DNA extraction, PCR amplification with 16S rDNA specific primers, purification of amplification products, DNA sequencing, and sequence alignment. It was named Bifidobacterium breve CCFM1078.

[0008] In one embodiment of the present invention, the colonies of the short-lived Bifidobacterium CCFM1078 on MRS medium are convex to cushion-shaped, with intact edges, soft, moist, white and glossy.

[0009] In one embodiment of the present invention, the drug comprises live cells, lyophilized cells, fermentation products, or lysates of Bifidobacterium breve CCFM1078.

[0010] In one embodiment of the present invention, the method for collecting the bacterial cells of Bifidobacterium breve CCFM1078 is as follows: the above-mentioned Bifidobacterium breve CCFM1078 is inoculated into the culture medium at an inoculation amount of 2-4% of the total mass of the culture medium, and cultured at 37°C for 24-48 h to obtain a culture solution; the culture solution is centrifuged to obtain bacterial cells.

[0011] In one embodiment of the present invention, the culture medium is MRS culture medium.

[0012] In one embodiment of the present invention, the infant colic is infant colic caused by a mother-infant separation model.

[0013] In one embodiment of the present invention, the drug contains the Bifidobacterium breve CCFM1078, a drug carrier, and / or pharmaceutical excipients.

[0014] In one embodiment of the present invention, the drug carrier includes one or more of the following commonly used in medicine: fillers, adhesives, wetting agents, disintegrants, lubricants, and flavoring agents.

[0015] In one embodiment of the present invention, the pharmaceutical excipient includes excipients and / or additives.

[0016] In one embodiment of the present invention, the drug is a microbial preparation of Bifidobacterium breve CCFM1078.

[0017] In one embodiment of the present invention, the number of *Bifidobacterium breve* CCFM1078 cells in the drug is not less than 1 × 10⁻⁶. 9 CFU / mL or 1×10 9 CFU / g.

[0018] In one embodiment of the present invention, the dosage form of the drug includes a liquid preparation, a solid preparation, or a semi-solid preparation.

[0019] In one embodiment of the present invention, the solid dosage form includes tablets, granules, powders, or blocks.

[0020] In one embodiment of the present invention, the liquid formulation includes an aqueous solution, a suspension, or an emulsion.

[0021] [Beneficial Effects] 1. This invention screened out a strain of Bifidobacterium breve (Bifidobacterium breve) Bifidobacterium breve CCFM1078, this Bifidobacterium breve 1078, has a relieving effect on infantile colic caused by mother-infant separation, specifically manifested in: (1) Affecting the visceral pain threshold in rats with intestinal colic; (2) Improved defecation frequency in rats with intestinal colic; (3) Improved the number of chromaffin cells and 5-HT content in the colon of rats with intestinal colic; (4) Increase the time spent in the central region of the open field experiment in rats with intestinal colic; (5) Reduce the relative expression level of adrenocorticotropic hormone-releasing factor mRNA in the hippocampus of rats with intestinal colic; (6) Improved the relative expression levels of colonic nerve growth factor and myosin receptor kinase A mRNA in rats with irritable bowel syndrome; (7) Reduce serum TNF-α and IL-1β levels in rats with colic; (8) Reduced the levels of TNF-α and IL-6 in the hippocampus of rats with colic; (9) Improves the content of short-chain fatty acids in rats with intestinal colic.

[0022] Therefore, Bifidobacterium breve CCFM1078 has great application potential in the preparation of medicines for the prevention and / or treatment of infantile colic.

[0023] 2. Bifidobacterium breve is a type of probiotic and has been included in the "List of Strains that can be Used in Food". Therefore, the Bifidobacterium breve CCFM1078 screened in this invention will not pose any potential safety risks to children with colic.

[0024] 3. The cultivation process of Bifidobacterium breve only requires culture medium and some control of culture conditions, and the cost is relatively low, which will not bring too much economic burden to the families of infants with colic. Attached Figure Description

[0025] Figure 1 : Visceral pain threshold levels in rats with intestinal colic in different groups.

[0026] Figure 2 Number of defecations in rats with intestinal colic in different groups.

[0027] Figure 3 : 5-HT content in the colon of rats with intestinal colic in different groups.

[0028] Figure 4 The number of chromaffin cells in the colon of rats with intestinal colic in different groups.

[0029] Figure 5 Time spent in the central region of rats with colic in different groups during the open field experiment.

[0030] Figure 6 The relative expression levels of adrenocorticotropic hormone-releasing factor mRNA in the hippocampus of rats with colic in different groups.

[0031] Figure 7 The relative expression levels of colonic nerve growth factor mRNA in rats with colic in different groups.

[0032] Figure 8 The relative expression levels of myosin receptor kinase A mRNA in rats with colic in different groups.

[0033] Figure 9 Serum TNF-α levels in rats with colic in different groups.

[0034] Figure 10 Serum IL-1β levels in rats with colic in different groups.

[0035] Figure 11 TNF-α levels in the hippocampus of rats with colic in different groups.

[0036] Figure 12 IL-6 levels in the hippocampus of rats with colic in different groups.

[0037] Figure 13 Propionic acid content in the cecal contents of rats with intestinal colic in different groups. Detailed Implementation

[0038] The present invention will be further described in conjunction with the following specific embodiments.

[0039] The Wistar mice used in the following examples were purchased from Beijing Spefol Biotechnology Co., Ltd., China; the Bifidobacterium breve used in the following examples ( Bifidobacterium longum subsp. infantisCCFM1078 was isolated by the Biotechnology Center of the School of Food Science and Technology, Jiangnan University, and has been disclosed in patent CN112111424B, with accession number GDMCC No: 61011; the ELISA kits for detecting IL-6, TNF-α and IL-1β involved in the following examples were purchased from Shanghai Enzyme Linked Laboratory.

[0040] The culture media involved in the following examples are as follows: MRS solid medium: peptone 10 g / L, beef extract 10 g / L, glucose 20 g / L, sodium acetate 2 g / L, yeast extract 5 g / L, diammonium citrate 2 g / L, K2PO4·3H2O 2.6 g / L, MgSO4·7H2O 0.1 g / L, MnSO4 0.05 g / L, Tween 80 1 mL / L, agar 15 g / L, cysteine ​​1 g / L.

[0041] MRS liquid culture medium: peptone 10 g / L, beef extract 10 g / L, glucose 20 g / L, sodium acetate 2 g / L, yeast extract 5 g / L, diammonium hydrogen citrate 2 g / L, K2PO4·3H2O 2.6 g / L, MgSO4·7H2O 0.1 g / L, MnSO4 0.05 g / L, Tween 80 1 mL / L, cysteine ​​1 g / L.

[0042] Example 1: Preparation of Bifidobacterium breve CCFM1078 bacterial suspension In all embodiments, the preparation methods of the Bifidobacterium breve CCFM1078 bacterial suspension are as follows: Streaking of *Bifidobacterium breve* CCFM1078 bacterial suspension onto MRS solid medium and incubating at 37°C for 48 h yielded single colonies. These single colonies were then inoculated into MRS liquid medium and incubated at 37°C for 24 h to obtain an activated culture. This activated culture was then inoculated into MRS liquid medium at a 1% (v / v) inoculum and incubated at 37°C for 24 h to obtain a primary seed culture. The primary seed culture was then inoculated into MRS liquid medium at a 1% (v / v) inoculum and incubated at 37°C for 24 h to obtain a secondary seed culture. The secondary seed culture was then inoculated into MRS liquid medium at a 1% (v / v) inoculum and incubated at 37°C for 24 h to obtain a bacterial suspension. The bacterial suspension was centrifuged at 6000 g for 15 min, and the precipitate was collected. The precipitate was washed twice with physiological saline buffer and then centrifuged again at 6000 g for 10 min to obtain bacterial cells. The *Bifidobacterium breve* bacterial cells were resuspended in physiological saline to a cell concentration of 1×10⁻⁶ cells. 9 CFU / mL was used to obtain a bacterial suspension of Bifidobacterium breve CCFM1078.

[0043] Example 2: Effect of Bifidobacterium breve CCFM1078 on visceral pain threshold in rats with intestinal colic The animal experiment design is as follows: Female pregnant Wistar rats, specific pathogen-free (SPF). All rats were housed in an SPF-grade barrier facility at the Animal Experiment Center of Jiangnan University, with a temperature of 22-26℃, relative humidity of 40-70%, 12-hour light-dark cycle, and random access to sterile water and standard feed. After birth, the control group received no treatment; the remaining rats were separated from birth on day 1, and separated into individual cages daily from 9:00 to 12:00 on days 2-14. Male pups were used for the experiment on day 21. Eight rats were in the control group. The remaining 16 rats were randomly divided into two groups: a model group and an experimental group. Starting from day 2, gavage was administered at a dose of 1*10... 9 CFU / animal / day; the control group and model group were administered the same volume of physiological saline by gavage. All groups were administered gavage until 4 weeks of age, and were then sacrificed after open field test and measurement of pain threshold.

[0044] Infantile colic is a functional gastrointestinal disorder, and visceral hypersensitivity is a core characteristic of functional gastrointestinal disorders. The visceral pain threshold reflects visceral sensitivity; high sensitivity indicates a lowered visceral pain threshold. The visceral pain threshold in rats was measured using the abdominal withdrawal reflex. A 5F catheter was connected to an inflatable balloon, and a three-way stopcock was used to connect it to a manometer. After anesthetizing the rats, the balloon was inserted 6 cm into the anus. After the rats had recovered and adapted for 10 minutes, the balloon was slowly inflated until the rats exhibited arched back (AWR=3). Inflation was immediately stopped, and the pressure value at this point was recorded as the rat's visceral pain threshold.

[0045] Appendix Figure 1 The results showed that, compared with the control group, the pain threshold of the model group decreased by 70.07% (p<0.05), and compared with the model group, the pain threshold of the CCFM1078 group increased by 1.28 times.

[0046] Example 3: Effect of Bifidobacterium breve CCFM1078 on defecation frequency in rats with intestinal colic See Example 1 and Example 2 for strain culture and animal experiments.

[0047] Mother-infant separation models can lead to abnormal intestinal motility, manifested as increased defecation frequency. Rats are placed in a fecal collection box under undisturbed conditions, and the number of fecal particles is counted after 1 hour to determine the defecation frequency. The frequency of defecation can reflect intestinal motility levels to some extent.

[0048] Appendix Figure 2 The results showed that, compared with the control group, the number of defecation frequency in the colon of the model group increased by 1.51 times, while the number of defecation frequency in the CCFM1078 group decreased by 26.76% compared with the model group, and was close to the normal level.

[0049] Example 4: Bifidobacterium breve CCFM1078 improves the number of chromaffin cells and 5-HT content in the colon of rats with intestinal colic. See Example 1 and Example 2 for strain culture and animal experiments.

[0050] Dissected colon tissue was used to measure 5-HT content using liquid chromatography-tandem mass spectrometry after sample pretreatment; another part of the colon was fixed with 4% paraformaldehyde for 24 h, dehydrated, embedded in paraffin, dewaxed to water in paraffin sections, antigen retrieval, serum blocking, addition of primary antibody, addition of secondary antibody, autofluorescence quenching, DAPI counterstaining of cell nuclei, mounting, and microscopic examination and photography.

[0051] EC cells regulate 5-HT concentration through their number and secretory activity. 5-HT, as a core signaling molecule, directly amplifies pain signals. Together, they constitute a key pathological pathway for visceral hypersensitivity.

[0052] Appendix Figure 3 The results showed that, compared with the control group (9.86 ng / mg), the 5-HT content in the colon of the model group was significantly increased (15.16 ng / mg) (p<0.05). Compared with the model group, intervention with Bifidobacterium breve CCFM1078 significantly reduced the 5-HT content (10.95 ng / mg) (p<0.05).

[0053] Appendix Figure 4 The results showed that, compared with the control group, the number of enterochromaffin cells in the colon of the model group increased significantly by 2 times (p<0.05), while the number of enterochromaffin cells in the CCFM1078 group decreased by 56.37% compared with the model group (p<0.05).

[0054] Example 5: Effect of Bifidobacterium breve CCFM1078 on the time spent in the central region of the open field test in rats with intestinal colic. See Example 1 and Example 2 for strain culture and animal experiments.

[0055] Rats were placed in the center of an open field experimental device made of black acrylic plates with their backs facing upwards, and cameras were used to track and record the rats' activity trajectories and distances in various areas over 5 minutes.

[0056] The open field test can assess spontaneous exploratory behavior and anxiety-like state in rats in an open environment. The length of time spent in the central region can measure the degree of "anxiety" in rats. Studies have shown that rats with functional gastrointestinal disorders caused by mother-infant separation have significantly reduced time spent in the central region.

[0057] Appendix Figure 5This indicates that, compared with the control group, the central region dwell time in the model group was reduced by 31.11%. Compared with the model group, the central region dwell time in the CCFM1078 group was significantly increased by 1.58 times (p<0.05).

[0058] Example 6: Bifidobacterium breve CCFM1078 reduced the relative expression level of adrenocorticotropic hormone-releasing factor mRNA in the hippocampus.

[0059] See Example 1 and Example 2 for strain culture and animal experiments.

[0060] Total RNA was extracted from a portion of the hippocampus using the Trizol method, reverse transcribed into cDNA, and then the content of adrenocorticotropic hormone-releasing factor was detected using quantitative PCR (qPCR).

[0061] Previous studies have shown that chronic stress or anxiety significantly increases the relative expression of corticotropin-releasing factor (CRF) mRNA in the hippocampus, thereby overactivating the hypothalamic-pituitary-adrenal axis and exacerbating stress-related neuroendocrine and behavioral responses. Sustained high expression of CRF can further affect mood regulation and cognitive function, and is closely related to anxiety and depressive-like behaviors. Reducing the relative expression of CRF mRNA in the hippocampus may help alleviate stress-induced mood disorders and related neurological dysfunctions.

[0062] Appendix Figure 6 This indicates that, compared with the control group, the relative expression level of corticotropin-releasing factor (FRG) mRNA in the hippocampus of the model group was significantly increased by 1.83-fold (p<0.05). Compared with the model group, the relative expression level of FRG mRNA in the hippocampus of the CCFM1078 group was decreased by 37.57% (p<0.05).

[0063] Example 7: Bifidobacterium breve CCFM1078 improves the relative expression levels of colonic nerve growth factor and myosin receptor kinase A mRNA in rats with intestinal colic. See Example 1 and Example 2 for strain culture and animal experiments.

[0064] Total RNA was extracted from a portion of the colon using the Trizol method, reverse transcribed into cDNA, and then the levels of nerve growth factor and myosin receptor kinase A were detected using quantitative PCR (qPCR).

[0065] Previous studies have shown that mother-infant separation stress leads to an increase in the relative expression levels of nerve growth factor and myosin receptor kinase A mRNA, which in turn leads to the differentiation of intestinal stem cells into enterochromaffin cells. Ultimately, this results in 5 Increased HT levels. Reduced nerve activity. The relative expression levels of growth factor and myosin receptor kinase A mRNA can alleviate functional gastrointestinal disorders.

[0066] Appendix Figure 7 This indicates that, compared with the control group, the relative expression level of colonic nerve growth factor mRNA in the model group increased significantly by 1.89-fold (p<0.05). Compared with the model group, the relative expression level of colonic nerve growth factor mRNA in the CCFM1078 group decreased by 37.04% (p<0.05).

[0067] Appendix Figure 8 This indicates that, compared with the control group, the relative expression level of myosin receptor kinase A mRNA in the model group was significantly increased by 2.77-fold (p<0.05). Compared with the model group, the relative expression level of myosin receptor kinase A mRNA in the CCFM1078 group was decreased by 28.92% (p<0.05).

[0068] Example 8: Effects of Bifidobacterium breve CCFM1078 on serum TNF-α and IL-1β levels in rats with intestinal colic See Example 1 and Example 2 for strain culture and animal experiments.

[0069] After measuring the visceral pain threshold, rats were euthanized, and their serum was collected. Serum TNF-α levels were measured using an enzyme-linked immunosorbent assay (ELISA) kit. TNF-α and IL-1β are key pro-inflammatory cytokines, and their levels can reflect the body's inflammatory state.

[0070] Appendix Figure 9 The results showed that, compared with the control group (212.25 pg / mL), the serum TNF-α level in the model group was significantly increased (276.62 pg / mL) (p<0.05). Compared with the model group, *Bifidobacterium breve* CCFM1078 significantly reduced the serum TNF-α level (223.15 pg / mL) (p<0.05). (Appendix) Figure 10 The results showed that, compared with the control group (359.30 pg / mL), the serum IL-1β level in the model group was not significantly different (382.52 pg / mL). Compared with the model group, Bifidobacterium breve CCFM1078 significantly reduced the serum IL-1β level (321.06 pg / mL) (p<0.05).

[0071] Example 9: Effects of Bifidobacterium breve CCFM1078 on hippocampal TNF-α and IL-6 levels in rats with intestinal colic See Example 1 and Example 2 for strain culture and animal experiments.

[0072] After measuring the visceral pain threshold, blood was collected from rats and they were euthanized. Then, a portion of the rat's hippocampal tissue was homogenized, and the supernatant was extracted. The levels of cytokines TNF-α and IL-6 were measured using an enzyme-linked immunosorbent assay kit.

[0073] Appendix Figure 11 The results showed that, compared with the control group (83.45 pg / mg protein), the hippocampal TNF-α level in the model group was significantly increased (179.28 pg / mg protein) (p<0.05). Compared with the model group, Bifidobacterium breve CCFM1078 significantly reduced the hippocampal TNF-α level (75.36 pg / mg protein) (p<0.05).

[0074] Appendix Figure 12 The results showed that, compared with the control group (0.80 pg / mg protein), the hippocampal IL-6 level in the model group was significantly increased (1.90 pg / mg protein) (p<0.05). Compared with the model group, Bifidobacterium breve CCFM1078 significantly reduced the hippocampal IL-6 level (0.98 pg / mg protein) (p<0.05).

[0075] Example 10: Effect of Bifidobacterium breve CCFM1078 on the content of short-chain fatty acids in rats with intestinal colic See Example 1 and Example 2 for strain culture and animal experiments.

[0076] After measuring the visceral pain threshold, rats were blooded and euthanized, and cecal contents were collected. The cecal contents were pretreated and then subjected to GC analysis. MS was used to measure the content of short-chain fatty acids.

[0077] Studies have shown that propionic acid can provide energy to intestinal mucosal cells to enhance the intestinal barrier, regulate intestinal peristalsis to relieve bloating, and balance intestinal immunity to reduce inflammation. (See attached image) Figure 13 The results showed that, compared with the control group (25.75 μmol / g), the propionic acid content in the cecal contents of the model group was significantly reduced (16.20 μmol / g) (p<0.05). Compared with the model group, Bifidobacterium breve CCFM1078 significantly increased the propionic acid content in the cecal contents (21.12 μmol / g) (p<0.05).

[0078] 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 The use of CCFM1078 in the preparation of medicines for the prevention and / or treatment of colic.

2. The application as described in claim 1, characterized in that, The drug includes live cells, lyophilized cells, fermentation products, or lysates of Bifidobacterium breve CCFM1078.

3. The application as described in claim 2, characterized in that, The drug contains the aforementioned Bifidobacterium breve CCFM1078, a drug carrier, and / or pharmaceutical excipients.

4. The application as described in claim 3, characterized in that, The drug carrier includes one or more of the following commonly used in medicine: fillers, adhesives, wetting agents, disintegrants, lubricants, and flavoring agents.

5. The application as described in claim 4, characterized in that, The pharmaceutical excipients include excipients and / or additives.

6. The application as described in claim 5, characterized in that, The drug is a microbial preparation containing Bifidobacterium breve CCFM1078.

7. The application as described in claim 6, characterized in that, In the aforementioned drug, the number of *Bifidobacterium breve* CCFM078 cells is not less than 1 × 10⁻⁶. 9 CFU / mL or 1×10 9 CFU / g.

8. The application as described in claim 7, characterized in that, The dosage forms of the medicine include liquid, solid, or semi-solid preparations.

9. The application as described in claim 8, characterized in that, The solid dosage form includes tablets, granules, powders, or blocks.

10. The application as described in claim 8, characterized in that, The liquid formulation includes aqueous solutions, suspensions, and emulsions.

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