Bifidobacterium breve for relieving irritable bowel syndrome and application thereof

Abstract

The application discloses a Bifidobacterium breve for relieving irritable bowel syndrome and application thereof, and belongs to the technical field of microorganisms. The application provides a Bifidobacterium breve CCFM1300 which has the effect of relieving irritable bowel syndrome, can improve the visceral pain threshold and reduce the defecation frequency, significantly reduces nerve growth factor and myosin receptor kinase A, and then reduces the number of enteric chromaffin cells and the content of 5-HT. The Bifidobacterium breve can be used for preparing medicines for preventing and treating irritable bowel syndrome, and can be used for preparing probiotic food and health products, and has a very wide application prospect.

Description

TECHNICAL FIELD

[0001] The present application relates to a Bifidobacterium breve for relieving irritable bowel syndrome and an application thereof, and belongs to the technical field of microorganisms. BACKGROUND

[0002] Irritable bowel syndrome is a common functional bowel disease with a high incidence rate, and the incidence rate is about 5%-10% worldwide. Irritable bowel syndrome is divided into diarrhea-predominant irritable bowel syndrome, constipation-predominant irritable bowel syndrome and mixed irritable bowel syndrome. Irritable bowel syndrome mainly manifests as abdominal pain, accompanied by changes in stool form, which seriously affects the life of patients and is prone to recurrence. According to statistics, the direct and indirect cost of IBS treatment exceeds 280 billion yuan per year. The causes of IBS are various, and different treatment methods are adopted in clinic according to different causes, but long-term drug use will cause significant adverse reactions and side effects.

[0003] Irritable bowel syndrome is closely related to intestinal flora. It is reported that the intestinal flora of patients with irritable bowel syndrome is disordered; in addition, the amount of metabolic products such as short-chain fatty acids and bile acids is different from that of healthy people. Abnormal changes of intestinal flora and its metabolites will cause a variety of adverse symptoms. Probiotics can regulate intestinal flora and its metabolites, so probiotics can be used to relieve irritable bowel syndrome. SUMMARY

[0004] The purpose of the present application is to overcome the defects of the prior art drugs, such as long-acting time and side effects, and to provide a Bifidobacterium breve CCFM1300 and a new use of the strain in relieving IBS caused by maternal and infant separation stress.

[0005] The present application provides a Bifidobacterium breve CCFM1300, which has been preserved in the Guangdong Microbial Culture Collection Center on February 16, 2023, and the preservation number is GDMCC No: 63174.

[0006] In one embodiment, the Bifidobacterium breve CCFM1300 has the following biological characteristics:

[0007] (1) Bacterial characteristics: milky yellow;

[0008] (2) Colony characteristics: milky yellow raised colonies on MRS solid plates, regular edges, gram-positive bacteria;

[0009] (3) Growth characteristics: cultured in MRS medium at 37℃ constant temperature and aerobic conditions for about 16h to reach the late logarithmic phase.

[0010] The present application also provides a composition containing the Bifidobacterium breve CCFM1300.

[0011] In one embodiment, the composition is a food, a drug or a health product.

[0012] In one embodiment, the number of viable Bifidobacterium breve CCFM1300 in the composition is not less than 1 x 10 9 CFU / g or 1 x 10 9 CFU / mL.

[0013] The present application also provides a method for preparing a drug for preventing and / or treating irritable bowel syndrome, wherein the Bifidobacterium breve CCFM1300 is used as the main active ingredient of the drug.

[0014] In one embodiment, the number of viable Bifidobacterium breve CCFM1300 in the drug is not less than 1 x 10 9 CFU / g or 1 x 10 9 CFU / mL.

[0015] In one embodiment, the drug contains the Bifidobacterium breve CCFM1300, a drug carrier and / or a pharmaceutical excipient.

[0016] The present application also provides the use of the Bifidobacterium breve CCFM1300 in the preparation of a drug for preventing and / or treating irritable bowel syndrome.

[0017] In one embodiment, the irritable bowel syndrome is caused by maternal separation.

[0018] In one embodiment, the use includes at least one of the following effects:

[0019] (1) reducing the number of defecation particles of an irritable bowel syndrome subject;

[0020] (2) increasing the pain threshold of an irritable bowel syndrome subject;

[0021] (3) reducing the number of intestinal chromaffin cells of an irritable bowel syndrome subject;

[0022] (4) reducing the 5-HT content of the colon of an irritable bowel syndrome subject;

[0023] (5) reducing the relative expression of mRNA of nerve growth factor and muscle receptor kinase A of an irritable bowel syndrome subject.

[0024] The present application also provides a drug for preventing and / or treating irritable bowel syndrome, wherein the drug contains the Bifidobacterium breve CCFM1300.

[0025] In one embodiment, the number of viable Bifidobacterium breve CCFM1300 in the drug is not less than 1 x 10 9 CFU / g.

[0026] In an embodiment, the medicine contains the above-mentioned Bifidobacterium breve CCFM1300, a pharmaceutical carrier and / or a pharmaceutical excipient.

[0027] The present application also provides a food or health product containing the Bifidobacterium breve CCFM1300.

[0028] In an embodiment, the food is a dairy product, a legume product, a meat product or a fruit and vegetable product produced by a starter culture containing the Bifidobacterium breve CCFM1300.

[0029] In an embodiment, the preparation method of the starter culture is inoculating the Bifidobacterium breve CCFM1300 into a culture medium at an inoculation amount of 1-5% of the total mass of the culture medium, culturing at 37°C for 18h to obtain a culture solution; centrifuging the culture solution to obtain bacterial cells; resuspending the bacterial cells with physiological saline to obtain the starter culture.

[0030] In an embodiment, the culture medium is MRS culture medium.

[0031] The present application also provides the use of the Bifidobacterium breve CCFM1300 in a food additive, in particular as a food starter culture.

[0032] Beneficial effects: The Bifidobacterium breve CCFM1300 screened in the present application can effectively reduce the number of defecation particles, increase the pain threshold, reduce the number of intestinal chromaffin cells, reduce the content of 5-HT in the colon, and reduce the relative expression of mRNA of nerve growth factor and muscle ball receptor kinase A, showing better treatment or prevention effect on irritable bowel syndrome, and thus can be used to prepare medicines for preventing and treating irritable bowel syndrome. The strain of the present application is a food safety strain, which can be used to prepare probiotic food and health products, and has very wide application prospects.

[0033] Biological material preservation

[0034] The Bifidobacterium breve CCFM1300 is classified as Bifidobacterium breve, which was preserved in the Guangdong Microbial Culture Collection Center on February 16, 2023, at 5th floor, No. 59 Building, Guangzhou, Guangdong, China, with a preservation number of GDMCC No: 63174. BRIEF DESCRIPTION OF DRAWINGS

[0035] Figure 1 The figure shows the change of the pain threshold of each group of rats, i.e. the airbag pressure in the body of the rat when reaching the arching state, indicating that the Bifidobacterium breve CCFM1300 reduces the pain threshold of the rat; in the figure, the letters above the column chart are different, indicating significant difference (p<0.05).

[0036] Figure 2 The number of feces in 10 minutes of each group of rats indicates that B. breve CCFM1300 reduces the number of defecation of rats; in the figure: the letters above the column chart are different, indicating significant difference (p<0.05).

[0037] Figure 3 The content of 5-HT in the colon tissue of each group indicates that B. breve CCFM1300 reduces the content of 5-HT in the colon tissue; in the figure: the letters above the column chart are different, indicating significant difference (p<0.05).

[0038] Figure 4 The colon tissue immunofluorescence staining sections of each group indicate that B. breve CCFM1300 significantly reduces the number of colon enterochromaffin cells of rats; in the figure: the letters above the column chart are different, indicating significant difference (p<0.05).

[0039] Figure 5 The change of the relative expression amount of the mRNA of nerve growth factor in the colon of each group indicates that B. breve CCFM1300 can reduce the expression amount of nerve growth factor in the colon of rats; in the figure: the letters above the column chart are different, indicating significant difference (p<0.05).

[0040] Figure 6 The change of the relative expression amount of the mRNA of muscle ball receptor kinase A in the colon of each group indicates that B. breve CCFM1300 can reduce the expression amount of muscle ball receptor kinase A in the colon; in the figure: the letters above the column chart are different, indicating significant difference (p<0.05). DETAILED DESCRIPTION

[0041] In the present application, unless otherwise specified, the "%" or percentage used for explaining the concentration or ratio is weight percentage.

[0042] Some embodiments of the present application are related to the following culture medium:

[0043] MRS liquid culture medium: 10 g of tryptone, 10 g of beef extract, 5 g of yeast powder, 20 g of glucose, 2 g of diammonium hydrogen citrate, 5 g of sodium acetate, 2 g of dipotassium hydrogen phosphate, 0.5 g of magnesium sulfate heptahydrate, 0.25 g of manganese sulfate monohydrate, 1 mL of Tween 80, and water to 1000 mL.

[0044] MRS solid culture medium is obtained by adding 1.5% agar based on the total weight of the liquid culture medium.

[0045] Example 1: Collection, isolation and identification of B. breve CCFM1300

[0046] Take 1 g of healthy infant fecal samples collected from Daqing, Heilongjiang, gradient dilution and coating on MRS solid medium, placed in anaerobic environment at 37°C for 72h, observation and record the colony morphology, pick the colony line purification, then in MRS liquid medium at 37°C for 48h, the resulting colonies were gram staining and record the strain morphology, discard the gram negative strains and gram positive cocci in the colony, select the gram positive bacilli, after hydrogen peroxide enzyme analysis, discard the hydrogen peroxide enzyme positive strains, retain the hydrogen peroxide enzyme negative strains, with fructose-6-phosphokinase detection to discard negative strains, the resulting 16S rDNA sequencing identified as Bifidobacterium breve, named FHLJDQ3M5, stored in Jiangnan University Food Biotechnology Center Culture Collection, accession number CCFM1300. The resulting Bifidobacterium breve was subcultured, the bacterial cells were collected and centrifuged at 3000 rpm for 10 min, washed, repeated 3 times, the resulting bacterial cells were added to the matrix protectant for freeze preservation, and preserved in Guangdong Microbial Culture Collection Center, accession number GDMCC No:63174.

[0047] 16S rDNA amplification conditions: 95℃ 5min; 35 cycles (95℃ 30s, 55℃ 30s, 72℃ 2min); 72℃ 10min. Amplification primers: 27F: (5'-AGAGTTTGATCCTGGCTCAG-3'), 1492R: (5'-TACGGCTACCTTGTTACGACT T-3') Amplification product purification and sequence alignment process according to the method described in the literature (Turroni F et al. Exploring the Diversity of the Bifidobacterial Population in the Human Intestinal Tract [J]. Appl Environ Microb. 2009; 75(6): 1534-45).

[0048] Example 2: Bifidobacterium breve CCFM1300 improves visceral hypersensitivity in rats with irritable bowel syndrome caused by maternal-infant separation

[0049] 1. Experimental animals

[0050] Male SPF Wistar pregnant rats were purchased from Beijing Sperbio Biotechnology Co., Ltd. The rats were raised in polypropylene cages, one rat per cage, with food and water in the cage, temperature control (22℃), relative humidity (50%±10%), free water, and fed with standard feed.

[0051] 2. Bifidobacterium breve CCFM1300 culture

[0052] (1) Activation culture:

[0053] MRS liquid medium, 37℃ anaerobic incubator static culture.

[0054] Culture goal: Take the frozen bacteria, pick single colony in liquid MRS liquid medium, and culture in 37℃ anaerobic incubator for about 24h to activate Bifidobacterium breve CCFM1300.

[0055] (2) Primary culture:

[0056] MRS liquid medium, 37℃ anaerobic incubator static culture.

[0057] Culture goal: Take the activated Bifidobacterium breve CCFM1300 and inoculate it into MRS liquid medium at 2% inoculation amount by volume of medium, and pass two generations.

[0058] (3) Secondary culture:

[0059] Culture medium and culture conditions are the same as primary culture.

[0060] Take the Bifidobacterium breve CCFM1300 after primary culture and inoculate it into 1L MRS liquid medium at 2% inoculation amount by volume of medium, and culture in 37℃ anaerobic incubator for about 24h, then collect the bacteria. Wash twice with physiological saline, then resuspend in 30% glycerol solution, and the viable count is 6×10 10 CFU / mL, or adjusted to 1×10 9 CFU / mL. When used in subsequent experiments, centrifuge and discard the glycerol, then resuspend in physiological saline after washing once with physiological saline.

[0061] The bacterial suspension of Bifidobacterium breve CCFM683 is prepared in the same way as above; Bifidobacterium breve CCFM683 is disclosed in the patent application document with publication number CN115624572A.

[0062] 3. Experimental method

[0063] (1) Establishment of rat model of irritable bowel syndrome caused by early life maternal-infant separation stress

[0064] After the birth of the young mice, the blank group was not treated; the rest was taken as day 0 on the day of birth, and the young mice were separated alone every day from 9:00 to 12:00 from day 2 to day 14, and the male young mice were used for experiments on day 21.

[0065] (2) Experimental grouping and administration

[0066] The blank group without treatment has 10 rats. The remaining 30 rats are randomly divided into three groups, namely the model group, the B. breve CCFM1300 treatment group and the B. breve CCFM683 group. From the 21st day, the rats are given intragastrical administration, and the intragastrical administration dose is 2 x 10 9 / rat / day; the blank group and the model group are given intragastrical administration of the same volume of normal saline. All groups are given intragastrical administration until the rats are 8 weeks old, and then the pain threshold is measured and the rats are sacrificed.

[0067] The most prominent feature of irritable bowel syndrome is abdominal pain, and many studies have shown that abdominal pain is caused by visceral hypersensitivity, i.e., the decrease of the visceral pain threshold. The measurement method of the visceral pain threshold is the abdominal wall withdrawal reflex: a small inflatable air bag is connected with a pressure gauge and an inflation device through a three-way joint. After the rats are anesthetized, the air bag is inserted into the anus for 6 cm. After the rats are stable after waking up, the air bag is slowly inflated until the rats arch their backs, and the pressure value at this moment is recorded as the visceral pain threshold of the rats.

[0068] Figure 1 It is shown that the pain threshold of the model group is significantly lower than that of the blank group (p<0.05), B. breve CCFM1300 can significantly improve the pain threshold (p<0.05) and is close to the normal level, and B. breve CCFM683 has no such effect.

[0069] Example 3: B. breve CCFM1300 improves the defecation frequency of irritable bowel syndrome rats caused by maternal separation

[0070] The strain culture and animal experiment are shown in Example 2.

[0071] Diarrhea-predominant irritable bowel syndrome can cause an increase in defecation frequency, affect the quality of life of patients, and reduce the defecation frequency is an important indicator for relieving the disease. The rats are placed alone in a square open field with a side length of 1 meter, and after 10 minutes without interference, the number of fecal particles of the rats is checked, i.e., the defecation frequency of the rats.

[0072] Figure 2 It is shown that the number of fecal particles of the model group is significantly increased by 5.3 times compared with the blank group (p<0.05) within 10 minutes, which proves that the defecation frequency of irritable bowel syndrome rats is increased, B. breve CCFM1300 can significantly reduce the defecation frequency (p<0.05) and is reduced by 64% compared with the model group; and B. breve CCFM683 is reduced, but still has a difference compared with the blank group.

[0073] Example 4: B. breve CCFM1300 improves the colon 5-HT content of irritable bowel syndrome rats caused by maternal separation

[0074] The strain culture and animal experiment are shown in Example 2.

[0075] Anatomical sections of colon tissue were prepared and 5-HT content was measured using high performance liquid chromatography after sample pre-treatment.

[0076] 5-HT accelerates intestinal peristalsis and speeds up fecal excretion. Meanwhile, a number of studies have shown that 5-HT abnormalities are positively correlated with visceral hypersensitivity. Figure 3 It is shown that the 5-HT of the model group is significantly increased to 3.2 times (p<0.05) compared with the blank group, and B. breve CCFM1300 can significantly reduce the 5-HT content (p<0.05), which is reduced by 52% compared with the model group; while B. breve CCFM683 has no such effect.

[0077] Example 5: B. breve CCFM1300 improves the number of enterochromaffin cells in the colon of rats with irritable bowel syndrome caused by maternal-infant separation

[0078] Strain culture and animal experiments are described in Example 2.

[0079] A portion of the colon was fixed with 4% paraformaldehyde for 24 hours, dehydrated, paraffin-embedded, paraffin section deparaffinized to water, antigen repaired, serum blocked, first antibody added, second antibody added, spontaneous fluorescence quenched, DAPI restained nucleus, mounted and examined by microscopy.

[0080] 95% of 5-HT in the human body is secreted by enterochromaffin cells. Figure 4 It is shown that the number of enterochromaffin cells in the model group is significantly increased to 2.2 times (p<0.05) compared with the blank group, and B. breve CCFM1300 can reduce the number of enterochromaffin cells (p<0.05), which is reduced by 37% compared with the model group; while B. breve CCFM683 has a decrease, but still has a significant difference compared with the blank group.

[0081] Example 6: B. breve CCFM1300 improves the relative expression of nerve growth factor mRNA in the colon of rats with irritable bowel syndrome caused by maternal-infant separation

[0082] Strain culture and animal experiments are described in Example 2.

[0083] A portion of the colon was used to extract total RNA using the Trizol method, and the content of nerve growth factor was detected by quantitative PCR (qPCR) after reverse transcription to cDNA.

[0084] Previous studies have shown that maternal-infant separation stress can increase the relative expression of nerve growth factor mRNA, which in turn leads to the differentiation of intestinal stem cells into enterochromaffin cells. Ultimately, it leads to an increase in 5-HT. Reducing the relative expression of nerve growth factor mRNA can alleviate irritable bowel syndrome.

[0085] Figure 5The results show that, compared with the blank group, the relative expression of colon nerve growth factor and muscle receptor kinase A mRNA in the model group increased significantly by 2.3 times (p<0.05), B. breve CCFM1300 can reduce the relative expression of colon nerve growth factor and muscle receptor kinase A mRNA (p<0.05), which is 30% lower than that of the model group; and B. breve CCFM683 has no such effect.

[0086] Example 7: B. breve CCFM1300 improves the relative expression of colon muscle receptor kinase A mRNA in rats with irritable bowel syndrome caused by maternal separation

[0087] Strain culture and animal experiment see Example 2.

[0088] A portion of the colon was used to extract total RNA using the Trizol method, and the content of muscle receptor kinase A was detected by quantitative PCR (qPCR) after reverse transcription into cDNA.

[0089] Previous studies have shown that maternal separation stress can increase the relative expression of muscle receptor kinase A mRNA, which in turn leads to differentiation of intestinal stem cells into intestinal chromaffin cells. Ultimately, it leads to an increase in 5-HT. Reducing the relative expression of muscle receptor kinase A mRNA can alleviate irritable bowel syndrome.

[0090] Figure 6 The results show that, compared with the blank group, the relative expression of muscle receptor kinase A mRNA in the model group increased significantly by 2.5 times (p<0.05), B. breve CCFM1300 can reduce the relative expression of colon muscle receptor kinase A mRNA (p<0.05), which is 40% lower than that of the model group; and B. breve CCFM683 has a decrease, but still has a significant difference compared with the blank group.

[0091] The above experimental results show that B. breve CCFM1300 can alleviate rat irritable bowel syndrome, specifically by reducing visceral pain threshold and reducing the number of defecations. In addition, the reason for the alleviating effect is that it reduces nerve growth factor and muscle receptor kinase A, reduces the abnormal increase in the number of intestinal chromaffin cells, and thus reduces the content of colon 5-HT.

[0092] Example 8: Application of B. breve CCFM1300 tablet preparation

[0093] The basic steps of the manufacturing process are as follows: strain activation → expansion culture → collection of bacterial bodies → preparation of bacterial suspension → freeze-drying → total mixing → tabletting:

[0094] 1. Bacterial activation: Bifidobacterium breve CCFM1300 was inoculated into MRS liquid medium at a volume ratio of 2% and incubated in an anaerobic incubator at 37°C. The culture was continuously activated for two generations.

[0095] 2. Expansion culture: The activated Bifidobacterium breve CCFM1300 was inoculated into 1L MRS liquid medium at a volume ratio of 2% and incubated in an anaerobic incubator at 37°C for 24 hours.

[0096] 3. Collection of bacterial cells and preparation of bacterial suspension: After the expansion culture, the bacterial cells were collected by centrifugation at 4°C, washed twice with PBS (pH=7.4), and then prepared into a bacterial suspension with a concentration of 10 10 CFU / mL using 13% skimmed milk solution by weight.

[0097] 4. Freeze-drying: The bacterial powder was prepared according to the conventional freeze-drying process.

[0098] 5. Total mixing: 2% stearic acid was added as a lubricant and 3% CMC-Na was added as a binder based on the total weight of the bacterial powder, and then mixed uniformly.

[0099] 6. Tabletting: The tablets were prepared by a tabletting machine according to the conventional tabletting process.

[0100] Although the present application has been disclosed in the preferred embodiments as above, it is not intended to limit the present application, and any person skilled in the art can make various modifications and modifications without departing from the spirit and scope of the present application. Therefore, the scope of protection of the present application should be defined by the claims.

Claims

1. Bifidobacterium breve ( Bifidobacterium breve The use of CCFM1300 in the preparation of drugs for the prevention and / or treatment of irritable bowel syndrome is characterized by, The Bifidobacterium breve CCFM1300 has been preserved in Guangdong Microbial Culture Collection Center on February 16, 2023, and the preservation number is GDMCC No: 63174.

2. Use according to claim 1, characterized in that, The viable cell number of B. infantis CCFM1300 in the medicine is not less than 1 x 10 9 CFU / g or 1 x 10 9 CFU / mL.

3. Use according to claim 2, characterized in that, The medicine contains the Bifidobacterium breve CCFM1300, and a medicine carrier and / or a pharmaceutical excipient.

4. The use according to any one of claims 1 to 3, characterized in that, The irritable bowel syndrome is an irritable bowel syndrome caused by maternal and infant separation.

5. Use according to claim 4, characterized in that, The application includes at least one of the following effects: (1) reducing the defecation particle number of an irritable bowel syndrome individual; (2) improving the pain threshold of an irritable bowel syndrome individual; (3) reducing the number of enteric chromaffin cells of an irritable bowel syndrome individual; (4) reducing the 5-HT content of the colon of an irritable bowel syndrome; (5) reducing the mRNA relative expression amount of nerve growth factor and muscle ball receptor kinase A of an irritable bowel syndrome.

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