Bifidobacterium bifidum capable of relieving postpartum constipation of mother generation and food allergy of offspring and application of bifidobacterium bifidum

Abstract

The invention discloses bifidobacterium bifidum capable of relieving postpartum constipation of a mother generation and food allergy of a child generation and application of the bifidobacterium bifidum, and belongs to the field of microorganisms. A strain of bifidobacterium bifidum CCFM1523 is screened out, postpartum constipation of female rats is relieved, specifically, the number and weight of black excrement grains in 5 h and the small intestine propulsion rate can be increased, and the first black excrement discharging time is shortened. When young rats take the probiotics bifidobacterium bifidum CCFM1523, the allergic symptom is relieved to a certain degree, and particularly, the apparent score of food allergic rats induced by OVA and the content of OVA-sIgE and mMCP-1 in bodies are reduced. The bifidobacterium bifidum CCFM1523 has the effect of relieving intractable constipation in a purgative colonic animal model constructed by using the folium sennae, specifically, after intervention of the bifidobacterium bifidum CCFM1523, the number of excrement grains of mice within 5 hours and the water content of the excrement are remarkably increased, and meanwhile, the time for discharging first black excrement is shortened.

Description

TECHNICAL FIELD

[0001] The present application relates to a bifidobacterium longum capable of relieving maternal postpartum constipation and infant food allergy, and an application thereof, and belongs to the technical field of microorganisms and the technical field of medicine. BACKGROUND

[0002] Constipation is a group of symptoms characterized by difficulty in defecation and / or decreased frequency of defecation, and hard stool. Difficulty in defecation includes straining, effort, incomplete evacuation, a sensation of anorectal blockage, time consumption, and the need for assistance in defecation. Decreased frequency of defecation refers to less than 3 times per week. The global prevalence of constipation varies by region, but it is widespread. Surveys show that the incidence of constipation in the Chinese population is as high as 27%, but the rate of seeking medical treatment is only 17%. With the aging of society, changes in dietary structure, and the influence of social, psychological and other factors, the incidence of constipation is showing a trend of increasing year by year. In addition, for postpartum women, constipation not only leads to difficulty in defecation, abdominal distension and intestinal discomfort, but also affects breast secretion, the mother-infant relationship and the overall recovery process of the puerpera. At present, the main clinical methods for relieving constipation are laxatives, intestinal lubricants or dietary regulation. However, long-term use of laxatives has problems of tolerance and side effects, and some drugs can even change the normal structure of intestinal flora, further aggravating intestinal dysfunction. Therefore, we need to find safe, effective and long-term regulatory interventions.

[0003] On the other hand, the incidence of food allergy in infants and young children is showing a trend of increasing year by year, especially for common food proteins (such as milk, eggs, peanuts, etc.), which often manifests as eczema, diarrhea, vomiting, respiratory symptoms, and even anaphylactic shock in severe cases. Food allergy in infants and young children not only affects growth and development, but also increases the risk of immune-related diseases such as asthma and allergic rhinitis in later life. At present, the treatment for food allergy is limited, mainly relying on strict avoidance of allergenic food and symptomatic treatment, but this approach is difficult to fundamentally improve the immune imbalance and has great limitations. Therefore, developing safe and effective interventions to reduce the risk of food allergy in infants and young children is also an important problem to be solved.

[0004] Probiotics have the effects of regulating intestinal flora balance, improving intestinal function and immune regulation, and have been widely used in the research of constipation and allergic diseases in recent years. Studies have shown that probiotics can improve constipation by promoting the production of short-chain fatty acids (SCFAs), enhancing intestinal peristalsis, regulating neurotransmitter (such as 5-hydroxytryptamine, 5-HT) levels, and repairing intestinal barrier function. At the same time, probiotics can relieve food allergy by regulating immune balance, inhibiting inflammatory response, and reducing IgE levels. However, existing researches are mostly focused on a single field of action, and strains with the dual functions of relieving constipation and food allergy are still rare.

[0005] In summary, the prior art still lacks a functional strain capable of simultaneously improving multiple types of constipation (postpartum constipation, cathartic colon constipation) and relieving food allergy in infants, and the strain provided by the present application has dual efficacy, high safety, and broad application prospects. It not only provides a new solution for the improvement of postpartum constipation and the relief of cathartic colon constipation, but also provides a new intervention idea for the relief of food allergy in infants, and has important research value and industrialization potential. SUMMARY

[0006] In view of the above deficiencies in the prior art, the present application provides a probiotic strain capable of relieving maternal postpartum constipation, intractable constipation, and infant food allergy, and applications thereof, aiming to solve the technical problem of the lack of probiotic strains capable of relieving multiple types of constipation (cathartic colon constipation, maternal postpartum constipation) and improving infant food allergy symptoms in the prior art.

[0007] The first technical solution provided by the present application is a Bifidobacterium bifidum (Bifidobacterium bifidum) strain CCFM1523. Bifidobacterium bifidum The Bifidobacterium bifidum CCFM1523 was deposited with the Guangdong Microbial Culture Collection Center on November 6, 2025, at the address of 59 Building, 5th Floor, Guangzhou Martyrs' Park, Guangzhou, with the deposit number of GDMCC No: 67227.

[0008] The second technical solution provided by the present application is a microbial inoculant containing the Bifidobacterium bifidum CCFM1523 of the first technical solution.

[0009] In some embodiments, the dosage form of the microbial inoculant includes a liquid preparation or a powdery solid preparation.

[0010] In some embodiments, the amount of the Bifidobacterium bifidum CCFM1523 added in the microbial inoculant is at least 1x10 6 CFU / mL or 1x10 6 CFU / g.

[0011] Further, the amount of the Bifidobacterium bifidum CCFM1523 added in the microbial inoculant is at least 5x10 10 CFU / mL or 5x10 10 CFU / g.

[0012] The third technical solution provided by the present application is a medicine containing the Bifidobacterium bifidum CCFM1523 of the first technical solution or the microbial inoculant of the second technical solution.

[0013] In some embodiments, the amount of Bifidobacterium bifidum CCFM1523 added in the drug product is at least 1×10 6 CFU / mL or 1×10 6 CFU / g.

[0014] Further, the amount of Bifidobacterium bifidum CCFM1523 added in the drug product is at least 5×10 10 CFU / mL or 5×10 10 CFU / g.

[0015] In some embodiments, the dosage form of the drug product comprises granules, capsules, tablets, pills, or oral solution.

[0016] In some embodiments, the drug product further comprises a pharmaceutically acceptable carrier.

[0017] In some embodiments, the carrier comprises one or more of fillers, binders, wetting agents, disintegrants, lubricants, and flavoring agents commonly used in medicine.

[0018] The fourth aspect of the present application provides use of the Bifidobacterium bifidum CCFM1523 of the first aspect or the microbial agent of the second aspect in the preparation of a drug product for relieving and / or alleviating postpartum constipation.

[0019] In some embodiments, the drug product has at least one of the following effects: (1) shortening the time for the first black stool of the postpartum individual, increasing the number of 5h black stool particles, the weight of black stool, and the small intestine propulsion rate of the postpartum individual, and promoting the intestinal peristalsis of the postpartum individual; (2) repairing the intestinal mechanical barrier of the postpartum individual; (3) regulating the level of inflammatory factors in the postpartum individual.

[0020] The fifth aspect of the present application provides use of the Bifidobacterium bifidum CCFM1523 of the first aspect or the microbial agent of the second aspect in the preparation of a drug product for preventing, relieving, and / or alleviating food allergy in infants and young children.

[0021] In some embodiments, the use comprises reducing the content of OVA-sIgE, mMCP 1 in the allergic individual.

[0022] The sixth aspect of the present application provides use of the Bifidobacterium bifidum CCFM1523 of the first aspect or the microbial agent of the second aspect in the preparation of a drug product for preventing, relieving, and / or alleviating cathartic colon constipation.

[0023] In some implementations, the application includes shortening the time to the first black stool, increasing the number of black stools, weight, and small intestinal propulsion rate within 5 hours, and promoting intestinal peristalsis.

[0024] In some embodiments, the pharmaceutical product further comprises a drug carrier and / or pharmaceutical excipients.

[0025] Compared with the prior art, the beneficial effects of the present invention are as follows: The probiotic strain disclosed in this invention was obtained by screening from fecal samples of healthy individuals and identified by molecular biological methods. The results showed that the strain was Bifidobacterium bifidum (Bifidobacterium bifidum). Bifidobacterium bifidum Bifidobacterium bifidum is a probiotic; when ingested in sufficient quantities, it can have beneficial effects on the host's health. The screening and functional verification animal experiments of the Bifidobacterium bifidum involved in this patent have all passed the ethics committee review, and it is listed in the "List of Microbial Strains that Can Be Used in Food".

[0026] This invention provides a probiotic strain that can alleviate various types of constipation (postpartum constipation, laxative-induced colonic constipation) and improve food allergy symptoms in offspring. The probiotic is *Bifidobacterium bifidum* CCFM1523. In animal experiments, *Bifidobacterium bifidum* CCFM1523 showed good alleviating effects in animals with postpartum constipation induced by loperamide in mother rats and in animals with constipation induced by laxative colonic constipation. Specifically, in the postpartum constipation model, compared with the model group, it shortened the time to first black stool passage (reduced by 11.51%), increased the number of black stool particles at 5 hours (increased by 125.93%), increased black stool weight (increased by 139.57%), and increased small intestinal propulsion rate (increased by 102.94%), thus promoting intestinal peristalsis. Furthermore, it showed a good alleviating effect in experiments on ovalbumin (OVA)-induced food allergy symptoms in young mice. Specifically, when young mice were given Bifidobacterium bifidum CCFM1523 probiotics, compared with the model group, the apparent scores of young mice regarding food allergy were significantly reduced, the content of OVA-sIgE in serum (reduced by 62.12%) and the content of mMCP-1 in jejunal tissue were reduced (reduced by 39.57%), thus alleviating the food allergy symptoms in young mice.

[0027] In animal experiments using a laxative constructed from senna leaves to treat colonic constipation, Bifidobacterium bifidum CCFM1523 showed a good alleviating effect, specifically by shortening the time to expel the first black stool (reduced by 42.78%), increasing the number of fecal pellets at 5 hours (increased by 122.22%), and increasing fecal water content (increased by 26.26%) compared to the model group.

[0028] Preservation of biological materials A strain of Bifidobacterium bifidum ( Bifidobacterium bifidum CCFM1523, taxonomic name: Bifidobacterium bifidumBifidobacterium bifidum CCFM1523 was preserved in Guangdong Microbial Culture Collection Center on November 6, 2025, at 5th floor, No. 59 Building, Guangzhou Martyrs' Park, 100 Xianlie Road, Guangzhou, with a preservation number of GDMCC No: 67227. BRIEF DESCRIPTION OF DRAWINGS

[0029] Figure 1 The postpartum constipation appearance index of the mother mouse intervened by pyrrolipeimine was constructed.

[0030] Figure 2 The mother mouse animal experiment flow chart.

[0031] Figure 3 The mother mouse appearance index.

[0032] Figure 4 The mother mouse inflammation factor index.

[0033] Figure 5 The mother mouse intestinal mechanical barrier related index.

[0034] Figure 6 The young mouse food allergy animal experiment flow chart.

[0035] Figure 7 The young mouse appearance score.

[0036] Figure 8 The young mouse OVA-sIgE and mMCP-1 content in vivo.

[0037] Figure 9 The laxative colon mouse constipation appearance index result. DETAILED DESCRIPTION

[0038] The preferred embodiments of the present application are described below, and it should be understood that the embodiments are used to better explain the present application and are not used to limit the present application.

[0039] Test method: The detection method of the expression amount of the gene involved in the following examples Real-time fluorescent quantitative polymerase chain reaction (qRT-PCR) is used to determine the expression amount of the gene. First, RNA is extracted from fresh tissue, and the specific method is as follows: Fresh tissue 0.2 g removed after dissection of mice was repeatedly ground in a mortar (180℃, 4 h high temperature enzyme inactivation) with added liquid nitrogen, 1 mL TrizoL reagent was further added to the mortar and ground, and after the liquid was basically clarified, it was collected into a 1.5 mL enzyme-free centrifuge tube, and room temperature was placed for 15 min, 200 μL of chloroform solution was added to the centrifuge tube, and shaken for 15 s, and room temperature was placed for 10 min, and 4℃, 12000 r / min centrifugation for 15 min, 600 μL of upper colorless aqueous phase was taken into another enzyme-free centrifuge tube, and 500 μL of isopropanol was added. Mix well by inverting up and down, and let stand at room temperature for 10 min, then centrifuge at 4℃, 12000 r / min for 10 min, discard the supernatant, and leave the white precipitate of RNA at the bottom of the centrifuge tube, add 1 mL of 75% ethanol solution prepared with DEPC water, vortex to resuspend, centrifuge at 4℃, 7500 r / min for 5 min, discard the supernatant, and dry at room temperature. Add 30 μL of RNase free water to the dried RNA, and measure the RNA concentration and purity with Nanodrop after dissolving the RNA, and detect the quality of the RNA by agarose gel electrophoresis. Reverse transcription to synthesize cDNA was carried out according to the operation steps of HiFiScript gDNA RemovaL RT MasterMix reverse transcription kit of Kangwei Century Company, and the cDNA was stored at -20℃.

[0040] The mouse gene and the reference gene β-action gene primer are shown in Table 1.

[0041] Table 1 Mouse gene primer sequence

[0042] qRT-PCR reaction system and reaction conditions: PCR amplification was performed with Bio-Rad CFX96TM real-time fluorescent quantitative PCR instrument, and the fluorescence signal was read.

[0043] Table 2 Reaction system

[0044] The reaction conditions are: 95℃ 30s; 95℃ 10s, 60℃ 30s, a total of 40 cycles. β-action gene as the reference gene, the results were analyzed by CFX96Manager software.

[0045] The materials used in the examples are as follows: The animals involved in the following examples and the information of the feeding environment are as follows: Male, male 8-week Balb / c mice were purchased from SPF, and were raised in a specific pathogen free (SPF) level experimental animal barrier, with a relative humidity of (50 ± 10) %, a relative temperature of (23 ± 2) °C, and a 12 h light / dark cycle.

[0046] The culture medium involved in the following examples is as follows: MRS liquid medium: beef extract 10 g; tryptone 10 g; yeast powder 5 g; glucose 20 g; anhydrous sodium acetate 5 g; MgSO4·7H2O 0.1 g; MnSO4·H2O 0.05 g; diammonium hydrogen citrate 2 g; K2HPO4·3H2O 2.6 g; Tween 80 1 mL; adjust pH to 6.8 ± 0.2; make up to 1 L. Autoclave at 115 °C for 20 min.

[0047] MRS solid medium: add 2% agar powder to the MRS liquid medium.

[0048] The 10% skimmed milk involved in the following examples is a 10% mass fraction skimmed milk powder aqueous solution, sterilized at 105 °C for 15 min.

[0049] Example 1: Screening and identification of Bifidobacterium bifidum CCFM1523 1. Isolation and screening of Bifidobacterium bifidum CCFM1523: (l) Use a disposable sterile sampling device to collect a fecal sample, dilute and spread on MRS (containing 0.1% cysteine hydrochloride) solid medium, and place in an anaerobic environment at 37 °C for 48 hours; observe and record the colony morphology, and pick the white, smooth-surfaced colonies for streaking and purification; culture in MRS (containing 0.1% cysteine hydrochloride) liquid medium at 37 °C for 24 hours, and subculture twice.

[0050] 2. Molecular biology identification of Bifidobacterium bifidum CCFM1523: (l) Single bacterial genome extraction: Take 1 mL of the bacterial suspension cultured in step 1 in a 1.5 mL centrifuge tube, centrifuge at 10000 rpm for 2 min, discard the supernatant to obtain the bacterial body; wash the bacterial body with 1 mL of sterile water, then centrifuge at 10000 rpm for 2 min, discard the supernatant to obtain the bacterial body; add 200 μL of SDS lysis solution, 80°C water bath for 30 min; add 200 μL of phenol-chloroform solution to the bacterial lysis solution, the composition and volume ratio of the phenol-chloroform solution are Tris saturated phenol: chloroform: isopropyl alcohol = 25:24:1, mix well, then centrifuge at 12000 rpm for 5-10 min, take 200 μL of the supernatant; add 400 μL of ice ethanol or ice isopropyl alcohol to 200 uL of the supernatant, stand at -20°C for 1 h, centrifuge at 12000 rpm for 5-10 min, discard the supernatant; resuspend the precipitate with 500 μL of 70% (volume percent) ice ethanol, centrifuge at 12000 rpm for 1-3 min, discard the supernatant; dry in a 60°C oven or air dry; redissolve the precipitate with 50 μL of ddH2O for PCR; (2) 16S rDNA PCR: A. Bacterial 16S rDNA 20 μL PCR reaction system: 5 μL; 27F, 0.5 μL; 1492R, 0.5 μL; Taq enzyme, 10 μL; template, 1 μL; ddH2O, 8 μL.

[0051] B. PCR conditions: 95°C for 5 min; 95°C for 10 s; 55°C for 30 s; 72°C for 30 s; step 2-4 34x; 72°C for 5 min; 12°C for 2 min; C. Prepare 1% agarose gel, then mix the PCR product with 10000x loading buffer, load 2 μL, run at 120V for 30 min, then image the gel; D. The obtained PCR product is sent to a professional sequencing company, and the obtained sequencing results are searched and similarity compared in GeneBank using BLAST, identified as Bifidobacterium bifidum, named Bifidobacterium bifidum CCFM1523, preserved at -80°C for standby.

[0052] Example 2: Confirmation of the dose of loperamide for constructing the postpartum constipation symptoms of the mother mice The specific steps are as follows: 1. Preparation of loperamide Loperamide solution: the administration dose is 10 mg / kg·BW and 20 mg / kg·BW, and the mother mice are gavaged on the 5th day after delivery, with 0.2 mL per mother mouse.

[0053] 2. Animal experiment - mother mouse animal experiment - determination of loperamide dose Take a certain number of 8-week-old healthy Balb / c female mice and male mice, 1:1 ratio of cage for 5 days to achieve natural mating. After mating, the male mice were removed, and the female mice were then regularly monitored for weight changes to determine their pregnancy. Screening 15 pregnant female mice, divided into blank group, drug 10 group, drug 20 group, and after gavage drug, the apparent index detection was carried out.

[0054] 3. Apparent index detection and its experimental results The first time of black stool and the number of 5h black stool particles were determined: The first time of black stool: mix gum powder and water in a ratio of 1:10, heat on the electromagnetic oven until transparent, stir constantly while heating, then add 10% (w / v) activated carbon powder, stir and boil until mixed evenly, and then store in a 4°C refrigerator. The first time of black stool was measured the day before the end of the experiment. Each female mouse was gavaged with 0.2 mL of ink, and the specific time of ink gavage was recorded. The mouse's defecation state was closely monitored, and the first activated carbon-containing feces was recorded. The time difference between the two was the first time of black stool in mice.

[0055] The number of 5h black stool particles was counted after gavaging the female mice with 0.2 mL of ink, and the number of black stool particles within 5h was counted, which was the number of 5h black stool particles.

[0056] The results are shown in Figure 1 For the first time of black stool, when the drug concentration was 10 mg / kg·BW, there was no significant difference between the drug group and the blank group. When the drug concentration was 20 mg / kg·BW, the first time of black stool in the drug group was significantly higher than that in the blank group ( P <0.01). For the number of 5h black stool particles, when the drug concentration was 10 mg / kg·BW, there was no significant difference between the drug group and the blank group. When the drug concentration was 20 mg / kg·BW, the number of 5h black stool particles in the drug group was significantly lower than that in the blank group ( P <0.0001).

[0057] Therefore, when using loperamide to construct a postpartum constipation model in mice, the drug dose of 10 mg / kg·BW cannot successfully construct a constipation model. When the drug dose reaches 20 mg / kg·BW, the time of the first black stool of the mice is significantly increased, and the number of black stool particles at 5 h is significantly reduced, successfully constructing a postpartum constipation model. Unlike the general use of loperamide to construct constipation (for example: the drug dose used in CN112980734A patent is 10 mg / kg·BW), the ordinary drug dose cannot meet the requirements, so the effect of Bifidobacterium bifidum CCFM1523 intervention may have better link effects compared with other probiotics in the patent.

[0058] Example 3: Bifidobacterium bifidum CCFM1523 improves postpartum constipation symptoms in mice The specific steps are as follows: 1. Preparation of gavage bacterial solution Preparation of Bifidobacterium bifidum CCFM1523 bacterial solution and Bifidobacterium bifidum CCFM1063 bacterial solution: After taking the Bifidobacterium bifidum from the -80℃ refrigerator, streak it on the MRS solid culture medium, and culture it at 37℃ for 48 h. Pick a single colony on the MRS liquid culture medium and culture it at 37℃ for 24 h to prepare the seed solution. Inoculate the prepared seed solution into new MRS liquid culture medium at an inoculation amount of 2% (v / v) and culture it at 37℃ for 18 h. Repeat the same way for another generation to prepare the Bifidobacterium bifidum fermentation broth (the culture medium used in this process contains 0.1% cysteine hydrochloride). Then centrifuge the prepared Bifidobacterium bifidum fermentation broth at 8000 r / min and 4℃ for 15 min, discard the supernatant and reserve the bacterial pellet. Add 30 mL of 60% glycerol to the pellet. Before use, centrifuge the glycerol bacterial solution to remove glycerol, reserve the bacterial pellet, and resuspend it with 10% skim milk to obtain a bacterial suspension, and adjust the concentration of the bacterial suspension to 5×10 10 CFU / mL.

[0059] 2. Preparation of loperamide Loperamide solution: The drug dose is 20 mg / kg·BW, and the mice are gavaged on the 5th day after delivery, with 0.2 mL of gavage per mouse.

[0060] 3. Animal experiment of mice Take a certain amount of (more than 20) 8-week-old healthy Balb / c female mice and male mice, cage them together at a ratio of 1:1 for 5 days to achieve natural mating. After mating, the male mice are removed, and then the body weight change of the female mice is regularly monitored to determine their pregnancy. Twenty pregnant female mice are selected and randomly divided into four groups after delivery, namely the blank group, the model group, and the Bifidobacterium bifidum CCFM1523 and Bifidobacterium bifidum CCFM1063 intervention groups, with 5 in each group. The day of delivery is recorded as day 0, and the intervention begins on day 5 and ends on day 20.

[0061] Modeling method: The blank group of female mice were given 0.2 mL of sterile water by gavage, and the other groups of female mice were given 0.2 mL of loperamide solution by gavage. The loperamide solution was prepared fresh; the blank group and the model group were given 0.2 mL of 10% sterilized skim milk by gavage every day, and the Bifidobacterium bifidum CCFM1523 and Bifidobacterium bifidum CCFM1063 intervention groups were given 0.2 mL of 10% sterilized skim milk resuspended with bacteria at a concentration of 5 x 10 10 CFU / mL of bacteria solution.

[0062] The specific grouping of the female mouse animal experiment is shown in Table 2, and the experimental procedure is shown in Figure 2 .

[0063] Table 3: Grouping of female mouse animal experiment

[0064] 4. Detection of apparent indicators and experimental results The apparent indicators were detected 2 days before the end of the animal experiment, and the detection indicators included: (1) 5h black stool particle number: timing starts when each female mouse is placed in a different cage, and the number of black stool particles in 5h is collected. The number of black stool in each cage is recorded as the 5h black stool particle number.

[0065] (2) Time of first black stool: mix gum powder and water at a ratio of 1:10, heat on an electric stove until transparent, stir constantly while heating, then add 10% (w / v) activated carbon powder, stir and boil until well mixed. Store the solution in a 4°C refrigerator after it cools. Measure the time of first black stool in the morning on the day before the end of the experiment. When measuring, each female mouse is given 0.2 mL of ink by gavage, and the specific time of ink gavage is recorded. Pay close attention to the defecation status of the mouse, and record the time when the first feces containing activated carbon is excreted. The time difference between the two is the time of first black stool of the mouse.

[0066] (3) Black stool weight: weigh the black stool collected from each female mouse in 5h, which is the black stool weight.

[0067] (4) Small intestinal propulsion rate: Before sacrifice, each mouse was gavaged with 0.2 mL of ink (see the ink treatment process above). After 30 min, the mice were sacrificed and dissected. The upper end from the lower end of the pylorus to the cecum was cut off. The total length of the small intestine was measured as the total length of the small intestine. The distance from the pylorus to the front of the ink was the ink propulsion length. The small intestinal propulsion rate is calculated using the following formula.

[0068]

[0069] The experimental results of the apparent indicators are as follows: Figure 3 As shown, compared with the control group, the time to excrete the first black feces in the female mice of the model group was significantly increased. P <0.0001), number of black stool particles in 5 hours ( P <0.0001) and a significant decrease in the weight of black stool ( P <0.001), and the small intestinal propulsion rate was significantly reduced ( P <0.001), specifically, the time to pass the first black stool increased by 47.34%, the number of black stools after 5 hours decreased by 60.22%, the weight of black stools decreased by 55.56%, and the small intestinal propulsion rate decreased by 53.89%. According to the positive result criteria for constipation relief in the "Methods for Functional Testing and Evaluation of Health Foods (2023 Edition)," the postpartum constipation state in female mice induced by loperamide was successful. After intervention with the probiotics Bifidobacterium bifidum CCFM1523 and CCFM1063, CCFM1523 showed an effect in relieving postpartum constipation in female mice. Detailed information includes: compared with the Model group, there was no statistically significant difference in the time to pass the first black stool in the CCFM1523-treated group (…). P >0.05), but still 11.51% lower than the model group, with the number and weight of black stool particles increasing by 125.93% and 139.57% respectively after 5 hours. P <0.001), the small intestinal propulsion rate was significantly increased ( P <0.01), an increase of 102.94%. This indicates that the probiotic CCFM1523 can alleviate postpartum constipation in female mice. Bifidobacterium bifidum CCFM1063 does not have the effect of relieving postpartum constipation; that is, not all Bifidobacterium bifidum strains are effective in relieving postpartum constipation.

[0070] Example 4: Bifidobacterium bifidum CCFM1523 reduces the expression of inflammatory factors IL-1β / IL-6 / IL-17 in female mice with postpartum constipation. Real-time quantitative polymerase chain reaction (qRT-PCR) was used to determine the expression levels of IL-1β, IL-6, and IL-17 genes. The detailed methods and reaction conditions are described above. IL-1β, IL-6, and IL-17 have strong pro-inflammatory characteristics and can induce the production of various pro-inflammatory mediators, ultimately leading to widespread inflammatory events. By detecting the gene expression levels of inflammatory factors in the colon tissues of the mother rats, the inflammatory conditions of the mother rats with postpartum constipation and the relief of inflammation by probiotic intervention can be determined.

[0071] The specific results are shown in Figure 4 Compared with the Control group, the expression levels of inflammatory factors in the colon of the Model group mice increased significantly, specifically, the expression level of IL-17 increased by 2.65 times ( P P < 0.01), the expression level of IL-1β increased significantly by 3.15 times ( P P < 0.01), and the expression level of IL-6 increased significantly by 2.48 times ( P P < 0.05). This indicates that when the mother rats are in a postpartum constipation state, the expression levels of inflammatory factors in the body increase significantly, and the body inflammation level is high. After intervention with probiotic CCFM1523, the gene expression levels of IL-17, IL-6, and IL-1β in the colon tissues decreased by 2.46 times, 2.67 times, and 3.71 times, respectively. Based on the above data analysis and comparison, it can be found that probiotic CCFM1523 can well reduce the expression levels of pro-inflammatory factors in the body of mother rats with postpartum constipation and relieve the body inflammation state.

[0072] Example 5: Bifidobacterium CCFM1523 maintains the intestinal barrier integrity of mother rats with postpartum constipation and increases the gene expression levels of ZO-1 / cla-3 / CDH1 Real-time quantitative polymerase chain reaction (qRT-PCR) was used to determine the expression levels of ZO-1, cla-3, and CDH1 genes in the colon tissues. The detailed methods and reaction conditions are described above. The intestinal epithelial barrier mainly relies on tight junctions and adherent junctions to maintain. ZO-1 (Zonula occludens-1) is a key scaffold molecule of tight junction proteins, responsible for connecting transmembrane proteins and cytoskeleton, ensuring the integrity of the barrier; CDH1 (E-cadherin) is an important component of adherent junctions, which regulates cell adhesion and signal transduction, and helps to maintain the polarity and homeostasis of epithelial cells; Claudin-3 (CLDN3) is one of the core transmembrane proteins of tight junctions, which directly participates in the regulation of epithelial permeability. The three work together to maintain the function of the colon barrier and prevent harmful substances from crossing the epithelium into the body. Figure 5The expression of ZO-1, cla-3 and CDH1 in the model group was reduced by 139.63%, 98.44% and 123.94% respectively compared with the control group (P<0.01), and the mechanical barrier of the intestinal tract was damaged. P After the intervention of probiotic CCFM1523, the expression of ZO-1, cla-3 and CDH1 in the model group was increased by 1.65 times (P<0.05), 2.76 times (P<0.0001) and 4.35 times (P<0.0001) respectively, and the expression of the related genes was close to or even higher than that of the control group. Therefore, probiotic CCFM1523 can restore the intestinal barrier damage in the model group to the normal level. P P P

[0073] The expression of AQP3 in the colon tissue was determined by real-time fluorescence quantitative polymerase chain reaction (qRT-PCR), and the detailed method and reaction conditions are described above. Aquaporins-3 (AQP3) is responsible for regulating water reabsorption in the intestinal tract and is mainly distributed in the basolateral membrane of the colon epithelial cells. Compared with the control group, the expression of AQP3 in the colon of the model group was significantly increased (P<0.0001), and the reabsorption of water in the colon contents was enhanced, which led to dry and hard feces, which was consistent with the result that the weight of the black feces in the model group was significantly lower than that in the control group within 5h. After the intervention of probiotic CCFM1523, the expression of AQP3 in the colon of the model group was significantly reduced (P<0.0001), and the reabsorption of water in the intestinal contents was reduced, which increased the water content of the feces, which was consistent with the result of the apparent index. P P

[0074] Example 6: Bifidobacterium CCFM1523 relieves food allergy in offspring experiment I. Preparation of gavage bacterial solution ​​​​​Preparation of Bifidobacterium bifidum CCFM1523 bacterial liquid: After Bifidobacterium bifidum CCFM1523 was taken out from the refrigerator at -80°C, it was streaked on MRS solid culture medium and cultured at 37°C for 48 h. A single colony was picked and cultured in MRS liquid medium at 37°C for 24 h to prepare seed liquid. The prepared seed liquid was inoculated into new MRS liquid medium at an inoculation amount of 2% (v / v) and cultured at 37°C for 18 h. The same culture was repeated once again to prepare Bifidobacterium bifidum CCFM1523 fermentation liquid. Then the prepared Bifidobacterium bifidum CCFM1523 fermentation liquid was centrifuged at 8000 r / min at 4°C for 15 min, and 30 mL of 60% glycerol was added. Before use, the glycerol was removed, resuspended with 10% skim milk to prepare bacterial suspension, and the concentration of the bacterial suspension was adjusted to 5×10 9 CFU / mL.

[0075] II. Preparation of sensitizing liquid Sensitizing liquid: Cholera toxin adjuvant (CT) and ovalbumin (OVA) were dissolved in a suitable solvent (sterile water) to prepare the sensitizing liquid, wherein the concentration of CT was 0.5 μg / g and the concentration of OVA was 100 μg / g. The pups were weighed from the fifth day, and the gavage was performed according to the body weight, with the ratio of body weight: reagent = 1:10. The sensitizing liquid was gavaged every 5 days.

[0076] Challenge liquid: the concentration of OVA challenge was 1200 μg / g, which was configured according to the body weight of the mice on the 20th day. The corresponding amount of OVA was dissolved in the corresponding amount of sterile water according to the ratio of the body weight of the mice and the reagent. Example 2.3 After the mother mice finished production, it was recorded as the 0th day of the pups. The gender of the pups was determined on the fifth day, and female pups were selected for subsequent OVA-induced food allergy and probiotic intervention experiments. The detailed grouping and experimental process are shown in Table 4 and Figure 6 The sensitization test of the pups was started on the 7th day, and the pups were divided into a blank group, a model group, and a CCFM1523 group. The pups in the blank group and the model group and the CCFM1523 group were all offspring of the model group mother mice in the mother mouse experiment. First, the pups were weighed and recorded every day; second, the CCFM1523 group pups were gavaged with probiotic bacterial liquid every day, and the concentration of the bacterial liquid was 5×10 9CFU / mL, the blank group and the model group of mice were given skim milk solution by gavage every day; in addition, the model group and the CCFM1523 group of young mice were given sensitizing solution by gavage on 7d, 12d, 17d respectively, and the blank group of young mice was given corresponding weight of sterile water by gavage (when the young mice were given for the first time, the operation should be careful to prevent damage to the young mice). On the 22nd day, the young mice were given challenge test, the blank group of young mice was given corresponding weight of sterile water by gavage, and the model group and the CCFM1523 group of young mice were given OVA challenge solution by gavage. Within 30 minutes after the operation, the young mice were given apparent score, and after the evaluation was completed, the young mice were taken blood and dissection operation.

[0077] Table 4: Grouping of young mice experiment

[0078] IV. Apparent score and results The apparent score content includes: "no allergic symptoms" is recorded as 0 points, "repeatedly scratching ears, mouth, and scratching the ear canal with the hind legs, tail with scratching marks, and hair not smooth" is recorded as 1 point, "reduced activity, reduced food intake, and disheveled hair" is recorded as 2 points, "long time still, rapid breathing rate, disheveled hair, and dull, and hair standing" is recorded as 3 points, "bulging eyes, conjunctival hyperemia, no response to stimulation, tremor, convulsion, and cyanotic lips" is recorded as 4 points, and "death" is recorded as 5 points. The score results are shown in Figure 7 .

[0079] The hair of the normal group of young mice was smooth and lustrous, and no allergic symptoms occurred; the hair of the model group of mice was disheveled and dull, and some young mice showed hair standing and rapid breathing rate symptoms; after the young mice took CCFM1523, the above food allergy symptoms were improved P <0.05).

[0080] Example 7: Bifidobacterium CCFM1523 on food allergy mice serum OVA specific IgE, mMCP 1 has a remission effect After the young mice were taken blood and killed, the young mouse blood was centrifuged at 3000r / min for 2h, and the young mouse serum was taken, and the content of OVA specific IgE in the mouse serum was determined by ELISA kit. The content of mMCP-1 in the jejunum tissue of the young mice was quantified by ELISA method. The specific method is as follows: the jejunum tissue was washed with pre-cooled PBS to remove residual blood, and the surrounding adipose tissue was removed, weighed, and then cut into pieces. The cut tissue was crushed with PBS solution according to the weight volume ratio of 1:9 on the high throughput tissue crusher, and finally the homogenate was centrifuged at 5000xg for 5 10 minutes, the supernatant was taken for detection, and the experiment was carried out according to the corresponding kit instruction, and the content of mMCP-1 in the tissue was calculated according to the standard curve. The specific results are shown inFigure 8 .

[0081] As shown in Table 6, compared with the Model group, the content of OVA-sIgE in the serum of the CCFM1523 group was significantly reduced (P<0.05), reduced by 3.84 times; the content of mMCP-1 in the jejunum tissue of the CCFM1523 group was significantly reduced (P<0.01), reduced by 1.65 times. In summary, when the young mice were fed with Bifidobacterium CCFM1523, the contents of OVA-sIgE and mMCP-1 in the OVA-induced food allergy mice were reduced. Figure 8 P P

[0082] Example 8: Bifidobacterium CCFM1523 improves the symptoms of constipation caused by laxatives in mice The specific steps are as follows: I. Preparation of gavage bacterial solution Preparation of Bifidobacterium CCFM1523 bacterial solution: After taking Bifidobacterium CCFM1523 from the -80°C refrigerator, streak on MRS solid culture medium, cultivate at 37°C for 48h, pick single colonies on MRS liquid culture medium, cultivate at 37°C for 24h, and prepare seed solution; inoculate the prepared seed solution into new MRS liquid culture medium at an inoculation amount of 2% (v / v), cultivate at 37°C for 18h, and cultivate again in the same way for one generation, and prepare Bifidobacterium CCFM1523 fermentation broth; then centrifuge the prepared Bifidobacterium CCFM1523 fermentation broth at 8000r / min, 4°C for 15min, resuspend a part of it with 10% skim milk to prepare bacterial suspension, and adjust the concentration of the bacterial suspension to 6x10 9 CFU / mL.

[0083] II. Treatment of senna extract The purchased senna extract (Xi'an Xuhuang Biotechnology Co., Ltd.) was prepared into different concentrations of senna extract solution with sterile water for use in animal experiments.

[0084] III. Animal experiment ​​​Eighteen 8-week-old male C57 / 6J mice were randomly divided into three groups (n=6 per group) after a one-week acclimatization period: a control group, a model group, and a CCFM1523 group. Except for the control group, all other groups were administered senna leaf solution via gavage to establish a laxative colon model. The senna leaf solution was prepared fresh for each use. One week after discontinuation of the drug, constipation-related indicators were measured to determine the success of the model establishment. After model establishment, a two-week intervention was initiated. The control and model groups were administered 0.2 mL of 10% sterile skim milk via gavage daily, while the CCFM1523 group was administered 0.2 mL of 10% sterile skim milk daily with a bacterial concentration of 6 × 10⁻⁶. 9 CFU / mL bacterial suspension.

[0085] The specific animal experiment procedure is shown in Table 4.

[0086] Table 5 Animal Experiment Procedure

[0087] IV. Apparent Indicator Testing Phenotypic indicators were tested two days before the end of the animal experiment. These indicators included: ① 5-hour fecal pellet count: Start timing when each mouse is placed in a different cage, collect feces for 5-6 hours, and record the number of feces in each cage to get the number of fecal pellets in 5-6 hours.

[0088] ② Time of first black stool excretion: Mix gum arabic powder and water at a ratio of 1:10 until homogeneous. Heat on an induction cooker until transparent, stirring constantly. Then add 10% (w / v) activated charcoal powder, stir, and boil until homogeneous. After cooling, store in a 4°C refrigerator. The time of the first black stool excreted by mice was measured on the morning before the end of the experiment. To ensure accuracy, mice were fasted overnight but allowed free access to water before the measurement. During the measurement, each mouse was gavaged with 0.2 mL of the above-mentioned ink, and the gavage time was recorded. The mice's defecation status was closely monitored. The time when the first stool containing activated charcoal was excreted was recorded. The time difference between the two recorded times is the time of the first black stool excreted by the mouse.

[0089] ③ Fecal moisture content: This indicator was measured 1 day before the end of the animal experiment. Each mouse was placed in a box lined with filter paper, and the feces of each mouse were collected individually. The weight of the feces before and after freeze-drying was recorded, and the fecal moisture content was calculated using the following formula:

[0090] V. Experimental Results like Figure 9 As shown, compared with the control group, the time to excrete the first black feces in the model group mice was significantly increased. P <0.01), fecal water content significantly decreased ( P<0.01), and the number of fecal particles in 5 hours was significantly reduced ( P <0.001), indicating that mice treated with senna leaf extract did indeed exhibit constipation symptoms, and the model was successful. Mice treated with the probiotic CCFM1523 showed varying degrees of change in the apparent indicators of constipation. Specifically, the time to pass the first black stool in mice was significantly shortened (…). P <0.01), shortened by 0.57 times; 5h fecal particle count and fecal moisture content increased significantly ( P <0.001, P <0.05), which increased by 2.22 times and 1.26 times respectively. According to the positive result judgment criteria for relieving constipation in the "Methods for Functional Testing and Evaluation of Health Foods (2023 Edition)", Bifidobacterium bifidum CCFM1523 can relieve the symptoms of colonic constipation caused by senna leaf laxative.

[0091] 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. A strain of Bifidobacterium bifidum ( Bifidobacterium bifidum CCFM1523, characterized in that, The Bifidobacterium bifidum CCFM1523 was deposited at the Guangdong Provincial Center for Microbial Culture Collection on November 6, 2025, with accession number GDMCCNo: 67227.

2. A microbial inoculant, characterized in that, The microbial agent contains Bifidobacterium bifidum CCFM1523 as described in claim 1.

3. The microbial agent according to claim 2, characterized in that, In the microbial inoculant, the amount of Bifidobacterium bifidum CCFM1523 added is at least 1×10⁻⁶. 6 CFU / mL or 1×10 6 CFU / g.

4. A medicine, characterized in that, The drug contains Bifidobacterium bifidum CCFM1523 as described in claim 1, or the microbial agent as described in claim 2 or 3.

5. The use of Bifidobacterium bifidum CCFM1523 as described in claim 1, or the microbial agent as described in claim 2 or 3, in the preparation of a medicine for relieving and / or alleviating postpartum constipation.

6. The application according to claim 5, characterized in that, The drug has at least one of the following effects: (1) Shorten the time for the first black stool to be expelled in postpartum individuals, increase the number of black stools, weight of black stools and small intestinal propulsion rate in postpartum individuals within 5 hours, and promote intestinal peristalsis in postpartum individuals; (2) Repairing the postpartum individual intestinal mechanical barrier; (3) Regulate the level of inflammatory factors in postpartum individuals.

7. The use of Bifidobacterium bifidum CCFM1523 as described in claim 1, or the microbial agent as described in claim 2 or 3, in the preparation of a medicine for preventing, alleviating and / or reducing food allergies in infants and young children.

8. The application according to claim 7, characterized in that, The application includes reducing OVA-sIgE and mMCP in allergic individuals. The content of 1.

9. The use of Bifidobacterium bifidum CCFM1523 as described in claim 1, or the microbial agent as described in claim 2 or 3, in the preparation of a medicine for preventing, relieving, and / or alleviating laxative constipation.

10. The application according to claim 9, characterized in that, The applications include shortening the time to pass the first black stool, increasing the number of black stools, weight, and small intestinal propulsion rate within 5 hours, and promoting intestinal peristalsis.

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