Bifidobacterium longum subsp. Longum capable of improving mother generation postpartum constipation and offspring food allergy through mother-infant vertical transfer mode and application of bifidobacterium longum subsp. Longum

Abstract

The invention discloses a bifidobacterium longum subsp. Longum for improving postpartum constipation of a mother generation and food allergy of a child generation in a mother-child vertical transfer mode and application of the bifidobacterium longum subsp. Longum, and belongs to the field of microorganisms. A strain of bifidobacterium longum subsp. Longum CCFM1522 is screened and has a relieving effect in a female mouse postpartum constipation model constructed by using loperamide after delivery of a female mouse, specifically, the number and weight of black feces grains of the female mouse within 5 hours can be increased, and the first black feces discharge time is shortened. Further, in experiments for inducing food allergy by using ovalbumin on young rats, when female rats take probiotics of the bifidobacterium longum subsp. Longum CCFM1522 after delivery or the young rats take the bifidobacterium longum subsp. Longum CCFM1522 independently, the allergy symptoms in a food allergy animal model can be relieved to a certain extent; specifically, the apparent score of the young rats and the content of OVA-sIgE, mMCP-1 and beta-Hex in the bodies are reduced.

Description

TECHNICAL FIELD

[0001] The present application relates to a long bifidobacterium longum subsp. longum strain for improving maternal postpartum constipation and infant food allergy through vertical transmission, and an application thereof, belonging to the field of microbial technology. BACKGROUND

[0002] Constipation is a group of symptoms characterized by difficulty in defecation, decreased frequency of bowel movements, or hard and dry stools. Postpartum constipation is a common problem for women during lactation, often occurring in the first 2-5 days after delivery, and gradually subsiding thereafter, but about 10% of women still have persistent symptoms. The causes include psychological barriers to defecation caused by perineal injury, large amounts of iron supplementation, long-term bed rest during the puerperium, high-calorie and low-fiber diet, and lack of sleep during lactation. Existing treatment drugs often have defects such as poor taste, limited effect, large side effects, strong dependence, and the like, and drug selection during lactation needs to be particularly cautious to avoid adverse effects on breast milk and infants, so it is particularly urgent to develop a safe and effective alternative solution. Probiotics are a class of active microorganisms that can bring health benefits after sufficient intake, and are concerned due to their safe and mild characteristics. In terms of relieving constipation, bifidobacterium and lactobacillus have been studied more, and the mechanisms of action mainly include regulating intestinal flora balance, promoting the growth of beneficial bacteria, enhancing intestinal peristalsis, and accelerating the discharge of residues; at the same time, their metabolites such as organic acids and short-chain fatty acids can lower the intestinal pH, soften the stool, and stimulate the secretion of digestive enzymes, thereby improving postpartum constipation symptoms.

[0003] Infant food allergy is a growing public health problem worldwide, with a prevalence of about 5%-8%, and common allergens include milk, eggs, soybeans, and peanuts. Allergies can cause skin itching, hives, respiratory symptoms, and digestive discomfort, and even induce anaphylactic shock in severe cases, which seriously affects the growth and development and quality of life of infants. Due to the immaturity of the immune system and the weakness of the gastrointestinal barrier, infants are more likely to have abnormal immune responses to dietary antigens. The current main intervention measures are food avoidance and drug treatment, but the former affects nutrition and development, and the latter can alleviate symptoms but is difficult to fundamentally correct immune abnormalities. Therefore, it is urgent to explore safe and effective new intervention strategies. Recent studies have shown that the intestinal microecology plays a key role in the development of food allergy. Probiotics can improve intestinal flora balance, enhance mucosal barrier function, and regulate immune response, showing potential for application. Lactobacillus and bifidobacterium have been proven to promote Treg differentiation, reduce Th2 response and IgE levels, and promote short-chain fatty acid production, thereby maintaining immune homeostasis, providing a theoretical basis and practical prospect for probiotic intervention in food allergy.

[0004] There is a complex and close physiological connection between mother and infant. Maternal status not only directly affects the process of pregnancy and delivery, but also profoundly shapes the health of the offspring through breastfeeding and other ways. In recent years, studies have shown that the metabolic products of maternal intestinal flora may be transmitted to breast milk through the 'gut-mammary gland axis', and then act on the immune development and disease susceptibility of infants. For example, the immune active factors, short-chain fatty acids and specific bacterial components in breast milk can regulate the colonization of infant intestinal flora, promote immune tolerance and reduce the risk of food allergy. Probiotics have a dual role in regulating intestinal function and immune homeostasis, and have become a potential intervention option. On the one hand, probiotics can improve maternal intestinal motility and flora balance and relieve postpartum constipation; on the other hand, they can also indirectly regulate the immune response of offspring by affecting the maternal immune environment and breast milk composition, thereby reducing the risk of food allergy. Therefore, screening a strain of probiotics that can improve maternal postpartum constipation while reducing the risk of food allergy in offspring not only helps to solve the common health needs of mother and infant, but also opens up a new direction for the application of probiotics in the field of maternal and infant health. SUMMARY

[0005] In view of the deficiencies in the prior art, the present application provides a Bifidobacterium longum subsp. longum strain for improving postpartum constipation of maternal generation and food allergy of offspring generation through vertical transmission between mother and infant, and application thereof, aiming to solve the technical problem in the prior art that there is a lack of probiotics that can relieve postpartum constipation of maternal generation and food allergy of offspring generation through maternal relief during lactation.

[0006] The first technical solution provided by the present application is a Bifidobacterium longum subsp. longum strain (CCFM1522). Bifidobacterium longum subsp. Longum Bifidobacterium longum subsp. Longum The Bifidobacterium longum subsp. longum strain was deposited with the Guangdong Microbial Culture Collection Center on November 6, 2025, at the address of No. 59 Building, 5th Floor, Guangzhou Xianlie Middle Road 100 Courtyard, and the deposit number is GDMCC No: 67226.

[0007] The second technical solution provided by the present application is a microbial agent containing the Bifidobacterium longum subsp. longum strain CCFM1522 of the first technical solution.

[0008] In some embodiments, the dosage form of the microbial agent includes a liquid preparation or a powdery solid preparation.

[0009] In some embodiments, the amount of the Bifidobacterium longum subsp. longum strain CCFM1522 added in the microbial agent is at least 1×10 6 CFU / mL or 1×10 6 CFU / g.

[0010] Further, the amount of the B. longum subsp. longum CCFM1522 added in the microbial agent is at least 5×10 10 CFU / mL or 5×10 10 CFU / g.

[0011] The third technical solution provided by the present application is a medicine containing the B. longum subsp. longum CCFM1522 of the first technical solution or the microbial agent of the second technical solution.

[0012] In some embodiments, the amount of the B. longum subsp. longum CCFM1522 added in the medicine is at least 1×10 6 CFU / mL or 1×10 6 CFU / g.

[0013] Further, the amount of the B. longum subsp. longum CCFM1522 added in the medicine is at least 5×10 10 CFU / mL or 5×10 10 CFU / g.

[0014] In some embodiments, the dosage form of the medicine includes granules, capsules, tablets, pills, or oral liquids.

[0015] In some embodiments, the medicine further comprises a pharmaceutically acceptable carrier.

[0016] In some embodiments, the carrier includes one or more of fillers, binders, wetting agents, disintegrants, lubricants, and flavoring agents commonly used in medicine.

[0017] The fourth technical solution provided by the present application is the use of the B. longum subsp. longum CCFM1522 of the first technical solution or the microbial agent of the second technical solution in the preparation of a medicine for relieving and / or reducing postpartum constipation.

[0018] In some embodiments, the medicine has at least one of the following effects: (1) shortening the time of 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 damage of the postpartum individual; (3) regulating the level of inflammatory factors in the postpartum individual.

[0019] The fifth technical solution provided by the present application is the use of the B. longum subsp. longum CCFM1522 of the first technical solution or the microbial agent of the second technical solution in the preparation of a medicine for preventing, relieving, and / or reducing food allergy of offspring.

[0020] In some implementations, the application includes alleviating food allergy symptoms in offspring through mother-to-child vertical transmission and reducing OVA-sIgE and mMCP levels in allergic individuals. 1. Content of β-Hex.

[0021] In some embodiments, the pharmaceutical product further comprises a drug carrier and / or pharmaceutical excipients.

[0022] Compared with the prior art, the beneficial effects of the present invention are as follows: This invention screened a probiotic strain of Bifidobacterium longum subsp. CCFM1522, which relieved constipation in a constipation model constructed using loperamide in female mice after giving birth, shortened the time to expel the first black stool (reduced by 29.69%), increased the number of black stools at 5 hours (increased by 173.64%), the weight of black stools (166.13%), and the small intestinal propulsion rate (31.11%), and promoted intestinal peristalsis.

[0023] Furthermore, in a food allergy experiment involving probiotic intervention in the construction of progeny ovalbumin (OVA), the probiotic showed a mitigating effect. Specifically, when young mice were given Bifidobacterium longum subsp. longum CCFM1522 probiotic alone, compared with the model group, the apparent scores of young mice regarding food allergy were significantly reduced, and the levels of OVA-sIgE in serum, mMCP-1 and β-Hex in jejunal tissue decreased by 60.04%, 42.80%, and 73.91%, respectively, alleviating the food allergy symptoms in young mice. In the mother-to-child vertical transmission pathway, compared with the model group, after mother mice were given Bifidobacterium longum subsp. longum CCFM1522, the apparent scores of lactating mice regarding food allergy were significantly reduced, and the levels of OVA-sIgE in serum, mMCP-1 and β-Hex in jejunal tissue decreased by 57.12%, 74.74%, and 80.73%, respectively, alleviating the food allergy symptoms in young mice.

[0024] 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 is *Bifidobacterium longum* subsp. *longum* (…). Bifidobacterium longum subsp. Longum Bifidobacterium longum 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 longum subsp. longum involved in this patent have all passed the ethics committee review and are listed in the "List of Microbial Strains that Can Be Used in Food".

[0025] Preservation of biological materials A strain of *Bifidobacterium longum* subspecies CCFM1522 is taxonomically named as follows: Bifidobacterium longum subsp. Longum Figure 1It was deposited on November 6, 2025 at the Guangdong Provincial Center for Microbial Culture Collection, located at 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, with accession number GDMCC No: 67226. Attached Figure Description

[0026] Figure 2 To investigate the epigenetic indicators of postpartum constipation in female rats treated with piperidinamide.

[0027] Figure 3 This is a flowchart of an experiment on a female mouse.

[0028] Figure 4 For female mice, the epigenetic indicators are...

[0029] Figure 5 These are indicators of inflammatory factors in female mice.

[0030] Figure 6 These are indicators related to the intestinal mechanical barrier in female mice.

[0031] Figure 7 Flowchart for animal experiments on food allergies in young mice.

[0032] Figure 8 Appearance scores were assigned to young mice.

[0033] Bifidobacterium longum subsp. Longum The values ​​represent the levels of OVA-sIgE, β-Hex, and mMCP-1 in young mice. Detailed Implementation

[0034] The preferred embodiments of the present invention are described below. It should be understood that the embodiments are for better explanation of the present invention and are not intended to limit the present invention.

[0035] Test method: The following examples illustrate methods for detecting gene expression levels. To determine gene expression levels using real-time quantitative polymerase chain reaction (qRT-PCR), RNA must first be extracted from fresh tissue. The specific method is as follows: 0.2g of fresh tissue taken from dissected mice was repeatedly ground in a mortar (180℃, 4 h high-temperature enzyme inactivation) with liquid nitrogen. Then, 1 mL of TrizoL reagent was added to the mortar, and grinding continued until the liquid was basically clear. The tissue was then collected into a 1.5 mL enzyme-free centrifuge tube and allowed to stand at room temperature for 15 min. 200 μL of chloroform solution was added to the centrifuge tube, and the tube was gently shaken for 15 s. The tube was allowed to stand at room temperature for 10 min, and then centrifuged at 4℃ and 12000 r / min for 15 min. 600 μL of the colorless upper aqueous phase was transferred to another enzyme-free centrifuge tube, and 500 μL of isopropanol was added. Invert the tube to mix thoroughly, let stand at room temperature for 10 min, then centrifuge at 12000 r / min for 10 min at 4℃. Discard the supernatant, leaving the white precipitate formed at the bottom of the centrifuge tube containing RNA. Add 1 mL of 75% ethanol solution prepared with DEPC water, vortex to resuspend, and centrifuge at 7500 r / min for 5 min at 4℃. Discard the supernatant and allow to evaporate and dry at room temperature. Add 30 μL of RNase-free water to the dried RNA. After the RNA dissolves, determine the RNA concentration and purity using Nanodrop, and assess the RNA quality by agarose gel electrophoresis. Using the extracted total RNA as a template, reverse transcribe cDNA according to the instructions of the Comvita HiFiScript gDNA RemovaL RT MasterMix reverse transcription kit and store at -20℃.

[0036] The primers for the mouse gene and the internal reference gene β-action are shown in Table 1.

[0037] Table 1. Mouse gene primer sequences

[0038] qRT-PCR reaction system and reaction conditions: PCR amplification was performed using the Bio-Rad® CFX96™ Real-Time PCR instrument, and the fluorescence signal was read.

[0039] Table 2 Reaction System

[0040] The reaction conditions are: The temperature was set at 95℃ for 30 seconds, followed by cycles of 95℃ for 10 seconds and 60℃ for 30 seconds, for a total of 40 cycles. The β-action gene was used as an internal reference, and the results were analyzed using CFX96Manager software.

[0041] Materials used in the examples: The animals and their rearing environment information involved in the following embodiments are as follows: Male Balb / c mice, aged 8 weeks, were purchased from Spefair Company and housed in a specific pathogen-free (SPF) laboratory animal barrier environment with a relative humidity of (50 ± 10)% and a relative temperature of (23 ± 2)℃, with a 12-hour alternation of light and darkness.

[0042] The culture media involved in the following examples are as follows: MRS liquid culture medium: 10g beef extract; 10g tryptone; 5g yeast extract; 20g glucose; 5g anhydrous sodium acetate; 0.1g MgSO4·7H2O; 0.05g MnSO4·H2O; 2g diammonium hydrogen citrate; 2.6g K2HPO4·3H2O; 1mL Tween 80; adjust pH to 6.8±0.2; bring volume to 1L. Autoclave at 115℃ for 20min.

[0043] MRS solid medium: 2% agar powder is added to the MRS liquid medium.

[0044] All culture media contained 0.1% cysteine ​​hydrochloride.

[0045] The 10% skim milk involved in the following examples is: a 10% skim milk powder aqueous solution, sterilized at 105°C for 15 minutes.

[0046] Example 1: Screening and identification of Bifidobacterium longum subspecies CCFM1522 1. Isolation and screening of Bifidobacterium longum subspecies CCFM1522: (l) Collect fecal samples using a disposable sterile fecal collection device, dilute and spread them on MRS (containing 0.1% cysteine ​​hydrochloride) solid medium, and incubate at 37°C for 48 hours under anaerobic conditions; observe and record the colony morphology, pick milky white, smooth colonies for streak purification; incubate at 37°C for 24 hours in MRS (containing 0.1% cysteine ​​hydrochloride) liquid medium, and subculture twice.

[0047] 2. Molecular biological identification of Bifidobacterium longum subspecies CCFM1522: (l) Single-cell genome extraction: Take 1 mL of the bacterial suspension from step 1 into a 1.5 mL centrifuge tube, centrifuge at 10000 rpm for 2 min, discard the supernatant to obtain bacterial cells; wash the bacterial cells with 1 mL of sterile water, centrifuge at 10000 rpm for 2 min, discard the supernatant to obtain bacterial cells; add 200 μL of SDS lysis buffer, incubate at 80℃ for 30 min; add 200 μL of phenol-chloroform solution to the bacterial cell lysis buffer, wherein the composition and volume ratio of the phenol-chloroform solution is Tris saturated phenol:chloroform:isoamyl alcohol = 25:24:1, mix by inversion, centrifuge at 12000 rpm for 5~10 min, take 200 μL of supernatant; add 400 μL of ice-cold ethanol or ice-cold isopropanol to 200 μL of supernatant, incubate at -20℃ for 1 h, centrifuge at 12000 rpm for 5~10 min, discard the supernatant; add 500 Resuspend the precipitate in 70% (v / v) ice-cold ethanol, centrifuge at 12000 rpm for 1-3 min, discard the supernatant; dry in a 60℃ oven or air dry; dissolve the precipitate again in 50 μL ddH2O for PCR preparation. (2) 16S rDNA PCR: A. Bacterial 16S rDNA PCR reaction system: 5 μL; 27F, 0.5 μL; 1492R, 0.5 μL; Taq enzyme, 10 μL; template, 1 μL; ddH2O, 8 μL.

[0048] B. PCR conditions: 95℃ for 5 min; 95℃ for 10 s; 55℃ for 30 s; 72℃ for 30 s; step 2-4 34×; 72℃ for 5 min; 12℃ for 2 min; C. Prepare a 1% agarose gel, then mix the PCR product with 10000× loading buffer, load 2 μL, run at 120V for 30 min, and then perform gel imaging; D. The obtained PCR product was sent to a professional sequencing company. The sequencing results were compared with those obtained by searching and similarity analysis in GeneBank using BLAST, and it was identified as *Bifidobacterium longum* subsp. *longum*. Figure 1 Figure 2 It was named Bifidobacterium longum subsp. CCFM1522 and stored at -80℃ for future use.

[0049] Example 2: Confirmation of the dosage of loperamide to treat postpartum constipation symptoms in female rats The specific steps are as follows: 1. Preparation of loperamide Loperamide solution: The dosage is 10 mg / kg BW and 20 mg / kg BW. Gavage should begin on the 5th day after the female mice give birth, with 0.2 mL administered to each female mouse.

[0050] 2. Animal experiments—Maternal rat animal experiments—Determination of loperamide dosage A certain number of healthy 8-week-old Balb / c female and male mice were housed together at a 1:1 ratio for 5 days to allow for natural mating. After mating, the male mice were removed, and the weight changes of the female mice were monitored regularly to determine their pregnancy status. Fifteen female mice that successfully became pregnant were selected and divided into a control group, a drug-treated group (group 10), and a drug-treated group (group 20). After the drugs were administered by gavage, phenotypic indicators were measured.

[0051] 3. Apparent index detection and experimental results Determination of time to first black stool and number of black stool particles within 5 hours: Time of first black feces excretion: Mix gum arabic powder and water at a ratio of 1:10 until homogeneous, heat on an induction cooker until transparent, stirring constantly during heating. 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 feces excreted by mice was measured on the morning before the end of the experiment. During measurement, each female mouse was gavaged with 0.2 mL of ink, and the specific time of gavage was recorded. The mice's defecation status was closely monitored, and the time when the first feces containing activated charcoal was excreted was recorded. The time difference between the two recorded times is the time of the first black feces excreted by the mouse.

[0052] After the mother mouse was given 0.2 mL of ink via gavage, the timing was started, and the number of black stools within 5 hours was counted to determine the number of black stools in 5 hours.

[0053] The results are as follows Figure 3 As shown in the figure. Regarding the time to first melena, when the drug concentration was 10 mg / kg BW, there was no significant difference between the drug-treated group and the control group; however, when the drug concentration was 20 mg / kg BW, the time to first melena in the drug-treated group was significantly longer than that in the control group (P < 0.01). Regarding the number of melena particles at 5 hours, when the drug concentration was 10 mg / kg BW, there was no significant difference between the drug-treated group and the control group; however, when the drug concentration was 20 mg / kg BW, the number of melena particles at 5 hours in the drug-treated group was significantly lower than that in the control group (P < 0.0001).

[0054] Therefore, it can be seen that when using loperamide to construct a postpartum constipation model in female mice, a drug dosage of 10 mg / kg BW is insufficient to successfully construct the constipation model. However, when the drug dosage reaches 20 mg / kg BW, the time to the first black stool in the female mice significantly increases, and the number of black stool particles within 5 hours significantly decreases, successfully constructing a postpartum constipation model. This differs from the common use of loperamide to construct constipation models (e.g., the drug dosage used in patent CN116083327A is 10 mg / kg BW), where ordinary drug dosages are insufficient.

[0055] Example 3: Bifidobacterium longum subsp. CCFM1522 improves postpartum constipation symptoms in female mice. The specific steps are as follows: 1. Preparation of oral bacterial solution Preparation of *Bifidobacterium longum* subsp. *longum* CCFM1522 and CCFM1240 bacterial cultures: *Bifidobacterium longum* subsp. *longum* CCFM1522 and CCFM1240 were removed from a -80℃ freezer and streaked onto MRS solid medium, incubated at 37℃ for 48 h. Single colonies were picked and incubated in MRS liquid medium at 37℃ for 24 h to prepare seed culture. The prepared seed culture was inoculated into fresh MRS liquid medium at an inoculum rate of 2% (v / v) and incubated at 37℃ for 18 h. This process was repeated for one generation to prepare fermentation broths of *Bifidobacterium longum* subsp. *longum* CCFM1522 and CCFM1240. The prepared fermentation broths were then centrifuged at 8000 r / min and 4℃ for 15 min. The supernatant was discarded, and the bacterial precipitate was retained. 30 mg of [unspecified substance] was added to the precipitate. 60% glycerol (mL). Before use, centrifuge the glycerol bacterial culture to remove the glycerol, retain the bacterial cells, and resuspend them in 10% skim milk to obtain a bacterial suspension. Adjust the concentration of the bacterial suspension to 5 × 10⁻⁶. 10 CFU / mL.

[0056] 2. Preparation of loperamide Loperamide solution: The dosage is 20 mg / kg BW. Gavage should begin on the 5th day after the female mice give birth, with 0.2 mL administered to each female mouse.

[0057] 3. Animal experiments with female rats A certain number (>20) of healthy 8-week-old Balb / c female and male mice were housed together at a 1:1 ratio for 5 days to allow for natural mating. After mating, the male mice were removed, and the weight changes of the female mice were monitored regularly to determine their pregnancy status. Fifteen female mice that successfully became pregnant were selected and randomly divided into three groups after giving birth: a control group, a model group, a Bifidobacterium longum subsp. CCFM1522 intervention group, and a Bifidobacterium longum CCFM1240 intervention group, with 5 mice in each group. The day of birth was recorded as day 0, and intervention began on day 5 and ended after 20 days.

[0058] Modeling method: Female mice in the control group were administered 0.2 mL of sterile water by gavage, while female mice in other groups were administered 0.2 mL of loperamide solution by gavage. The loperamide solution was prepared fresh for each use. Both the control and model groups were administered 0.2 mL of 10% sterile skim milk by gavage daily. The intervention groups of *Bifidobacterium longum* subsp. *longum* CCFM1522 and *Bifidobacterium longum* CCFM1240 were administered 0.2 mL of 10% sterile skim milk by gavage daily, with a bacterial concentration of 5 × 10⁻⁶. 10CFU / mL bacterial suspension.

[0059] The specific experimental groups for the female mice are shown in Table 3, and the experimental procedure is as follows: Figure 4 As shown.

[0060] Table 3 Grouping of female rats in experiments

[0061] 4. Apparent index detection and experimental results Phenotypic indicators were tested two days before the end of the animal experiment. These indicators included: (1) Number of black feces in 5 hours: Start timing when each female mouse is placed in a different cage, collect the number of black feces in 5 hours, and record the number of black feces in each cage as the number of black feces in 5 hours.

[0062] (2) Time of first black stool: Mix gum arabic powder and water at a ratio of 1:10, heat on an induction cooker until transparent, stirring constantly during heating. Then add 10% (w / v) activated charcoal powder, stir and boil until well mixed. After the solution cools, store it in a refrigerator at 4°C. The time of the first black stool in mice was measured on the morning before the end of the experiment. During the measurement, each female mouse was gavaged with 0.2 mL of ink, and the specific time of gavage was recorded. The defecation status of the mice was monitored at all times. The time when the first stool containing activated charcoal was expelled was recorded. The time difference between the two times was the time of the first black stool in mice.

[0063] (3) Weight of black feces: The weight of black feces collected from each female mouse within 5 hours is the weight of black feces.

[0064] (4) Small intestinal propulsion rate: Before sacrifice, each mouse was given 0.2 mL of ink by gavage (see the ink treatment process above). After 30 minutes, the mice were sacrificed and dissected. The upper end of the small intestine from the lower end of the pylorus to the cecum was cut off, and the total length of the small intestine was measured as the total length of the small intestine. The distance from the pylorus to the ink tip is the length of ink propulsion. Calculate the small intestinal propulsion rate using the following formula:

[0065] The experimental results of the apparent indicators are as follows: Figure 5 As shown, compared with the control group, the time for the first black feces to pass in the model group female mice was significantly increased. P <0.001), number of black stool particles in 5 hours ( P <0.01) and a significant decrease in the weight of black stool ( P <0.05), and the small intestinal propulsion rate was significantly reduced ( P<0.001), specifically, the time to first black stool increased by 89.91%, the number of black stools after 5 hours decreased by 60.22%, the weight of black stools decreased by 55.86%, and the small intestinal propulsion rate decreased by 53.90%. According to the positive result criteria for relieving constipation in the "Methods for Functional Testing and Evaluation of Health Foods (2023 Edition)," the use of loperamide to construct a postpartum constipation state in female mice is feasible. After intervention with *Bifidobacterium longum* subsp. *longum* CCFM1522 and *Bifidobacterium longum* CCFM1240, compared with the model group, the time to first black stool in the *Bifidobacterium longum* subsp. *longum* CCFM1522-treated group decreased by 29.69% ( P <0.01), the number and weight of black stool particles after 5 hours increased by 173.64% and 166.13%, respectively. P <0.01), although there was no statistically significant difference in small intestinal propulsion rate ( P >0.05), but still improved by 31.11% compared to the model group. Bifidobacterium longum CCFM1240 only significantly improved small intestinal propulsion rate ( P <0.05), but it did not significantly improve the time to first black stool, the number of black stools after 5 hours, or the weight of black stools. Therefore, CCFM1240 did not relieve postpartum constipation in female mice.

[0066] This indicates that Bifidobacterium longum subsp. CCFM1522 can alleviate postpartum constipation in female mice, and its effect on improving the quantity and weight of black stools within 5 hours is particularly significant.

[0067] Example 4: Bifidobacterium longum subsp. CCFM1522 reduced the expression of inflammatory factors IL-1β / IL-6 / IL-17 in female mice with postpartum constipation. The expression levels of IL-1β, IL-6, and IL-17 genes were determined using quantitative real-time polymerase chain reaction (qRT-PCR). Detailed methods and reaction conditions are described above. IL-1β, IL-6, and IL-17 all exhibit strong pro-inflammatory characteristics, inducing the production of various pro-inflammatory mediators and ultimately leading to widespread inflammatory events. By detecting the gene expression levels of inflammatory factors in the colonic tissue of maternal mice, the inflammatory status of postpartum constipation in maternal mice and the effectiveness of probiotic intervention in alleviating inflammation were assessed. Specific results are shown below. Figure 6 Compared with the control group, the expression levels of inflammatory factors in the colon of mice in the model group were significantly increased, specifically, the expression level of IL-17 increased by 2.649 times. P <0.01), the expression level of IL-1β was significantly increased by 3.151 times ( P <0.01), the expression level of IL-6 was significantly increased by 2.484 times ( PA value <0.05 indicates that when female mice are in a state of postpartum constipation, the expression levels of inflammatory factors in their bodies are significantly increased, indicating a high level of inflammation. After intervention with Bifidobacterium longum subsp. CCFM1522, the gene expression levels of IL-17, IL-6, and IL-1β in colon tissue decreased by 64.00%, 60.20%, and 71.39%, respectively.

[0068] Based on the above data analysis and comparison, it can be found that Bifidobacterium longum subspecies CCFM1522 can effectively reduce the expression of pro-inflammatory factors in the bodies of female mice with postpartum constipation and alleviate the inflammatory state of the body.

[0069] Example 5: Bifidobacterium longum subsp. CCFM1522 maintains intestinal barrier integrity in postpartum constipation in female mice and increases gene expression levels of ZO-1 / cla-3 / CDH1. The expression levels of ZO-1, cla-3, and CDH1 genes in colon tissue were determined using real-time quantitative polymerase chain reaction (qRT-PCR). Detailed methods and reaction conditions are described above. The intestinal epithelial barrier is mainly maintained by tight junctions and adhesion junctions. ZO-1 (Zonula occludens-1), as a key scaffold molecule of tight junction proteins, is responsible for connecting transmembrane proteins to the cytoskeleton, ensuring the integrity of the barrier. CDH1 (E-cadherin) is an important component of adhesion junctions, regulating intercellular adhesion and signal transduction, and helping to maintain epithelial cell polarity and homeostasis. Claudin-3 (CLDN3) is one of the core transmembrane proteins of tight junctions, directly involved in regulating epithelial permeability. These three work synergistically to maintain colonic barrier function and prevent harmful substances from crossing the epithelium into the body. Figure 7 As shown, compared with the control group, the gene expression levels of ZO-1, cla-3, and CDH1 in the model group female mice decreased by 139.625%, 98.44%, and 123.94%, respectively (P < 0.01), indicating disruption of the intestinal barrier integrity. After intervention with Bifidobacterium longum subsp. CCFM1522, the expression levels of ZO-1, cla-3, and CDH1 in the female mice increased by 2.96-fold (P < 0.01). P <0.001), 2.29 times ( P <0.01), 2.39 times ( P <0.01), the expression levels of related genes were similar to those of the control group, or even increased.

[0070] This indicates that Bifidobacterium longum subsp. CCFM1522 can restore the intestinal barrier damage in female mice with postpartum constipation to normal levels.

[0071] The expression level of AQP3 gene in colonic tissue was determined by real-time quantitative polymerase chain reaction (qRT-PCR). Detailed methods and reaction conditions are described above. Aquaporins-3 (AQP3) are responsible for regulating water reabsorption in the intestine and are mainly distributed on the basolateral membrane of colonic epithelial cells. Compared with the control group, the expression level of AQP3 in the colon of model group mother mice was significantly increased. P <0.001), enhanced intestinal reabsorption of water from colonic contents led to constipation, which is consistent with the observation that the weight of black stool within 5 hours was significantly lower in the model group than in the control group. After intervention with *Bifidobacterium longum* subsp. *CCCFM1522*, the expression level of AQP3 in the colon of female mice was significantly reduced ( P <0.01), which reduces the reabsorption of water from the intestinal contents, resulting in increased fecal water content, which is consistent with the results of the apparent indicators.

[0072] Example 6: Experiment on the maternal relief of offspring food allergies by Bifidobacterium longum subsp. CCFM1522 I. Preparation of oral bacterial solution Preparation of *Bifidobacterium longum* subsp. *longum* CCFM1522 and CCFM1240 bacterial cultures: *Bifidobacterium longum* subsp. *longum* CCFM1522 and CCFM1240 were removed from a -80℃ freezer and streaked onto MRS solid medium. The cultures were incubated at 37℃ for 48 h. Single colonies were picked and incubated in MRS liquid medium at 37℃ for 24 h to obtain seed culture. The seed culture was inoculated into fresh MRS liquid medium at a 2% (v / v) inoculum and incubated at 37℃ for 18 h. This process was repeated for one generation to obtain fermentation broths of *Bifidobacterium longum* CCFM1522 and CCFM1240. The fermentation broth of *Bifidobacterium longum* subsp. *longum* CCFM1522 was then centrifuged at 8000 r / min and 4℃ for 15 min, and 30 mL of 60% glycerol was added. Before use, remove the glycerin, resuspend the bacteria in 10% skim milk to obtain a bacterial suspension, and adjust the concentration of the bacterial suspension to 5 × 10⁻⁶. 10 CFU / mL and 5×10 9 CFU / mL.

[0073] II. Preparation of sensitizing solution The sensitization solution was prepared by dissolving cholerae vibriin adjuvant (CT) and ovalbumin (OVA) in a suitable solvent (sterile water), with a CT concentration of 0.5 μg / g and an OVA concentration of 100 μg / g. Pups were weighed starting on day 5 and administered the solution via gavage at a ratio of body weight to reagent of 1:10. The sensitization solution was administered via gavage every 5 days.

[0074] Activation solution: The concentration of OVA for activation is 1200 μg / g, which is prepared according to the body weight of mice on day 20. By calculating the ratio of mouse body weight to reagent, the corresponding amount of OVA is dissolved in the corresponding amount of sterile water.

[0075] III. Experimental Protocol for Young Rats In Example 2.3, the animal protocol was defined as day 0 of the pups after the mother mouse gave birth. The sex of the pups was determined on day 5, and female pups were selected for subsequent OVA-induced food allergy and probiotic intervention experiments. Detailed grouping and experimental procedures are shown in Table 4 and [Table data missing]. Figure 8 As shown. Sensitization tests were conducted on pups starting on day 7, divided into a blank group, a model group, a *Bifidobacterium longum* subsp. *longum* CCFM1522 group-A (vertical transfer), a *Bifidobacterium longum* subsp. *longum* CCFM1522 group-B (probiotic intervention), a *Bifidobacterium longum* CCFM1240-A group, and a *Bifidobacterium longum* CCFM1240-B group. The pups in the blank group, model group, *Bifidobacterium longum* subsp. *longum* CCFM1522-B group, and *Bifidobacterium longum* CCFM1240-B group were all offspring of mothers in the model group of the maternal experiments. The pups in the *Bifidobacterium longum* subsp. *longum* CCFM1522-A group and the *Bifidobacterium longum* CCFM1240-A group were offspring of mothers in the *Bifidobacterium longum* subsp. *longum* CCFM1522 and *Bifidobacterium longum* CCFM1240-A group, respectively, were offspring of mothers in the *Bifidobacterium longum* subsp. *longum* CCFM1522 and *Bifidobacterium longum* CCFM1240 treatment groups of the maternal experiments. First, the young mice were weighed and their weight recorded daily. Second, the young mice in the *Bifidobacterium longum* subsp. *longum* CCFM1522-B group and the *Bifidobacterium longum* CCFM1240-B group were administered their respective bacterial solutions by gavage daily according to their weight. The young mice in the control group, model group, *Bifidobacterium longum* subsp. *longum* CCFM1522-A group, and *Bifidobacterium longum* CCFM1240-A group were administered skim milk by gavage daily according to their weight. Additionally, on days 7, 12, and 17, the young mice were administered sensitizing solutions by gavage. The control group mice were administered sterile water corresponding to their body weight, while the model group and intervention groups (groups A and B) mice were administered OVA sensitizing solution by gavage (care should be taken during the first gavage to prevent injury to the young mice). On day 22, a challenge test was performed on the young mice. The control group mice were administered sterile water corresponding to their body weight, while the model group and intervention groups (groups A and B) mice were administered OVA challenge solution by gavage. Within 30 minutes after the operation, the pups were assessed for their appearance. After the assessment, blood was drawn and the pups were dissected.

[0076] Table 4

[0077] IV. Appearance Scoring and Results The appearance assessment includes: 0 points for "no allergic symptoms"; 1 point for "repeatedly scratching ears and mouth, scratching ear canals with hind legs, scratching marks on the tail, and rough fur"; 2 points for "reduced activity level, decreased appetite, and disheveled fur"; 3 points for "prolonged immobility, increased respiratory rate, disheveled and dull fur, and piloerection"; 4 points for "protruding eyes, conjunctival congestion, no response to stimulation, tremors, convulsions, and cyanosis of the lips"; and 5 points for "death". The assessment results are shown below. Figure 8 .

[0078] In the normal group, the young mice had smooth and shiny fur and showed no allergic symptoms; in the model group, the mice had messy and dull fur, and some young mice exhibited piloerection; Bifidobacterium longum subsp. CCFM1522-A group ( P <0.05) and Bifidobacterium longum subsp. CCFM1522-B group ( P Both (<0.001) could improve food allergy symptoms in young mice, but in terms of overall phenotypic indicators, the reduction in food allergy symptoms was more significant in the CCFM1522-B group. However, Bifidobacterium longum CCFM1240 did not alleviate the phenotypic symptoms of food allergies in either group A or B.

[0079] This means that *Bifidobacterium longum* subsp. *CCFM1522* can alleviate allergy symptoms in young mice both through maternal administration and through the administration of the probiotic itself. This also demonstrates that not all *Bifidobacterium longum* strains possess the same efficacy as *Bifidobacterium longum* subsp. *CCFM1522*.

[0080] Example 7: Effects of Bifidobacterium longum subsp. CCFM1522 on serum OVA-specific IgE and mMCP in food-allergic mice 1. β-Hex has a relieving effect. After euthanizing young mice by enucleation and blood collection, the blood was allowed to stand for 2 hours and then centrifuged at 3000 rpm. Serum was collected, and the OVA-specific IgE content in the mouse serum was measured using an ELISA kit. The levels of mMCP-1 and β-Hex in the jejunal tissue of young mice were quantified using ELISA. The specific method was as follows: the jejunal tissue was rinsed with pre-cooled PBS to remove residual blood and surrounding adipose tissue. After weighing, the tissue was minced. The minced tissue was mixed with PBS solution at a weight-to-volume ratio of 1:9 and homogenized using a high-throughput tissue homogenizer. Finally, the homogenate was centrifuged at 5000×g for 5 minutes. After 10 minutes, collect the supernatant for analysis. Perform the experiment according to the corresponding kit instructions, and calculate the levels of mMCP-1 and β-Hex in the tissue based on the standard curve. See below for detailed results. ​ .

[0081] Depend on ​As shown, for the OVA-sIgE index, compared with the model group of young mice, the serum OVA-sIgE content of young mice in the Bifidobacterium longum subsp. CCFM1522-A group was significantly lower. P <0.05), a decrease of 57.12%, and the OVA-sIgE content in the serum of young mice in the Bifidobacterium longum subsp. ciliate CCFM1522-B group was significantly reduced ( P <0.01), a decrease of 64.04%. In summary, the food allergy symptoms of the young mice in both CCFM1522-A and CCFM1522-B groups were alleviated. The levels of mMCP-1 and β-Hex in the jejunal tissue of young mice in the Bifidobacterium longum subsp. CCFM1522-A group were significantly lower than those in the model group. P <0.01, P <0.0001), representing reductions of 74.74% and 80.73%, respectively. In the jejunal tissue of young mice in the *Bifidobacterium longum* subsp. *CCFM1522-B* group, the contents of mMCP-1 and β-Hex were lower than in the model group, decreasing by 42.80% and 73.91%, respectively, with a significant decrease in β-Hex. P <0.05) Overall, administering Bifidobacterium longum subsp. CCFM1522 probiotics to mother mice after giving birth or administering Bifidobacterium longum subsp. CCFM1522 to pups alone reduced the levels of OVA-sIgE, mMCP-1, and β-Hex in OVA-induced food allergy mice.

[0082] 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 longum subsp. ( Bifidobacterium longum subsp. Longum CCFM1522, characterized in that, The *Bifidobacterium longum* subspecies was deposited at the Guangdong Provincial Center for Microbial Culture Collection on November 6, 2025, with accession number GDMCC No: 67226.

2. A microbial inoculant, characterized in that, The microbial agent contains the long subspecies of Bifidobacterium longum CCFM1522 as described in claim 1.

3. The microbial agent according to claim 2, characterized in that, In the microbial inoculant, the amount of *Bifidobacterium longum* subsp. *CCFM1522* 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 longum subsp. CCFM1522 as described in claim 1, or the microbial agent as described in claim 2.

5. The pharmaceutical product according to claim 4, characterized in that, The amount of *Bifidobacterium longum* subsp. *CCFM1522* added to the drug is at least 1 × 10⁻⁶. 6 CFU / mL or 1×10 6 CFU / g.

6. The use of the Bifidobacterium longum subsp. CCFM1522 as described in claim 1 or the microbial agent as described in any one of claims 2 to 3 in the preparation of a medicine for relieving and / or alleviating postpartum constipation.

7. The application according to claim 6, 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 postpartum individual intestinal mechanical barrier damage; (3) Regulate the level of inflammatory factors in postpartum individuals.

8. The use of the Bifidobacterium longum subsp. CCFM1522 as described in claim 1 or the microbial agent as described in any one of claims 2 to 3 in the preparation of a medicine for preventing, alleviating and / or reducing food allergies in offspring.

9. The application according to claim 8, characterized in that, The applications include alleviating food allergy symptoms in offspring through mother-to-child vertical transmission and reducing OVA-sIgE and mMCP levels in allergic individuals.

1. Content of β-Hex.

10. The application according to any one of claims 6 to 9, characterized in that, The drug also contains a drug carrier and / or pharmaceutical excipients.

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