Application of bifidobacterium longum subsp. Longum capable of relieving constipation and improving anxiety

Abstract

The invention discloses application of bifidobacterium longum subsp. Longum capable of relieving constipation and improving anxiety, and belongs to the field of microorganisms. According to the invention, a strain of Bifidobacterium longum CCFM1467 is screened, and plays a role in relieving constipation in a mouse diarrhea colon model, specifically, the number of defecation grains and the water content of excrement of a mouse are increased, and the first black excrement discharge time of the mouse is shortened; furthermore, crowd experiments are carried out on the bifidobacterium longum subsp. Longum CCFM1467, and it is found that the probiotics CCFM1467 can have a remarkable relieving effect on the constipation symptom of a patient, can relieve anxiety and has great application value.

Description

Technical Field

[0001] The present invention relates to the application of a strain of Bifidobacterium longum subsp. longum capable of relieving constipation and improving anxiety, and belongs to the field of microorganisms. Background Art

[0002] Constipation is a group of symptoms, manifested as difficult defecation and / or decreased frequency of defecation, and hard stools. Difficult defecation includes straining during defecation, difficult evacuation, a feeling of incomplete defecation, a sense of anorectal blockage, time-consuming defecation, and the need for assisted defecation. Decreased frequency of defecation refers to less than 3 bowel movements 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 medical treatment rate is only 17%. Moreover, with the aging of society, the change of diet structure, and the influence of social and psychological factors, the incidence of constipation shows an increasing trend year by year. In a study on functional constipation in the elderly, it was found that constipation patients often have anxiety, but their underlying mechanisms are different. For example, the causes of constipation include: slow intestinal motility, excessive water absorption of intestinal contents, nervous system and endocrine factors, intestinal flora imbalance, etc. The formation factors of anxiety and depression include: stress and the imbalance of the hypothalamic-pituitary-adrenal axis (HPA axis), changes in neurotransmitters, psychosocial factors, immune system and inflammatory responses, etc. In addition, long-term constipation not only has a great negative impact on the mental health of patients, but also poses a huge disease risk to the body, and even leads to cancer.

[0003] At present, the drugs used to treat constipation have more or less problems such as poor taste, weak effect, large toxic and side effects, poor patient adaptability, and easy drug dependence. Therefore, it is urgent to find a safe and effective drug substitute. Probiotics refer to living microorganisms that can have a beneficial effect on the host after ingestion of sufficient quantities. At present, probiotics are gradually being used in various fields due to their safety and mild characteristics. The application of probiotics to relieve constipation is mainly concentrated in Bifidobacterium and Lactobacillus, which mainly regulate the balance of intestinal flora and promote the growth of beneficial bacteria; enhance intestinal peristalsis and accelerate the discharge of food residues; in addition, the organic acids and short-chain fatty acids produced by probiotics can reduce the pH value of the intestine and soften the stool; stimulate the intestine to produce digestive enzymes, help food digestion and absorption, and reduce the occurrence of constipation. The three probiotics studied in CN202410827431.9 can regulate the intestinal flora and increase the relative abundance of beneficial bacteria, thereby relieving constipation. The composite probiotics evaluated in CN202410874628.8 significantly improve intestinal peristalsis and relieve constipation symptoms. However, almost all patents on probiotics for relieving constipation currently involve animal experiments and lack clinical verification. Animal experiments are suitable for preliminary exploration of substances and mechanisms and cannot represent the results of human experiments. For example, aloin has a good laxative effect in animal experiments, but in human experiments, some people experience side effects such as gastrointestinal discomfort and diarrhea. Therefore, human experiments are the gold standard for verifying the effects of human use and can provide more direct and reliable data on human health and disease status.

[0004] Probiotic products exist in many forms, such as capsules, tablets, powders, liquid beverages, etc. However, these products face some challenges during storage and transportation: first, most probiotic products need to be stored under refrigerated conditions to ensure their activity; second, even if stored under appropriate conditions, the activity of probiotics will gradually decrease over time. Since most probiotic products can only work best when they are in the live state, once the strains die, their effectiveness may be greatly reduced. Compared with live bacteria, dead bacteria do not require strict refrigeration conditions and have excellent stability and long shelf life. Therefore, if dead bacteria can also show certain health benefits, it can greatly reduce production and logistics costs and have great application potential in the food industry. Therefore, if it can be proved that dead bacteria are equally effective, this will provide more flexibility and economy for the production and distribution of probiotic products. However, there are few probiotic products in the prior art that have the same constipation relief effect as live and dead bacteria.

[0005] In summary, the prior art lacks probiotics that can relieve constipation with both live and dead bacteria, and that can relieve constipation and anxiety in clinical trials. Summary of the invention

[0006] [Technical issues]

[0007] In the prior art, there is a lack of probiotics that can relieve constipation and anxiety in clinical experiments.

[0008] [Technical Solution]

[0009] The present invention provides a probiotic strain that can relieve constipation. In addition to having the effect of relieving constipation in animals, it also has the effect of relieving constipation clinically and can effectively relieve anxiety. The probiotic is Bifidobacterium longum subsp. longum CCFM1467. In animal experiments, CCFM1467 has a good alleviating effect on cathartic colon induced by senna leaf extract. Specifically, compared with the model group, it can increase the fecal water content (by 13.4%), the number of defecations in 5 hours (by 68%), and shorten the time to defecate the first black stool (by 26.7%); in a multi-center, double-blind, randomized controlled human experiment, CCFM1467 can relieve constipation, improve the fecal characteristics (91.06%), reduce the impact of constipation on the quality of life of patients (42.91%), and the total effective rate is 63.6%. It can even relieve the anxiety and depression emotions of constipation patients to a certain extent (37.62%, 16.67%). The product forms include but are not limited to microbial preparations, fermented foods, health products, etc.

[0010] The present invention provides a strain of Bifidobacterium longum subsp. longum CCFM1467, which was deposited at the Guangdong Provincial Culture Collection Center of Microorganisms on February 28, 2025, with the deposit number GDMCC No: 65852.

[0011] The present invention also provides a microbial agent, which contains live cells, dead cells or fermentation supernatant of the above-mentioned Bifidobacterium longum subsp. longum CCFM1467.

[0012] In one embodiment, the dosage form of the microbial agent includes liquid preparation or powder solid preparation.

[0013] In one embodiment, when the microbial agent contains cells of Bifidobacterium longum subsp. longum CCFM1467, the cell count of Bifidobacterium longum subsp. longum CCFM1467 is at least 1×10 9 CFU / mL or 1×10 9 CFU / g.

[0014] The present invention also provides a product, which contains the above-mentioned Bifidobacterium longum subsp. longum CCFM1467 or the above-mentioned microbial agent, and the product is a food, a drug or a health product.

[0015] In one embodiment, the dosage form of the drug includes granules, capsules, tablets, pills or oral liquids.

[0016] In one embodiment, the food includes dairy products, soy products, fruit and vegetable products or meat products.

[0017] The present invention also provides the application of the microbial inoculum in the preparation of a drug for relieving constipation and / or relieving depression, and the drug has at least one of the following functions:

[0018] (1) Reducing the constipation symptoms of patients, shortening the toilet time, increasing the defecation frequency of constipated patients, and relieving the hardening of feces;

[0019] (2) Significantly improving the quality of life of constipated patients and reducing the impact of constipation on patients' lives;

[0020] (3) Significantly reducing the occurrence of abdominal cramps, defecation pain and other conditions in patients;

[0021] (4) Relieving the anxiety and depression emotions of constipated patients.

[0022] In one embodiment, the drug further contains a drug carrier and / or pharmaceutical excipients.

[0023] The present invention also provides the application of the Bifidobacterium longum subsp. longum CCFM1467 or the microbial inoculum in the preparation of a health product helpful for moistening the intestine and relieving constipation.

[0024] Beneficial effects:

[0025] The present invention screened a probiotic Bifidobacterium longum subsp. longum CCFM1467, which has the effect of relieving constipation in a mouse cathartic colon model, specifically manifested as increasing the number of defecations and the water content of feces in mice, and shortening the time for mice to defecate the first black feces; further conducting a human experiment on Bifidobacterium longum subsp. longum CCFM1467, it was found that the probiotic CCFM1467 can significantly relieve the constipation symptoms of patients, specifically manifested as: reducing the impact of constipation on patients' lives (42.91%), improving the fecal characteristics of constipated patients (91.01%), alleviating the constipation symptoms of patients (34.67%), and alleviating the anxiety (37.62%) and depression emotions (16.67%) of constipated patients, and the overall effective rate is 63.60%.

[0026] Biological material preservation

[0027] A strain of Bifidobacterium longum subsp. longum CCFM1467, with its taxonomic name being Bifidobacterium longum, was deposited at the Guangdong Microbial Culture Collection Center on February 28, 2025, with the deposit number GDMCC No: 65852. Description of the Drawings

[0028] Figure 1 : Effects of Bifidobacterium longum CCFM1467 on the apparent indexes of constipation; * indicates p < 0.05, ** indicates p < 0.01, *** indicates p < 0.001, **** indicates p < 0.0001 (compared with the model group CC); # indicates p < 0.05, ## indicates p < 0.01, indicates p < 0.001, # indicates p < 0.0001 (compared with the blank group NC);

[0029] Figure 2 : Effects of Bifidobacterium longum CCFM1319 on the apparent indexes of constipation;

[0030] Figure 3 : Results of the population experiment questionnaire; Note: Different groups of data were compared using Mann - Whitney unpaired comparison. * indicates significant difference between the bacterial powder group and the placebo group, * indicates p < 0.05, ** indicates p < 0.01, *** indicates p < 0.001. Detailed Implementation Manner

[0031] The information on animals and breeding environments involved in the following examples is as follows:

[0032] Male 8 - week - old C57 / 6J mice were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. and were raised in a specific pathogen - free (SPF) - level experimental animal barrier. The breeding environment had a relative humidity of (50 ± 10)% and a relative temperature of (23 ± 2)°C, with a 12 - hour alternation of light and darkness.

[0033] The media involved in the following examples are as follows:

[0034] MRS liquid medium: Beef extract 10g; Tryptone 10g; Yeast powder 5g; Glucose 20g; Sodium acetate anhydrous 5g; MgSO4·7H2O 0.1g; MnSO4·H2O 0.05g; Ammonium citrate dibasic 2g; K2HPO4·3H2O 2.6g; Tween 80 1mL; Adjust the pH to 6.8 ± 0.2; Make up the volume to 1L. Autoclave at 115°C for 20 minutes.

[0035] MRS solid medium: Add 2% agar powder to the basis of MRS liquid medium.

[0036] Food-grade MRS medium ( / L): Edible yeast extract 10 g (purchased from Angel Yeast Co., Ltd.), edible glucose 20 g (purchased from Shandong Hairong Biotechnology Co., Ltd.), soy peptone 10 g (Henan Wanbang Chemical Technology Co., Ltd.), edible magnesium sulfate 0.1 g (purchased from Laizhou Laiyu Chemical Co., Ltd.), edible manganese sulfate 0.05 g (purchased from Lianyungang Longtaiwei Food Ingredients Co., Ltd.), edible sodium acetate 5 g (purchased from Jiangsu Kelunduo Food Ingredients Co., Ltd.), edible ammonium citrate 2 g (purchased from Jiangsu Kelunduo Food Ingredients Co., Ltd.), edible dipotassium hydrogen phosphate 2 g (purchased from Xuzhou Fengrui Biotechnology Co., Ltd.).

[0037] The 10% skim milk involved in the following examples is an aqueous solution of skim milk powder with a mass fraction of 10%, sterilized at 105 °C for 15 min.

[0038] Example 1: Screening and identification of Bifidobacterium longum subsp. longum CCFM1467

[0039] 1. Isolation and screening of Bifidobacterium longum subsp. longum CCFM1467:

[0040] (l) Collect fecal samples from a 78-year-old elderly person using a disposable sterile stool collector, dilute and spread them on MRS (containing 1% cysteine hydrochloride) solid medium, and culture at 37 °C for 48 hours in an anaerobic environment; observe and record the colony morphology, pick colonies that are milky white and have a smooth surface for streak purification; culture in MRS (containing 1% cysteine hydrochloride) liquid medium at 37 °C for 24 hours and passage 2 times.

[0041] 2. Molecular biological identification of Bifidobacterium longum subsp. longum CCFM1467:

[0042] (l) Single-bacterium genomic DNA extraction: Take 1 mL of the bacterial suspension cultured in Step 1 into a 1.5 mL centrifuge tube, centrifuge at 10,000 rpm for 2 min, and discard the supernatant to obtain the bacterial cells; after rinsing the bacterial cells with 1 mL of sterile water, centrifuge at 10,000 rpm for 2 min, and discard the supernatant to obtain the bacterial cells; add 200 μL of SDS lysis solution, and incubate in a water bath at 80 °C for 30 min; add 200 μL of phenol-chloroform solution to the bacterial cell lysate, where the composition and volume ratio of the phenol-chloroform solution is Tris-saturated phenol:chloroform:isoamyl alcohol = 25:24:1, invert and mix well, then centrifuge at 12,000 rpm for 5 - 10 min, and take 200 μL of the supernatant; add 400 μL of ice ethanol or ice isopropanol to 200 μL of the supernatant, let stand at -20 °C for 1 h, centrifuge at 12,000 rpm for 5 - 10 min, and discard the supernatant; add 500 μL of 70% (volume percentage) ice ethanol to resuspend the precipitate, centrifuge at 12,000 rpm for 1 - 3 min, and discard the supernatant; dry in an oven at 60 °C or air dry naturally; dissolve the precipitate with 50 μL of ddH2O for PCR;

[0043] (2) 16S rDNA PCR:

[0044] A. 20 μL PCR reaction system for bacterial 16S rDNA: 5 μL; 27F, 0.5 μL; 1492R, 0.5 μL; Taq enzyme, 10 μL; template, 1 μL; ddH2O, 8 μL.

[0045] 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 for 34 cycles; 72 °C for 5 min; 12 °C for 2 min;

[0046] C. Prepare 1% agarose gel, then mix the PCR product with 10,000×loading buffer, load 2 μL, run at 120 V for 30 min, and then perform gel imaging;

[0047] D. Send the obtained PCR product to a professional sequencing company, search and perform similarity alignment with the obtained sequencing results using BLAST in GeneBank, identify it as Bifidobacterium longum subsp. longum, name it Bifidobacterium longum subsp. longum CCFM1467, and store it at -80 °C for standby.

[0048] Example 2: Improvement of laxative-induced constipation symptoms in mice by Bifidobacterium longum subsp. longum CCFM1467

[0049] The specific steps are as follows:

[0050] I. Preparation of the gavage bacterial solution

[0051] Preparation of Bifidobacterium longum subsp. longum CCFM1467 bacterial liquid: After taking out Bifidobacterium longum subsp. longum CCFM1467 from the -80°C refrigerator, streak it on MRS solid medium and culture it at 37°C for 48 h. Pick a single colony and inoculate it into MRS liquid medium, then culture it at 37°C for 24 h to prepare a seed liquid; inoculate the prepared seed liquid into a new MRS liquid medium at an inoculation amount of 2% (v / v), culture it at 37°C for 18 h, and culture it for another generation in the same way to prepare the fermentation liquid of Bifidobacterium longum subsp. longum CCFM1467; then centrifuge the prepared fermentation liquid of Bifidobacterium longum subsp. longum CCFM1467 at 8000 r / min and 4°C for 15 min. Resuspend a part of it with 10% skim milk to prepare a bacterial suspension, and adjust the concentration of the bacterial suspension to 6×10 9 CFU / mL.

[0052] Preparation of heat-killed Bifidobacterium longum subsp. longum CCFM1467 bacterial liquid: Pasteurize the live bacteria with a concentration of 6×10 9 CFU / mL and repeatedly freeze-thaw it in the -80°C refrigerator. Take 1 tube and perform viable bacteria counting by the plate pour method. After determining that there is no viable bacteria growth, store it frozen in the -80°C refrigerator.

[0053] II. Treatment of senna leaf extract

[0054] Prepare different concentrations of senna leaf extract solutions with sterile water from the purchased finished product of senna leaf extract (Xi'an Xuhuang Biotechnology Co., Ltd.) for use in animal experiments.

[0055] III. Animal experiments

[0056] After 32 male C57 / 6J mice at 8 weeks old were acclimated for one week, they were randomly divided into 4 groups, namely the blank control group (NC), the model group (CC), the Bifidobacterium longum live bacteria CCFM1467 intervention group (CCFM1467), and the heat-killed Bifidobacterium longum CCFM1467 intervention group (HKCCFM1467), with 8 mice in each group.

[0057] Except for the blank group, mice in other groups were gavaged with senna leaf solution to construct a cathartic colon model, and the senna leaf solution needed to be prepared freshly before use. One week after stopping the drug, constipation-related indicators were measured to determine whether the model was successfully constructed. After the modeling was completed, an intervention was carried out for two weeks. The blank group and the model group were gavaged with 0.2 mL of 10% sterilized skim milk every day. The Bifidobacterium longum live bacteria CCFM1467 intervention group was gavaged with 0.2 mL of a bacterial suspension with a bacterial concentration of 6×10 9 CFU / mL resuspended in 10% sterilized skim milk. The heat-killed Bifidobacterium longum CCFM1467 intervention group was gavaged with 0.2 mL of the inactivated bacteria with a concentration of 6×10 9Bacterial suspension of CFU / mL.

[0058] The specific animal experiment process is shown in Table 1.

[0059] Table 1 Animal experiment process

[0060]

[0061] IV. Detection of apparent indexes

[0062] The detection of apparent indexes was completed 2 days before the end of the animal experiment. The detection indexes include:

[0063] ① Number of fecal pellets in 5 h: Start timing when each mouse is placed in a different cage. Collect the feces within 5 - 6 h, and record the number of feces in each cage as the number of fecal pellets within 5 - 6 h.

[0064] ② Time to first black feces: Mix gum arabic powder and water at a ratio of 1:10 evenly, heat it to transparency on an induction cooker, stir constantly during heating, then add 10% (w / v) activated carbon powder, stir and boil until evenly mixed, and store the solution in a 4°C refrigerator after cooling. Measure the time to first black feces of mice on the morning of the day before the end of the experiment. To ensure the accuracy of the results, mice need to fast overnight without water before measurement. During measurement, each mouse is gavaged with 0.2 mL of the above-mentioned ink, and the gavage time is recorded. Keep an eye on the defecation status of the mouse at all times. When the first feces containing activated carbon is excreted, record the time. The time difference between the two is the time to first black feces of the mouse.

[0065] ③ Fecal moisture content: This index 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 separately. Record the weights of the feces of the mouse before and after freeze-drying, and calculate the fecal moisture content according to the following formula:

[0066]

[0067] V. Experimental results

[0068] As Figure 1As shown, compared with the normal group, the time for the first black stool excretion in the model group of mice was significantly increased (P < 0.0001), the fecal water content was significantly decreased (P < 0.0001), and the number of feces in 5 h was significantly reduced (P < 0.001). This indicates that the mice treated with senna leaf extract did indeed exhibit symptoms of constipation, and the model was successful. After treatment with probiotics and heat-killed bacteria, the apparent indicators of constipation in the mice changed to varying degrees. According to the positive result determination criteria for relieving constipation in the "Functional Test and Evaluation Methods for Health Foods (2023 Edition)", both the viable and heat-killed bacteria of probiotic CCFM1467 can relieve constipation to a certain extent, and the viable bacteria of probiotic CCFM1467 have the best effect in relieving constipation, specifically manifested as follows: compared with the model group, it can increase the fecal water content (by 13.4%), the number of defecations in 5 h (by 68.0%), and shorten the time for the first black stool excretion (by 26.7%).

[0069] As Figure 2 shown, when viable Bifidobacterium longum subsp. CCFM1319 (CCFM1319) was intervened, it could significantly reduce the intestinal transit time of constipated mice, and significantly increase the defecation frequency and fecal water content; after intervention with heat-killed CCFM1319 (HKCCFM1319), only the defecation frequency of constipated mice was increased. It can be seen that viable CCFM1319 can relieve constipation, but heat-killed CCFM1319 does not have the effect of relieving constipation.

[0070] In contrast, the advantages of CCFM1467 are further highlighted, that is, both heat-killed and viable CCFM1467 have the effect of relieving constipation. This indicates that during the production process of CCFM1467, even if the viability of probiotics decreases or even fails due to long transportation time or improper storage, it still has the effect of relieving constipation, which provides a lot of convenience for the actual production process.

[0071] Example 3: Preparation of probiotic powder

[0072] Bacterial sludge: Bifidobacterium longum subsp. CCFM1467 was cultured in a food-grade MRS medium at 37 °C for 24 h, centrifuged to discard the supernatant, and the bacterial sludge was collected. After washing with sterile water and centrifuging again to discard the supernatant, the bacterial sludge was collected.

[0073] Freeze-drying protectant: skim milk powder 200 g / L, mannitol 300 g / L. The ratio of bacterial sludge to freeze-drying protectant is 1:3 w / w.

[0074] Freeze-drying: After mixing the bacterial sludge and the protectant evenly, it was pre-cooled at -80 °C overnight and then placed in a freeze-dryer for freeze-drying.

[0075] CCFM1467 powder: 0.2 g of bacterial sludge, 1.8 g of maltodextrin, and each gram of powder contains 10 9Live bacteria of CFU, 2 g of bacterial powder per bag.

[0076] Example 4: Population experiment on probiotics for improving constipation

[0077] I. Volunteer recruitment and experimental process

[0078] Inclusion criteria for volunteer recruitment: (1) Unrestricted by gender and race, aged 18 or above; (2) Must meet the diagnostic criteria for Rome IV functional constipation and exclude functional defecation disorders; (3) Have not used any drugs affecting gastrointestinal motility in the past week; (4) Have not taken probiotic products in the past week; (5) Good compliance, able to comply with the regulations according to the requirements of the clinical trial; (6) The subject or guardian has signed the informed consent form; (7) The patient fills in the written informed consent form, basic situation questionnaire, dietary structure and living condition questionnaire, and promises to cooperate with the research throughout the trial period.

[0079] 1. The diagnostic criteria for functional constipation are as follows:

[0080] (1) At least meet 2 or more of the following criteria:

[0081] a. Straining during defecation or a sense of urgency to defecate (more than 25% of the time);

[0082] b. Passing lumpy or hard stools (more than 25% of the time) (Bristol Stool Form Scale types 1-2);

[0083] c. A feeling of incomplete evacuation (more than 25% of the time);

[0084] d. A feeling of obstruction or blockage in the anorectum (more than 25% of the time);

[0085] e. Needing manual maneuvers to assist defecation (such as digital evacuation, supporting the pelvic floor) (more than 25% of the time);

[0086] f. Fewer than 3 spontaneous bowel movements per week.

[0087] (2) Rarely have loose stools without taking laxatives;

[0088] (3) Not yet meeting the diagnosis of irritable bowel syndrome (IBS);

[0089] (4) Symptoms have been present for ≥6 months before diagnosis and meet the above diagnostic criteria in the past 3 months.

[0090] 2. The diagnostic criteria for functional defecation disorders (must include all of the following conditions):

[0091] (1) The patient must meet the diagnostic criteria for functional constipation and / or constipation-predominant irritable bowel syndrome

[0092] (2) Characteristic decreased elimination function during repeated attempts to defecate, as confirmed by two of the following three tests:

[0093] a. Abnormal balloon ejection test;

[0094] b. Abnormal anorectal defecation pattern shown by pressure measurement or perianal electromyography;

[0095] c. Etiology examination shows decreased rectal emptying ability.

[0096] (3) Symptoms occurred for at least 6 months before diagnosis and met the above diagnostic criteria in the past 3 months.

[0097] 3. Volunteer exclusion criteria: (1) Those who need to take other laxatives at the same time; (2) Patients who refuse to sign the informed consent form, or whose compliance is estimated to be poor and the possibility of follow-up is poor; (3) Those who have taken probiotic products within 2 weeks; (4) Those who have taken antibiotics within 2 weeks; (5) Subjects who are considered by the researchers to be unsuitable for inclusion.

[0098] A total of 93 volunteers were recruited at the time of enrollment and randomly divided into two groups, 48 ​​in the placebo group and 45 in the probiotic group. The trial period was 4 weeks. The placebo group took 2 placebos per day, and the probiotic intervention group took 2 probiotic powders per day. Both groups took the medicine half an hour to one hour after breakfast and dinner. They can be taken directly or with warm water (not exceeding 37°C). During the entire experiment, feces were sampled and questionnaires were filled out 3 times (before enrollment, 2 weeks after enrollment, and 4 weeks after enrollment). After the 4-week experiment, a total of 35 people in the placebo group completed the experiment, and 40 people in the probiotic group completed the experiment.

[0099] 2. Observation Project

[0100] All volunteers collected feces on the 0th day, the second weekend, and the fourth weekend of the experiment, and filled out scales to record changes in various indicators.

[0101] (1) Fecal collection: After signing the informed consent form, distribute fecal sampling cups to volunteers and ask them to collect feces. Please inform the volunteers to collect samples within the specified time, collect about 1 / 3 of the bottle of samples, and place them in a low-temperature environment.

[0102] (2) The scale includes (a total of 5 assessment scales, the higher the score, the more severe the constipation):

[0103] ① The Constipation Severity Scale (CSS) describes the typical symptoms of constipation, including decreased bowel movement frequency, longer toilet time, the need for assistance to defecate, and the urge to defecate but inability to defecate.

[0104] ② Symptom Index of Constipation Patients (PAC-SYM): PAC-SYM is used to describe these symptoms and reflect the defecation status of patients. Because in addition to reduced defecation frequency and hard stools, typical symptoms of constipation also include abdominal cramps, pain during defecation, anal burning sensation, etc.

[0105] ③ Quality of Life Score (PAC-QOL): It is used to describe the impact of constipation on patients' daily lives. For example, due to constipation, the appetite may decrease, and patients may feel nervous, depressed, and restless. The higher the value, the greater the negative impact of constipation on patients' lives.

[0106] ④ Self-Rating Anxiety Scale is used to describe the anxiety status of constipation patients. A SAS score of 1.00 ≤ SAS score < 1.40 indicates mild anxiety, 1.40 ≤ SAS score < 1.80 indicates moderate anxiety, and 1.80 ≤ SAS score indicates severe anxiety.

[0107] ⑤ Self-Rating Depression Scale is used to describe the depression status of constipation patients. A SDS score of 1.12 ≤ SDS score < 1.52 indicates mild depression, 1.52 ≤ SDS score < 1.92 indicates moderate depression, and 1.92 ≤ SDS score indicates severe depression.

[0108] The Symptom Self-Rating Form for Constipation Patients (PAC-SYM), Constipation Symptom Score (CSS), Quality of Life Score for Constipation Patients (PAC-QOL), Psychological Anxiety Status Score (SAS), and Psychological Depression Status Score (SDS) were all in the form of online questionnaires. The analysis results are shown in Figure 3 as follows; The information sheets of the subjects with anxiety and depression before enrollment are shown in Table 2 and Table 3 respectively.

[0109] Table 2 Information Sheet of Subjects with Anxiety Symptoms before Enrollment

[0110]

[0111] Table 3 Information Sheet of Subjects with Depression Problems before Enrollment

[0112]

[0113] The higher the fecal form score, the softer the feces, and the better the effect of relieving constipation.

[0114] (3) Analysis of the Effective Rate

[0115] The calculation method of the effective rate was formulated with reference to the efficacy evaluation criteria in the "Rome IV Criteria for Functional Gastrointestinal Disorders" and the "Guiding Principles for Clinical Research of New Chinese Medicines" in 2016.

[0116] Efficacy Index = [(Total Score before Treatment - Total Score after Treatment) / Total Score before Treatment] × 100%.

[0117] 30% ≤ efficacy index < 70% indicates effective cases; 70% ≤ efficacy index indicates markedly effective cases.

[0118] Total effective rate = [(number of effective cases + number of markedly effective cases) / total number of cases] × 100%

[0119] We mainly measured the effect of the bacterial powder on relieving constipation through 5 indicators: the Patient Assessment of Constipation - Symptom (PAC - SYM), Constipation Symptom Score (CSS), Patient Assessment of Constipation - Quality of Life (PAC - QOL), Self - rating Anxiety Scale (SAS), and Self - rating Depression Scale (SDS). By adding up the scores of these 5 indicators in the questionnaire survey, we focused on the improvement effect of the bacterial powder on these indicators to measure its effective rate in relieving constipation.

[0120] The results are shown in Table 4. In terms of relieving constipation symptoms, the effective rate of the placebo was 8.8%, and the effective rate of the CCFM1467 bacterial powder was 63.6%. There was a highly significant difference between the effective rate of the CCFM1467 bacterial powder and that of the placebo (p < 0.0001).

[0121] Table 4 Effective rates of each group in relieving constipation symptoms

[0122]

[0123] III. Experimental Results

[0124] In the population experiment, the baseline period refers to the severity of constipation of the subjects at the time of enrollment. The evaluation indicators mainly come from 5 questionnaire surveys (Patient Assessment of Constipation - Symptom (PAC - SYM), Constipation Symptom Score (CSS), Patient Assessment of Constipation - Quality of Life (PAC - QOL), Self - rating Anxiety Scale (SAS), and Self - rating Depression Scale (SDS)). The study found that there was no significant difference in the scores of the constipation symptom evaluation indicators of the subjects in the placebo group and the bacterial treatment group at the baseline period, indicating that the severity of constipation of the two groups of subjects was the same. For the same treatment group, comparing before and after the intervention (for example, before vs. after placebo intervention; before vs. after CCFM1467 intervention), for the placebo: there was no significant difference in the scores of the 5 constipation symptom evaluation scales before and after the intervention, indicating that the placebo had no effect on relieving constipation. However, after the intervention with Bifidobacterium longum subsp. CCFM1467, there were significant differences in the scores of the above 5 constipation symptom evaluation scales, such as Figure 3As shown, after 2 weeks of CCFM1467 probiotic intervention, the constipation severity index (CSS) of the patients showed a significant decrease (P < 0.001, 66.13%), and the fecal form changed significantly (from dry and hard to soft and loose) (P < 0.01, 46.34%). At the same time, in the analysis results of the Patient Assessment of Constipation - Symptom (PAC - SYM) and Patient Assessment of Constipation - Quality of Life (PAC - QOL) for constipation patients, it was also found that after taking the probiotic powder of Bifidobacterium longum subsp. CCFM1467 for 2 weeks, the defecation situation of constipation patients could be significantly changed (P < 0.001, 37.66%), and the quality of life of the patients was significantly improved (P < 0.001, 40.28%). Considering that constipation may have a certain impact on the patients' psychology, we mainly re - analyzed the Self - Rating Anxiety Scale (SAS) of 14 subjects with anxiety symptoms and the Self - Rating Depression Scale (SDS) of 24 subjects with depressive symptoms at the time of enrollment. It was found that after taking the CCFM1467 probiotic powder for 2 weeks, the depression and anxiety conditions of the patients were improved to a certain extent (P < 0.05, 12.69%, P < 0.05, 30.95%), especially the effect on improving the anxiety of constipation patients was more significant.

[0125] After the 4 - week experiment of probiotic intervention, compared with before the intervention, the scores of the Patient Assessment of Constipation - Symptom of the patients decreased significantly (P < 0.001, 34.67%), the scores of the quality of life decreased significantly (P < 0.001, 42.91%), the constipation severity index decreased significantly (P < 0.01, 26.08%), the fecal form changed significantly (from dry and hard to soft and loose) (P < 0.001, 91.06%), the self - rating anxiety score decreased significantly (P < 0.01, 37.62%), and the self - rating depression score decreased significantly (P < 0.05, 16.67%). This indicates that there is a certain positive correlation between the extension of the time of taking probiotics and the improvement effect of the patients' quality of life.

[0126] Finally, by calculating the effective rate, it was found that the effective rate of CCFM1467 probiotic in relieving constipation was 63.60%. The current data all show that taking CCFM1467 probiotics for a period of time (at least 2 weeks) can relieve the symptoms of constipation patients and can, to a certain extent, soothe the mental health of the patients.

[0127] Although the present invention has been disclosed above with preferred embodiments, it is not intended to limit the present invention. Anyone familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the protection scope of the present invention should be defined by the claims.

Claims

1. A Bifidobacterium longum microorganism CCFM1467, characterized in that, The Bifidobacterium longum subsp. longum was deposited at the Guangdong Microbial Culture Collection Center on February 28, 2025, with the deposit number GDMCC No: 65852.

2. A microbial inoculant, characterized in that, The microbial inoculum contains live cells, dead cells or fermentation supernatants of the Bifidobacterium longum subsp. longum CCFM1467 described in claim 1.

3. The microbial inoculum according to claim 2, wherein The dosage form of the microbial inoculum includes liquid preparations or powder solid preparations.

4. The microbial inoculant according to claim 2 or 3, characterized in that, When the microbial inoculum contains cells of Bifidobacterium longum subsp. longum CCFM1467, the cell count of Bifidobacterium longum subsp. longum CCFM1467 is at least 1×10 9 CFU / mL or 1×10 9 CFU / g.

5. A product, characterized in that, The product contains the Bifidobacterium longum subsp. longum CCFM1467 described in claim 1, or the microbial inoculum described in any one of claims 2 to 4, and the product is a food, a drug or a health product.

6. The product according to claim 5, wherein The dosage form of the drug includes granules, capsules, tablets, pills or oral liquids.

7. The product according to claim 6, wherein The food includes dairy products, soy products, fruit and vegetable products or meat products.

8. Use of Bifidobacterium longum subsp. longum CCFM1467 according to claim 1, or the microbial agent according to any one of claims 2 to 4, in the preparation of a medicament for relieving constipation and / or relieving depression, characterized in that, The drug has at least one of the following functions: (1) Reducing the constipation symptoms of patients, shortening the toilet time, increasing the defecation frequency of constipated patients, and relieving the hardening of feces; (2) Significantly improving the quality of life of constipated patients and reducing the impact of constipation on patients' lives; (3) Significantly reducing the occurrence of abdominal cramps, defecation pain and other conditions in patients; (4) Relieving the anxiety and depression of constipated patients.

9. The application according to claim 8, wherein The drug also contains a drug carrier and / or pharmaceutical excipients.

10. Use of the Bifidobacterium longum subsp. longum CCFM1467 described in claim 1, or the microbial inoculum described in any one of claims 2 to 4, in the preparation of a health product helpful for moistening the intestines and relieving constipation.

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