A compound probiotic for relieving constipation and its application

Abstract

This invention relates to a compound probiotic for relieving constipation and its application, wherein the compound probiotic includes Bifidobacterium animalis subsp. lactis (GDMCC No: 65455). Bifidobacterium animal subsp. lactis Strain C-2 and Lactococcus lactis subsp. lactis with accession number CGMCC No. 15805 Lactococcus lactis subsp. lactis Strain 4021. There is a potential interaction between the two strains, enabling them to cooperate and synergistically enhance each other's effects, significantly improving constipation. Specifically, this is manifested in increasing fecal water content, improving small intestinal transit efficiency, regulating the levels of gastrointestinal regulatory peptides, and modulating inflammatory factor levels. This indicates that this compound probiotic has excellent application prospects in the preparation of formulations that regulate the intestinal environment.

Description

Technical Field

[0001] This invention belongs to the field of probiotic technology and relates to a compound probiotic for relieving constipation and its application. Background Technology

[0002] With changes in dietary structure, the increased intake of high-oil, high-fat, and low-fiber foods, coupled with the mental stress brought about by a fast-paced lifestyle, has significantly affected the normal function of the intestines, leading to slowed intestinal peristalsis and even causing gastrointestinal diseases such as constipation. Constipation seriously affects people's quality of life. Long-term constipation not only causes adverse reactions such as abdominal discomfort and loss of appetite, but also causes many harms to health. For example, severe cases may lead to anorectal diseases such as hemorrhoids and anal fissures, and further increase the risk of irritable bowel syndrome and colon cancer in people with constipation.

[0003] Currently, traditional methods for treating constipation have significant limitations. While commonly used osmotic laxatives and laxatives can alleviate related symptoms to some extent, they are often accompanied by adverse reactions such as diarrhea, electrolyte imbalance, and easy relapse after discontinuation of the medication. These side effects not only bring new troubles to patients, but may also cause other health problems.

[0004] As beneficial members of the gut microbiota, probiotics are increasingly recognized for their importance in maintaining normal intestinal function and improving the gut microecological environment in gut health research. Constipation patients often exhibit a significant imbalance in their gut microbiota composition, along with a marked decrease in gut microbial diversity. Probiotics, as an effective means of regulating the gut microbiota, can restore gut microbiota balance, improve the gut microecological environment, alleviate constipation and other gastrointestinal diseases, and enhance the body's metabolic level. Therefore, developing a probiotic product that can effectively relieve constipation has significant application value. Summary of the Invention

[0005] In view of the shortcomings of the existing technology, the purpose of this invention is to provide a compound probiotic for relieving constipation and its application.

[0006] To achieve this objective, the present invention adopts the following technical solution: In a first aspect, the present invention provides a compound probiotic for relieving constipation, wherein the compound probiotic includes Bifidobacterium animalis subsp. lactis (GDMCC No: 65455). Bifidobacterium animalis subsp. lactis Strain C-2 and *Lactococcus lactis* subsp. *lactococcus* with accession number CGMCC No. 15805 Lactococcus lactis subsp.lactis 4021 strain.

[0007] This invention develops a novel probiotic compounding method and a novel strategy for relieving constipation, namely, Bifidobacterium animalis subsp. lactis. Bifidobacterium animalis subsp. lactis Strain C-2 and Lactococcus lactis subsp. lactis of CGMCC No. 15805 Lactococcus lactis subsp.lactis A combination of strain 4021 was conducted, revealing a potential interaction between the two strains. They were found to work synergistically, with the same bacterial count. Compared to intervention methods lacking either strain, the combination significantly improved the relief of constipation-related symptoms, specifically by: (1) increasing fecal water content; (2) improving small intestinal transit efficiency; (3) regulating the levels of gastrointestinal regulatory peptides; and (4) modulating inflammatory factor levels. Therefore, this compound probiotic has promising applications in preparing formulations that regulate the intestinal environment. Furthermore, since both strains are probiotics, their use in preparing products with relevant efficacy is safe and unlikely to induce dependence.

[0008] The preparation method of the compound probiotics can be carried out using conventional techniques in the art. For example, it can be as follows: after activating the two strains, they are separately inoculated into a culture medium for cultivation to obtain a culture solution; the culture solution is centrifuged, and the bacterial suspension is resuspended to obtain a bacterial suspension; the two bacterial suspensions are mixed according to the required live bacteria ratio. Alternatively, a protective agent can be added for freeze-drying to obtain a freeze-dried bacterial powder product.

[0009] Preferably, the culture medium includes MRS medium or TPY medium.

[0010] Preferably, the MRS culture medium comprises, in concentration: 8-12 g / L peptone, 8-12 g / L beef extract, 15-25 g / L glucose, 1-3 g / L sodium acetate, 3-7 g / L yeast extract, 1-3 g / L diammonium citrate, 2-3 g / L K₂PO₄·3H₂O, 0.05-0.2 g / L MgSO₄·7H₂O, 0.01-0.1 g / L MnSO₄, 0.5-2 mL / L Tween 80, and 0.1-1 g / L cysteine ​​hydrochloride.

[0011] Preferably, the TPY culture medium comprises, in concentration: 8-12 g / L hydrolyzed casein, 3-7 g / L soybean peptone, 1-3 g / L yeast extract, 3-7 g / L glucose, 0.1-1 g / L L-cysteine, 1-4 g / L dipotassium hydrogen phosphate, 0.1-1 g / L magnesium chloride, 0.1-1 g / L zinc sulfate, 0.05-0.5 g / L calcium chloride, 0.0001-0.01 mg / L ferric chloride, and 0.5-2 mL / L Tween 80.

[0012] Preferably, the ratio of viable bacteria count of strain C-2 to strain 4021 is 1:10-10:1, for example, it can be 1:10, 1:9, 1:8, 1:7, 1:6, 1:5, 1:4, 1:3, 1:2, 1:1, 2:1, 3:1, 5:1, 6:1, 8:1, 10:1, etc. Other specific values ​​within this range can be selected, and will not be elaborated here.

[0013] Based on the potential interaction between the two strains, the present invention has also found that when the two strains are used in combination at the specific live bacteria ratio described above, they have a more significant effect in relieving constipation-related symptoms.

[0014] In a second aspect, the present invention provides a probiotic agent for relieving constipation, wherein the strains in the probiotic agent include the compound probiotics described in the first aspect.

[0015] Preferably, the total live bacteria content in the probiotic agent is not less than 1×10⁻⁶. 9 CFU / mL or 1×10 9 CFU / g, for example, could be 1×10 9 CFU / mL (CFU / g), 2×10 9 CFU / mL (CFU / g), 5×10 9 CFU / mL (CFU / g), 8×10 9 CFU / mL (CFU / g), 1×10 10 CFU / mL (CFU / g), 5×10 10 CFU / mL (CFU / g), 1×10 11 CFU / mL (CFU / g), etc. Other specific point values ​​within this range can be selected, which will not be elaborated here.

[0016] Preferably, the dosage form of the probiotic agent includes solution, lyophilized powder, capsule, tablet or granule.

[0017] Preferably, the probiotic agent further includes a freeze-drying protectant.

[0018] Preferably, the freeze-drying protectant includes any one or a combination of at least two of the following: skim milk, sucrose, lactose, trehalose, dextran, gelatin, dextrin, gum arabic, sodium alginate, polyvinylpyrrolidone, sorbitol, or xylitol.

[0019] Preferably, the probiotic agent is in the form of a solution, which is prepared by the following method: Strain C-2 and strain 4021 were inoculated into the culture medium and activated and fermented sequentially to obtain fermentation broth. The fermentation broth was centrifuged and resuspended in solvent to obtain C-2 bacterial suspension and 4021 bacterial suspension. The C-2 bacterial suspension and 4021 bacterial suspension were mixed according to the live bacteria ratio to obtain the probiotic agent.

[0020] Preferably, the probiotic agent is in the form of a lyophilized powder, which is prepared by the following method: Strain C-2 and strain 4021 were inoculated into culture medium and activated and fermented sequentially to obtain fermentation broth. The fermentation broth was centrifuged, mixed with a protectant, and then freeze-dried to obtain C-2 bacterial powder and 4021 bacterial powder. The C-2 bacterial powder and 4021 bacterial powder were mixed according to the live bacteria ratio to obtain the probiotic agent.

[0021] Thirdly, the present invention provides the use of the compound probiotics according to the first aspect or the probiotic agent according to the second aspect in the preparation of formulations for regulating the intestinal environment.

[0022] Preferably, the formulation further includes pharmaceutically acceptable excipients.

[0023] Preferably, the excipients include any one or a combination of at least two of the following: fillers, pH adjusters, antioxidants, binders, wetting agents, disintegrants, emulsifiers, cosolvents, solubilizers, osmotic pressure regulators, colorants, antibacterial agents, or buffers.

[0024] Compared with the prior art, the present invention has the following beneficial effects: This invention develops a novel probiotic compounding method and a novel strategy for relieving constipation, namely, Bifidobacterium animalis subsp. lactis. Bifidobacterium animalis subsp. lactis Strain C-2 and Lactococcus lactis subsp. lactis of CGMCC No. 15805 Lactococcus lactis subsp.lactis A combination of strain 4021 was conducted, revealing a potential interaction between the two strains. They were found to work synergistically, with the same bacterial count. Compared to intervention methods lacking either strain, the combination significantly improved the relief of constipation-related symptoms, specifically by: (1) increasing fecal water content; (2) improving small intestinal transit efficiency; (3) regulating the levels of gastrointestinal regulatory peptides; and (4) modulating inflammatory factor levels. Therefore, this compound probiotic has promising applications in preparing formulations that regulate the intestinal environment. Furthermore, since both strains are probiotics, their use in preparing products with relevant efficacy is safe and unlikely to induce dependence.

[0025] The C-2 strain involved in this invention is classified and named as follows: Bifidobacterium animalis subsp. lactisThe depositary institution is Guangdong Provincial Center for Microbial Culture Collection, with accession number GDMCC No: 65455, deposit date November 8, 2024, and deposit address 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou.

[0026] The strain 4021 involved in this invention is classified as *Lactococcus lactis* subsp. *lactococcus*. Lactococcus lactis subsp. lactis The depositary institution is the China General Microbiological Culture Collection Center (CGMCC), with accession number CGMCC No. 15805, deposit date May 24, 2018, and deposit address No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing. Attached Figure Description

[0027] Figure 1 This is a graph showing the statistical results of the weight analysis of mice in each experimental group.

[0028] Figure 2 This is a graph showing the statistical results of the water content of mouse feces in each experimental group.

[0029] Figure 3 This is a graph showing the statistical results of the time when mice in each experimental group excreted their first black feces.

[0030] Figure 4 This is a graph showing the statistical results of small intestinal transit rate in mice from each experimental group.

[0031] Figure 5 This is a graph showing the statistical results of the levels of excitatory and inhibitory gastrointestinal regulatory peptides in the serum of mice in each experimental group.

[0032] Figure 6 This is a graph showing the statistical results of short-chain fatty acid content in mice from each experimental group.

[0033] Figure 7 This is a graph showing the statistical results of the expression levels of colonic mucin and tight junction protein in mice of each experimental group.

[0034] Figure 8 This is a graph showing the statistical results of the content of inflammatory factors in the colon tissue of mice in each experimental group.

[0035] Figure 9 This is a graph showing the statistical results of the levels of inflammatory factors in the serum of mice in each experimental group. Detailed Implementation

[0036] The technical solution of the present invention will be further illustrated below through specific embodiments. Those skilled in the art should understand that the embodiments described are merely illustrative of the present invention and should not be construed as limiting the invention in any way.

[0037] The following information pertains to the bacterial species: ① The C-2 strains involved in the following examples are classified and named as follows: Bifidobacterium animalis subsp. lactis The accession number is GDMCC No: 65455; ② The strain 4021 involved in the following examples is classified as Lactococcus lactis subsp. lactis. Lactococcus lactis subsp. lactis The accession number is CGMCC No. 15805; ③ The following examples involve ATCC 27673 strain, which is Bifidobacterium animalis subsp. lactis ATCC 27673 strain.

[0038] The test results below show the differences between groups using the abcd alphabetical notation method. The same letter indicates that the difference between groups is not significant, and different letters indicate that the difference is significant. The significance level is α=0.05.

[0039] Example This embodiment explores the effect of the compound probiotics provided by the present invention on improving various indicators in constipated mice: (1) 6-week-old BALB / c mice (weight 19±1g) were kept in an environment of 22℃, 55% humidity, 12 hours of lighting, and were acclimatized for one week (free access to food and water).

[0040] (2) Animal grouping, modeling and intervention methods ① After one week of acclimatization, mice were randomly divided into 9 groups: S1 (C-2 bacterial suspension), S2 (4021 bacterial suspension), S3 (C-2 bacterial suspension + 4021 bacterial suspension, live bacteria ratio 10:1), S4 (C-2 bacterial suspension + 4021 bacterial suspension, live bacteria ratio 1:1), S5 (C-2 bacterial suspension + 4021 bacterial suspension, live bacteria ratio 1:10), S6 (ATCC 27673 bacterial suspension + 4021 bacterial suspension, live bacteria ratio 10:1), S7 (model group, saline), S8 (positive control group, lactulose, 20 mg / kg), and S9 (blank control group, saline). Each group contained 8 mice. The total live bacteria concentration in each of groups S1-S6 was 1×10⁻⁶. 9 CFU / mL.

[0041] ② Intervention method: During the first week of the experiment, mice in groups S1 to S8 were given loperamide hydrochloride at a dose of 20 mg / kg to establish a constipation model, while mice in group S9 were given sterile saline at a dose of 0.2 mL / mouse. During the second to fourth weeks of the experiment, mice in each group were given the corresponding bacterial suspension or preparation by gavage once a day, 0.2 mL each time, according to the grouping in ①.

[0042] (3) Analysis of various indicators in constipated mice ① Mouse body weight analysis During the experiment, the weight of mice was monitored every 7 days. The weight statistics for each group of mice after the bacterial suspension gavage experiment are as follows: Figure 1 As shown in the figure, the statistical results show that the weight of mice in group S7 (model group) was significantly lower than that in group S9 (blank control group), while the weight of mice in groups S3 to S5 was significantly increased after probiotic intervention. It can be seen that the compound probiotics provided by the present invention can effectively alleviate the weight loss trend caused by constipation.

[0043] ② Fecal moisture content analysis After the experiment, mice in each group were transferred to clean, empty cages, and fecal samples were collected 3 hours later. The fecal samples were collected in tubes and weighed for wet and dry weight. The fecal water content was determined using the following formula, and the results were statistically analyzed as follows. Figure 2 As shown.

[0044] Fecal moisture content (%) = (Fecal wet weight - Fecal dry weight) / Fecal wet weight × 100% Fecal water content is an important indicator of the degree of constipation. Statistical results in the figure show that the fecal water content of mice in group S7 (model group) was significantly lower than that of mice in group S9 (blank control group), indicating that after intervention with loperamide hydrochloride by gavage, the fecal water content decreased, resulting in hard, dry feces, thus indicating the successful establishment of the constipation mouse model. After probiotic intervention, the fecal water content of mice in groups S3 to S5 was significantly higher than that of group S7 (model group), demonstrating that the compound probiotics provided in this invention can effectively improve the dry stool symptoms in constipated mice.

[0045] ③ Analysis of the time of first black stool excretion and small intestinal transit rate Time of first black feces excretion: After the experiment, each group of mice was orally administered 0.2 mL of 20% ink suspension and placed individually in a clean cage. The time at which the mouse excreted its first black feces was recorded as the start time. The time after the mouse excreted its first black feces was recorded as the end time. The interval between these times was the time of first black feces excretion. The statistical results are as follows: Figure 3 As shown.

[0046] Small intestinal transit rate: After the experiment, mice in each group were fasted overnight but allowed free water. Each group of mice was orally administered 0.2 mL of 20% ink suspension. Thirty minutes later, the mice were euthanized by cervical dislocation. The euthanized mice were placed supine on a wooden board and secured with nails. The stomach was located in the left upper abdomen, and the gastroesophageal junction was severed at the pylorus. The intestines were separated, and the small intestine was severed from the colon. The separated intestinal tract was placed on a tray, and the small intestine was gently stretched into a straight line. The length of the intestinal tract was measured as the "total length of the small intestine." The distance from the pylorus to the ink front was measured as the "small intestinal transit rate." The small intestinal transit rate was calculated using the following formula, and the statistical results are as follows: Figure 4 As shown.

[0047] Small intestinal transit rate (%) = Ink propulsion length / Total small intestinal length × 100% Depend on Figure 3 , Figure 4 Statistical results showed that, compared with the S9 group (blank control group), the S7 group (model group) mice had a significantly increased first-fecal-passing time and a significantly decreased small intestinal transit rate. However, after probiotic intervention, the first-fecal-passing time of mice in groups S3 to S5 was significantly shortened, and the small intestinal transit rate was significantly improved. This demonstrates that the compound probiotics composed of strains C-2 and 4021 provided in this invention can effectively improve constipation-related symptoms.

[0048] ④ Analysis of gastrointestinal regulatory peptide levels After the experiment, mouse serum was collected and centrifuged at 571 g for 15 min at 4°C. The levels of excitatory gastrointestinal regulatory peptides motilin (MTL) and gastrin (Gas), as well as inhibitory gastrointestinal regulatory peptides vasoactive intestinal peptide (VIP) and somatostatin (SS), were determined according to the ELISA kit instructions. The results are as follows: Figure 5 As shown.

[0049] The statistical results in the figure show that, compared with group S9 (blank control group), the serum levels of GAS and MTL in mice in group S7 (model group) decreased significantly, while the levels of VIP and SS increased significantly. This indicates that loperamide hydrochloride has an inhibitory effect on excitatory gastrointestinal regulatory peptides in mice, and constipation leads to changes in gastrointestinal bioactive peptides, inhibiting intestinal muscle motility and thus weakening intestinal peristalsis. After probiotic intervention, the levels of gastrointestinal regulatory peptides in mouse serum were correspondingly improved. Among them, the relevant indicators of mice in groups S3 to S5 were significantly better than those in group S7 (model group) and tended to be similar to those in group S9 (blank control group).

[0050] ⑤ Short-chain fatty acid level analysis During week 4 of the experiment, fecal samples from each group of mice were collected. The levels of short-chain fatty acids (SCFAs) in the feces were quantitatively analyzed by gas chromatography-mass spectrometry (GC-MS). Specifically, 20 mg of fecal sample was weighed into a centrifuge tube, immersed in a saturated sodium chloride solution, and stirred until no obvious lumps remained. Then, 20 μL of 10% H₂SO₄ solution was added for acidification. Next, 500 μL of anhydrous diethyl ether was added for extraction. The extracted mixture was centrifuged at 18000 g for 10 min to obtain the supernatant. This supernatant was transferred to a tube containing 0.25 g of anhydrous Na₂SO₄ for drying. After standing for 30 min, it was centrifuged at 12000 g for 5 min to obtain the upper ether phase, which was then transferred to a sample vial for GC analysis. The results are as follows: Figure 6 As shown.

[0051] The statistical results in the figure show that, compared with the S9 group (blank control group), the levels of acetic acid, propionic acid, isobutyric acid, butyric acid, isovaleric acid, and valeric acid in the feces of the S7 group (model group) mice were significantly reduced. After probiotic intervention, the levels of related short-chain fatty acids in the mouse fecal samples all showed varying degrees of increase. The group using a combination of strains C-2 and 4021 showed the best intervention effect. Therefore, the compound probiotics provided by this invention can effectively alleviate the problem of decreased short-chain fatty acid levels caused by constipation.

[0052] ⑥ Analysis of expression levels of colon tissue-related proteins After the experiment, colon tissues from mice in each group were collected, and RNA was extracted from the mouse colon tissues according to the instructions of the animal tissue / cell RNA extraction kit. The RNA was reverse transcribed into cDNA, and then real-time quantitative PCR was performed according to the kit instructions to analyze the expression levels of mucin (MUC2) and three tight junction proteins (ZO-1, Claudin-1, and Occudin) in the mouse intestinal tissue. The results are as follows: Figure 7 As shown.

[0053] Constipation damages the intestinal mucosal layer, leading to an inflammatory response that affects tight junction proteins between epithelial cells and alters intestinal permeability. Statistical results in the figure show that the expression levels of four proteins in the colon of mice in group S7 (model group) were lower than those in group S9 (blank control group). After probiotic intervention, the levels of related proteins in the colon of mice in each intervention group showed varying degrees of upregulation. Among them, the compound probiotic intervention in groups S3 to S5 showed the best effect. This demonstrates that the compound probiotic provided by this invention has a positive effect on improving intestinal mucosal barrier function. It can repair the intestinal mucosal structure and function damaged by constipation by promoting the expression of related proteins, thus maintaining the stability of the intestinal environment.

[0054] ⑦ Analysis of colonic inflammatory marker levels After the experiment, colon tissue and serum were collected from mice in each group. Real-time quantitative PCR was used to detect inflammatory factors in the colon tissue, including tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), and interleukin-10 (IL-10). The results are shown below. Figure 8 and Figure 9 As shown.

[0055] TNF-α and IL-1β are two highly potent pro-inflammatory cytokines that play important roles in inflammatory responses. IL-1β can guide inflammatory cells into lesion sites, promote the expression of vascular leukocyte adhesion molecules, and induce an inflammatory response. TNF-α mainly induces various cells to produce other inflammatory factors in the early stages of inflammation, generating important synergistic effects. IL-10 is a multicellular, multifunctional cytokine that plays an important regulatory role in the immune system, regulating cell growth and differentiation, and participating in inflammatory and immune responses.

[0056] The statistical results in the figure show that, compared with group S9 (blank control group), the levels of TNF-α and IL-1β, two pro-inflammatory factors, in the colon tissue and serum of mice in group S7 (model group) were significantly increased, while the relative expression level of the anti-inflammatory factor IL-10 was significantly decreased. This indicates that constipation can lead to an inflammatory response in mice. After probiotic intervention, the inflammatory response was improved, demonstrating that the compound probiotics provided by this invention can effectively reduce the release of inflammatory factors in the body and alleviate intestinal damage.

[0057] The applicant declares that the technical solution of this invention is illustrated by the above embodiments, but this invention is not limited to the above embodiments, that is, it does not mean that this invention must rely on the above embodiments to be implemented. Those skilled in the art should understand that any improvements to this invention, equivalent substitutions of raw materials for the products of this invention, addition of auxiliary components, selection of specific methods, etc., all fall within the protection scope and disclosure scope of this invention.

[0058] The preferred embodiments of the present invention have been described in detail above. However, the present invention is not limited to the specific details in the above embodiments. Within the scope of the technical concept of the present invention, various simple modifications can be made to the technical solution of the present invention, and these simple modifications all fall within the protection scope of the present invention.

[0059] It should also be noted that the various specific technical features described in the above specific embodiments can be combined in any suitable manner without contradiction. In order to avoid unnecessary repetition, the present invention will not describe the various possible combinations separately.

Claims

1. A compound probiotic for relieving constipation, characterized in that, The compound probiotics include Bifidobacterium animalis subsp. lactis, GDMCC No: 65455. Bifidobacterium animalissubsp. lactis Strain C-2 and *Lactococcus lactis* subsp. *lactococcus* with accession number CGMCC No. 15805 Lactococcus lactis subsp.lactis 4021 strain.

2. The compound probiotic for relieving constipation according to claim 1, characterized in that, The ratio of viable bacteria counts of strain C-2 to strain 4021 is 1:10-10:

1.

3. A probiotic agent for relieving constipation, characterized in that, The strains in the probiotic agent include the compound probiotics described in claim 1 or 2.

4. The probiotic agent for relieving constipation according to claim 3, characterized in that, The total live bacteria content in the probiotic agent is not less than 1×10⁻⁶. 9 CFU / mL or 1×10 9 CFU / g.

5. The probiotic agent for relieving constipation according to claim 3, characterized in that, The dosage forms of the probiotic agent include solutions, lyophilized powders, capsules, tablets, or granules.

6. The probiotic agent for relieving constipation according to claim 3, characterized in that, The probiotic agent also includes a freeze-drying protectant.

7. The probiotic agent for relieving constipation according to claim 6, characterized in that, The freeze-drying protectant includes any one or a combination of at least two of the following: skim milk, sucrose, lactose, trehalose, dextran, gelatin, dextrin, gum arabic, sodium alginate, polyvinylpyrrolidone, sorbitol, or xylitol.

8. The probiotic agent for relieving constipation according to claim 5, characterized in that, The probiotic agent is in the form of a solution, which is prepared by the following method: Strain C-2 and strain 4021 were inoculated into the culture medium and activated and fermented sequentially to obtain fermentation broth. The fermentation broth was centrifuged and resuspended in solvent to obtain C-2 bacterial suspension and 4021 bacterial suspension. The C-2 bacterial suspension and 4021 bacterial suspension were mixed according to the live bacteria ratio to obtain the probiotic agent.

9. The probiotic agent for relieving constipation according to claim 5, characterized in that, The probiotic agent is in the form of a lyophilized powder, which is prepared by the following method: Strain C-2 and strain 4021 were inoculated into culture medium and activated and fermented sequentially to obtain fermentation broth. The fermentation broth was centrifuged, mixed with a protectant, and then freeze-dried to obtain C-2 bacterial powder and 4021 bacterial powder. The C-2 bacterial powder and 4021 bacterial powder were mixed according to the live bacteria ratio to obtain the probiotic agent.

10. The use of a compound probiotic according to claim 1 or 2 or a probiotic agent according to any one of claims 3-9 in the preparation of a formulation for regulating the intestinal environment.

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