Probiotic compositions for improving constipation and their uses

Abstract

This invention provides a probiotic composition for improving constipation and its uses. The probiotic composition of this invention comprises space-occupying bacteria, organic acid-producing bacteria, and oxygen-depleting bacteria, wherein the oxygen-depleting bacteria are *Weizmannii*, the organic acid-producing bacteria are *Lactobacillus* and / or *Lactobacillus lactis*, and the space-occupying bacteria are *Bifidobacterium*. The probiotic composition of this invention can effectively improve constipation and therefore can be used to prevent and / or treat constipation-related gastrointestinal diseases.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology and relates to a probiotic composition for improving constipation and its use in the preparation of products for the prevention and / or treatment of constipation. Background Technology

[0002] In the field of gastrointestinal dysfunction, constipation is a highly prevalent disease that seriously affects quality of life. Its pathological mechanism is closely related to the dysfunction of the "gut-brain axis" and the imbalance of the intestinal microecology.

[0003] Currently, first-line clinical treatment for constipation mainly relies on chemical drugs, such as osmotic laxatives (lactulose) and 5-HT4 receptor agonists (prucalopride). However, long-term use of these drugs can easily lead to electrolyte imbalances, drug dependence, and adverse reactions such as abdominal pain and palpitations. Overall, traditional drugs have single targets and are difficult to restore the inherent functional homeostasis of the intestine.

[0004] Given the limitations of chemical drugs, probiotics and other microecological regulators have become a research hotspot, but existing technologies still face significant bottlenecks.

[0005] First, the efficacy of single strains has limitations. Most studies focus on evaluating the efficacy of single strains, but single strains have limited targets and cannot systematically and comprehensively regulate the gut environment.

[0006] Secondly, the synergistic effect of simple bacterial conjugates is insufficient. For constipation, existing technologies disclose certain single Lactobacillus or Bifidobacterium strains, as well as conjugates or postbiotic preparations composed of them. However, these schemes are mostly simple combinations of strains or applications of single-type products, lacking systematic research and verification of synergistic effects between different bacterial genera and strains.

[0007] Therefore, providing a probiotic composition that can effectively improve functional gastrointestinal disorders, especially constipation, is a technical problem that urgently needs to be solved. Summary of the Invention

[0008] Based on the above-mentioned prior art, the purpose of this invention is to provide a probiotic composition for improving constipation and its uses.

[0009] In a first aspect, the present invention provides a probiotic composition for improving constipation, wherein the probiotic composition comprises space-occupying bacteria, organic acid-producing bacteria, and oxygen-depleting bacteria.

[0010] In this invention, the term "oxygen-depleting bacteria" refers to a class of probiotics that can effectively consume oxygen through their own metabolism in the gastrointestinal environment, thereby reducing the local redox potential (Eh) and creating and maintaining a suitable anaerobic environment for the growth of beneficial anaerobic bacteria.

[0011] In this invention, the oxygen-depleting bacteria are preferably Weizmann's bacteria.

[0012] According to some embodiments of the present invention, the Weizmania is selected from Weizmannia coagulans.

[0013] In this invention, the term "organic acid-producing bacteria" refers to a class of probiotics that can produce and secrete organic acids (e.g., lactic acid, acetic acid, propionic acid, butyric acid, etc.) by metabolizing carbohydrates in the gastrointestinal environment. Organic acid-producing bacteria lower the pH value of the intestinal environment by secreting organic acids, thereby directly or indirectly inhibiting the growth of harmful bacteria and regulating the balance of the intestinal flora.

[0014] In this invention, the organic acid-producing bacteria are preferably Lactobacillus and / or Lactobacillus lactis.

[0015] According to some embodiments of the present invention, the lactobacillus is selected from *Lactobacillus acidophilus*, *Lactobacillus casei*, *Lactobacillus paracasei*, *Lactobacillus crispatus*, *Lactobacillus delbrueckii* ssp. bulgaricus*, *Lactobacillus fermentum*, *Lactobacillus gasseri*, *Lactobacillus shelveticus*, *Lactobacillus johnsonii*, *Lactobacillus plantarum*, *Lactobacillus reuteri*, *Lactobacillus rhamnosus*, and *Lactobacillus salivarius*. One or more of *Lactobacillus salivarius*, *Lactobacillus sakei*, and *Lactobacillus curvus*.

[0016] According to some embodiments of the present invention, the lactobacillus is selected from one or more of Lactiplantibacillus plantarum, Lactiplantibacillus plantarum subsp. argentoratensis, Lactiplantibacillus pentosus, Lactiplantibacillus paraplantarum, Lactiplantibacillus fabifermentans, Lactiplantibacillus tropicalus, and Lactiplantibacillus nakhonrathomensis.

[0017] In this invention, the term "occupying bacteria" refers to a class of probiotics that can competitively adhere to intestinal mucosal epithelial cells and occupy intestinal ecological sites, thereby preventing or reducing the colonization of pathogenic and opportunistic pathogens.

[0018] In this invention, the occupant bacteria is preferably Bifidobacterium.

[0019] According to some embodiments of the present invention, the Bifidobacterium is selected from one or more of Bifidobacterium animalis subsp. lactis, Bifidobacterium longum subsp. infantis, Bifidobacterium adolescentis, Bifidobacterium animalis, Bifidobacterium bifidum, Bifidobacterium breve, Bifidobacterium infantis, and Bifidobacterium longum.

[0020] According to some embodiments of the present invention, the probiotic composition comprises Weizmann's coagulans, Lactobacillus acidophilus, Lactobacillus plantarum, and Bifidobacterium.

[0021] Preferably, the probiotic composition comprises *Weizmannii coagulans*, *Lactobacillus acidophilus*, *Lactobacillus plantarum*, and *Bifidobacterium* in an effective live bacteria concentration ratio of (0.6-1.4):(0.6-9.4):(5-20):(0.6-11.4).

[0022] More preferably, the probiotic composition comprises *Weizmannii coagulans*, *Lactobacillus acidophilus*, *Lactobacillus plantarum*, and *Bifidobacterium* in an effective live bacteria concentration ratio of (0.6-1.4):(0.6-8):(5-20):(0.6-10).

[0023] According to some embodiments of the present invention, the Bifidobacterium is Bifidobacterium longum infantis subsp. and / or Bifidobacterium animalis lactis subsp.

[0024] Preferably, the probiotic composition comprises *Weizmannii coagulans*, *Lactobacillus acidophilus*, *Lactobacillus plantarum*, *Bifidobacterium longum* subsp. *infantii*, and *Bifidobacterium animalis* subsp. *lactobacterium* in an effective live bacteria concentration ratio of (0.6-1.4):(0.6-8):(5-20):(0.6-1.4):(4-10).

[0025] More preferably, the probiotic composition comprises *Weizmannii coagulans*, *Lactobacillus acidophilus*, *Lactobacillus plantarum*, *Bifidobacterium longum* subsp. *infantii* and *Bifidobacterium animalis* subsp. *lactobacterium* in an effective live bacteria concentration ratio of (0.6-1.4):(0.6-8):(5-20):(0.6-1.4):(4-8).

[0026] In this invention, the effective viable bacterial concentration refers to the number of viable bacteria contained in a unit mass or volume of sample, expressed as CFU / g or CFU / ml.

[0027] According to some embodiments of the present invention, the Lactobacillus acidophilus includes a first Lactobacillus acidophilus and a second Lactobacillus acidophilus, wherein the first Lactobacillus acidophilus and the second Lactobacillus acidophilus are different strains from each other.

[0028] Preferably, the probiotic composition comprises *Weizmannii coagulans*, *Lactobacillus acidophilus* var. *mongolica*, *Lactobacillus acidophilus* var. *mongolica*, *Lactobacillus plantarum*, *Bifidobacterium longum* subsp. *infantii*, and *Bifidobacterium animalis* subsp. *lactobacterium* in an effective live bacteria concentration ratio of (0.6-1.4):(2-8):(0.6-1.4):(5-20):(0.6-1.4):(4-10).

[0029] According to some embodiments of the present invention, the *Weizmannia coagulans* is selected from at least one of *Weizmannia coagulans* BC99, *Weizmannia coagulans* HY08866, *Weizmannia coagulans* HY08867, *Weizmannia coagulans* MP08976, and *Weizmannia coagulans* HY08874.

[0030] It should be noted that *Weizmannia coagulans* and *Heyndrickxia coagulans* are names of the same bacterium from different classification and nomenclature periods; therefore, they have the same meaning in this invention. For example, *Weizmannia coagulans* HY08866 is also *Heyndrickxia coagulans* HY08866, and both refer to the same strain.

[0031] According to some embodiments of the present invention, the first Lactobacillus acidophilus and the second Lactobacillus acidophilus are each independently selected from at least one of Lactobacillus acidophilus LA-5, Lactobacillus acidophilus CS003, Lactobacillus acidophilus DDS-1, Lactobacillus acidophilus HY00768, Lactobacillus acidophilus MP01046, Lactobacillus acidophilus HY01039, Lactobacillus acidophilus HY00760 and Lactobacillus acidophilus HY01043.

[0032] According to some embodiments of the present invention, the *Lactobacillus plantarum* is selected from at least one of *Lactobacillus plantarum* HY02946, *Lactobacillus plantarum* MP02908, *Lactobacillus plantarum* MP00442, *Lactobacillus plantarum* HY05181, *Lactobacillus plantarum* HY00050, and *Lactobacillus plantarum* MP00134.

[0033] According to some embodiments of the present invention, the *Bifidobacterium longum* subsp. infantis is selected from at least one of *Bifidobacterium longum* subsp. infantis CS004, *Bifidobacterium longum* subsp. infantis MP08420, and *Bifidobacterium longum* subsp. infantis HY07708.

[0034] According to some embodiments of the present invention, the *Bifidobacterium lactis* subsp. *animal* is selected from at least one of *Bifidobacterium lactis* subsp. *animal* BB-12, *Bifidobacterium lactis* UABla-12, *Bifidobacterium lactis* subsp. *animal* HY09302, *Bifidobacterium lactis* subsp. *animal* MP08129, *Bifidobacterium lactis* subsp. *animal* MP04382 and *Bifidobacterium lactis* MP03316.

[0035] According to some embodiments of the present invention, the probiotic composition comprises Lactobacillus plantarum HY02946, Bifidobacterium longum subsp. infantis CS004, Weizmannii coagulans BC99 or Weizmannii coagulans HY08866 and Lactobacillus acidophilus CS003.

[0036] Preferably, the probiotic composition comprises *Lactobacillus plantarum* HY02946, *Bifidobacterium longum* subsp. *infantica* CS004, *Weizmannii coagulans* BC99 or *Weizmannii coagulans* HY08866, and *Lactobacillus acidophilus* CS003 in an effective live bacteria concentration ratio of (5-20):(0.6-1.4):(0.6-1.4):(0.6-1.4).

[0037] More preferably, the probiotic composition comprises *Lactobacillus plantarum* HY02946, *Bifidobacterium longum* subsp. *infant* CS004, *Weizmannii coagulans* BC99 or *Weizmannii coagulans* HY08866, and *Lactobacillus acidophilus* CS003 in an effective live bacteria concentration ratio of 15:1:1:1.

[0038] According to some embodiments of the present invention, the probiotic composition comprises Lactobacillus plantarum HY02946, Bifidobacterium animalis subsp. lactis BB-12, Bifidobacterium longum subsp. infantis CS004, Weizmannii coagulans BC99 or HY08866, and Lactobacillus acidophilus CS003.

[0039] Preferably, the probiotic composition comprises *Lactobacillus plantarum* HY02946, *Bifidobacterium animalis* subsp. *lactobacter* BB-12, *Bifidobacterium longum* subsp. *infant* CS004, *Weizmannii coagulans* BC99 or HY08866, and *Lactobacillus acidophilus* CS003 in an effective live bacteria concentration ratio of (5-20):(4-10):(0.6-1.4):(0.6-1.4):(0.6-1.4).

[0040] More preferably, the probiotic composition comprises *Lactobacillus plantarum* HY02946, *Bifidobacterium animalis* subsp. *lactobacter* BB-12, *Bifidobacterium longum* subsp. *infant* CS004, *Weizmannii coagulans* BC99 or HY08866, and *Lactobacillus acidophilus* CS003 in an effective live bacteria concentration ratio of 15:6:1:1:1.

[0041] According to a preferred embodiment of the present invention, the probiotic composition comprises *Lactobacillus plantarum* HY02946, *Bifidobacterium animalis* subsp. *lactobacter* BB-12, *Lactobacillus acidophilus* LA-5, *Bifidobacterium longum* subsp. *infant* CS004, *Weizmannii coagulans* BC99 or *Weizmannii coagulans* HY08866, and *Lactobacillus acidophilus* CS003.

[0042] Preferably, the probiotic composition comprises *Lactobacillus plantarum* HY02946, *Bifidobacterium animalis* subsp. *lactobacter* BB-12, *Lactobacillus acidophilus* LA-5, *Bifidobacterium longum* subsp. *infantica* CS004, *Weizmannii coagulans* BC99 or *Weizmannii coagulans* HY08866, and *Lactobacillus acidophilus* CS003 in an effective live bacteria concentration ratio of (5-20): (4-10): (2-8): (0.6-1.4): (0.6-1.4): (0.6-1.4).

[0043] More preferably, the probiotic composition comprises *Lactobacillus plantarum* HY02946, *Bifidobacterium animalis* subsp. *lactobacter* BB-12, *Lactobacillus acidophilus* LA-5, *Bifidobacterium longum* subsp. *infantica* CS004, *Weizmannii coagulans* BC99 or *Weizmannii coagulans* HY08866, and *Lactobacillus acidophilus* CS003 in an effective live bacteria concentration ratio of (5-20): (4-8): (4-8): (0.6-1.4): (0.6-1.4): (0.6-1.4).

[0044] More preferably, the probiotic composition comprises *Lactobacillus plantarum* HY02946, *Bifidobacterium animalis* subsp. *lactobacter* BB-12, *Lactobacillus acidophilus* LA-5, *Bifidobacterium longum* subsp. *infant* CS004, *Weizmannii coagulans* BC99 or *Weizmannii coagulans* HY08866, and *Lactobacillus acidophilus* CS003 in an effective live bacteria concentration ratio of 15:6:6:1:1:1.

[0045] According to another preferred embodiment of the present invention, the probiotic composition comprises *Lactobacillus plantarum* HY02946, *Bifidobacterium animalis* subsp. *lactamase* UABla-12, *Lactobacillus acidophilus* DDS-1, *Bifidobacterium longum* subsp. *infant* CS004, *Weizmannii coagulans* BC99 or *Weizmannii coagulans* HY08866, and *Lactobacillus acidophilus* CS003.

[0046] Preferably, the probiotic composition comprises *Lactobacillus plantarum* HY02946, *Bifidobacterium animalis* subsp. *lactobacter* UABla-12, *Lactobacillus acidophilus* DDS-1, *Bifidobacterium longum* subsp. *infantica* CS004, *Weizmannii coagulans* BC99 or *Weizmannii coagulans* HY08866, and *Lactobacillus acidophilus* CS003 in an effective live bacteria concentration ratio of (5-20): (4-10): (2-8): (0.6-1.4): (0.6-1.4): (0.6-1.4).

[0047] More preferably, the probiotic composition comprises *Lactobacillus plantarum* HY02946, *Bifidobacterium animalis* subsp. *lactobacter* UABla-12, *Lactobacillus acidophilus* DDS-1, *Bifidobacterium longum* subsp. *infantica* CS004, *Weizmannii coagulans* BC99 or *Weizmannii coagulans* HY08866, and *Lactobacillus acidophilus* CS003 in an effective live bacteria concentration ratio of (5-20): (6-10): (2-6): (0.6-1.4): (0.6-1.4): (0.6-1.4): (0.6-1.4) respectively.

[0048] More preferably, the probiotic composition comprises *Lactobacillus plantarum* HY02946, *Bifidobacterium animalis* subsp. *lactobacter* UABla-12, *Lactobacillus acidophilus* DDS-1, *Bifidobacterium longum* subsp. *infant* CS004, *Weizmannii coagulans* BC99 or *Weizmannii coagulans* HY08866, and *Lactobacillus acidophilus* CS003 in an effective live bacteria concentration ratio of 10:8:4:1:1:1.

[0049] In a second aspect, the present invention provides a product for preventing, relieving, improving and / or treating constipation, comprising the probiotic composition according to the first aspect of the present invention.

[0050] According to some embodiments of the present invention, the product further comprises excipients; the excipients are selected from one or more of pharmaceutical excipients, food excipients, and health product excipients.

[0051] In this invention, the terms "pharmaceutical excipient," "pharmaceuticalally acceptable excipient," or similar terms generally refer to substances that facilitate administration of the active agent to a subject and absorption by the subject and can be included in the medicament of this invention without causing significant adverse toxicological effects on the patient.

[0052] In this invention, the term "food additives" or similar terms generally refer to auxiliary materials added during food processing, other than the main raw materials, to improve the quality, processing technology, preservation performance, or nutritional value of the food. They typically do not constitute the main body of the food, but play a crucial role in the final product's appearance, taste, stability, and safety.

[0053] In this invention, the term "health supplement excipient" or similar terms generally refer to auxiliary materials other than the active ingredient in health foods (dietary supplements), used to transform the active ingredient into a dosage form suitable for consumer use and to ensure the stability, safety and effectiveness of the product.

[0054] According to some embodiments of the present invention, non-limiting examples of the excipients may be selected from one or more of excipients, fillers, flow aids, diluents, instant solvents, and disintegrants. Specifically, they are selected from water, NaCl, physiological saline solutions, lactated Ringer's solution, ordinary sucrose, ordinary glucose, binders, fillers, disintegrants, lubricants, coatings, sweeteners, flavorings, salt solutions, alcohols, oils, gelatin, carbohydrates such as lactose, amylose or starch, fatty acid esters, hydroxymethyl cellulose, polyvinylpyrrolidone, and dyes, etc. Such articles can be sterilized, and those skilled in the art will recognize that other excipients may also be used in the present invention.

[0055] According to some embodiments of the present invention, the constipation is selected from functional constipation, slow transit constipation, outlet obstruction constipation, drug-induced constipation, metabolic-endocrine constipation, low volume constipation, dysbiosis constipation, and diarrhea-predominant irritable bowel syndrome.

[0056] According to some embodiments of the present invention, the product is selected from one or more of pharmaceuticals, food, and health products.

[0057] Thirdly, the present invention provides the use of the probiotic composition according to the first aspect of the present invention in the preparation of products for the prevention and / or treatment of constipation.

[0058] According to some embodiments of the present invention, the constipation is selected from functional constipation, slow transit constipation, outlet obstruction constipation, drug-induced constipation, metabolic-endocrine constipation, low volume constipation, dysbiosis constipation, and diarrhea-predominant irritable bowel syndrome.

[0059] According to some embodiments of the present invention, the product is selected from one or more of pharmaceuticals, food, and health products.

[0060] Compared with the prior art, the present invention has at least the following beneficial effects:

[0061] This invention overcomes the technical biases of existing probiotic products, such as single strain function, limited target, and lack of synergy, and develops a probiotic composition suitable for various complex constipation.

[0062] This invention determined the effect of lysates of 28 strains with potential to improve constipation on 5-HT secretion in RIN-14B cells through in vitro experiments. Combining 5-HT-promoting effects with species diversity, the effects on 5-HT secretion in RIN-14B cells were measured after 1:1 mixtures of single strains, dual strains, triple strains, quadrivalent strains, pentavalent strains, and hexavalent strains. The results showed that a quadrivalent probiotic composition containing *Lactobacillus acidophilus*, *Bifidobacterium longum*, *Bifidobacterium animalis*, *Lactobacillus plantarum*, and *Weizmann's coagulans*; a pentavalent probiotic composition containing *Lactobacillus acidophilus*, *Bifidobacterium longum* subsp. *inf.*, *Bifidobacterium animalis* subsp. *liquid.*, *Lactobacillus plantarum*, and *Weizmann's coagulans*; and a hexavalent composition containing two different strains of *Lactobacillus acidophilus*, *Bifidobacterium longum* subsp. *inf.*, *Bifidobacterium animalis* subsp. *liquid.*, *Lactobacillus plantarum*, and *Weizmann's coagulans* were superior to other conjugate compositions in promoting 5-HT production.

[0063] Based on the six-component combination, this invention, through further screening experiments, finally determined two optimal six-component combination schemes: a six-component probiotic composition consisting of *Lactobacillus plantarum* HY02946, *Bifidobacterium animalis* subsp. *lactobacter* BB-12, *Lactobacillus acidophilus* LA-5, *Bifidobacterium longum* subsp. *infant* CS004, *Weizmannii coagulans* BC99, and *Lactobacillus acidophilus* CS003; and a six-component probiotic composition consisting of *Lactobacillus plantarum* HY02946, *Bifidobacterium animalis* subsp. *lactobacter* UABla-12, *Lactobacillus acidophilus* DDS-1, *Bifidobacterium longum* subsp. *infant* CS004, *Weizmannii coagulans* BC99, and *Lactobacillus acidophilus* CS003.

[0064] This invention also demonstrated through in vitro experiments that the hexabiotic composition of this invention can effectively improve defecation behavior in mice, reduce the time to first black stool, average fecal weight, and average fecal water content, and has a good effect on improving functional constipation. Furthermore, the hexabiotic composition of this invention can also promote small intestinal peristalsis in mice with functional constipation, thereby relieving constipation. Attached Figure Description

[0065] The embodiments of the present invention will now be described in detail with reference to the accompanying drawings, wherein:

[0066] Figure 1 The standard curve of the 5-HT standard sample in the embodiment of the present invention is shown. Detailed Implementation

[0067] The present invention will be further described in detail below with reference to specific embodiments. The embodiments given are only for illustrating the present invention and are not intended to limit the scope of the present invention.

[0068] The method for detecting viable bacteria count in this example is as follows:

[0069] ① F1 generation breeding:

[0070] Mycelium powder: Weigh 0.1g of mycelium powder into a 15mL centrifuge tube, add 4.9mL of MRS or TPY broth and mix well;

[0071] Glycerin bacteria: Take glycerin bacteria stored at -80℃, thaw them, and inoculate them into MRS or TPY broth at an inoculation rate of 4% (Lactobacillus or Lactobacillus lactis) or 10% (Bifidobacterium or Weizmannii) and mix well. Incubate anaerobically at 37℃ for 24-48 hours.

[0072] ② F2 generation culture: After mixing the F1 generation bacterial culture, transfer it to MRS or TPY broth at the same inoculation amount and anaerobic static culture at 37℃ for 24h-48h.

[0073] ③ Preparation of 1% solid culture medium: Weigh 52.24g of MRS broth and 36.65g of TPY broth into 1L beakers, add 10g of agar powder to each, and add 1L of distilled water. Mix well, dispense into 400mL glass bottles, autoclave at 118℃ for 15min, and then place in a 50℃ water bath for later use.

[0074] ④ Dilution and Sample Addition: Add 450 μL of PBS to each well of a 96-well plate. After mixing the F2 generation bacterial culture, perform a tenfold serial dilution with PBS (i.e., add 50 μL of bacterial culture to 450 μL of PBS). Take 10 μL of the diluted sample. 5 10 6 and 10 7 Add 100 μL of each dilution solution to the center of a sterile Petri dish, pour in about 15 mL of MRS or TPY semi-solid culture medium and shake well. After drying, incubate upside down at 37°C for 48-72 h.

[0075] ⑤ After incubation, select plates with colony counts between 30-300 CFU for counting to determine the viable count:

[0076] viable bacteria count = average colony count × dilution factor

[0077] Example 1: Screening of Functional Bacteria

[0078] 1. Candidate strains

[0079] Candidate strains are shown in Table 1.

[0080] The applicant's (Hangzhou Yuanda) proprietary strains were stored at -80°C in preservation tubes containing 15% (v / v) glycerol. Commercially purchased strains were in powder form or isolated from products.

[0081]

[0082]

[0083] 2. Determination of 5-HT content

[0084] Experimental methods:

[0085] (1) Reagent preparation

[0086] Preparation of RPMI 1640 complete medium: Add 10 mL FBS and 1 mL double antibiotic to 89 mL RPMI 1640 medium, mix well, and store at 4℃.

[0087] TPY broth preparation: Weigh 26.4g of TPY liquid culture medium, dissolve it in 1000mL of water, autoclave at 121℃ for 15min, and store at 4℃ for later use.

[0088] MRS broth preparation: Weigh 52.24g of MRS broth and dissolve it in 1000mL of water. Autoclave at 121℃ for 15min and store at 4℃ for later use.

[0089] (2) Cell culture

[0090] 1) RIN-14B cell resuscitation

[0091] ① Take out one RIN-14B cell cryopreservation tube stored in the liquid nitrogen tank;

[0092] ② Thaw the cell cryopreservation tubes in a 37°C water bath, remove all the cell slurry and place it in 5 mL of complete RPMI 1640 medium, mix well by pipetting, centrifuge (1000 rpm, 5 min), and discard the supernatant.

[0093] ③ Add 5 mL of complete RPMI 1640 medium, mix thoroughly by pipetting, resuspend the cells, and after the cells are completely mixed and resuspended, take 200 μL, stain with trypan blue, and count the viable cells.

[0094] ④ Inoculate 1.0E+06 cells into a T25 flask, add 5mL of culture medium, and incubate at 37℃ in a 5% CO2 incubator for 48-72h.

[0095] 2) Passaging of RIN-14B cells

[0096] ① Remove the cell culture flask and observe the cell adhesion and confluence under a microscope to see if it reaches more than 80%;

[0097] ② Discard the culture medium and add 3 mL of PBS to the culture flask to gently wash the cells twice;

[0098] ③ Discard the PBS, add 1 mL of trypsin-EDTA, digest in an incubator for 3 min, and add 2 mL of complete culture medium to stop the digestion;

[0099] ④ Centrifuge at 1000 rpm for 5 min, discard the supernatant, add 5 mL of culture medium to resuspend, and take 200 μL, stain with trypan blue, and count the viable cells.

[0100] ⑤ Inoculate 3.0E+06 cells into a T75 flask, add 5mL of culture medium, and incubate at 37℃ in a 5% CO2 incubator for 48-72h.

[0101] 3) RIN-14B cell plate

[0102] ① Aspirate the culture medium from the T75 cell flask, wash twice with 3 mL PBS, add 3 mL 0.25% trypsin-EDTA, and digest the adherent cells;

[0103] ② After digestion for 2 min, add 3 mL of complete culture medium to stop digestion, disperse by pipetting, and centrifuge (1000 rpm, 5 min).

[0104] ③ Discard the supernatant, add 5 mL of complete culture medium and disperse by pipetting, take 200 μL, stain with trypan blue and count the viable cells;

[0105] ④ Seed 1.0E+05 cells per well in a 24-well cell culture plate, 1 mL per well, and incubate overnight in a CO2 incubator.

[0106] (3) Strain culture

[0107] ① F1 generation: Take the glycerol tube of the test strain out of the -80℃ freezer. After the glycerol tube is thawed, mix the bacterial solution and add the bacterial solution to MRS or TPY liquid medium at an inoculation rate of 4%-10% (v / v). Incubate at 37℃ in an anaerobic workstation for 24-48 hours.

[0108] ② F2 generation: After gently mixing the cultured F1 generation bacterial solution, transfer it to 6 mL of MRS or TPY liquid medium at the same inoculation amount, and incubate at 37℃ in an anaerobic workstation for 24-48 hours.

[0109] (4) Take the F2 generation bacterial suspension of each strain, dilute it with MRS or TPY liquid medium, and set up single-strain groups, dual-strain groups, triple-strain groups, quadri-strain groups, penta-strain groups and hexa-strain groups respectively. The components of the multi-strain groups are from different species and are combined in equal proportions. Set up 3 final concentrations for each group: 1E+07, 1E+08, 1E+09 CFU / mL. Take 1 mL and sonicate it for later use.

[0110] (5) Strain-cell interaction

[0111] ① Take out the RIN-14B cells that have been resting overnight. Add 200 μL of bacterial lysis buffer to each well of a 24-well plate for each experimental group, and add an equal volume of bacterial culture medium to the control group. Each strain is replicated in 3 copies. After mixing gently, incubate in a carbon dioxide incubator for 24 hours.

[0112] ② After co-incubation, transfer the sample from each well to a 1.5 mL centrifuge tube, centrifuge (8000 rpm, 5 min, 4 °C), take the supernatant into a new centrifuge tube, filter it through a 0.22 μm filter membrane to remove impurities, and store it temporarily in a -80 °C refrigerator.

[0113] (6) Sample 5-HT determination test

[0114] ① Standard curve determination: Take 5-HT standard sample and prepare a 1000 ng / mL standard solution using RPMI 1640 medium. Dilute the solution to 500, 250, and 100 ng / mL standard solutions, and determine and plot the standard curve (see...). Figure 1 ).

[0115] ② Sample determination: After thawing the co-incubation supernatant sample at room temperature, the 5-HT content was determined by HPLC. The specific parameters are shown below:

[0116]

[0117] Experimental results:

[0118] As can be seen from the results of 5-HT content of single strains in Table 3, Lactobacillus acidophilus, Weizmann's coagulans, Lactobacillus plantarum, Bifidobacterium (Bifidobacterium longum subsp. infantis and Bifidobacterium animalis subsp. lactis) and Lactobacillus rhamnosus all showed a certain ability to promote cell secretion of 5-HT.

[0119] To further determine the ability of compositions prepared from different strains to promote 5-HT production by cells, the inventors randomly combined the single strains listed in Table 3 to prepare compositions containing dual, triple, quadrivalent, pentavalent, and hexavalent probiotics, and tested the ability of different compositions to promote 5-HT secretion by cells. Statistical analysis of the 5-HT production levels promoted by the dual to hexavalent compositions revealed that the dual and triple compositions did not show a significant increase in 5-HT production compared to single strains. However, the quadrivalent, pentavalent, and hexavalent probiotic compositions showed a positive correlation with the increase in the number of probiotic strains, exhibiting a certain dose-dependent effect. The quadrivalent probiotic composition promoted 5-HT production in the range of 500-2100 ng / mL, the pentavalent composition in the range of 500-2200 ng / mL, and the hexavalent composition in the range of 500-2400 ng / mL.

[0120] The inventors further compared and analyzed the ability of different strains in quadrivalent to hexavalent combinations to promote 5-HT production. They found that the quadrivalent, pentavalent, and hexavalent probiotic combinations containing Lactobacillus rhamnosus had a lower ability to promote 5-HT production than those without Lactobacillus rhamnosus. The 5-HT production levels of the quadrivalent, pentavalent, and hexavalent probiotic combinations containing Lactobacillus rhamnosus ranged from 500 to 1200 ng / mL, and no significant effect was observed in increasing the secretion of 5-HT. The combination of Lactobacillus rhamnosus with other bacteria did not show a better synergistic effect. The 5-HT content of the quadruple probiotic combination without Lactobacillus rhamnosus is about 800-2100 ng / mL, the 5-HT content of the pentavalent probiotic combination is about 900-2200 ng / mL, and the 5-HT content of the hexavalent probiotic combination is about 1100-2400 ng / mL. The synergistic effect between the strains is good, which can better promote the production of 5-HT by cells.

[0121]

[0122] 3. Organic acid production capacity and adhesion rate

[0123] Experimental methods:

[0124] (1) Acid production capacity test

[0125] ① Experimental instruments

[0126] The main instrument information for the experiment is shown in Table 4.

[0127]

[0128] ② Main reagents for the experiment

[0129] The main reagents used in the experiment are shown in Table 5.

[0130]

[0131] ③ Acid production test experiment

[0132] Adjust the viable bacteria count in each group to 10 10 Take 0.75 g of the CFU / g culture medium and place it in 35 g of MRS medium. Gently shake until the bacterial powder is completely dissolved. Insert a glass electrode, add 5 mm of liquid paraffin, and place the container in a 37°C water bath. Turn on the pH meter to monitor pH and ORP changes in real time. At the experimental endpoint, determine the total acid value of the MRS and the total amount of organic acids produced.

[0133] (2) Adhesion rate detection test method

[0134] ① Experimental instruments

[0135] The main instruments used in the experiment are shown in Table 6.

[0136]

[0137] ② Main reagents for the experiment

[0138] The main reagents used in the experiment are shown in Table 7.

[0139]

[0140] ③ Preparation of Caco-2 cell monolayers

[0141] Cell resuscitation and passage: Resuscitate Caco-2 cells and seed them in DMEM medium (containing 1% penicillin-streptomycin) with 20% FBS, and culture at 37°C and 5% CO2 until 80% confluence. After trypsin digestion, passage at a 1:3 ratio, with medium changed every 2 days.

[0142] Monolayer cell seeding: Digest cells and adjust density to 1.5 × 10⁶ cells / year. 5 10 cells / mL, seeded into 12-well plates (1 mL per well), and cultured for 2 days until a tight monolayer is formed.

[0143] ④ Preparation of bacterial suspension

[0144] Strain activation: Take a glycerol tube and inoculate it at 4% into MRS liquid medium, and anaerobically culture at 37°C for 24 h. Mix well, transfer it to MRS liquid medium at 4% and anaerobically culture at 37°C for 20 h.

[0145] Cell washing: Centrifuge at 6000×g for 5 min, collect the cells, wash twice with sterile PBS, and resuspend in DMEM complete medium without antibiotics to a final concentration of 2×10⁻⁶. 8 CFU / mL.

[0146] ⑤ Adhesion test

[0147] Co-incubation of bacterial strain and cells: Aspirate the culture medium from the Caco-2 cell wells and gently wash twice with preheated PBS. Add 1 mL of bacterial suspension to each well, and add an equal volume of cell culture medium to the control group. Perform two replicates per group. Incubate at 37°C and 5% CO2 for 2 hours (simulating intestinal adhesion time).

[0148] Remove any unattached bacteria, aspirate the bacterial suspension, and gently wash three times with preheated PBS, adding it slowly along the well wall each time to avoid disrupting the cell monolayer.

[0149] Cell lysis and collection: Add 1 mL of 1% Triton X-100 (dissolved in PBS) to each well, lyse at room temperature for 10 minutes, and gently shake to promote lysis.

[0150] Gradual dilution and plating: Take the lysis buffer and perform a 10-fold serial dilution (10... ⁻2 Up to 10 ⁻4 Take 100 μL of each dilution and spread it onto MRS agar plates, with two plates for each dilution. Incubate anaerobically at 37°C for 48 hours, and then count the colonies.

[0151] ⑥ Adhesion rate calculation

[0152] The formula for calculating adhesion rate is: Adhesion rate = Number of bacteria / Number of cells

[0153] Experimental results:

[0154] The experimental results are shown in Table 8. All strains in the table below have the ability to produce organic acids within 2 hours, but Lactobacillus and Lactobacillus showed better acid production ability. All strains in the table below have a certain degree of adhesiveness, but Bifidobacterium showed better cell adhesion.

[0155]

[0156] Through research on the production of organic acids and cell adhesion rates of different bacterial strains, the inventors found that *Lactobacillus plantarum* and *Lactobacillus acidophilus* have a greater advantage in the production of organic acids compared to other bacterial species. Among them, *Lactobacillus plantarum* HY02946 produced 25.07 mg of organic acids at 2 hours. In terms of adhesion rate, *Bifidobacterium* has a greater advantage in cell adhesion rate compared to other bacterial species. Among them, *Bifidobacterium longum* subsp. infantis CS004 achieved a cell adhesion rate of 9.2%.

[0157] Example 2: Screening of the proportion of probiotic compositions

[0158] 1. Experimental Design

[0159] The inventors further screened more preferred probiotic combinations from the quadruple, pentad, and hexapod compositions screened in Example 1. These included a quadruple probiotic composition containing Lactobacillus acidophilus, Bifidobacterium, Lactobacillus plantarum, and Weizmann's coagulans; a pentad probiotic composition containing Lactobacillus acidophilus, Bifidobacterium longum subsp. infantis, Bifidobacterium animalis subsp. lactis, Lactobacillus plantarum, and Weizmann's coagulans; and a hexapod probiotic composition containing two different strains of Lactobacillus acidophilus, Bifidobacterium longum subsp. infantis, Bifidobacterium animalis subsp. lactis, Lactobacillus plantarum, and Weizmann's coagulans. These combinations showed the best ability to promote 5-HT production in cells compared to other combinations. Therefore, based on the above quadruple, pentad, and hexapod compositions, the proportions of different strains were further studied.

[0160] The strains with the best single-strain effects, HY02946, BB-12, LA-5, CS004, BC99, CS003, UABla-12, and DDS-1, were selected and their proportions were set to 0.1, 0.2, 0.4, 0.6, 0.8, 1, 1.2, 1.4, 1.8, 2, 4, 6, 8, 10, 15, 20, 25, and 30, respectively, to form the aforementioned quadrivalent, pentavalent, and hexavalent strains. Experimental results showed that when the ratio of HY02946 was set to 1-30, the ratios of BB-12 and LA-5 were both set to 2-10, the ratio of UABla-12 was set to 4-12, the ratio of DDS-1 was set to 1-8, and the ratios of CS004, BC99, and CS003 were set to 0.2-1.8, the 5-HT secretion was greater than 1800 ng / ml. The six-combination HY02946, BB-12, LA-5, CS004, BC99, and CS003, and the six-combination HY02946, UABla-12, DDS-1, CS004, BC99, and CS003, showed superior effects at all ratios, with 5-HT secretion exceeding 2000 ng / ml, generally outperforming other combinations. Therefore, further optimization of the combination ratios was conducted on these two six-combination compositions. The ratio settings for each strain are shown in Table 9 below. In addition, the acid production capacity and adhesion rate of all screened combinations were tested, using the same experimental methods as in Example 1.

[0161]

[0162] The 5-HT content of each formulation sample was detected using the same method as in Example 1. The experimental results were analyzed using TTEST.

[0163] 2. Experimental Results

[0164] Experimental results showed that in the hexa-combination (HY02946, BB-12, LA-5, CS004, BC99, CS003), when the ratio of HY02946 was set at 5-20, the ratio of BB-12 at 4-10, the ratio of LA-5 at 2-8, and the ratio of CS004, BC99, and CS003 at 0.6-1.4, the 5-HT secretion was above 2400 ng / mL. Among them, when the ratio of HY02946:BB-12:LA-5:CS004:BC99:CS003 was 15:6:6:1:1:1, the 5-HT concentration could reach 2920.94 ng / mL.

[0165] In the composition HY02946, UABla-12, DDS-1, CS004, BC99, and CS003, when the ratio of HY02946 was set at 5-20, the ratio of UABla-12 at 4-10, the ratio of DDS-1 at 2-8, and the ratio of CS004, BC99, and CS003 at 0.6-1.4, the 5-HT secretion level was above 2400 ng / mL. When the ratio of HY02946:UABla-12:DDS-1:CS004:BC99:CS003 was 10:8:4:1:1:1, the 5-HT concentration reached 2817.05 ng / mL.

[0166]

[0167]

[0168]

[0169] When using other strains besides the hexavalent combination to verify the ability of different hexavalent combinations to promote cell production of 5-HT, this example uses Lactobacillus rhamnosus as an example to replace each strain and then test the ability of the hexavalent combination to promote cell production of 5-HT. The experimental results are shown in Table 11.

[0170]

[0171] The results of organic acid production from the probiotic composition (Table 12) show that, through optimization of the proportions of each probiotic in the composition, when the proportion of *Lactobacillus plantarum* HY02946 was set at 5-20, the proportion of *Bifidobacterium animalis* subsp. *lactamella* UABla-12 / BB12 was set at 4-10, the proportion of *Lactobacillus acidophilus* DDS-1 / LA-5 was set at 2-8, and the proportions of *Bifidobacterium longum* subsp. *infant* CS004, *Weizmannii coagulans* BC99, and *Lactobacillus acidophilus* CS003 were set at 0.6-1.4, the amount of organic acid produced in 1-3 hours showed a significant advantage compared to compositions with other proportion ranges. When co-cultured for 4 hours, the organic acid content reached approximately 100-200 mg. Compared to commercially available products, the probiotic composition of this invention, at the 4th hour of culture, achieved a maximum organic acid production (186.30 mg), which was 2.3 times higher than the organic acid production (80.08 mg) of commercially available products. Regarding the cell adhesion rate, within the preferred range for each strain, the cell adhesion rate of each probiotic composition is higher than that of compositions in other ratio ranges, reaching approximately 20-45. Compared with commercially available products, the probiotic composition of this invention has the highest cell adhesion rate (44.5), which is about 3 times higher than the cell adhesion rate of commercially available products (15.0).

[0172]

[0173]

[0174]

[0175] Example 3: Experimental Study on Oxygen Deprivation and Production Promotion of Probiotic Composition

[0176] I. Oxygen Deprivation and Growth Promotion Experiment

[0177] 1. Experimental Methods

[0178] 1.1 Main reagents for the experiment

[0179] The main reagents used in the experiment are shown in Table 13.

[0180]

[0181] 1.2 Experimental Group Information

[0182] The experimental groups and information on the strains / compositions in each group are shown in the table below.

[0183]

[0184] 1.3 Preparation of experimental reagents

[0185] MRS liquid culture medium: Weigh 52.24 g of MRS broth and dissolve it in 1000 mL of water, then autoclave at 115°C for 20 min and store at 4°C.

[0186] 0.1% Resazurin stock solution: Weigh 0.1g of Resazurin and dissolve it in 100mL of water. Filter the solution through a 0.22μm filter membrane for sterilization and store at 4℃.

[0187] MRS+Resazurin medium: Add 90 μL of 0.1% Resazurin stock solution to 30 mL of MRS liquid medium, mix well and store at 4℃.

[0188] 1.4 Preparation of bacterial suspension

[0189] ① F1 generation culture: According to Table 14, weigh 0.1g of bacterial powder for each formula combination into a 15mL centrifuge tube, add 9.9mL MRS and mix well, and anaerobic culture at 37℃ for 24h.

[0190] ② F2 generation culture: After mixing the F1 generation bacterial culture, inoculate 4% into 3mL MRS medium and incubate anaerobically at 37℃ for 24h.

[0191] ③ Bacterial culture treatment: Mix the bacterial culture after the culture is completed, and dilute it with PBS solution to a concentration of 1×10⁻⁶. 7 CFU / mL bacterial suspension is available for use.

[0192] 1.5 Determination of growth curve

[0193] ① In the anaerobic workstation, the diluted bacterial suspensions of each group were inoculated into 3 mL of MRS + Resazurin medium at a 4% inoculation rate. A blank medium was set up as the control group, and this was recorded as T0. The culture was then incubated anaerobically at 37℃. 100 μL of the bacterial suspension from each group's T0 was added to a 96-well plate, with two replicates for each group. The color change was photographed and the OD600 value was measured using a microplate reader.

[0194] ② In a biosafety cabinet, inoculate the diluted bacterial suspensions of each group into 3 mL of MRS + Resazurin medium at a 4% inoculation rate. A blank medium is set up as the control group; this is recorded as T0. Incubate at 37°C in a constant temperature incubator. Add 100 μL of the bacterial suspension from each group's T0 well to a 96-well plate, with two replicates per group. Photograph the color changes and measure the OD600 value using a microplate reader.

[0195] ③ Take out the bacterial suspensions at 3h, 6h, 9h and 12h of anaerobic and aerobic culture. Set up a blank culture medium as the control group. The anaerobic group was operated in the anaerobic workstation and the aerobic group was operated in the biosafety cabinet. Take 100μL of bacterial suspension from each group and add it to a 96-well plate. Make two replicates for each group. Take pictures to record the color change and detect the OD600 value with an ELISA reader.

[0196] 2. Experimental Results

[0197] The experimental results are shown in the table below.

[0198]

[0199] In their research experiments on oxygen deprivation and growth promotion of probiotic compositions, the inventors discovered that *Weizmannii* in the probiotic compositions of this invention exhibits a better oxygen deprivation and growth-promoting effect on the probiotic compositions under aerobic conditions than other bacteria. As shown in Table 15, the experimental results indicate that under anaerobic conditions, the presence or absence of *Weizmannii* in the composition has virtually no effect on the growth of the strains. However, under aerobic conditions, the addition of *Weizmannii* significantly promotes the growth of the compositions. For example, in groups G18 (containing BC99) and G18-2 (HY08866), after 12 hours of incubation, the OD values ​​of both groups were significantly higher. 600 The values ​​were 0.03051 and 0.02963, respectively. Compared to G18-1 (which does not contain Weizmannia), the OD values ​​of the composition were... 600 The value was only 0.01782; for example, in groups G43 (including BC99) and G43-2 (HY08866), after 12 hours of incubation, the OD values ​​of both groups were only 0.01782. 600 The values ​​were 0.03432 and 0.03267, respectively. Compared to group G43-1 (which does not contain Weizmannia), the OD values ​​of the composition were [missing information].600 The value was only 0.01432. Furthermore, when other strains, such as *Lactobacillus rhamnosus*, were used to replace *Weizmannii* in the probiotic composition, the composition's growth was not promoted under aerobic conditions. For example, the OD600 value of G18-3 after 12 hours of incubation was not significantly different from that of G18-1, and the OD600 value of G43-3 after 12 hours of incubation was essentially the same as that of G43-1. This indicates that *Weizmannii* in the composition can effectively exert its oxygen-depleting effect and promote the synergistic growth of other probiotics in the composition.

[0200] II. Dissolved Oxygen Saturation Detection Experiment

[0201] 1. Experimental Methods

[0202] 1.1 Experimental Conditions

[0203] The dissolved oxygen and dissolved oxygen saturation of the probiotic composition were measured using a Hach 1130DO. The specific experimental conditions are shown in the table below:

[0204]

[0205] 1.2 Experimental Grouping

[0206] The experimental groups are shown in the table below.

[0207]

[0208] 2. Experimental Results

[0209]

[0210] The experimental results showed that when the compositions in each experimental group were cultured for 60 minutes, the composition containing *Weizmannia* reduced dissolved oxygen by about 60%-80%, while the composition without *Weizmannia* reduced dissolved oxygen by only about 32%-34%. When the compositions in each experimental group were cultured for 95 minutes, the composition containing *Weizmannia* reduced dissolved oxygen by about 93%-99%, while the composition without *Weizmannia* reduced dissolved oxygen by only about 63%-64%. This indicates that *Weizmannia* has a significant biological oxygen-consuming effect and can effectively promote the probiotic composition to exert its oxygen-depleting effect in an aerobic environment, effectively promoting the synergistic growth of other probiotics in the probiotic composition.

[0211] Example 4: Efficacy verification of probiotic composition for constipation

[0212] 1. Experimental Methods

[0213] 1.1 Experimental Materials

[0214] (1) Key experimental reagents are shown in Table 19.

[0215]

[0216] (2)Test substance

[0217] The animal grouping information is shown in Table 20.

[0218]

[0219] 1.2 Animal system

[0220] (1)Experimental animals

[0221] SPF - level Balb / c mice, male, 7 - 8 weeks old, weighing 20g to 22g, were purchased from Zhejiang Vital River Laboratory Animal Technology Co., Ltd., license number: SCXK(Zhe)2024 - 0001, animal certificate number: 20251016Abzz06199990627.

[0222] (2)Environmental adaptation

[0223] After the animals were placed in the room, they were adaptively fed for 3 days before the experiment began. The main inspection contents during the adaptation period were: whether there were abnormal appearances; whether the general condition was normal. Unqualified animals were not included in the experiment.

[0224] (3)Feeding management

[0225] They were housed in the barrier system of Hangzhou Mosslite Biotechnology Co., Ltd., experimental animal use license number: SYXK(Zhe)2022 - 0032.

[0226] (4)Experimental design and grouping

[0227] The animal modeling methods for each of the above groups are shown in Table 21.

[0228]

[0229] (5)Animal administration

[0230] Treatment started on the first day of modeling. The treatment groups were respectively given the corresponding test articles by gavage, and the negative control group and the model control group were respectively given an equal volume of PBS by gavage, once a day until the end of the experiment.

[0231] 1.3 Observation and index detection

[0232] (1)Body weight monitoring

[0233] After the adaptive feeding of the animals ended, the body weight of the mice was weighed once a week, and the changes in the body weight of the mice were recorded.

[0234] (2)Detection of fecal moisture content

[0235] Weigh the empty glass bottle and record the bottle weight as W 空Mice were placed in metabolic cages, and the number of fecal pellets was recorded. At the same time, mouse feces were collected in glass bottles. After 5 hours of collection, the weight of the feces, including the bottle, was measured as W1. The mouse feces were then placed in a forced-air drying oven at 90°C for 5 hours and the weight of the feces, including the bottle, was measured as W2.

[0236] Fecal wet weight = wet weight - empty bottle weight = W1 - W 空

[0237] Fecal dry weight = Dry weight - Empty bottle weight = W2 - W 空 .

[0238] Fecal moisture content = (W1 - W2) / W1 × 100%

[0239] (3) Defecation test

[0240] The mice were fasted but allowed free access to water the night before the test. Sixteen hours later, normal drug administration procedures were performed, including administration of loperamide hydrochloride and the test bacteria. Forty-five minutes after drug administration, each mouse was given 0.2 mL of 10% carbon dioxide suspension by gavage, and the carbon dioxide administration time T1 was recorded. At the same time, mouse feces were collected 5 hours later using a metabolic cage. If the mouse excreted black feces, the excretion time T2 was recorded. The time of the first black feces excreted by each mouse was calculated (T2-T1).

[0241] (4) Small intestine propulsion experiment

[0242] Mice were fasted for 16 hours the night before the experiment, followed by normal drug administration procedures, including administration of loperamide hydrochloride and the test bacteria. Forty-five minutes later, each mouse was administered 0.2 mL of a 10% carbon dioxide suspension by gavage. Twenty-five minutes later, the mice were injected intraperitoneally with a solution of 50 acetaminophen. Five minutes later, the entire intestinal tract from the stomach to the cecum was harvested, and the length of the small intestine and the distance the carbon dioxide was propelled were measured. The carbon dioxide propulsion rate was calculated using the formula.

[0243] Carbon ink propulsion rate (%) = Carbon ink propulsion distance (cm) / Total length of small intestine (cm) × 100%

[0244] (5) Statistical analysis

[0245] All data from this experiment were entered into Excel for corresponding calculations. GraphPad Prism software was used for statistical analysis of the experimental data. P < 0.05 was considered statistically significant. The experimental results are expressed as Mean ± SEM (standard error).

[0246] 2. Experimental Results

[0247] (1) Evaluation of the time index for the first black stool excretion

[0248] The results are shown in Table 22. The results indicate that compared with the normal control group (NC), the time to excretion of the first black stool in the model group (MC) mice was significantly prolonged (P < 0.0001), indicating significant gastrointestinal motility disorders in the constipation model mice, demonstrating successful model establishment. The defecation time in each treatment group (quadrivalent group TB, pentavalent group FB, and several hexavalent groups) was significantly shorter than that in the model control group (MC), with the AB, DU, and DH groups showing particularly significant effects (P < 0.01), while the TB, FB, and AH groups also showed significant improvement (P < 0.05), exhibiting better effects than the positive control group. Notably, there was no statistically significant difference between the two hexavalent control groups (AR and DR) and the model control group (MC), indicating that replacing the bacterial strains in the composition of this invention with other strains could not achieve the same therapeutic effect. In summary, this experiment demonstrates that specific combinations can effectively alleviate constipation, significantly shorten the time to excretion of the first black stool, and are more effective than positive control drugs. The quadrivalent, pentavalent, and hexavalent bacterial combinations of this invention exhibit synergistic effects, and their effectiveness cannot be achieved simply by stacking or increasing the number of bacterial strains.

[0249]

[0250] (2) Evaluation of mouse fecal weight and fecal moisture content

[0251] The results are shown in Table 23. It can be seen that compared with the normal control group (NC), the fecal weight of the model control group (MC) was significantly reduced (0.11±0.03 g vs 0.49±0.06 g), and the water content was also significantly reduced (40.35±3.25% vs 57.91±1.06%), indicating that the constipation model was successfully established and the animals exhibited obvious constipation symptoms. The fecal weight and water content of all treatment groups were significantly higher than those of the model control group and the experimental control group (AR, DR). Group AB showed the most significant effect in increasing fecal weight (P<0.001) and the highest degree of water content recovery. Groups PC, TB, FB, DU, AH, and DH showed significant improvement in both indicators, while the AR and DR groups showed relatively smaller improvements. These results indicate that replacing the bacterial strains in the composition of this invention with other strains cannot achieve the same therapeutic effect. The quadruple, pentavalent, and hexavalent bacterial combinations of this invention exhibit synergistic effects, and their effects cannot be achieved simply by piling up or increasing the number of strains.

[0252]

[0253] (3) Evaluation of carbon ink propulsion rate index

[0254] The results are shown in Table 24. It can be seen that compared with the normal control group (NC, 62.02±2.17%), the carbon propulsion rate of the model control group (MC) was significantly reduced to 17.58±1.01% (P<0.0001), indicating that the intestinal peristalsis function of the constipation model mice was severely impaired. The carbon propulsion rate of all treatment groups was significantly higher than that of the model control group. The DU group showed extremely significant effects (P<0.001), while the PC, TB, FB, AB, AH, and DH groups showed significant (P<0.05) or extremely significant (P<0.01) effects. However, there was no statistically significant difference between the AR and DR groups and the model control group. This indicates that various treatment interventions, and specific combinations of quadruple, quintuple, and hexaple therapies, can effectively promote intestinal peristalsis, significantly increase the carbon propulsion rate, and thus alleviate constipation symptoms.

[0255]

[0256] The results in Tables 22 to 24 show that specific combinations of quadruple, pentavalent, and hexavalent probiotics (especially AB and DU) can effectively relieve constipation through multiple targets (improving intestinal motility, increasing stool volume and moisture, etc.), and their effects are superior to conventional drugs. Furthermore, comparisons of different hexavalent bacterial combinations also demonstrate that this invention achieves a synergistic effect by combining specific strains; such an effect cannot be achieved simply by piling up strains or increasing the number of strains.

[0257] The above descriptions are merely several exemplary embodiments of the present invention and are not intended to limit the present invention in any way. Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Any equivalent or related embodiments obtained by those skilled in the art through some modifications or variations made to the above-disclosed technical content without departing from the scope of the present invention are within the scope of the present invention.

Claims

1. A probiotic composition for improving constipation, wherein, The probiotic composition comprises oxygen-depleting bacteria, organic acid-producing bacteria, and space-occupying bacteria; Preferably, the oxygen-depleting bacteria is Weizmann's bacterium; Preferably, the organic acid-producing bacteria are Lactobacillus and / or Lactobacillus lactis; Preferably, the occupant bacteria is Bifidobacterium.

2. The probiotic composition according to claim 1, wherein: The Weizmania bacteria mentioned are selected from Weizmannia coagulans. The lactobacillus is selected from one or more of the following: Lactobacillus acidophilus, Lactobacillus casei, Lactobacillus paracasei, Lactobacillus crispatus, Lactobacillus delbrueckii ssp. bulgaricus, Lactobacillus fermentum, Lactobacillus gasseri, Lactobacillus helveticus, Lactobacillus johnsonii, Lactobacillus plantarum, Lactobacillus reuteri, Lactobacillus rhamnosus, Lactobacillus salivarius, Lactobacillus sakei, and Lactobacillus curvus. The Lactiplantibacillus is selected from one or more of the following: Lactiplantibacillus plantarum, Lactiplantibacillus plantarum subsp. argentoratensis, Lactiplantibacillus pentosus, Lactiplantibacillus paraplantarum, Lactiplantibacillus fabifermentans, Lactiplantibacillus tropicus, and Lactiplantibacillus nakhonrathomensis. The Bifidobacterium is selected from one or more of the following: Bifidobacterium animalis subsp. lactis, Bifidobacterium longum subsp. infantis, Bifidobacterium adolescentis, Bifidobacterium animalis, Bifidobacterium bifidum, Bifidobacterium breve, Bifidobacterium infantis, and Bifidobacterium longum.

3. The probiotic composition according to claim 2, wherein, The probiotic composition contains *Weizmannii coagulans*, *Lactobacillus acidophilus*, *Lactobacillus plantarum*, and *Bifidobacterium*. Preferably, the probiotic composition comprises *Weizmannii coagulans*, *Lactobacillus acidophilus*, *Lactobacillus plantarum*, and *Bifidobacterium* in an effective live bacteria concentration ratio of (0.6-1.4):(0.6-9.4):(5-20):(0.6-11.4). More preferably, the probiotic composition comprises *Weizmannii coagulans*, *Lactobacillus acidophilus*, *Lactobacillus plantarum*, and *Bifidobacterium* in an effective live bacteria concentration ratio of (0.6-1.4):(0.6-8):(5-20):(0.6-10).

4. The probiotic composition according to claim 3, wherein, The Bifidobacteria are selected from Bifidobacterium longum subsp. infantis and / or Bifidobacterium animalis subsp. lactis. Preferably, the probiotic composition comprises *Weizmannii coagulans*, *Lactobacillus acidophilus*, *Lactobacillus plantarum*, *Bifidobacterium longum* subsp. *infantii*, and *Bifidobacterium animalis* subsp. *lactobacterium* in an effective live bacteria concentration ratio of (0.6-1.4):(0.6-8):(5-20):(0.6-1.4):(4-10); More preferably, the probiotic composition comprises *Weizmannii coagulans*, *Lactobacillus acidophilus*, *Lactobacillus plantarum*, *Bifidobacterium longum* subsp. *infantii* and *Bifidobacterium animalis* subsp. *lactobacterium* in an effective live bacteria concentration ratio of (0.6-1.4):(0.6-8):(5-20):(0.6-1.4):(4-8).

5. The probiotic composition according to claim 4, wherein, The Lactobacillus acidophilus includes Lactobacillus acidophilus I and Lactobacillus acidophilus II, wherein the Lactobacillus acidophilus I and Lactobacillus acidophilus II are different strains from each other; Preferably, the probiotic composition comprises *Weizmannii coagulans*, *Lactobacillus acidophilus* var. *mongolica*, *Lactobacillus acidophilus* var. *mongolica*, *Lactobacillus plantarum*, *Bifidobacterium longum* subsp. *infantii*, and *Bifidobacterium animalis* subsp. *lactobacterium* in an effective live bacteria concentration ratio of (0.6-1.4):(2-8):(0.6-1.4):(5-20):(0.6-1.4):(4-10).

6. The probiotic composition according to any one of claims 2 to 5, wherein, The *Wickemium coagulans* is selected from at least one of *Wickemium coagulans* BC99, *Wickemium coagulans* HY08866, *Wickemium coagulans* HY08867, *Wickemium coagulans* MP08976, and *Wickemium coagulans* HY08874; and / or, The first and second *Lactobacillus acidophilus* are each independently selected from at least one of *Lactobacillus acidophilus* LA-5, *Lactobacillus acidophilus* CS003, *Lactobacillus acidophilus* DDS-1, *Lactobacillus acidophilus* HY00768, *Lactobacillus acidophilus* MP01046, *Lactobacillus acidophilus* HY01039, *Lactobacillus acidophilus* HY00760, and *Lactobacillus acidophilus* HY01043; and / or, The *Lactobacillus plantarum* is selected from at least one of *Lactobacillus plantarum* HY02946, *Lactobacillus plantarum* MP02908, *Lactobacillus plantarum* MP00442, *Lactobacillus plantarum* HY05181, *Lactobacillus plantarum* HY00050, and *Lactobacillus plantarum* MP00134; and / or, The *Bifidobacterium longum* infant subsp. *longum* is selected from at least one of *Bifidobacterium longum* infant subsp. *CS004*, *Bifidobacterium longum* infant subsp. *MP08420*, and *Bifidobacterium longum* infant subsp. *HY07708*; and / or, The *Bifidobacterium lactis* subsp. *animal* is selected from at least one of *Bifidobacterium lactis* subsp. *animal* BB-12, *Bifidobacterium lactis* UABla-12, *Bifidobacterium lactis* subsp. *animal* HY09302, *Bifidobacterium lactis* subsp. *animal* MP08129, *Bifidobacterium lactis* subsp. *animal* MP04382, and *Bifidobacterium lactis* MP03316.

7. The probiotic composition according to claim 6, wherein, The probiotic composition contains Lactobacillus plantarum HY02946, Bifidobacterium longum subsp. infantis CS004, Weizmannii coagulans BC99 or Weizmannii coagulans HY08866 and Lactobacillus acidophilus CS003. Preferably, the probiotic composition comprises *Lactobacillus plantarum* HY02946, *Bifidobacterium longum* subsp. *infantica* CS004, *Weizmannii coagulans* BC99 or *Weizmannii coagulans* HY08866, and *Lactobacillus acidophilus* CS003 in an effective live bacteria concentration ratio of (5-20):(0.6-1.4):(0.6-1.4):(0.6-1.4). More preferably, the probiotic composition comprises *Lactobacillus plantarum* HY02946, *Bifidobacterium longum* subsp. *infant* CS004, *Weizmannii coagulans* BC99 or *Weizmannii coagulans* HY08866, and *Lactobacillus acidophilus* CS003 in an effective live bacteria concentration ratio of 15:1:1:

1.

8. The probiotic composition according to claim 6, wherein, Lactobacillus plantarum HY02946, Bifidobacterium animalis subsp. lactis BB-12, Bifidobacterium longum subsp. infantis CS004, Weizmann's coagulans BC99 or HY08866, Lactobacillus acidophilus CS003. Preferably, the probiotic composition comprises *Lactobacillus plantarum* HY02946, *Bifidobacterium animalis* subsp. *lactobacter* BB-12, *Bifidobacterium longum* subsp. *infant* CS004, *Weizmannii coagulans* BC99 or HY08866, and *Lactobacillus acidophilus* CS003 in an effective live bacteria concentration ratio of (5-20):(4-10):(0.6-1.4):(0.6-1.4):(0.6-1.4). More preferably, the probiotic composition comprises *Lactobacillus plantarum* HY02946, *Bifidobacterium animalis* subsp. *lactobacter* BB-12, *Bifidobacterium longum* subsp. *infant* CS004, *Weizmannii coagulans* BC99 or HY08866, and *Lactobacillus acidophilus* CS003 in an effective live bacteria concentration ratio of 15:6:1:1:

1.

9. The probiotic composition according to claim 6, wherein, The probiotic composition comprises Lactobacillus plantarum HY02946, Bifidobacterium animalis subsp. lactis BB-12, Lactobacillus acidophilus LA-5, Bifidobacterium longum subsp. infantis CS004, Weizmannii coagulans BC99 or Weizmannii coagulans HY08866 and Lactobacillus acidophilus CS003. Preferably, the probiotic composition comprises *Lactobacillus plantarum* HY02946, *Bifidobacterium animalis* subsp. *lactobacter* BB-12, *Lactobacillus acidophilus* LA-5, *Bifidobacterium longum* subsp. *infantica* CS004, *Weizmannii coagulans* BC99 or *Weizmannii coagulans* HY08866, and *Lactobacillus acidophilus* CS003 in an effective live bacteria concentration ratio of (5-20): (4-10): (2-8): (0.6-1.4): (0.6-1.4): (0.6-1.4). More preferably, the probiotic composition comprises *Lactobacillus plantarum* HY02946, *Bifidobacterium animalis* subsp. *lactobacter* BB-12, *Lactobacillus acidophilus* LA-5, *Bifidobacterium longum* subsp. *infantica* CS004, *Weizmannii coagulans* BC99 or *Weizmannii coagulans* HY08866, and *Lactobacillus acidophilus* CS003 in an effective live bacteria concentration ratio of (5-20): (4-8): (4-8): (0.6-1.4): (0.6-1.4): (0.6-1.4). More preferably, the probiotic composition comprises *Lactobacillus plantarum* HY02946, *Bifidobacterium animalis* subsp. *lactobacter* BB-12, *Lactobacillus acidophilus* LA-5, *Bifidobacterium longum* subsp. *infant* CS004, *Weizmannii coagulans* BC99 or *Weizmannii coagulans* HY08866, and *Lactobacillus acidophilus* CS003 in an effective live bacteria concentration ratio of 15:6:6:1:1:

1.

10. The probiotic composition according to claim 6, wherein, The probiotic composition contains Lactobacillus plantarum HY02946, Bifidobacterium animalis subsp. lactis UABla-12, Lactobacillus acidophilus DDS-1, Bifidobacterium longum subsp. infantis CS004, Weizmannia coagulans BC99 or Weizmannia coagulans HY08866 and Lactobacillus acidophilus CS003. Preferably, the probiotic composition comprises *Lactobacillus plantarum* HY02946, *Bifidobacterium animalis* subsp. *lactobacter* UABla-12, *Lactobacillus acidophilus* DDS-1, *Bifidobacterium longum* subsp. *infantica* CS004, *Weizmannii coagulans* BC99 or *Weizmannii coagulans* HY08866, and *Lactobacillus acidophilus* CS003 in an effective live bacteria concentration ratio of (5-20): (4-10): (2-8): (0.6-1.4): (0.6-1.4): (0.6-1.4). More preferably, the probiotic composition comprises *Lactobacillus plantarum* HY02946, *Bifidobacterium animalis* subsp. *lactobacter* UABla-12, *Lactobacillus acidophilus* DDS-1, *Bifidobacterium longum* subsp. *infantica* CS004, *Weizmannii coagulans* BC99 or *Weizmannii coagulans* HY08866, and *Lactobacillus acidophilus* CS003 in an effective live bacteria concentration ratio of (5-20): (6-10): (2-6): (0.6-1.4): (0.6-1.4): (0.6-1.4): (0.6-1.4) respectively. More preferably, the probiotic composition comprises *Lactobacillus plantarum* HY02946, *Bifidobacterium animalis* subsp. *lactobacter* UABla-12, *Lactobacillus acidophilus* DDS-1, *Bifidobacterium longum* subsp. *infant* CS004, *Weizmannii coagulans* BC99 or *Weizmannii coagulans* HY08866, and *Lactobacillus acidophilus* CS003 in an effective live bacteria concentration ratio of 10:8:4:1:1:

1.

11. A product for preventing, relieving, improving and / or treating constipation, comprising a probiotic composition according to any one of claims 1 to 10.

12. The product according to claim 11, wherein, The product also includes excipients; the excipients are selected from one or more of pharmaceutical excipients, food excipients, and health product excipients; Preferably, the excipients are selected from one or more of excipients, fillers, flow aids, diluents, quick-dissolving agents, and disintegrants.

13. Use of the probiotic composition according to any one of claims 1 to 10 in the preparation of products for the prevention, relief, improvement and / or treatment of constipation.

14. The product according to claim 11 or 12, or the use according to claim 13, wherein, The constipation is selected from functional constipation, slow transit constipation, outlet obstruction constipation, drug-induced constipation, metabolic-endocrine constipation, low volume constipation, constipation-predominant irritable bowel syndrome, and mixed irritable bowel syndrome.

15. The product according to claim 11 or 12 or the use according to claim 13, wherein, The product is selected from one or more of the following: pharmaceuticals, food, and health products.

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