Probiotic formulations and uses thereof

Abstract

The present application provides a probiotic preparation and its use. The probiotic preparation of the present application comprises active ingredients and adjuvants, wherein the active ingredients comprise oxygen scavengers, organic acid producers and place holders; the adjuvants comprise flavoring agents, oligosaccharides and other additives; wherein the oligosaccharides are not lactulose, stachyose, raffinose, oligogentiobiose and oligochitosan, and the other additives are not theaflavins, ethylenediaminetetraacetic acid disodium salt and sorbic acid. The present application effectively promotes the metabolic activity of probiotics through a specific combination of adjuvants, rather than just providing physical protection or nutritional support. Experimental data show that the adjuvant combination of the present application can significantly and continuously improve the oxidation-reduction potential and Na + K + ATPase activity of probiotics. Moreover, the probiotic preparation of the present application can effectively improve functional gastrointestinal diseases and has a bidirectional regulation function, which is suitable for different intestinal motility disorders, such as diarrhea and constipation.

Description

Technical Field

[0001] This invention relates to the field of biological products. Specifically, this invention relates to a probiotic preparation and its use; more specifically, this invention relates to a probiotic preparation for improving functional gastrointestinal disorders and its use in the preparation of products for the prevention and / or treatment of functional gastrointestinal disorders. Background Technology

[0002] Probiotics, as live microorganisms, can exert beneficial effects on the host's health when ingested in sufficient quantities, and are now widely used in the food, health product, and pharmaceutical fields. However, the actual efficacy of probiotic products highly depends on their ability to rapidly recover and perform metabolic functions in the complex gastrointestinal environment after ingestion.

[0003] In the development of probiotic formulations, the selection of excipients is crucial. They are not only used for shaping, flavoring, and stabilizing products, but also profoundly affect the survival status and metabolic activity of probiotics. Traditional excipient screening focuses on physical stability, taste, or basic protection of the bacteria (such as freeze-drying protection), typically using simple sugars, starches, or colloids. However, this conventional excipient system has significant limitations: it often only provides an inert physical support or basic nutritional environment, failing to actively intervene in and activate probiotics at the metabolic level.

[0004] Specifically, the excipient combinations in many existing probiotic preparations may lead to the following adverse effects: (1) Slow metabolic initiation: After probiotics come into contact with the preparation medium, they need to go through a process of “activation” or “restart” metabolism. An unsuitable excipient environment cannot provide effective metabolic trigger signals and substrates, resulting in slow bacterial recovery and delaying the performance of their physiological functions; (2) Low energy metabolism level: The key functions of probiotics, such as colonization, antagonism of pathogens, and production of beneficial metabolites (such as short-chain fatty acids), all require a large amount of energy (ATP). If the excipient system cannot effectively support its mitochondrial function or transmembrane ion transport and other energy production processes, it will directly limit its overall metabolic intensity and therapeutic potential; (3) Redox homeostasis imbalance: The activation and metabolism of probiotics in an aerobic environment are accompanied by dynamic changes in redox potential. Inappropriate excipients may not be able to help the bacteria establish a favorable reduced intracellular environment or cope with external oxidative pressure, resulting in low efficiency of metabolic pathways and even accelerating bacterial death.

[0005] Therefore, providing a formulation that can effectively enhance the redox metabolism level and energy generation capacity of probiotics during the formulation activation stage is of great significance for enhancing the onset speed, stability and final efficacy of probiotic products. Summary of the Invention

[0006] To address the aforementioned technical problems, the purpose of this invention is to provide a probiotic preparation and its uses. The probiotic preparation of this invention effectively promotes the metabolic activity of probiotics through the use of specific excipient combinations, rather than merely providing physical protection or nutritional support.

[0007] In a first aspect, the present invention provides a probiotic preparation comprising active ingredients and excipients, wherein the active ingredients comprise aerobic bacteria, organic acid-producing bacteria, and space-occupying bacteria; the excipients comprise flavoring agents, oligosaccharides, and other additives; wherein the oligosaccharides are not lactulose, stachyose, raffinose, gentiosaccharides, or chitosan oligosaccharides; and the other additives are not theaflavins, disodium EDTA, or sorbic acid.

[0008] According to some embodiments of the present invention, the flavoring agent is selected from one or more of D-mannitol, alitane, aspartame, advansame, acesulfame potassium, erythritol, glycyrrhizate, mogroside, maltitol, xylitol, neotame, lactitol, sucralose, sorbitol, sematrandole, asparagine methyl acesulfame, steviol glycosides, cyclamate, isomerized lactose, isomaltulose, arabinose, citric acid, lactic acid, phosphoric acid, tartaric acid, malic acid, metatartaric acid, acetic acid, hydrochloric acid, adipic acid, fumaric acid, sodium hydroxide, potassium carbonate, sodium carbonate, sodium citrate, potassium citrate, trisodium bicarbonate, monosodium citrate, edible flavorings, edible fragrances, and concentrated fruit juice; preferably, the flavoring agent is selected from one or more of xylitol, arabinose, and concentrated fruit juice.

[0009] According to some embodiments of the present invention, the oligosaccharide is selected from one or more of stachyose, raffinose, isomaltulose, lactulose, fructooligosaccharide, xylooligosaccharide, galactooligosaccharide, isomaltooligosaccharide, isomaltooligosaccharide, gentiosaccharide, soybean oligosaccharide, and chitosan oligosaccharide; preferably, the oligosaccharide is selected from one or more of fructooligosaccharide, galactooligosaccharide, isomaltooligosaccharide, soybean oligosaccharide, and isomaltooligosaccharide.

[0010] According to some embodiments of the present invention, the other additives are selected from one or more of vitamin C, D-isoascorbic acid and its sodium salt, ascorbic acid, calcium ascorbate, sodium ascorbate, zinc sulfate, citric acid, vitamin E, vitamin B6, nicotinamide, zinc gluconate, disodium pyrroloquinoline quinone, aloe vera gel, calcium lactate, and vitamin E acetate; preferably, the other additives are selected from one or more of vitamin C, vitamin B6, nicotinamide, zinc gluconate, disodium pyrroloquinoline quinone, aloe vera gel, calcium lactate, and vitamin E acetate.

[0011] According to some embodiments of the present invention, the probiotic preparation has a live bacteria count of not less than 100 million CFU / g, preferably 1-100 billion CFU / g, and more preferably 80-100 billion CFU / g.

[0012] According to some embodiments of the present invention, the probiotic preparation comprises, by weight, 0.2-1 parts of active ingredient and 0.3-2.5 parts of excipients.

[0013] Preferably, the probiotic preparation comprises, by weight, the following:

[0014] The active ingredient is 0.2-1 part, preferably 0.5-0.7 parts;

[0015] The seasoning is used in an amount of 0.24-2.1 parts, preferably 0.5-1.7 parts;

[0016] The oligosaccharide content is 0.041-0.16 parts, preferably 0.079-0.121 parts;

[0017] Other additives: 0.052-0.23 parts, preferably 0.11-0.13 parts.

[0018] More preferably, the probiotic preparation comprises, by weight, the following:

[0019] The active ingredient is 0.2-1 part, preferably 0.5-0.7 parts, and more preferably 0.6 parts;

[0020] The xylitol content is 0.2-1 part, preferably 0.5-0.7 parts, and more preferably 0.6 parts;

[0021] The concentrated fruit juice is 0.04-0.1 parts, preferably 0.06-0.08 parts, and more preferably 0.07 parts;

[0022] The amount of fructooligosaccharides is 0.02-0.06 parts, preferably 0.03-0.05 parts, and more preferably 0.04 parts;

[0023] The amount of isomaltooligosaccharide is 0.02-0.08 parts, preferably 0.04-0.06 parts, and more preferably 0.05 parts;

[0024] The amount of galactooligosaccharides is 0.001-0.02 parts, preferably 0.009-0.011 parts, and more preferably 0.01 parts;

[0025] Vitamin C 0.00001-0.0002 parts, preferably 0.00009-0.00011 parts, more preferably 0.0001 parts;

[0026] Vitamin B6: 0.00001-0.00002 parts, preferably 0.000014-0.000016 parts, more preferably 0.000015 parts;

[0027] Nicotinamide: 0.0001-0.0002 parts, preferably 0.00014-0.00016 parts, more preferably 0.00015 parts;

[0028] The zinc gluconate is 0.0004-0.001 parts, preferably 0.0006-0.0008 parts, and more preferably 0.0007 parts;

[0029] The disodium salt of pyrroloquinoline quinone is 0.0000001-0.000001 parts, preferably 0.0000004-0.0000006 parts, and more preferably 0.0000005 parts;

[0030] The amount of aloe vera gel is 0.05-0.2 parts, preferably 0.09-0.11 parts, and more preferably 0.1 parts;

[0031] The amount of calcium lactate is 0.001-0.02 parts, preferably 0.0121-0.0123 parts, and more preferably 0.0122 parts.

[0032] More preferably, the probiotic preparation comprises, by weight, the following:

[0033] The active ingredient is 0.2-1 part, preferably 0.5-0.7 parts, and more preferably 0.6 parts;

[0034] The amount of arabinose is 1-2 parts, preferably 1.4-1.6 parts, and more preferably 1.5 parts;

[0035] The concentrated fruit juice is 0.05-0.1 parts, preferably 0.07-0.09 parts, and more preferably 0.085 parts;

[0036] The amount of fructooligosaccharides is 0.02-0.06 parts, preferably 0.03-0.05 parts, and more preferably 0.04 parts;

[0037] The amount of isomaltooligosaccharide is 0.02-0.08 parts, preferably 0.04-0.06 parts, and more preferably 0.05 parts;

[0038] The amount of galactooligosaccharides is 0.001-0.02 parts, preferably 0.009-0.011 parts, and more preferably 0.01 parts;

[0039] Vitamin E acetate: 0.0002-0.0008 parts, preferably 0.0005-0.0007 parts, more preferably 0.00056 parts;

[0040] Vitamin C 0.00001-0.0002 parts, preferably 0.00009-0.00011 parts, more preferably 0.0001 parts;

[0041] Vitamin B6: 0.00001-0.00002 parts, preferably 0.000014-0.000016 parts, more preferably 0.000015 parts;

[0042] Nicotinamide: 0.0001-0.0002 parts, preferably 0.00014-0.00016 parts, more preferably 0.00015 parts;

[0043] The zinc gluconate is 0.0004-0.001 parts, preferably 0.0006-0.0008 parts, and more preferably 0.0007 parts;

[0044] The disodium salt of pyrroloquinoline quinone is 0.0000001-0.000001 parts, preferably 0.0000004-0.0000006 parts, and more preferably 0.0000005 parts;

[0045] The amount of aloe vera gel is 0.05-0.2 parts, preferably 0.09-0.11 parts, and more preferably 0.1 parts.

[0046] According to some embodiments of the present invention, the probiotic preparation further comprises a solvent; preferably, the solvent content in the probiotic preparation is 8-10 kg / 10 L, more preferably 8.5-9.5 kg / 10 L; preferably, the solvent is water.

[0047] 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.

[0048] In this invention, the oxygen-depleting bacteria are preferably Weizmann's bacteria.

[0049] According to some embodiments of the present invention, the Weizmania is selected from Weizmannia coagulans.

[0050] 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.

[0051] In this invention, the organic acid-producing bacteria are preferably Lactobacillus and / or Lactobacillus lactis.

[0052] 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*.

[0053] 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.

[0054] 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.

[0055] In this invention, the occupant bacteria is preferably Bifidobacterium.

[0056] 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.

[0057] According to some embodiments of the present invention, the active ingredient comprises Weizmann's coagulation bacteria, Lactobacillus acidophilus, Lactobacillus plantarum, and Bifidobacterium.

[0058] Preferably, the active ingredient 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):(10-20):(0.6-9.4).

[0059] More preferably, the Bifidobacteria include Bifidobacterium longum subsp. infantis and Bifidobacterium animalis subsp. lactis.

[0060] More preferably, the active ingredient 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):(0.6-9.4):(10-20):(0.6-1.4):(4-8).

[0061] More preferably, 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.

[0062] Particularly preferably, the active ingredient comprises *Weizmannii coagulans*, *Lactobacillus acidophilus* var. *mongolica*, *Lactobacillus acidophilus* var. *mongolica*, *Lactobacillus plantarum*, *Bifidobacterium longum* subsp. *infantii*, and *Bifidobacterium animalis* subsp. *milk* in an effective live bacteria concentration ratio of (0.6-1.4):(4-8):(0.6-1.4):(10-20):(0.6-1.4):(4-8) respectively.

[0063] According to some embodiments of the present invention, the *Weizmannii coagulans* is selected from at least one of *Weizmannii coagulans* BC99, *Weizmannii coagulans* HY08866, *Weizmannii coagulans* HY08867, and *Weizmannii coagulans* HY08874; and / or,

[0064] 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* HY01039, *Lactobacillus acidophilus* HY00760, and *Lactobacillus acidophilus* HY01043; and / or,

[0065] The *Lactobacillus plantarum* is selected from at least one of *Lactobacillus plantarum* HY02946, *Lactobacillus plantarum* HY05181, and *Lactobacillus plantarum* HY00050; and / or,

[0066] The *Bifidobacterium longum* infant subsp. *longum* is selected from at least one of *Bifidobacterium longum* infant subsp. *CS004*, *Bifidobacterium longum* infant subsp. *MP09089*, and *Bifidobacterium longum* infant subsp. *MP09270*; and / or,

[0067] 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* HY11786, *Bifidobacterium lactis* subsp. *animal* MP15099, *Bifidobacterium lactis* subsp. *animal* HY09279 and *Bifidobacterium lactis* HY10036.

[0068] According to some embodiments of the present invention, the active ingredients comprise *Lactobacillus plantarum* HY02946, *Bifidobacterium longum* subsp. *infant* CS004, *Weizmannii coagulans* BC99 or *Weizmannii coagulans* HY08866, and *Lactobacillus acidophilus* CS003.

[0069] Preferably, the active ingredient 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 (10-20):(0.6-1.4):(0.6-1.4):(0.6-1.4).

[0070] More preferably, the active ingredient 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.

[0071] or,

[0072] The active ingredients include Lactobacillus plantarum HY02946, Bifidobacterium animalis subsp. lactis BB-12, Bifidobacterium longum subsp. infantis CS004, Weizmannii coagulans BC99 or HY08866, and Lactobacillus acidophilus CS003.

[0073] Preferably, the active ingredient 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 (10-20): (4-8): (0.6-1.4): (0.6-1.4): (0.6-1.4).

[0074] More preferably, the active ingredients comprise Lactobacillus plantarum HY02946, Bifidobacterium animalis subsp. lactis BB-12, Bifidobacterium longum subsp. infantis CS004, Weizmannii coagulans BC99 or HY08866, and Lactobacillus acidophilus CS003 in an effective live bacteria concentration ratio of 15:6:1:1:1.

[0075] or,

[0076] The active ingredients include 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.

[0077] Preferably, the active ingredient 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 (10-20): (4-8): (4-8): (0.6-1.4): (0.6-1.4): (0.6-1.4).

[0078] More preferably, the active ingredient 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.

[0079] Or more preferably, the probiotic composition comprises *Lactobacillus plantarum* HY00050, *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.

[0080] Or 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 20:8:8:1.4:1.4:1.4.

[0081] According to some embodiments of the present invention, the active ingredient comprises Weizmann's coagulation bacteria, Lactobacillus acidophilus, Lactobacillus plantarum, and Bifidobacterium.

[0082] Preferably, the active ingredient 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 active ingredient 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).

[0083] More preferably, the Bifidobacterium may be selected from Bifidobacterium longum subsp. infantis and / or Bifidobacterium animalis subsp. lactis.

[0084] More preferably, the active ingredient comprises *Weizmannii coagulans*, *Lactobacillus acidophilus*, *Lactobacillus plantarum*, *Bifidobacterium longum* subsp. *infantii*, and *Bifidobacterium animalis* subsp. *milk* in an effective live bacteria concentration ratio of (0.6-1.4):(0.6-8):(5-20):(0.6-1.4):(4-10); even more preferably, the probiotic composition comprises *Weizmannii coagulans*, *Lactobacillus acidophilus*, *Lactobacillus plantarum*, *Bifidobacterium longum* subsp. *infantii*, and *Bifidobacterium animalis* subsp. *milk* in an effective live bacteria concentration ratio of (0.6-1.4):(0.6-8):(5-20):(0.6-1.4):(4-8).

[0085] More preferably, 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.

[0086] Particularly 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).

[0087] According to some embodiments of the present invention, the *Weizmannii coagulans* is selected from at least one of *Weizmannii coagulans* BC99, *Weizmannii coagulans* HY08866, *Weizmannii coagulans* HY08867, *Weizmannii coagulans* MP08976, and *Weizmannii coagulans* HY08874; and / or,

[0088] 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,

[0089] 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,

[0090] 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,

[0091] 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.

[0092] According to some embodiments of the present invention, the active ingredients comprise *Lactobacillus plantarum* HY02946, *Bifidobacterium longum* subsp. *infant* CS004, *Weizmannii coagulans* BC99 or *Weizmannii coagulans* HY08866, and *Lactobacillus acidophilus* CS003.

[0093] Preferably, the active ingredient 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).

[0094] More preferably, the active ingredient 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.

[0095] or,

[0096] The active ingredients include Lactobacillus plantarum HY02946, Bifidobacterium animalis subsp. lactis BB-12, Bifidobacterium longum subsp. infantis CS004, Weizmannii coagulans BC99 or HY08866, and Lactobacillus acidophilus CS003.

[0097] Preferably, the active ingredient 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).

[0098] More preferably, the active ingredients comprise Lactobacillus plantarum HY02946, Bifidobacterium animalis subsp. lactis BB-12, Bifidobacterium longum subsp. infantis CS004, Weizmannii coagulans BC99 or HY08866, and Lactobacillus acidophilus CS003 in an effective live bacteria concentration ratio of 15:6:1:1:1.

[0099] or,

[0100] The active ingredients include *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.

[0101] Preferably, the active ingredient 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).

[0102] More preferably, the active ingredient 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).

[0103] More preferably, the active ingredient 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.

[0104] According to some embodiments of the present invention, the active ingredients comprise *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.

[0105] Preferably, the active ingredient 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).

[0106] 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.

[0107] 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.

[0108] According to some embodiments of the present invention, the dosage form of the probiotic preparation is a liquid preparation.

[0109] In a second aspect, the present invention provides the use of the probiotic preparation according to the first aspect of the present invention in the preparation of products for the prevention, relief, improvement and / or treatment of functional gastrointestinal disorders.

[0110] Preferably, the functional gastrointestinal disorder is diarrhea and / or constipation.

[0111] More preferably, the diarrhea is selected from one or more of diarrhea-predominant irritable bowel syndrome, acute diarrhea, chronic diarrhea, osmotic diarrhea, secretory diarrhea, exudative diarrhea, and motility diarrhea.

[0112] More preferably, 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.

[0113] Preferably, the product is selected from one or more of pharmaceuticals, food, and health products.

[0114] Compared with the prior art, the present invention has at least the following beneficial effects:

[0115] (1) This invention effectively promotes the metabolic activity of probiotics through a specific combination of excipients, rather than merely providing physical protection or nutritional support. Experimental data show that the excipient combination of this invention can significantly and continuously increase the redox potential (Eh) and Na+ of probiotics. + K +-ATPase activity, the absence or replacement of any single or partial excipient cannot achieve the same effect, which proves that the excipient combination is a functional system with internal synergistic relationship that has been carefully screened, rather than a simple accumulation of ingredients.

[0116] (2) The sustained and significant increase in redox potential indicates that the excipient combination of the present invention can effectively promote the aerobic metabolic activity of probiotics, accelerate their recovery from dormant state and their entry into an active physiological state. Na + K + The significant increase in ATPase activity proves that the excipient system of this invention can greatly enhance the energy production capacity of probiotic cells, which is the basis for probiotics to achieve all probiotic functions such as colonization, antagonism of pathogens, and production of beneficial metabolites.

[0117] (3) This invention overcomes the limitations of existing probiotic products, such as single strain function, limited target, and lack of synergistic technology, and develops a probiotic composition suitable for a variety of complex gastrointestinal disorders.

[0118] (4) This invention determined the inhibition rate of the fermentation broth of strains with the potential to alleviate diarrhea against Escherichia coli and Staphylococcus aureus through in vitro experiments. Considering the overall antibacterial effect and species richness, the antibacterial rate was measured for single strains, dual-strain combinations, and hexa-strain combinations. The results showed that as the number of strains in the combination increased, the overall antibacterial effect gradually increased. Most of the combined strains showed a similar inhibitory trend against Staphylococcus aureus and Escherichia coli, with the hexa-strain combination exhibiting the best overall antibacterial effect. This invention also demonstrated through in vitro experiments that the tetra-, penta-, and hexa-strain combinations of this invention can reduce the average fecal water content in mice and have a significant ameliorative effect on antibiotic-associated diarrhea and infectious diarrhea.

[0119] (5) This invention uses in vitro experiments to determine the effect of lysates of strains with potential to improve constipation on the 5-HT secretion of RIN-14B cells. Combining the 5-HT-promoting effect and species diversity, the effects on the 5-HT secretion of RIN-14B cells were determined by mixing single strains, dual strains, triple strains, quadrivalent strains, pentavalent strains, and hexavalent strains in a 1:1 ratio. The results showed that the quadrivalent probiotic composition containing Lactobacillus acidophilus, Bifidobacterium longum, Lactobacillus plantarum, and Weizmann's coagulans; the pentavalent probiotic composition containing Lactobacillus acidophilus, Bifidobacterium longum subsp. infantis, Bifidobacterium animalis subsp. lactis, Lactobacillus plantarum, and Weizmann's coagulans; and the hexavalent composition containing two different strains of Lactobacillus acidophilus, Bifidobacterium longum subsp. infantis, Bifidobacterium animalis subsp. lactis, Lactobacillus plantarum, and Weizmann's coagulans were better than other conjugate compositions in promoting 5-HT production. 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.

[0120] (6) The present invention also demonstrated through in vitro experiments that the hexabiotic composition of the present 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. In addition, the hexabiotic composition of the present invention can also promote small intestinal peristalsis in mice with functional constipation, thereby relieving constipation. Attached Figure Description

[0121] The embodiments of the present invention will now be described in detail with reference to the accompanying drawings, wherein:

[0122] Figure 1 The standard curve of 5-HT (5-hydroxytryptamine) standard sample in the embodiments of the present invention is shown. Detailed Implementation

[0123] 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.

[0124] The method for detecting viable bacteria count in this example is as follows:

[0125] ① F1 generation breeding:

[0126] Mycelium powder: Weigh 0.1g of mycelium powder into a 15mL centrifuge tube, add 4.9mL of MRS or TPY broth and mix well;

[0127] 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.

[0128] 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.

[0129] ③ Preparation of 1% solid culture medium: Weigh 52.24g and 36.65g of MRS broth and TPY broth into 1L beakers respectively, add 10g of agar powder to each, add 1L of distilled water and mix well. Dispense into 400mL glass bottles, autoclave at 118℃ for 15min and then place in a 50℃ water bath for later use.

[0130] 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.

[0131] ⑤ After incubation, select plates with colony counts between 30-300 CFU for counting to determine the viable count:

[0132] viable count = average colony count × dilution factor.

[0133] Example 1: Screening of Functional Bacteria

[0134] 1. Candidate strains

[0135] Candidate strains are shown in Table 1. Among them, the applicant's (Hangzhou Yuanda) proprietary strains were stored at -80°C in preservation tubes containing 15% (v / v) glycerol. Commercially available strains were either in powder form or isolated from products.

[0136] Table 1. Strains Information Table

[0137]

[0138]

[0139]

[0140] 2. Determination of 5-HT content

[0141] Experimental methods:

[0142] (1) Reagent preparation

[0143] 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℃.

[0144] 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.

[0145] 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.

[0146] (2) Cell culture

[0147] 1) RIN-14B cell resuscitation

[0148] ① Take out one RIN-14B cell cryopreservation tube stored in the liquid nitrogen tank;

[0149] 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.

[0150] 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 for trypan blue staining and perform viable cell counting.

[0151] 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.

[0152] 2) Passaging of RIN-14B cells

[0153] ① Remove the cell culture flask and observe the cell adhesion and confluence under a microscope to see if it reaches more than 80%;

[0154] ② Discard the culture medium and add 3 mL of PBS to the culture flask to gently wash the cells twice;

[0155] 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;

[0156] ④ Centrifuge at 1000 rpm for 5 min, discard the supernatant, add 5 mL of culture medium to resuspend, and take 200 μL for trypan blue staining and live cell counting.

[0157] 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.

[0158] 3) RIN-14B cell plate

[0159] ① Discard the culture medium in the T75 cell flask, wash twice with 3 mL PBS, add 3 mL 0.25% trypsin-EDTA, and digest the adherent cells;

[0160] 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).

[0161] 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;

[0162] 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.

[0163] (3) Culture of strains

[0164] 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.

[0165] 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.

[0166] (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.

[0167] (5) Strain-cell interaction

[0168] 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.

[0169] 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.

[0170] (6) Sample 5-HT determination test

[0171] 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 ).

[0172] 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:

[0173] Table 2 HPLC determination of 5-HT parameters

[0174]

[0175] Experimental results:

[0176] The results of 5-HT content of single strains showed that 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.

[0177] To further determine the ability of compositions prepared from different strains to promote 5-HT production by cells, the inventors randomly combined single strains 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 dual to hexavalent compositions revealed that 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 and exhibited 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.

[0178] 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.

[0179] 3. Antibacterial experiments against Escherichia coli and Staphylococcus aureus

[0180] (1) Culture of strains

[0181] 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.

[0182] ② F2 generation: Gently mix the cultured F1 generation bacterial suspension and transfer it to 6 mL of MRS or TPY liquid medium at the same inoculum size. Incubate at 37℃ in an anaerobic workstation for 24-48 hours. After two generations of continuous activation, each candidate strain is combined into single-strain, dual-strain, triple-strain, quadri-strain, pentad, and hexa-strain groups. The components of the multi-strain groups are from different species and combined in equal proportions. The final concentration of each group composition is 1E+0.8 CFU / mL.

[0183] (2) Antibacterial test

[0184] Escherichia coli:

[0185] ① Preparation of Escherichia coli plates

[0186] Pathogenic bacteria culture: Escherichia coli was activated and transferred to BHI liquid medium at a 4% inoculum to the early stable stage. The concentration was adjusted to 1.0E+06 CFU / mL. 100 μL was spread on BHI solid plates and dried for later use.

[0187] ②Drilling holes in flat plates

[0188] Use a 1mL pipette tip to make three holes evenly on each E. coli plate, and four holes on each CK plate.

[0189] ③ Interaction experiment with Escherichia coli

[0190] Add 100 μL of the experimental group to each well of the experimental plate, with 3 replicates per group. Add MRS liquid medium and TPY liquid medium to the CK plate, with 2 replicates for each. Let stand for 1 hour to allow complete infiltration into the plate, then place it upright in an incubator at 37°C and incubate aerobically for 24 hours.

[0191] ④ Inhibition zone determination

[0192] The diameter of the inhibition zone on the experimental plate and the control plate was measured using a ruler, and the inhibition rate of the target bacteria against Escherichia coli was calculated.

[0193] The relative inhibition rate (%) of the target bacteria against Escherichia coli = (diameter of the inhibition zone of the target bacteria - diameter of the inhibition zone of the control group) / diameter of the inhibition zone of the control group

[0194] Staphylococcus aureus:

[0195] ①Preparation of Staphylococcus aureus bacterial culture

[0196] At an inoculation rate of 4%, Staphylococcus aureus was activated and transferred to BHI liquid medium until the early stable phase. The concentration was adjusted to 1.0E+04 CFU / mL, and 5 mL was transferred to a shaker tube for later use.

[0197] ②Preparation of Staphylococcus aureus culture medium

[0198] Weigh 67.4g of Staphylococcus aureus chromogenic medium powder, add 1L of distilled water or deionized water, stir and heat to boiling until completely dissolved, and place in a 50℃ water bath for later use.

[0199] ③ Interaction experiment with Staphylococcus aureus

[0200] Add 100 μL of the experimental group to the shake tube containing Staphylococcus aureus and mix well. Repeat the process three times per group. Add an equal volume of MRS or TPY liquid medium to the CK tube. Incubate at 37°C for 20 min. Mix well again. Take 50 μL of the mixture from each shake tube and add it to the center of a petri dish. Pour about 15 mL of Staphylococcus aureus chromogenic medium into each dish, shake well, air dry, and invert the dish to incubate aerobically at 37°C for 24 h.

[0201] ④ Determination of antibacterial rate

[0202] Viable bacteria counts were performed on Staphylococcus aureus plates to calculate the inhibition rate of the target bacteria against Staphylococcus aureus.

[0203] Relative inhibition rate against Staphylococcus aureus (%) = 1 - viable count of experimental plate / viable count of CK plate

[0204] (3) Experimental results

[0205] The results of single-strain inhibition rates against pathogenic bacteria showed that Lactobacillus acidophilus, Weizmann's coagulans, Lactobacillus plantarum, Bifidobacterium (Bifidobacterium longum subsp. infantis and Bifidobacterium animalis subsp. lactis) and Lactobacillus rhamnosus all showed certain inhibitory effects on Escherichia coli and Staphylococcus aureus.

[0206] To further determine the inhibitory effects of different strain combinations on *Escherichia coli* and *Staphylococcus aureus*, the inventors randomly combined single strains to prepare compositions containing dual, triple, quadrivalent, pentavalent, and hexavalent probiotics and tested the inhibitory effects of different compositions on pathogenic bacteria. Statistical analysis of the inhibition rates of the dual to hexavalent compositions on pathogenic bacteria revealed that dual and triple compositions did not show a significant increase in the inhibition rate 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 and exhibited a certain dose-dependent effect. The quadrivalent probiotic composition showed inhibition rates of 50-92% against both *Staphylococcus aureus* and *Escherichia coli*, the pentavalent composition showed inhibition rates of 50-93%, and the hexavalent composition showed inhibition rates of 50-95%.

[0207] The inventors further compared and analyzed the inhibitory effects of different strains combined in quadrivalent to hexavalent probiotic combinations on pathogenic bacteria. They found that the quadrivalent, pentavalent, and hexavalent probiotic combinations containing *Lactobacillus rhamnosus* all showed inhibition rates of 50%-65% against both pathogenic bacteria, without demonstrating a significant improvement in pathogenic bacteria inhibition. The combination of *Lactobacillus rhamnosus* with other bacteria did not show a better synergistic effect. The quadrivalent probiotic combinations without *Lactobacillus rhamnosus* showed inhibition rates of approximately 85-92% against *Staphylococcus aureus* and *Escherichia coli*, the pentavalent probiotic combinations showed inhibition rates of approximately 86-93%, and the hexavalent probiotic combinations showed inhibition rates of approximately 87-95%. The synergistic effect between the strains was good, resulting in better inhibition of *Escherichia coli* and *Staphylococcus aureus*.

[0208] Based on the combined results of 5-HT and the inhibition rate against Escherichia coli and Staphylococcus aureus, nine strains with high 5-HT content and high inhibition rate were finally screened out, as shown in Table 3, for subsequent experiments.

[0209] Table 3. Information on bacterial strains with high 5-HT content and high antibacterial rate.

[0210]

[0211] Example 2: Screening of the proportion of probiotic compositions

[0212] 1. Study on the production of 5-HT by synergistic bacteria

[0213] 1.1 Experimental Design

[0214] 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 coagulant; a pentad probiotic composition containing Lactobacillus acidophilus, Bifidobacterium longum subsp. infantis, Bifidobacterium animalis subsp. lactis, Lactobacillus plantarum, and Weizmann's coagulant; 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 coagulant. These combinations showed the best ability to promote 5-HT production in cells compared to other co-combined compositions. Therefore, based on the above quadruple, pentad, and hexapod compositions, further research was conducted on the ratio of different strains.

[0215] Select the strains with the best single-strain effects: HY02946, BB-12, LA-5, CS004, BC99 (or HY08866), CS003, UABla-12, and DDS-1. Set their proportions 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, and combine them 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, overall better than other combinations. Therefore, further optimization of the combination ratios of these two six-combination assemblies was conducted. The ratio settings for each strain are shown in Table 4 below.

[0216] Table 4 Screening of the proportion of each strain in the hexavalent combination

[0217]

[0218] 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.

[0219] 1.2 Experimental Results

[0220] 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.

[0221] 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.

[0222] Table 5. Detection results of 5-HT content in different hexavalent compositions.

[0223]

[0224]

[0225] When replacing the six-component combination with other strains to verify its ability to promote 5-HT production, this example uses *Lactobacillus rhamnosus* as an example. After replacing each strain, the ability of the six-component combination to promote 5-HT production was tested, and the experimental results are shown in Table 6. The results show that after replacing any strain in the six-component combination with *Lactobacillus rhamnosus*, the ability of the combination to produce 5-HT decreased significantly. This indicates that the strains in the six-component combination of the present invention have a synergistic effect.

[0226] Table 6. Detection results of 5-HT content in different hexavalent compositions

[0227]

[0228] 2. Study on combined bacterial inhibition

[0229] 2.1 Experimental Design

[0230] 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 coagulant; a pentad probiotic composition containing Lactobacillus acidophilus, Bifidobacterium longum subsp. infantis, Bifidobacterium animalis subsp. lactis, Lactobacillus plantarum, and Weizmann's coagulant; 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 coagulant. These combinations showed stronger inhibitory effects on pathogenic bacteria compared to other co-combined compositions. Therefore, based on the above quadruple, pentad, and hexapod compositions, further research was conducted on the ratio of different strains.

[0231] Selecting single-strain strains with good efficacy, such as HY02946, BB-12, LA-5, CS004, BC99, CS003, and HY08866 (or HY08866), and setting proportions of 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 tetrad, pentad, and hexademic strains. Experimental results showed that when the proportion of HY02946 was set to 1-25, the proportions of BB-12 and LA-5 were both set to 2-10, and the proportions of CS004, BC99 (or HY08866), and CS003 were set to... When the concentration was set between 0.2 and 1.8, the inhibition rates against Staphylococcus aureus and Escherichia coli both reached 90% or higher. Among these, the six-strain combinations HY02946, BB-12, LA-5, BC99, CS003, and CS004, and HY02946, BB-12, LA-5, HY08866, CS003, and CS004, showed superior effects at various ratios. The inhibition rates against Staphylococcus aureus ranged from 90% to 98%, and against Escherichia coli from 90% to 99%, demonstrating overall superiority over other combinations. Therefore, further optimization of the combination ratios for these two six-strain combinations was conducted. The ratio settings for each strain are shown in Table 7 below.

[0232] Table 7 Screening of the proportion of each strain in the hexavalent combination

[0233]

[0234] The inhibitory effects of each formulation sample on Escherichia coli and Staphylococcus aureus were tested using the same methods as in Example 1. The experimental results were analyzed using TTEST.

[0235] 2.2 Experimental Results

[0236] Experimental results showed that when the ratio of the six-component combination HY02946 was set at 10-20, the ratio of BB-12 at 4-8, the ratio of LA-5 at 4-8, and the ratio of CS004, BC99, and CS003 at 0.6-1.4, the inhibition rate against Staphylococcus aureus was above 95%, and the inhibition rate against Escherichia coli was above 96%. When the ratio of HY02946:BB-12:LA-5:CS004:BC99:CS003 was 15:6:6:1:1:1, the inhibition rate against Staphylococcus aureus reached 97.13%, and the inhibition rate against Escherichia coli reached 98.33%. When the ratio of HY02946:BB-12:LA-5:CS004:HY08866:CS003 is 15:6:6:1:1:1, the inhibition rate of Staphylococcus aureus reaches 96.84%, and the inhibition rate of Escherichia coli reaches 97.13%. When the ratio of HY02946:BB-12:LA-5:BC99:CS003:CS004 is 20:8:8:1.4:1.4:1.4, the inhibition rate of Staphylococcus aureus reaches 96.19%, and the inhibition rate of Escherichia coli reaches 97.67%. When the ratio of HY02946:BB-12:LA-5:HY08866:CS003:CS004 is 20:8:8:1.4:1.4:1.4, the inhibition rate of Staphylococcus aureus reaches 96.42%, and the inhibition rate of Escherichia coli reaches 97.33%.

[0237] Table 8. Inhibition rates of different hexavalent bacteria against pathogenic bacteria

[0238]

[0239]

[0240] The inhibitory effects of different hexavalent compositions on pathogenic bacteria were verified by replacing them with strains other than those in the hexavalent composition. In this example, *Lactobacillus rhamnosus* was used as an example. After replacing each strain, the pathogenic bacteria inhibition rate of the hexavalent composition was tested, and the experimental results are shown in Table 9. The results show that after replacing any strain in the hexavalent composition with *Lactobacillus rhamnosus*, the antibacterial ability of the composition decreased significantly. The results in Table 9 further demonstrate the synergistic effect between the strains in the hexavalent composition of the present invention.

[0241] Table 9. Inhibition rates of different hexabiotic combinations against pathogenic bacteria

[0242]

[0243] Example 3: Efficacy verification of probiotic composition for constipation

[0244] 1. Experimental Methods

[0245] 1.1 Experimental Materials

[0246] (1) The key experimental reagents are shown in Table 10

[0247] Table 10 Key Experimental Reagents

[0248]

[0249] (2) Test substances

[0250] The animal grouping information is shown in Table 11.

[0251] Table 11 Animal Experiment Grouping

[0252]

[0253] 1.2 Animal System

[0254] (1) Experimental animals

[0255] SPF-grade Balb / c mice, male, 7 - 8 weeks old, weighing 20 g to 22 g, were purchased from Zhejiang Vital River Laboratory Animal Technology Co., Ltd., license number: SCXK(Zhe) 2024 - 0001, animal certificate number: 20251016Abzz06199990627.

[0256] (2) Environmental adaptation

[0257] After the animals were placed in the room, they were adaptively raised for 3 days before starting the experiment. The main inspection contents during the adaptation period were: whether there were abnormal appearances; whether the general conditions were normal. Unqualified animals were not included in the experiment.

[0258] (3) Feeding management

[0259] They were raised in the barrier system of Hangzhou Mosslite Biotechnology Co., Ltd., experimental animal use license number: SYXK(Zhe) 2022 - 0032.

[0260] (4) Experimental design and grouping

[0261] The animal modeling methods for each of the above groups are shown in Table 12.

[0262] Table 12 Animal Modeling Methods for Each Group

[0263]

[0264] Note: IP*Q3D: Intraperitoneal injection, once every 3 days; QD*10 day: Administer once a day for 10 consecutive days.

[0265] (5) Animal administration

[0266] Treatment began on the first day of modeling. The treatment group was given the corresponding test sample by gavage, while the negative control group and the model control group were given an equal volume of PBS by gavage. The drugs were administered once a day until the end of the experiment.

[0267] 1.3 Observation and Indicator Testing

[0268] (1) Weight monitoring

[0269] After the animals have completed their acclimatization period, the mice were weighed weekly and their weight changes were recorded.

[0270] (2) Fecal moisture content test

[0271] Weigh the empty glass bottle and record its 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.

[0272] Fecal wet weight = wet weight - empty bottle weight = W1 - W 空 ;

[0273] Fecal dry weight = Dry weight - Empty bottle weight = W2 - W 空 ;

[0274] Fecal moisture content = (W1-W2) / W1×100%.

[0275] (3) Defecation test

[0276] 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).

[0277] (4) Small intestine propulsion experiment

[0278] 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.

[0279] Carbon ink propulsion rate (%) = Carbon ink propulsion distance (cm) / Total length of small intestine (cm) * 100% (5) Statistical analysis

[0280] 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).

[0281] 2. Experimental Results

[0282] (1) Evaluation of the time index for the first black stool excretion

[0283] The results are shown in Table 13. 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.

[0284] Table 13 Results of the time index for the first black stool excretion (mean ± standard deviation)

[0285]

[0286] Note: Statistical analysis was performed using the T-test method; comparisons were made with the normal control group. #### P<0.0001; compared with the model control group, *P<0.05, **P<0.01.

[0287] (2) Evaluation of mouse fecal weight and fecal moisture content

[0288] The results are shown in Table 14. 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 four-, five-, and six-strain bacterial combinations of this invention exhibit synergistic effects, and their effects cannot be achieved simply by piling up strains or increasing the number of strains.

[0289] Table 14 Results of stool weight test (mean ± standard deviation)

[0290]

[0291] Note: Statistical analysis was performed using the T-test method; comparisons were made with the normal control group. ### P<0.001, #### P<0.0001; compared with the model control group, *P<0.05, **P<0.01, ***P<0.001.

[0292] (3) Evaluation of carbon ink propulsion rate index

[0293] The results are shown in Table 15. 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, with the DU group showing extremely significant effects (P<0.001), and the PC, TB, FB, AB, AH, and DH groups showing 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.

[0294] Table 15 Carbon Graphite Propulsion Results

[0295]

[0296] Note: Statistical analysis was performed using the T-test method; comparisons were made with the normal control group. #### P<0.0001; compared with the model control group, *P<0.05, **P<0.01, ***P<0.001.

[0297] The results in Tables 13 to 15 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.

[0298] Example 4: Efficacy verification of probiotic composition against antibiotic-associated diarrhea (AAD)

[0299] 1. Experimental Methods

[0300] 1.1 Experimental Materials

[0301] (1) Key experimental reagents are shown in Table 16, triple antibiotic preparation information is shown in Table 17, and strain information is shown in Table 18.

[0302] Table 16 Key Experimental Reagents

[0303]

[0304] Table 17 Information on the preparation of triple antibiotics

[0305]

[0306] Table 18 Strain Information Table

[0307]

[0308] (2) Experimental grouping

[0309] Animal grouping information is shown in Table 19.

[0310] Table 19 Animal Experiment Grouping

[0311]

[0312] 1.2 Animal System

[0313] (1) Laboratory animals

[0314] C57BL / 6J male mice, 6 weeks old. Purchased from Zhejiang Vital River Laboratory Animal Technology Co., Ltd., license number: SCXK(Zhe) 2024-0001, animal certificate number: 20251016Abzz01699990630.

[0315] (2)Drug administration plan

[0316] An AAD mouse model was constructed by combining three antibiotics (clindamycin + ampicillin + streptomycin) for modeling. After successfully constructing the model, different test samples were used for therapeutic administration, and antibiotics were not discontinued during the treatment period to observe the effects of the test substances on AAD mice. The specific grouping design is as follows:

[0317] ① NC group: normal control group, gavaged with 0.9% normal saline;

[0318] ② MC group: model control group, gavaged with a mixed solution of triple antibiotics (10 mL / kg·BW / day) throughout the process;

[0319] ③ AW group: antibiotic withdrawal group, gavaged with a mixed solution of triple antibiotics (10 mL / kg·BW / day) for 3 days and then treatment was discontinued;

[0320] ④ In all other experimental groups, a mixed solution of triple antibiotics (10 mL / kg·BW / day) was gavaged throughout the process.

[0321] Treatment started 5 hours after modeling every day. The treatment groups were respectively gavaged with the corresponding test samples, and the NC group and MC group were gavaged with an equal volume of normal saline. Administration was once a day until the end of the experiment.

[0322] 1.3 Observation and index detection

[0323] (1)Body weight monitoring

[0324] After the adaptive feeding of the animals was completed, the body weight of the mice was measured daily to guide the dosage of the animals.

[0325] (2)Detection of fecal water content

[0326] Weigh the empty glass bottle and record the bottle weight as W 空 . Place the mouse in a metabolic cage, record the number of feces of the mouse, and at the same time collect the mouse feces in the glass bottle. After 1 hour of collection, weigh the feces together with the bottle as W1, and then place the mouse feces in a forced air drying oven at 90 °C for 5 hours and weigh the feces together with the bottle as W2.

[0327] Fecal wet weight = wet weight - empty bottle weight = W1 - W 空 ;

[0328] Fecal dry weight = dry weight - empty bottle weight = W2 - W 空 ;

[0329] Fecal moisture content = (W1-W2) / W1×100%.

[0330] (3) Stool characteristics score

[0331] The stool examination scoring criteria are as follows:

[0332] ① The feces are oval-shaped, with distinct particles, hard in texture, and brownish in color, with a score of 1;

[0333] ② The feces are sausage-shaped, smooth, soft, and yellowish in color, with a score of 2;

[0334] ③ The feces have blurred edges or no fixed shape, are soft and wet, and are yellowish in color, with a score of 3.

[0335] 1.4 Data Statistics

[0336] 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).

[0337] 2. Experimental Results

[0338] 2.1 Improvement of clinical symptoms in mice by the test sample

[0339] Before administration, the model group mice exhibited typical diarrhea symptoms. Throughout the administration period, the fecal characteristics score of the model control group was consistently higher than that of the normal control group, indicating that the IBS-D model was stable and suitable for efficacy evaluation.

[0340] Table 20 Effects of the test sample on fecal morphology scores in mice

[0341]

[0342] Note: Compared with the normal control group, ####P<0.0001; compared with the model control group, *P<0.05, **P<0.01 for each drug administration group.

[0343] Overall, the AB1, AB2, AB3, AB4, and AH groups showed better results than the quadrivalent and pentavalent strains, indicating that the more strains present, the better the synergistic effect. Furthermore, the TB1, TB2, FB, AB1, AB2, AB3, AB4, and AH groups all showed significant differences compared to the model group, while the antibiotic discontinuation group did not show a significant reduction in fecal characteristic scores. This suggests that the quadrivalent, pentavalent, and hexavalent strains of this invention all have excellent effects in relieving diarrhea, with the hexavalent strain showing the best effect. The study also found that when any strain other than *Lactobacillus plantarum*, *Bifidobacterium*, *Lactobacillus acidophilus*, and *Weizmannii coagulans* were arbitrarily replaced in the probiotic composition, taking the replacement of *Lactobacillus rhamnosus* as an example, the AR group results showed that replacing the strains in the composition with *Lactobacillus rhamnosus* HY02078 resulted in a fecal characteristic score of only 1.68 points, which was not significantly different from the model group.

[0344] 2.2 Effect of the test sample on the water content of mouse feces

[0345] The results of fecal moisture content testing are shown in Table 21. The results showed that on day 8 after administration, the fecal moisture content of the model control group was significantly higher than that of the normal control group. On day 8 after administration, the fecal moisture content of all treatment groups except AR group was significantly lower than that of the model control group (P < 0.05).

[0346] Table 21 Effect of the test sample on the water content of mouse feces

[0347]

[0348] Note: Compared with the normal control group, the model control group showed ####P<0.0001. Compared with the model control group, each drug-treated group showed *P<0.05.

[0349] The data in Table 21 show that the fecal water content of the tetravalent, pentavalent, and hexavalent strains of the present invention was significantly lower than that of the model group (p < 0.05). This indicates that probiotics can not only improve the "clinical manifestations" of diarrhea (fecal characteristics score), but also fundamentally improve intestinal physiological function, promote water absorption, and reduce fecal water content. Although the AR group showed a decrease, there was no statistically significant difference compared with the model group. Overall, the data in Table 21 show that the fecal water content study obtained results consistent with the fecal characteristics score study.

[0350] The results in Tables 20 and 21 together demonstrate that the probiotic composition prepared from *Lactobacillus plantarum*, *Bifidobacterium*, *Lactobacillus acidophilus*, and *Weizmannii coagulans* used in this invention exhibits good synergy among the various strains, achieving a good therapeutic effect in treating diarrhea.

[0351] Example 5: Efficacy verification of probiotic composition against infectious diarrhea

[0352] 1. Experimental Methods

[0353] 1.1 Experimental Materials

[0354] The experimental strain was the same as that in Example 4.

[0355] 1.2 Laboratory Animals

[0356] Select C57BL / 6N baby mice, SPF grade, male, 8-10g / mouse. Animal housing conditions should be set at room temperature 20-22℃, humidity 40-70%, with a 12-hour light / dark cycle. Bedding should be changed at least twice a week, along with the feeding box; replace the feeding box immediately if any abnormal conditions are observed. Water bottles and stoppers should be changed and sterilized daily, and cages should be sterilized every two weeks. All washed cages should be autoclaved after cleaning.

[0357] 1.3 Experimental grouping and dosing regimen

[0358] As shown in Table 22.

[0359] The treatment procedures for each group of mice are as follows:

[0360] Pretreatment of young mice: Except for the normal control group, the other young mice were given a quadruple antibiotic drinking water (gentamicin, vancomycin, metronidazole and polymyxin) for 4 days before E. coli infection to disrupt the resident microbiota, and then replaced with normal drinking water to remove the antibiotics.

[0361] Young mice were inoculated with *E. coli*: Except for the normal control group, the remaining young mice were inoculated with *E. coli* (standard strain, CICC10413). Each young mouse was orally administered 100 μL (4 × 10⁶ μL) via gavage. 9 (cfu / mL), administered once, with the vaccination time recorded as Day 0.

[0362] Administration to young mice: The probiotic group and the cefixime group were given the drug 1 day after infection, once a day for 5 consecutive days.

[0363] Table 22 Experimental Groups and Dosing Regimens

[0364]

[0365] 1.4 Experimental Indicators

[0366] Fecal scoring: During treatment, the fecal condition of the young mice was observed and scored daily (feces were graded into 6 levels: Grade 1 was normal stool, Grade 2 was yellow, formed, soft stool, Grade 3 was yellow, pasty stool, Grade 4 was yellow, watery, mucous stool, Grade 5 was yellow, egg-soup-like stool, and Grade 6 was completely yellow, watery stool. Grade 3 (yellow, pasty stool) and above indicated intestinal infection, Grades 4-5 indicated mild infection, and Grade 6 indicated severe infection. Symptom assessment was performed by the same person). Data are expressed as mean ± standard deviation (Mean ± SD).

[0367] Colorectal length: After the last administration, the young mice were sacrificed and the length of their colorectal length was measured.

[0368] Statistical analysis: p-values ​​were obtained according to the significance test method, where p<0.05 was considered statistically significant and p<0.01 was considered statistically significant.

[0369] 2. Experimental Results

[0370] 2.1 Stool score

[0371] Table 23 Stool scores of different experimental groups

[0372]

[0373] Note: Statistical analysis was performed using the T-test method; compared with the normal control group, ##P<0.01; compared with the model control group, *P<0.05, **P<0.01.

[0374] The results showed that the probiotic compositions of the present invention began to take effect on the third day of administration. The fecal scores of the AB1 and AB3 groups were significantly lower than those of the model group on the fourth day. On the fifth day, the effects of the AB1, AB2, AB3, AB4 and AH groups were all highly significant, which were better than the positive control group, demonstrating the superiority of rapid onset and strong efficacy.

[0375] However, the AR group had the slowest onset of action and the worst recovery throughout the treatment course, which again proves that replacing the probiotic composition of the present invention with other strains cannot achieve the same therapeutic effect. Furthermore, although the AR group consisted of six strains, from day 3 onwards, the effects of the present invention's quadrivalent strains TB1 and TB2, as well as the pentavalent strain FB, already reached or even exceeded those of the AR group. This further illustrates that the effectiveness of the present invention lies in the synergy between specific strains, and cannot be achieved simply by increasing the number of strains.

[0376] Example 6: Experimental Study on Formulation Activation

[0377] 1. Experimental Materials

[0378] 1.1 Probiotic Powder

[0379] The probiotic powder used in this embodiment comes from the following sources:

[0380] Lactobacillus plantarum HY02946 (500 billion CFU / g), Lactobacillus acidophilus CS003 (500 billion CFU / g), and Bifidobacterium longum subsp. infantis CS004 (500 billion CFU / g) are strains owned by the applicant and manufactured by Qingdao Nuosen Biotechnology Co., Ltd.

[0381] The *Vibrio coagulans* BC99 (200 billion CFU / g) was purchased from Jiangsu Weikang Biotechnology Co., Ltd.

[0382] Bifidobacterium animalis subsp. lactis BB-12 (55 billion CFU / g), Lactobacillus acidophilus DSM13241 (55 billion CFU / g), Lactobacillus acidophilus DSM34415 (250 billion CFU / g), and Bifidobacterium animalis subsp. lactis DSM34438 (500 billion CFU / g) were purchased from Chr. Hansen (Beijing) Trading Co., Ltd.

[0383] 1.2 Excipients

[0384] The sources of some of the auxiliary materials used in this embodiment are as follows:

[0385] Fructooligosaccharides were purchased from Shandong Bailong Chuangyuan Biotechnology Co., Ltd., product number P95;

[0386] Galacto-oligosaccharides were purchased from Shandong Bailong Chuangyuan Biotechnology Co., Ltd., product number P90;

[0387] Isomaltooligosaccharide was purchased from Shandong Bailong Chuangyuan Biotechnology Co., Ltd., product number P900;

[0388] L-arabinose was purchased from Jinan Shengquan Tanghe Tang Biotechnology Co., Ltd., CAS No. 5328-37-0;

[0389] Xylitol was purchased from Zhejiang Huakang Pharmaceutical Co., Ltd., CAS No. 16277-71-7;

[0390] Vitamin C was purchased from CSPC Pharmaceutical Group Co., Ltd., CAS No. 50-81-7.

[0391] 1.3 Experimental Instruments and Main Equipment

[0392] The experimental instruments and main equipment used in this embodiment are shown in Tables 24 and 25.

[0393] Table 24 Main Instruments for the Experiment

[0394]

[0395] Table 25 Main Reagents for the Experiment

[0396]

[0397] 1.4 Experimental Grouping

[0398] 1.4.1 Composition of Activation Solution

[0399] Table 26 Composition of Activation Solution 1

[0400]

[0401]

[0402] Note: The oligosaccharides of the present invention are replaced by lactulose, stachyose, raffinose, oligogentisose, and oligochitosan. Table 26, group 11 uses oligogentisose as an example. The antioxidant additives of the present invention are replaced by theaflavins, disodium EDTA, and sorbic acid. Group 12 uses theaflavins as an example.

[0403] Table 27 Composition of Activation Solution II

[0404]

[0405]

[0406] Note: The oligosaccharides of the present invention are replaced by lactulose, stachyose, raffinose, oligogentisose, and oligochitosan. Table 26, group 11 uses oligogentisose as an example. The antioxidant additives of the present invention are replaced by theaflavins, disodium EDTA, and sorbic acid. Group 12 uses theaflavins as an example.

[0407] 2. Experimental Procedure

[0408] 2.1 Detection of the redox potential of the activated hexabiotic probiotic preparation

[0409] Probiotic composition 1 was prepared according to the effective live bacteria concentration ratio of Lactobacillus plantarum HY02946: Bifidobacterium animalis subsp. lactis BB-12: Lactobacillus acidophilus DSM13241: Weizmannii coagulans BC99: Lactobacillus acidophilus CS003: Bifidobacterium longum subsp. infantis CS004 15:6:6:1:1:1.

[0410] Weigh 1.8 g of the six-component probiotic composition 1 and add it to 30 mL of preheated (37℃) excipient-containing solution 1 (activation solution 1) for activation (the water content in activation solution 1 is 9.34 kg / 10L). Set up one parallel group for each group. Mix well and place in a 37℃ water bath. Use the electrode method to detect the redox potential at 0h. Before measurement, rinse the electrode with deionized water, blot dry with filter paper, immerse it in the test solution, and count after the reading stabilizes. Rinse the electrode with deionized water, blot dry with filter paper, and immerse it in the preservation solution. Measure the redox potential of the sample at 0h, 30min, 1h, and 2h.

[0411] Probiotic composition 2 was prepared according to the effective live bacteria concentration ratio of Lactobacillus plantarum HY02946: Bifidobacterium animalis subsp. lactis DSM34438: Lactobacillus acidophilus DSM34415: Weizmannii coagulans BC99: Lactobacillus acidophilus CS003: Bifidobacterium longum subsp. infantis CS004 of 10:8:4:1:1:1.

[0412] Weigh 1.8 g of the six-component probiotic composition 2 and add it to 30 mL of preheated (37℃) excipient-containing solution 2 (activation solution 2) for activation (the water content in activation solution 2 is 8.804 kg / 10L). Set up one parallel group for each group. Mix well and place in a 37℃ water bath. Use the electrode method to detect the redox potential at 0h. Before measurement, rinse the electrode with deionized water, blot dry with filter paper, immerse it in the test solution, and count after the reading stabilizes. Rinse the electrode with deionized water, blot dry with filter paper, and immerse it in the preservation solution. Measure the redox potential of the sample at 0h, 30min, 1h, and 2h.

[0413] 2.2 Na+ in activated hexaprobiotic formulations + K + - Detection of ATPase activity

[0414] Weigh 0.18g of the six-in-one probiotic composition 1, add 3 mL of preheated (37℃) excipient-containing solution 1 (activation solution 1) for activation, mix well, and incubate at 37℃. Weigh 0.18g of the six-in-one probiotic composition 2, add 3 mL of preheated (37℃) excipient-containing solution 2 (activation solution 2) for activation, mix well, and incubate at 37℃.

[0415] Two samples were taken at time points of 0h, 15min, 30min, 1h, and 2h for Na analysis. + K + -ATPase activity assay, i.e., setting up a set of parallel assays.

[0416] The testing procedures follow Solarbio's Na + K+ The ATPase activity assay kit instructions are as follows:

[0417] ① Preparation of test solutions: Dissolve one vial of reagent 3 in 1 mL of distilled water immediately before use. Dissolve reagents 6 and 7 in 5 mL of distilled water each. Prepare the phosphorus determinant solution according to the ratio of H2O: reagent 6: reagent 7: reagent 8 = 2:1:1:1 (volume ratio), and prepare it immediately before use.

[0418] ② Sample processing: Take 1 mL of sample into a centrifuge tube, centrifuge at 8000g at 4℃ for 10 min, discard the supernatant, add 1 mL of reagent I, and sonicate the cells (power 200W, sonication for 3 seconds, interval 10 seconds, repeated 30 times). Centrifuge at 8000g at 4℃ for 10 min, collect the supernatant, and place on ice for testing.

[0419] ③ Preparation of standards: Take 0.1 mL of 10 μmol / mL standard phosphorus stock solution and add it to 1.9 mL of distilled water. Mix thoroughly to prepare a 0.5 μmol / mL standard phosphorus solution. Then dilute it successively to prepare standards of 0.25, 0.125, 0.0625, 0.03125 and 0 μmol / mL for plotting standard curves.

[0420] ④ Enzymatic reaction: Add the following reagents to the EP tube according to the table below and proceed accordingly.

[0421] Table 28 Enzyme-catalyzed reaction steps

[0422]

[0423] ⑤ Phosphorus determination: Add the following reagents to the EP tube according to the table below and proceed accordingly.

[0424] Vortex to mix thoroughly, then place in a 40℃ water bath for 10 min. Add 200 μL of the mixture to a 96-well plate and record the absorbance at 660 nm. These values ​​are recorded as A (blank), A (standard), A (control), and A (determined). The calculation formula is:

[0425] Na + K + -ATPase activity (U / 10¹⁰ cell) = 7.5 × (A assay - A control) / (A standard - A blank) × sample dilution factor.

[0426] 3. Experimental Results

[0427] 3.1 Detection results and analysis of redox potential of activated hexabiotic probiotic preparation

[0428] Table 29. Detection results of the redox potential of the six-in-one probiotic preparation using Activation Solution 1.

[0429]

[0430] Table 30. Detection results of the redox potential of the six-in-one probiotic preparation using Activation Solution II.

[0431]

[0432] Experimental results showed that when the activating liquid (excipients) contained xylitol, lemon concentrate, fructooligosaccharides, isomaltooligosaccharides, galactooligosaccharides, vitamin C, vitamin B6, nicotinamide, zinc gluconate, disodium pyrroloquinoline quinone, aloe vera gel concentrate (weight ratio 10:1), and calcium lactate (pentahydrate), or when the activating liquid contained arabinose, fructooligosaccharides, isomaltooligosaccharides, galactooligosaccharides, lemon concentrate, vitamin E acetate microcapsules, vitamin C, vitamin B6, nicotinamide, zinc gluconate, disodium pyrroloquinoline quinone, and aloe vera gel (weight ratio 10:1), the probiotics in the six-in-one probiotic preparation were activated in an aerobic environment, resulting in an increase in the redox potential. This result indicates that the accumulation of oxidized substances due to enhanced aerobic metabolism, the consumption of reducing substances by the antioxidant system, and the nutrient-promoting oxidation reaction of the activating liquid all contribute to this process. The activating solution promotes the metabolic activity of probiotics, leading to the accumulation of oxidized substances and the consumption of reduced substances, thereby increasing the redox potential. This increase in potential is a direct result of metabolic activation and can be seen as a marker of the probiotics transitioning from a dormant to an active state. The increased potential not only reflects the activated state but also further enhances the function of probiotics by activating the antioxidant system and optimizing energy metabolism. However, when the activating solution components are missing or replaced with other substances, the redox potential does not show a significant increase.

[0433] 3.2 Na+ in activated hexaprobiotic formulations + K + -ATPase activity detection results

[0434] Table 31 Results of Standard Curve Determination after Gradual Dilution of Standard Phosphorus Solution

[0435]

[0436] Table 32 Na + K + -ATPase activity detection results

[0437]

[0438] As can be seen from the data in Table 32, in the activation solution of the present invention, the enzyme activity continued to rise and then tended to stabilize within 1 hour, indicating that the six-component probiotics were activated and reached a steady state within 1 hour in the excipients with a specific composition.

[0439] This invention demonstrates, by monitoring the redox potential and Na⁺K⁺-ATPase activity of probiotic formulations, that a hexabiotic strain is activated within one hour in a specific excipient composition. Data shows that enzyme activity continuously increases and then stabilizes within one hour, while the redox potential increases synchronously, indicating successful activation and steady-state of bacterial metabolism.

[0440] 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 modifications or alterations made by those skilled in the art to the above-disclosed technical content without departing from the scope of the present invention to obtain equivalent or equivalent embodiments are within the scope of the present invention.

Claims

1. A probiotic preparation comprising an active ingredient and excipients, wherein, The active ingredients include oxygen-depleting bacteria, organic acid-producing bacteria, and space-occupying bacteria; The excipients include flavoring agents, oligosaccharides, and other additives; Wherein, the oligosaccharide is not lactulose, stachyose, raffinose, oligogentiosaccharide, or oligochitosan; and the other additives are not theaflavins, disodium EDTA, or sorbic acid. Preferably, the oxygen-depleting bacteria are Weizmannia; Preferably, the organic acid-producing bacteria are Lactobacillus and / or Lactobacillus lactis; Preferably, the occupant is Bifidobacterium.

2. The probiotic preparation according to claim 1, wherein: The flavoring agent is selected from one or more of the following: D-mannitol, alitane, aspartame, advans, acesulfame potassium, erythritol, glycyrrhizate, mogroside, maltitol, xylitol, neotame, lactitol, sucralose, sorbitol, sematrandezidine, asparagine methyl ester acesulfame, steviol glycosides, cyclamate, isomerized lactose, isomaltulose, arabinose, citric acid, lactic acid, phosphoric acid, tartaric acid, malic acid, metatartaric acid, acetic acid, hydrochloric acid, adipic acid, fumaric acid, sodium hydroxide, potassium carbonate, sodium carbonate, sodium citrate, potassium citrate, trisodium bicarbonate, monosodium citrate, edible flavorings, edible fragrances, and concentrated fruit juice. Preferably, the flavoring agent is selected from one or more of xylitol, arabinose, and concentrated fruit juice; Preferably, the oligosaccharide is selected from one or more of stachyose, raffinose, isomaltulose, lactulose, fructooligosaccharides, xylooligosaccharides, galactooligosaccharides, isomaltulose, gentiosaccharides, soybean oligosaccharides, and chitosan oligosaccharides. More preferably, the oligosaccharide is selected from one or more of fructooligosaccharides, galactooligosaccharides, isomaltooligosaccharides, soybean oligosaccharides, and isomaltulose; Preferably, the other additives are selected from one or more of vitamin C, D-isoascorbic acid and its sodium salt, ascorbic acid, calcium ascorbate, sodium ascorbate, zinc sulfate, citric acid, vitamin E, vitamin B6, nicotinamide, zinc gluconate, disodium pyrroloquinoline quinone, aloe vera gel, calcium lactate, and vitamin E acetate. More preferably, the other additives are selected from one or more of vitamin C, vitamin B6, nicotinamide, zinc gluconate, disodium pyrroloquinoline quinone, aloe vera gel, calcium lactate, and vitamin E acetate.

3. The probiotic preparation according to claim 1 or 2, wherein: The probiotic preparation has a live bacteria count of not less than 100 million CFU / g, preferably 1-100 billion CFU / g, and more preferably 80-100 billion CFU / g; Preferably, the probiotic preparation comprises, by weight, 0.2-1 parts of active ingredient and 0.3-2.5 parts of excipients; More preferably, the probiotic preparation comprises, by weight, the following: The active ingredient is 0.2-1 part, preferably 0.5-0.7 parts; The seasoning is used in an amount of 0.24-2.1 parts, preferably 0.5-1.7 parts; The oligosaccharide content is 0.041-0.16 parts, preferably 0.079-0.121 parts; Other additives: 0.052-0.23 parts, preferably 0.11-0.13 parts.

4. The probiotic preparation according to any one of claims 1 to 3, wherein: The probiotic preparation comprises, by weight, the following: The active ingredient is 0.2-1 part, preferably 0.5-0.7 parts, and more preferably 0.6 parts; The xylitol content is 0.2-1 part, preferably 0.5-0.7 parts, and more preferably 0.6 parts; The concentrated fruit juice is 0.04-0.1 parts, preferably 0.06-0.08 parts, and more preferably 0.07 parts; The amount of fructooligosaccharides is 0.02-0.06 parts, preferably 0.03-0.05 parts, and more preferably 0.04 parts; The amount of isomaltooligosaccharide is 0.02-0.08 parts, preferably 0.04-0.06 parts, and more preferably 0.05 parts; The amount of galactooligosaccharides is 0.001-0.02 parts, preferably 0.009-0.011 parts, and more preferably 0.01 parts; Vitamin C 0.00001-0.0002 parts, preferably 0.00009-0.00011 parts, more preferably 0.0001 parts; Vitamin B6: 0.00001-0.00002 parts, preferably 0.000014-0.000016 parts, more preferably 0.000015 parts; Nicotinamide: 0.0001-0.0002 parts, preferably 0.00014-0.00016 parts, more preferably 0.00015 parts; The zinc gluconate is 0.0004-0.001 parts, preferably 0.0006-0.0008 parts, and more preferably 0.0007 parts; The disodium salt of pyrroloquinoline quinone is 0.0000001-0.000001 parts, preferably 0.0000004-0.0000006 parts, and more preferably 0.0000005 parts; The amount of aloe vera gel is 0.05-0.2 parts, preferably 0.09-0.11 parts, and more preferably 0.1 parts; The amount of calcium lactate is 0.001-0.02 parts, preferably 0.0121-0.0123 parts, and more preferably 0.0122 parts.

5. The probiotic preparation according to any one of claims 1 to 3, wherein: The probiotic preparation comprises, by weight, the following: The active ingredient is 0.2-1 part, preferably 0.5-0.7 parts, and more preferably 0.6 parts; The amount of arabinose is 1-2 parts, preferably 1.4-1.6 parts, and more preferably 1.5 parts; The concentrated fruit juice is 0.05-0.1 parts, preferably 0.07-0.09 parts, and more preferably 0.085 parts; The amount of fructooligosaccharides is 0.02-0.06 parts, preferably 0.03-0.05 parts, and more preferably 0.04 parts; The amount of isomaltooligosaccharide is 0.02-0.08 parts, preferably 0.04-0.06 parts, and more preferably 0.05 parts; The amount of galactooligosaccharides is 0.001-0.02 parts, preferably 0.009-0.011 parts, and more preferably 0.01 parts; Vitamin E acetate: 0.0002-0.0008 parts, preferably 0.0005-0.0007 parts, more preferably 0.00056 parts; Vitamin C 0.00001-0.0002 parts, preferably 0.00009-0.00011 parts, more preferably 0.0001 parts; Vitamin B6: 0.00001-0.00002 parts, preferably 0.000014-0.000016 parts, more preferably 0.000015 parts; Nicotinamide: 0.0001-0.0002 parts, preferably 0.00014-0.00016 parts, more preferably 0.00015 parts; The zinc gluconate is 0.0004-0.001 parts, preferably 0.0006-0.0008 parts, and more preferably 0.0007 parts; The disodium salt of pyrroloquinoline quinone is 0.0000001-0.000001 parts, preferably 0.0000004-0.0000006 parts, and more preferably 0.0000005 parts; The amount of aloe vera gel is 0.05-0.2 parts, preferably 0.09-0.11 parts, and more preferably 0.1 parts.

6. The probiotic preparation according to any one of claims 1 to 5, wherein: The probiotic preparation also contains a solvent; Preferably, the solvent content in the probiotic preparation is 8-10 kg / 10 L, more preferably 8.5-9.5 kg / 10 L; Preferably, the solvent is water.

7. The probiotic preparation according to any one of claims 1 to 6, 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.

8. The probiotic preparation according to any one of claims 1 to 7, wherein, The active ingredients include *Weizmannii coagulans*, *Lactobacillus acidophilus*, *Lactobacillus plantarum*, and *Bifidobacterium*. Preferably, the active ingredient 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):(10-20):(0.6-9.4); More preferably, the Bifidobacteria include Bifidobacterium longum subsp. infantis and Bifidobacterium animalis subsp. lactis; More preferably, the active ingredient 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):(0.6-9.4):(10-20):(0.6-1.4):(4-8); More preferably, 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; Particularly preferably, the active ingredient comprises *Weizmannii coagulans*, *Lactobacillus acidophilus* var. *mongolica*, *Lactobacillus acidophilus* var. *mongolica*, *Lactobacillus plantarum*, *Bifidobacterium longum* subsp. *infantii*, and *Bifidobacterium animalis* subsp. *milk* in an effective live bacteria concentration ratio of (0.6-1.4):(4-8):(0.6-1.4):(10-20):(0.6-1.4):(4-8) respectively.

9. The probiotic preparation according to claim 8, wherein, The *Wickemium coagulans* is selected from at least one of *Wickemium coagulans* BC99, *Wickemium coagulans* HY08866, *Wickemium coagulans* HY08867, 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* 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* HY05181, and *Lactobacillus plantarum* HY00050; and / or, The *Bifidobacterium longum* infant subsp. *longum* is selected from at least one of *Bifidobacterium longum* infant subsp. *CS004*, *Bifidobacterium longum* infant subsp. *MP09089*, and *Bifidobacterium longum* infant subsp. *MP09270*; 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* HY11786, *Bifidobacterium lactis* subsp. *animal* MP15099, *Bifidobacterium lactis* subsp. *animal* HY09279 and *Bifidobacterium lactis* HY10036.

10. The probiotic preparation according to claim 9, wherein, The active ingredients include Lactobacillus plantarum HY02946, Bifidobacterium longum subsp. infantis CS004, Weizmannii coagulans BC99 or Weizmannii coagulans HY08866 and Lactobacillus acidophilus CS003. Preferably, the active ingredient 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 (10-20):(0.6-1.4):(0.6-1.4):(0.6-1.4). More preferably, the active ingredient 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. or, The active ingredients include Lactobacillus plantarum HY02946, Bifidobacterium animalis subsp. lactis BB-12, Bifidobacterium longum subsp. infantis CS004, Weizmannii coagulans BC99 or HY08866, and Lactobacillus acidophilus CS003. Preferably, the active ingredient 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 (10-20): (4-8): (0.6-1.4): (0.6-1.4): (0.6-1.4). More preferably, the active ingredients comprise Lactobacillus plantarum HY02946, Bifidobacterium animalis subsp. lactis BB-12, Bifidobacterium longum subsp. infantis CS004, Weizmannii coagulans BC99 or HY08866, and Lactobacillus acidophilus CS003 in an effective live bacteria concentration ratio of 15:6:1:1:

1. or, The active ingredients include 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 active ingredient 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 (10-20): (4-8): (4-8): (0.6-1.4): (0.6-1.4): (0.6-1.4). More preferably, the active ingredient 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. Or more preferably, the probiotic composition comprises *Lactobacillus plantarum* HY00050, *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. Or 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 20:8:8:1.4:1.4:1.

4.

11. The probiotic preparation according to any one of claims 1 to 7, wherein, The active ingredients include *Weizmannii coagulans*, *Lactobacillus acidophilus*, *Lactobacillus plantarum*, and *Bifidobacterium*. Preferably, the active ingredient 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 active ingredient 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). More preferably, the Bifidobacterium may be selected from Bifidobacterium longum subsp. infantis and / or Bifidobacterium animalis subsp. lactis; More preferably, the active ingredient comprises *Weizmannii coagulans*, *Lactobacillus acidophilus*, *Lactobacillus plantarum*, *Bifidobacterium longum* subsp. *infantii*, and *Bifidobacterium animalis* subsp. *lactobacter*, with an effective live bacteria concentration ratio of (0.6-1.4):(0.6-8):(5-20):(0.6-1.4):(4-10); even more preferably, the probiotic composition comprises *Weizmannii coagulans*, *Lactobacillus acidophilus*, *Lactobacillus plantarum*, *Bifidobacterium longum* subsp. *infantii*, and *Bifidobacterium animalis* subsp. *lactobacter*, with an effective live bacteria concentration ratio of (0.6-1.4):(0.6-8):(5-20):(0.6-1.4):(4-8). More preferably, 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; Particularly 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).

12. The probiotic preparation according to claim 11, 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.

13. The probiotic preparation according to claim 12, wherein, The active ingredients include Lactobacillus plantarum HY02946, Bifidobacterium longum subsp. infantis CS004, Weizmannii coagulans BC99 or Weizmannii coagulans HY08866 and Lactobacillus acidophilus CS003. Preferably, the active ingredient 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 active ingredient 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. or, The active ingredients include Lactobacillus plantarum HY02946, Bifidobacterium animalis subsp. lactis BB-12, Bifidobacterium longum subsp. infantis CS004, Weizmannii coagulans BC99 or HY08866, and Lactobacillus acidophilus CS003. Preferably, the active ingredient 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 active ingredients comprise Lactobacillus plantarum HY02946, Bifidobacterium animalis subsp. lactis BB-12, Bifidobacterium longum subsp. infantis CS004, Weizmannii coagulans BC99 or HY08866, and Lactobacillus acidophilus CS003 in an effective live bacteria concentration ratio of 15:6:1:1:

1. or, The active ingredients include *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. Preferably, the active ingredient 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 active ingredient 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 active ingredient 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.

14. The probiotic preparation according to claim 12, wherein, The active ingredients include *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. Preferably, the active ingredient 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.

15. The probiotic preparation according to any one of claims 1 to 14, wherein, The probiotic preparation is in liquid form.

16. Use of the probiotic preparation according to any one of claims 1 to 15 in the preparation of products for the prevention, relief, improvement and / or treatment of functional gastrointestinal disorders; Preferably, the functional gastrointestinal disorder is diarrhea and / or constipation; More preferably, the diarrhea is selected from one or more of diarrhea-predominant irritable bowel syndrome, acute diarrhea, chronic diarrhea, osmotic diarrhea, secretory diarrhea, exudative diarrhea, motility-related diarrhea, antibiotic-associated diarrhea, and infectious diarrhea; More preferably, the constipation is selected from one or more of 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. Preferably, the product is selected from one or more of pharmaceuticals, food, and health products.

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