A composite probiotic composition and a preparation method and application thereof

Abstract

This invention discloses a compound probiotic composition, its preparation method, and its applications. The compound probiotic composition comprises compound probiotics and compound compounds, wherein the components exert a synergistic effect in the body, significantly shortening the righting reflex recovery time after alcohol intake, effectively alleviating the physiological burden caused by alcohol consumption, and reducing alcohol-induced liver oxidative stress and lipid peroxidation levels. In other words, the compound probiotic composition has a hangover-relieving and liver-protecting effect. Furthermore, the compound probiotic composition helps improve and maintain the homeostasis of the gut-liver axis in alcohol- or non-alcohol-related sub-health states (such as sleep disorders), improving liver damage, regulating gut microbiota structure, and improving intestinal barrier damage. It is suitable for individuals requiring liver function regulation and nutritional intervention.

Description

Technical Field

[0001] This invention belongs to the field of biopharmaceutical manufacturing technology. More specifically, it relates to a compound probiotic composition, its preparation method, and its application. Background Technology

[0002] With the accelerated pace of modern life and increasing work pressure, irregular work and rest habits and unhealthy dietary structures are becoming increasingly common, leaving more and more people in a state of stress due to alcohol intake and digestive system dysfunction. The liver, as the body's core metabolic and detoxification organ, bears a long-term burden from the combined effects of diet, environment, and lifestyle. Numerous epidemiological studies show that approximately 30% of the global population suffers from fatty liver disease or abnormal liver function, including both alcoholic liver disease related to alcohol consumption and a large number of non-alcoholic gut-liver axis dysfunctions and sub-health conditions. Liver problems have become a serious global challenge threatening public health, urgently requiring the development of functional products that regulate gut-liver axis homeostasis to effectively alleviate liver damage caused by unhealthy lifestyles or alcohol intake.

[0003] The intestines and liver have a broad and bidirectional synergistic effect in terms of both anatomy and physiological function. Connected by the portal vein and bile ducts, they form the gut-hepatic axis, maintaining metabolic and immune homeostasis in the digestive system. Increasing research indicates that various liver diseases, including alcoholic liver disease, are accompanied by impaired intestinal barrier function and gut-hepatic axis dysfunction. Maintaining the integrity of the intestinal barrier can reduce the entry of bacterial metabolites and endotoxins into the liver via the gut-hepatic axis, thereby alleviating liver inflammation and metabolic burden. Therefore, maintaining intestinal barrier function and gut-hepatic homeostasis is one of the important strategies for intervening in abnormal liver function and maintaining liver health.

[0004] Currently, many hangover remedies and liver-protecting compositions incorporate probiotics to address the adverse effects of alcohol on the body. However, these compositions often focus on enhancing the activity of alcohol dehydrogenase and / or aldehyde dehydrogenase to achieve their hangover remedy and liver-protecting effects, or on providing the liver with antioxidant and other protective functions. They do not address the impact of the gut-liver axis on liver damage and intestinal barrier damage in non-alcohol-related sub-health states, nor do they adequately consider or address the functional stability of probiotics in the alcohol-exposed gut environment and their sustained physiological regulatory capacity in actual drinking scenarios. They lack a systematic design for the overall regulation of the gut-liver axis. For example, a hangover remedy and liver-protecting composition comprising probiotics (including Lactobacillus rhamnosus, Pediococcus pentosus, Lactobacillus acidophilus, Bifidobacterium animalis, and Lactobacillus casei), plant extracts, and prebiotics can increase the activity of alcohol dehydrogenase and aldehyde dehydrogenase, but it is unclear whether it can improve and maintain the homeostasis of gut-liver axis-related physiological functions in alcohol- or non-alcohol-related sub-health states.

[0005] In summary, there is an urgent need to develop products that can maintain stable functional output under ethanol exposure and gut-liver axis-related stress environments, while simultaneously regulating gut microbiota, gut barrier, and overall gut-liver axis homeostasis, in order to achieve long-term stability of liver function and support in multiple scenarios. Summary of the Invention

[0006] To address the shortcomings of the prior art, this invention provides a compound probiotic composition, its preparation method, and its application. This compound probiotic composition not only has hangover-relieving and liver-protecting effects, but it can also improve and maintain the homeostasis of the gut-liver axis in alcohol- or non-alcohol-related sub-health states, and improve liver damage and intestinal barrier damage.

[0007] The first objective of this invention is to provide a compound probiotic composition.

[0008] A second objective of this invention is to provide a method for preparing the compound probiotic composition.

[0009] A third objective of the present invention is to provide a formulation containing the aforementioned compound probiotic composition.

[0010] A fourth objective of this invention is to provide the use of the compound probiotic composition or the preparation in the preparation of hangover relief and liver protection products.

[0011] A fifth object of the present invention is to provide the use of the compound probiotic composition or the preparation in the preparation of products that improve liver damage.

[0012] A sixth object of the present invention is to provide the use of the compound probiotic composition or the preparation in the preparation of products that improve intestinal barrier damage.

[0013] The above-mentioned objective of this invention is achieved through the following technical solution:

[0014] This invention provides a compound probiotic composition comprising compound probiotics and a compound compound; wherein the mass ratio of the compound probiotics to the compound compound is (3-9):(1-2). The compound probiotics are composed of *Pediococcus pentosaceus* strain PP-01, *Pediococcus pentosaceus* strain PP-02, *Lactobacillus rhamnosus*, *Lactobacillus plantarum*, *Bifidobacterium bifidum*, *Saccharomyces boulardii*, and *Akermansia muciniphila* in a colony-forming unit ratio of (4–6):(4–6):(2–4):(1–3):(1–2):(1–2):(2–4), and the total number of live bacteria in the compound probiotics is at least 1 × 10⁻⁶. 10 CFU / g; The compound is composed of resveratrol, S-adenosylmethionine p-toluenesulfonic acid sulfate, curcumin, inulin, resistant starch, fructooligosaccharides, galactooligosaccharides, xylooligosaccharides and aloe polysaccharides in a mass ratio of (1-2):(4-6):(1-2):(3-5):(3-5):(3-5):(3-5):(1-2):(1-2).

[0015] Specifically, the mass ratio of the compound probiotics to the compound compound is 3 to 9:1.

[0016] More specifically, the mass ratio of the compound probiotics to the compound compound is 3:1.

[0017] Specifically, the compound probiotics are composed of Pediococcus pentosaceus PP-01 strain, Pediococcus pentosaceus PP-02 strain, Lactobacillus rhamnosus, Lactobacillus plantarum, Bifidobacterium bifidum, Saccharomyces boulardii, and Akkermansia myxotroph in a colony-forming unit ratio of (5-6):(5-6):(3-4):(2-3):(1-2):(1-2):(3-4).

[0018] More specifically, the compound probiotics are composed of Pediococcus pentosaceus PP-01 strain, Pediococcus pentosaceus PP-02 strain, Lactobacillus rhamnosus, Lactobacillus plantarum, Bifidobacterium bifidum, Saccharomyces boulardii, and Akkermansia myxotroph in a colony-forming unit ratio of 5:5:3:2:1:1:3.

[0019] Specifically, the composite compound is composed of resveratrol, S-adenosylmethionine p-toluenesulfonic acid sulfate, curcumin, inulin, resistant starch, fructooligosaccharides, galactooligosaccharides, xylooligosaccharides and aloe polysaccharides in a mass ratio of (1-2):(4-5):(1-2):(3-4):(3-4):(3-4):(3-4):(1-2):(1-2).

[0020] More specifically, the composite compound is composed of resveratrol, S-adenosylmethionine p-toluenesulfonic acid sulfate, curcumin, inulin, resistant starch, fructooligosaccharides, galactooligosaccharides, xylooligosaccharides and aloe polysaccharides in a mass ratio of 1:5:1:4:4:4:4:1:1.

[0021] Specifically, the *Pediococcus pentosaceus* PP-01 strain was deposited at the China General Microbiological Culture Collection Center (CGMCC) on April 25, 2025, with accession number CGMCC No. 34357; and the *Pediococcus pentosaceus* PP-02 strain was deposited at the same center on April 25, 2025, with accession number CGMCC No. 34358.

[0022] The present invention also provides a method for preparing the aforementioned compound probiotic composition, comprising the following steps: S1. Prepare freeze-dried bacterial powders of *Pediococcus pentosaceus* strain PP-01, *Pediococcus pentosaceus* strain PP-02, *Lactobacillus rhamnosus*, *Lactobacillus plantarum*, *Bifidobacterium bifidum*, *Saccharomyces boulardii*, and *Ackermania muscaria*, respectively. Mix the obtained freeze-dried bacterial powders of *Pediococcus pentosaceus* strain PP-01, *Pediococcus pentosaceus* strain PP-02, *Lactobacillus rhamnosaceus*, *Lactobacillus plantarum*, *Bifidobacterium bifidum*, *Saccharomyces boulardii*, and *Ackermania muscaria* uniformly according to the colony-forming unit ratio to obtain a product containing at least 1 × 10⁻⁶ viable bacteria. 10 CFU / g of compound probiotics; S2. The resveratrol, S-adenosylmethionine p-toluenesulfonate sulfate, curcumin, inulin, resistant starch, fructooligosaccharides, galactooligosaccharides, xylooligosaccharides and aloe polysaccharides are uniformly mixed according to the mass ratio to obtain a composite compound; Alternatively, resveratrol, S-adenosylmethionine p-toluenesulfonate sulfate, curcumin, inulin, resistant starch, fructooligosaccharides, galactooligosaccharides, xylooligosaccharides, and aloe polysaccharides may be weighed according to the aforementioned mass ratios and dissolved or dispersed in solvents to obtain concentrated solutions of each compound; wherein, resveratrol and curcumin are dissolved in propylene glycol, and S-adenosylmethionine p-toluenesulfonate sulfate, inulin, resistant starch, fructooligosaccharides, galactooligosaccharides, xylooligosaccharides, and aloe polysaccharides are dissolved or dispersed in sterile phosphate buffer; S3. The obtained compound probiotics and compound compound are mixed evenly according to the mass ratio to obtain the compound probiotic composition; Alternatively, the total mass of the compound probiotics and the concentrated solutions of each compound can be mixed according to the stated mass ratio, and sterile phosphate buffer can be added to prepare a homogeneous suspension. After freeze-drying, the compound probiotic composition can be obtained.

[0023] Specifically, in S1, *Pediococcus pentosaceus* PP-01, *Pediococcus pentosaceus* PP-02, *Lactobacillus rhamnosus*, *Lactobacillus plantarum*, *Bifidobacterium bifidum*, *Saccharomyces boulardii*, and *Ackermania muscaria* are cultured to the logarithmic growth phase, centrifuged, the supernatant is discarded, and the bacterial cells are collected to obtain wet bacterial sludge of each bacterium; the obtained wet bacterial sludge of each bacterium is mixed with a freeze-drying protectant, and after being mixed evenly, it is placed in a vacuum freeze-drying chamber for freeze-drying treatment to obtain freeze-dried bacterial blocks of each bacterium; the obtained freeze-dried bacterial blocks of each bacterium are pulverized and sieved to obtain freeze-dried bacterial powder of each bacterium.

[0024] The present invention also provides a formulation containing the aforementioned compound probiotic composition.

[0025] Optionally, the formulation may further contain a pharmaceutically or nutritionally acceptable carrier and / or excipients for the compound probiotic composition.

[0026] This invention seeks protection for the use of the compound probiotic composition or the preparation in the preparation of hangover relief and liver protection products.

[0027] This invention also claims protection for the use of the compound probiotic composition or the preparation in the preparation of products that improve liver damage.

[0028] Specifically, the liver damage was caused by alcohol or sleep disorders.

[0029] Specifically, the liver damage is caused by oxidative stress resulting from alcohol or sleep disorders.

[0030] The present invention also claims protection for the use of the compound probiotic composition or the preparation thereof in the preparation of products that improve intestinal barrier damage.

[0031] Specifically, the intestinal barrier damage is caused by alcohol or sleep disorders.

[0032] Optionally, the products include pharmaceuticals, health supplements, and functional foods.

[0033] The present invention has the following beneficial effects: This invention provides a compound probiotic composition, which comprises compound probiotics and compound compounds. The components exert a synergistic effect in the body, significantly shortening the righting reflex recovery time after alcohol intake, effectively alleviating the physiological burden caused by alcohol consumption, and reducing alcohol-induced liver oxidative stress and lipid peroxidation levels. In other words, the compound probiotic composition has a hangover-relieving and liver-protecting effect. Furthermore, the compound probiotic composition helps improve and maintain the homeostasis of the gut-liver axis in alcohol- or non-alcohol-related sub-health states (such as sleep disorders), improving liver damage, regulating gut microbiota structure, and improving intestinal barrier damage. It is suitable for individuals requiring liver function regulation and nutritional intervention. Attached Figure Description

[0034] Figure 1 The relative content of ethanol in the system after the compound probiotic composition described in Example 1 was incubated with ethanol under in vitro conditions; **** p <0.0001.

[0035] Figure 2 The relative survival rates of *Pediococcus pentosaceus* PP-01, PP-02, and *Lactobacillus plantarum* strains under different concentrations of ethanol and their comparison results; **** p <0.0001, no significant difference in ns.

[0036] Figure 3The results of comparing the righting reflex recovery time of mice after alcohol intake in the negative control group, model group, compound probiotic combination group, and Haiwang Jinzun group;* p <0.05, no significant difference in ns.

[0037] Figure 4 The effects of the control group, model group, and compound probiotic combination group on alcohol-induced liver morphology, liver function damage, oxidative stress, and related molecular indicators in mice, and their comparative results;* p <0.05,** p <0.01, *** p <0.001, no significant difference in ns.

[0038] Figure 5 The effects of the control group, model group, and compound probiotic combination group on alcohol-induced intestinal barrier-related molecular indicators in mice and their comparative results;* p <0.05,** p <0.01, *** p <0.001.

[0039] Figure 6 The results show the effects of the control group, model group, and compound probiotic combination group on the level distribution of intestinal flora in the feces of alcohol-induced mice.

[0040] Figure 7 The effects of the control group, control intervention group, model group, and model intervention group on liver function and intestinal barrier-related molecular markers in sleep-deprived mice, and their comparative results;* p <0.05,** p <0.01, *** p <0.001, no significant difference in ns. Detailed Implementation

[0041] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, but the embodiments do not limit the present invention in any way. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in this technical field.

[0042] Unless otherwise specified, all reagents and materials used in the following examples are commercially available.

[0043] The *Pediococcus pentosaceus* used in the embodiments of this invention ( Pediococcus pentosaceusBoth strains PP-01 and PP-02 were deposited on April 25, 2025, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing. The accession number for strain PP-01 is CGMCC No. 34357, and the accession number for strain PP-02 is CGMCC No. 34358.

[0044] The *Akermansia muciniphila* used in the embodiments of this invention (… Akkermansia muciniphila The number is BNCC341917; the Lactobacillus rhamnosus used in the examples ( Lactobacillus rhamnosus Lactobacillus plantarum ( Lactiplantibacillus plantarum Bifidobacterium bifidum ( Bifidobacterium bifidum ) and Saccharomyces boulardii ( Saccharomyces boulardii All of them are derived from commercially available probiotics, with no specific strain or brand requirements. The probiotics mentioned are all purchased from the Henan Provincial Engineering Technology Research Center for Industrial Microbial Strains.

[0045] The Pediococcus pentosaceus used in the comparative examples of this invention ( Pediococcus pentosaceus CTCC 24444 was purchased from the China Industrial Microbial Culture Collection Center.

[0046] The raw materials used in the preparation of the composite compounds in this invention are all derived from commercially available food-grade raw materials that comply with regulations, without specific model or brand requirements. In the examples and comparative examples, the raw materials used to prepare the composite compounds, including inulin, fructooligosaccharides, and galactooligosaccharides, were purchased from Jiangsu Ruikanglai Technology Co., Ltd.; xylooligosaccharides were purchased from Suixian Youbaojia Food Co., Ltd.; resistant starch, tea polyphenols, and rosemary extract were purchased from Xinmi Xiaoyao Food Store; and aloe polysaccharides were purchased from Xi'an Youlanda Biotechnology Co., Ltd.

[0047] Example 1: Preparation of a compound probiotic composition The compound probiotic composition described in this embodiment includes 75 parts of compound probiotics and 25 parts of compound compound; wherein, the compound probiotics are composed of *Pediococcus pentosaceus* PP-01, *Pediococcus pentosaceus* PP-02, *Lactobacillus rhamnosus*, *Lactobacillus plantarum*, *Bifidobacterium bifidum*, *Saccharomyces boulardii*, and *Aklermus obliterans* in a colony-forming unit (CFU) ratio of 5:5:3:2:1:1:3, and the total number of viable bacteria is 1×10⁻⁶. 10 CFU / g; The composite compound is composed of resveratrol, S-adenosylmethionine p-toluenesulfonic acid sulfate, curcumin, inulin, resistant starch, fructooligosaccharides, galactooligosaccharides, xylooligosaccharides and aloe polysaccharides in a mass ratio of 1:5:1:4:4:4:4:1:1.

[0048] The preparation method of the compound probiotic composition is as follows: S1. Preparation of compound probiotic powder; *Pediococcus pentosaceus* PP-01, *Pediococcus pentosaceus* PP-02, *Lactobacillus rhamnosus*, *Lactobacillus plantarum*, *Bifidobacterium bifidum*, *Saccharomyces boulardii*, and *Ackermania muscaria* were cultured to the logarithmic growth phase, centrifuged, the supernatant was discarded, and the bacterial cells were collected to obtain wet bacterial sludge of each bacterium; the wet bacterial sludge of each bacterium was mixed with a freeze-drying protectant, and after being mixed evenly, it was placed in a vacuum freeze-drying chamber for freeze-drying treatment to obtain freeze-dried bacterial blocks of each bacterium; the freeze-dried bacterial blocks of each bacterium were pulverized and sieved to obtain freeze-dried bacterial powder of each bacterium; the freeze-dried bacterial powders of *Pediococcus pentosaceus* PP-01, *Pediococcus pentosaceus* PP-02, *Lactobacillus rhamnosaceus*, *Lactobacillus plantarum*, *Bifidobacterium bifidum*, *Saccharomyces boulardii*, and *Ackermania muscaria* were mixed to prepare a mixture containing a total viable count of 1×10⁻⁶. 10 The compound probiotic powder contains CFU / g of Pediococcus pentosaceus PP-01, Pediococcus pentosaceus PP-02, Lactobacillus rhamnosus, Lactobacillus plantarum, Bifidobacterium bifidum, Saccharomyces boulardii, and Akkermansia myxotroph in a CFU ratio of 5:5:3:2:1:1:3. S2. Preparation of composite compound powder: Resveratrol, S-adenosylmethionine p-toluenesulfonic acid sulfate, curcumin, inulin, resistant starch, fructooligosaccharides, galactooligosaccharides, xylooligosaccharides and aloe polysaccharides are mixed evenly in a mass ratio of 1:5:1:4:4:4:4:1:1 and then sieved to obtain composite compound powder; S3. Preparation of compound probiotic composition: The obtained compound probiotic powder and compound compound powder are mixed evenly in a mass ratio of 75:25, and after sieving, the compound probiotic composition powder is obtained.

[0049] In addition to the methods described above, the compound probiotic composition can also be prepared by the following methods: S1. Preparation of compound probiotic powder; same as above; S2. Preparation of concentrated compounds: Resveratrol, S-adenosylmethionine p-toluenesulfonate sulfate, curcumin, inulin, resistant starch, fructooligosaccharides, galactooligosaccharides, xylooligosaccharides, and aloe polysaccharides were weighed in a mass ratio of 1:5:1:4:4:4:4:1:1 and dissolved or dispersed in solvents to prepare concentrated solutions of each compound; wherein, resveratrol and curcumin were dissolved in propylene glycol, and S-adenosylmethionine p-toluenesulfonate sulfate, inulin, resistant starch, fructooligosaccharides, galactooligosaccharides, xylooligosaccharides, and aloe polysaccharides were dissolved or dispersed in sterile phosphate buffer. S3. Preparation of compound probiotic composition: The obtained compound probiotic powder and the concentrated liquid of each compound (by mass of feed) are mixed in a mass ratio of 75:25, and an appropriate amount of sterile phosphate buffer is added to prepare a uniform suspension of compound probiotic composition. After freeze-drying, compound probiotic composition powder is obtained.

[0050] The compound probiotic composition powder can be directly used in subsequent formulation processing.

[0051] Example 2 Preparation of Compound Probiotic Composition The preparation method of the compound probiotic composition described in this embodiment is the same as that in Embodiment 1, except that the mass ratio of the compound probiotic powder to the compound compound powder is 60:40.

[0052] Example 3 Preparation of Compound Probiotic Composition The preparation method of the compound probiotic composition described in this embodiment is the same as that in Embodiment 1, except that the mass ratio of the compound probiotic powder to the compound compound powder is 90:10.

[0053] Comparative Example 1: Preparation of a compound probiotic composition The preparation method of the compound probiotic composition described in this comparative example is the same as that in Example 1, except that the compound probiotic powder does not contain Pediococcus pentosaccharis PP-01. The missing mass fraction due to the absence of this bacterium is made up with a freeze-drying protectant so that the total mass fraction of the compound probiotic composition is consistent with that in Example 1.

[0054] The compound probiotics described in this comparative example consist of *Pediococcus pentosaceus* PP-02, *Lactobacillus rhamnosus*, *Lactobacillus plantarum*, *Bifidobacterium bifidum*, *Saccharomyces boulardii*, and *Akermansia myxotropica* in a CFU ratio of 5:3:2:1:1:3, comprising 56.25 parts by weight, with a total viable count of 1 × 10⁻⁶. 10 CFU / g; The missing mass fraction (18.75 parts) in the compound probiotic due to the absence of Pediococcus pentosaccharis PP-01 was replenished with a freeze-drying protectant to the same mass fraction as in Example 1, and then uniformly mixed with 25 parts of the compound compound to obtain the compound probiotic composition.

[0055] Comparative Example 2: Preparation of the Compound Probiotic Composition The preparation method of the compound probiotic composition described in this comparative example is the same as that in Example 1, except that the compound probiotic powder does not contain Pediococcus pentosacchari PP-02. The missing mass fraction due to the absence of this bacterium is made up with a freeze-drying protectant so that the total mass fraction of the compound probiotic composition is consistent with that in Example 1.

[0056] The compound probiotics described in this comparative example consist of *Pediococcus pentosaceus* PP-01, *Lactobacillus rhamnosus*, *Lactobacillus plantarum*, *Bifidobacterium bifidum*, *Saccharomyces boulardii*, and *Akermansia myxotropica* in a CFU ratio of 5:3:2:1:1:3, comprising 56.25 parts by weight, with a total viable count of 1 × 10⁻⁶. 10CFU / g; The missing mass fraction (18.75 parts) in the compound probiotic due to the absence of Pediococcus pentosaccharis PP-01 was replenished with a lyophilization protectant to the same mass fraction as in Example 1, and then uniformly mixed with 25 parts of the compound compound to obtain the compound probiotic composition.

[0057] Comparative Example 3: Preparation of the Compound Probiotic Composition The preparation method of the compound probiotic composition described in this comparative example is the same as that in Example 1, except that the compound probiotic powder does not contain Lactobacillus rhamnosus, Lactobacillus plantarum, Bifidobacterium bifidum, Saccharomyces boulardii, and Akkermansia myxotroph. The mass fraction missing due to the absence of these bacteria is made up with a freeze-drying protectant so that the total mass fraction of the compound probiotic composition is consistent with that in Example 1.

[0058] The compound probiotic described in this comparative example consists of Pediococcus pentosaceus PP-01 and Pediococcus pentosaceus PP-02 in a CFU ratio of 1:1, with a mass fraction of 37.5 parts and a total viable count of 1×10⁻⁶. 10 CFU / g; The missing mass fraction (37.5 parts) in the compound probiotic due to the absence of the bacteria was replenished with a freeze-drying protectant to the same mass fraction as in Example 1, and then uniformly mixed with 25 parts of the compound compound to obtain the compound probiotic composition.

[0059] Comparative Example 4: Preparation of a compound probiotic composition The preparation method of the compound probiotic composition described in this comparative example is the same as that in Example 1, except that the compound probiotic powder does not contain Pediococcus pentosacchari PP-01 and Pediococcus pentosacchari PP-02. The missing mass fraction due to the absence of these bacteria is made up with a freeze-drying protectant so that the total mass fraction of the compound probiotic composition is consistent with that in Example 1.

[0060] The compound probiotics described in this comparative example consist of *Lactobacillus rhamnosus*, *Lactobacillus plantarum*, *Bifidobacterium bifidum*, *Saccharomyces boulardii*, and *Akermansia myxotropica* in a CFU ratio of 3:2:1:1:3, comprising 37.5 parts by weight, with a total viable count of 1 × 10⁻⁶. 10 CFU / g; The missing mass fraction (37.5 parts) in the compound probiotic due to the absence of the bacteria was replenished with a freeze-drying protectant to the same mass fraction as in Example 1, and then uniformly mixed with 25 parts of the compound compound to obtain the compound probiotic composition.

[0061] Comparative Example 5: Preparation of the Compound Probiotic Composition The preparation method of the compound probiotic composition described in this comparative example is the same as that in Example 1, except that the compound probiotic powder does not contain *Pediococcus pentosaceus* PP-01 and *Pediococcus pentosaceus* PP-02, but is replaced by an equal amount of *Pediococcus pentosaceus* CTCC 24444, so that the total mass fraction of the compound probiotic composition remains consistent with that in Example 1. That is, the compound probiotic in this comparative example is composed of *Pediococcus pentosaceus* CTCC 24444, *Lactobacillus rhamnosus*, *Lactobacillus plantarum*, *Bifidobacterium bifidum*, *Saccharomyces boulardii*, and *Akkermansia myxotropica* in a CFU ratio of 10:3:2:1:1:3, and its total viable count is 1×10⁻⁶. 10 CFU / g.

[0062] Comparative Example 6: Preparation of a compound probiotic composition The preparation method of the compound probiotic composition described in this comparative example is the same as that in Example 1, except that it does not contain compound compounds, and the missing mass fraction is made up with a freeze-drying protectant so that the total mass fraction of the compound probiotic composition is consistent with that in Example 1.

[0063] Comparative Example 7: Preparation of a compound probiotic composition The preparation method of the compound probiotic composition described in this comparative example is the same as that in Example 1, except that the compound does not contain resveratrol, S-adenosylmethionine p-toluenesulfonate sulfate and curcumin. The missing mass fraction due to the absence of the above compounds is made up with a freeze-drying protectant so that the total mass fraction of the compound probiotic composition is consistent with that in Example 1.

[0064] The composite compound described in this comparative example is composed of inulin, resistant starch, fructooligosaccharides, galactooligosaccharides, xylooligosaccharides, and aloe polysaccharides in a mass ratio of 4:4:4:4:1:1.

[0065] Comparative Example 8: Preparation of a compound probiotic composition The preparation method of the compound probiotic composition described in this comparative example is the same as that in Example 1, except that the compound does not contain inulin, resistant starch, fructooligosaccharides, galactooligosaccharides, xylooligosaccharides, and aloe polysaccharides, but is replaced by an equal amount of glucose, so that the total mass of the compound probiotic composition remains consistent with that in Example 1. That is, the compound described in this comparative example is composed of resveratrol, S-adenosylmethionine p-toluenesulfonic acid sulfate, curcumin, and glucose in a mass ratio of 1:5:1:18.

[0066] Comparative Example 9: Preparation of a compound probiotic composition The preparation method of the compound probiotic composition described in this comparative example is the same as that in Example 1, except that the compound does not contain inulin, resistant starch, fructooligosaccharides, galactooligosaccharides, xylooligosaccharides, and aloe polysaccharides, but is replaced by an equal amount of maltodextrin, so that the total mass fraction of the compound probiotic composition remains consistent with that in Example 1. That is, the compound composition described in this comparative example is composed of resveratrol, S-adenosylmethionine p-toluenesulfonic acid sulfate, curcumin, and maltodextrin in a mass ratio of 1:5:1:18.

[0067] Comparative Example 10: Preparation of a Compound Probiotic Composition The preparation method of the compound probiotic composition described in this comparative example is the same as that in Example 1, except that the compound does not contain inulin, resistant starch, fructooligosaccharides, galactooligosaccharides, xylooligosaccharides, and aloe polysaccharides. The missing mass fraction due to the absence of these compounds is made up with a freeze-drying protectant to ensure that the total mass fraction of the compound probiotic composition remains consistent with that in Example 1. That is, the compound composition described in this comparative example is composed of resveratrol, S-adenosylmethionine p-toluenesulfonic acid sulfate, and curcumin in a mass ratio of 1:5:1.

[0068] Comparative Example 11: Preparation of a compound probiotic composition The preparation method of the compound probiotic composition described in this comparative example is the same as that in Example 1, except that the compound does not contain resveratrol, S-adenosylmethionine p-toluenesulfonate sulfate and curcumin, but is replaced by an equal amount of tea polyphenols, so that the total mass of the compound probiotic composition is consistent with that in Example 1.

[0069] The composite compound described in this comparative example is composed of tea polyphenols, inulin, resistant starch, fructooligosaccharides, galactooligosaccharides, xylooligosaccharides, and aloe polysaccharides in a mass ratio of 7:4:4:4:4:1:1.

[0070] Comparative Example 12: Preparation of a compound probiotic composition The preparation method of the compound probiotic composition described in this comparative example is the same as that in Example 1, except that the compound does not contain resveratrol, S-adenosylmethionine p-toluenesulfonate sulfate and curcumin, but is replaced by an equal amount of rosemary extract, so that the total mass of the compound probiotic composition is consistent with that in Example 1.

[0071] The composite compound described in this comparative example is composed of rosemary extract, inulin, resistant starch, fructooligosaccharides, galactooligosaccharides, xylooligosaccharides, and aloe polysaccharides in a mass ratio of 7:4:4:4:4:1:1.

[0072] Comparative Example 13: Preparation of a compound probiotic composition The preparation method of the composite probiotic composition described in this comparative example is the same as that in Example 1, except that it only contains a composite compound composed of resveratrol, S-adenosylmethionine p-toluenesulfonate sulfate, and curcumin in a mass ratio of 1:5:1. The missing mass parts are supplemented with a lyoprotectant to keep the total mass parts of the composite probiotic composition consistent with that in Example 1.

[0073] Comparative Example 14 Preparation of Composite Probiotic Composition The preparation method of the composite probiotic composition described in this comparative example is the same as that in Example 1, except that it only contains a composite compound composed of inulin, resistant starch, fructooligosaccharide, galactooligosaccharide, xylooligosaccharide, and aloe polysaccharide in a mass ratio of 4:4:4:4:1:1. The missing mass parts are supplemented with a lyoprotectant to keep the total mass parts of the composite probiotic composition consistent with that in Example 1.

[0074] Test Example 1 Alleviating Effect of Composite Probiotic Composition on the Metabolic Burden Caused by Ethanol Intake The composite probiotic composition described in the present invention can be divided into three modules, namely the probiotic module, the antioxidant module, and the prebiotic module. The present invention sorted out the composition of the composite probiotic compositions described in Examples 1-3 and Comparative Examples 1-14 according to different modules, as shown in Table 1.

[0075] Table 1 Composition of the Composite Probiotic Compositions Described in Examples 1-3 and Comparative Examples 1-14

[0076] The present invention evaluated the effect of the composite probiotic compositions described in Examples 1-3 and Comparative Examples 1-14 on alleviating the metabolic burden of the body caused by ethanol intake by testing the effects of the compositions on the blood ethanol exposure level, blood ammonia concentration, and lactate / pyruvate ratio in ethanol-induced mice.

[0077] 1. Experimental Materials (1) Experimental Animals 114 male C57BL / 6 mice aged 7-8 weeks with a body weight of 20-25 g were purchased from the Experimental Animal Center of Sun Yat-sen University. The production license number of the experimental animals is SCXK (Guangdong) 2021-0029. The mice were housed in the Experimental Animal Center of Sun Yat-sen University, and the license number for the use of experimental animals is SYXK (Guangdong) 2023-0112 of Sun Yat-sen University.

[0078] (2) Experimental Reagents Normal saline, Hongxing Erguotou, ethanol colorimetric assay kit, blood ammonia colorimetric assay kit, L-lactic acid colorimetric assay kit, pyruvic acid colorimetric assay kit (Wuhan Elite Biotechnology Co., Ltd.).

[0079] 2. Experimental Methods (1) Establishment of an acute ethanol exposure model in mice After one week of acclimatization, the male C57BL / 6 mice were randomly divided into 19 groups: a blank control group, a model control group, and treatment groups for Examples 1-3 and Comparative Examples 1-14, with 6 mice per group. All mice were fasted for 12 hours before the experiment, but allowed free access to water. The example and comparative treatment groups were administered 100 mg / mouse by gavage a freshly prepared suspension of the corresponding compound powder in physiological saline (compound probiotic composition suspension). The blank control group and the model control group were administered an equal volume of physiological saline by gavage. Thirty minutes after gavage, except for the blank control group, all other groups were administered 10 mL / kg of 56-degree Red Star Erguotou (a type of Chinese liquor) by gavage to establish an acute ethanol exposure model. The blank control group was administered an equal volume of physiological saline by gavage. After gavage, small amounts of blood were collected from the tail vein at 0, 0.5, 1, 2, and 4 h. Blood was collected from the orbital cavity at 6 h, and all mice were euthanized by dislocation. All blood samples were stored in centrifuge tubes containing anticoagulant, centrifuged at 3500 r / min for 10 min at 4°C, and the supernatant was collected to obtain plasma, which was used to determine the concentration of ethanol, ammonia, and L-lactic acid / pyruvate in the plasma.

[0080] (2) Determination of plasma ethanol concentration curve AUC, ammonia concentration and lactate / pyruvate ratio Using the corresponding test kits, the ethanol concentration in plasma at different time points, the concentrations of ammonia, L-lactic acid, and pyruvate in plasma at 6 h were measured according to the instructions. Based on these results, the AUC of the ethanol concentration curve, the blood ammonia concentration, and the L-lactic acid / pyruvate ratio of each group of mice were calculated using the trapezoidal method.

[0081] 3. Experimental Results The average AUC of ethanol concentration curves, the average blood ammonia concentration, and the average L-lactic acid / pyruvate ratio of each group of mice are shown in Table 2.

[0082] Table 2. Average values ​​of ethanol concentration curve AUC, blood ammonia concentration, and L-lactic acid / pyruvate ratio.

[0083] As shown in Table 2, under the same ethanol exposure conditions, the compound probiotic compositions described in Examples 1-3 were significantly superior to the comparative groups in reducing blood ammonia levels. The compound probiotic composition described in Example 1 was significantly superior to Examples 2 and 3 and the comparative groups in reducing plasma ethanol concentration curve AUC, blood ammonia levels, and L-lactic acid / pyruvate ratio (all differences were statistically significant). p<0.05), and the relevant indicators were close to the level of the blank control group. Among them, the absence of Pediococcus pentosacchari PP-01 and / or PP-02 in Comparative Examples 1 and 2 resulted in a significant deterioration of the above indicators; the simplification of the bacterial community structure in Comparative Examples 3 and 4 also led to a significant deterioration of the above indicators; after using the same strain as Pediococcus pentosacchari to replace Pediococcus pentosacchari PP-01 and PP-02 in Comparative Example 5, the effect of Example 1 could not be restored; after replacing the prebiotic module of the present invention with glucose or maltodextrin in Comparative Examples 7 to 9, the improvement effect on the above indicators was significantly reduced; after replacing the antioxidant module of the present invention with tea polyphenols or rosemary extract in Comparative Examples 11 and 12, the effect of Example 1 could not be achieved; and Comparative Examples 6, 13 and 14 only contained some modules, and could not achieve the comprehensive improvement effect of Example 1. The above results indicate that the specific strain combination, prebiotic module, and antioxidant module in the compound probiotic composition of the present invention synergistically reduce the AUC of the ethanol concentration curve, blood ammonia level, and L-lactic acid / pyruvate ratio, thus exhibiting an unexpected metabolic burden relief effect under ethanol stress conditions.

[0084] Test Example 2: Effect of Compound Probiotic Composition on Ethanol Content under In Vitro Ethanol Incubation Conditions Taking the compound probiotic composition described in Example 1 as an example, this invention tested its effect on the relative content of ethanol in the system after incubation with ethanol under in vitro conditions.

[0085] 1. Experimental Materials Sterile phosphate buffer (pH 7.2); containing a total viable count of 2 × 10⁻⁶. 10 CFU-based compound probiotic composition; modified MRS medium (1 L medium contains 10 g tryptone, 8 g beef meal, 4 g yeast powder, 10 g glucose, 2 g dipotassium hydrogen phosphate, 2 g diammonium hydrogen citrate, 5 g sodium acetate, 0.2 g magnesium sulfate, 0.04 g manganese sulfate, 1 g Tween 80, 2 g porcine gastric mucoprotein, 0.5 g L-cysteine, diluted with distilled water to 1000 mL, pH adjusted to 6.2). 0.2, autoclaved at 121℃ for 20 min); anhydrous ethanol; ethanol colorimetric test kit (Wuhan Yilairuit Biotechnology Co., Ltd.).

[0086] 2. Experimental Methods The total number of live bacteria was 2×10 10The CFU-containing probiotic composition was dissolved in an appropriate amount of sterile phosphate buffer and incubated at 37°C for 15 min. Then, an equal volume of modified MRS medium was added to the suspension, and the mixture was incubated at 37°C for 2 h to restore bacterial activity. After incubation, the mixture was centrifuged at 4000 rpm for 10–15 min, the supernatant was discarded, and the suspension was resuspended in an appropriate amount of sterile phosphate buffer to prepare a solution containing approximately 1 × 10⁻⁶ viable bacteria. 10 A CFU / mL compound probiotic suspension was prepared, with sterile phosphate buffer as the control suspension.

[0087] A control group and a compound probiotic composition group were set up, with five replicate samples in each group. The reaction system for each sample was as follows: 1.7 mL of sterile phosphate buffer and 0.1 mL of anhydrous ethanol were mixed, and 0.2 mL of the corresponding suspension for each group was added to prepare a reaction solution with a total reaction volume of 2.0 mL; the final volume fraction of ethanol was approximately 5% (v / v). The reaction solutions of each group were incubated at 37℃ and 150 rpm for 4 h with shaking. After incubation, the mixture was centrifuged at 12000 rpm for 5 min, and the supernatant was collected. The ethanol concentration in each group of reaction solutions was determined according to the instructions of a commercial ethanol colorimetric assay kit. The relative ethanol concentration of the compound probiotic composition group was calculated with the ethanol concentration of the control group at 4 h as a reference.

[0088] 3. Experimental Results After the compound probiotic composition is incubated with ethanol under in vitro conditions, the relative content of ethanol in the system is as follows: Figure 1 As shown. By Figure 1 It can be seen that, compared with the control group, the relative ethanol concentration of the compound probiotic composition group was significantly lower.

[0089] Test Example 3: Determination of Ethanol Tolerance of Certain Strains in a Compound Probiotic Composition This invention uses *Lactobacillus plantarum* as a control strain to test the tolerance of *Pediococcus pentosaceus* strains PP-01 and PP-02 to ethanol.

[0090] 1. Experimental Materials MRS medium (1 L of medium contains 10 g tryptone, 5 g beef meal, 4 g yeast extract, 20 g glucose, 2 g dipotassium hydrogen phosphate, 2 g diammonium hydrogen citrate, 5 g sodium acetate, 0.2 g magnesium sulfate, 0.05 g manganese sulfate, and 1 g Tween 80, diluted with distilled water to a final volume of 1000 mL, and adjusted to pH 5.7). 0.2, autoclaved at 121℃ for 20 min); Pediococcus pentosaceus PP-01 strain (CGMCC No. 34357); Pediococcus pentosaceus PP-02 strain (CGMCC No. 34358); control strain Lactobacillus plantarum; anhydrous ethanol.

[0091] 2. Experimental methods Pediococcus pentosaceus PP-01, PP-01, and Lactobacillus plantarum strains were anaerobically cultured in MRS medium without ethanol until the logarithmic growth phase. The OD was adjusted, and each strain was subjected to plate counting to calculate the viable CFU. Each strain in the logarithmic growth phase was inoculated into MRS medium containing 0%, 5%, 8%, 10%, and 15% (v / v) ethanol at a certain quantity. After anaerobic culture at 37°C for 24 h, each strain was subjected to plate counting to calculate the viable CFU, and the relative survival rate and relative growth rate of each strain in different ethanol concentrations were calculated. Each experimental condition was independently repeated at least five times, and the obtained results were averaged and the standard deviation was calculated to compare the relative survival and relative growth abilities of different strains at different ethanol concentrations.

[0092] Relative survival = viable bacteria count at each concentration after 24 h / viable bacteria count at each concentration at 0 h 3. Experimental results The relative survival of Pediococcus pentosaceus PP-01, PP-02, and Lactobacillus plantarum strains under different ethanol concentrations and the comparison results are as Figure 2 shown. It can be Figure 2 seen that the proliferation ability of the control strain Lactobacillus plantarum was significantly weakened in MRS medium with 5% ethanol, and the survival rate was significantly reduced in MRS medium containing 8% ethanol or higher concentration. Compared with the control strain, strains PP-01 and PP-02 still maintained a high survival rate and proliferation ability in MRS medium containing 5%, 8%, and 10% ethanol, but their survival was significantly inhibited in MRS medium containing 15% ethanol. The results show that Pediococcus pentosaceus PP-01 and PP-02 strains have good ethanol tolerance, which is beneficial to maintaining activity under alcohol intake conditions, providing a strain basis for the application effect of the composite probiotic composition described in this invention under the condition of drinking alcohol.

[0093] Test Example 4 Protective effect of the composite probiotic composition on alcohol-induced liver-intestinal injury in mice Taking the composite probiotic composition described in Example 1 of this invention as an example, its protective effect on alcohol-induced liver-intestinal injury in mice was tested.

[0094] 1. Experimental materials (1) Experimental animals Forty-two 7-8-week-old male C57BL / 6 mice with a body weight of 20-25 g were purchased from the Experimental Animal Center of Sun Yat-sen University. The experimental animal production license number is SCXK (Guangdong) 2021-0029. The mice were raised in the Experimental Animal Center of Sun Yat-sen University, and the experimental animal use license number is SYXK (Guangdong) 2023-0112 of Sun Yat-sen University.

[0095] (2) Experimental reagents Triglyceride (TG), lipopolysaccharide (LPS), glutathione (GSH), malondialdehyde (MDA), D-lactate, and adenosine triphosphate (ATP) assay kits (Wuhan Yilairui Biotechnology Co., Ltd.); Diamine oxidase (DAO), alanine aminotransferase (ALT), aspartate aminotransferase (AST), alcohol dehydrogenase (ADH), acetaldehyde dehydrogenase (ALDH), superoxide dismutase (SOD), and catalase (CAT) activity assay kits (Wuhan Yilairui Biotechnology Co., Ltd.); Formalin, xylene, hematoxylin reagent, eosin reagent, hydrochloric acid ethanol, and resin mounting medium (Shanghai Sangon Biotech Co., Ltd.); RT-PCR kit and RT-qPCR kit (Hunan Aikerui Biotechnology Co., Ltd.); Haiwang brand Jinzun tablets (Shenzhen Haiwang Health Technology Development Co., Ltd.); 56-degree Hongxing Erguotou (Beijing Hongxing Co., Ltd.).

[0096] (3) Primer sequence Primers used in quantitative real-time PCR experiments include SREBP-1c , FAS , SCD1 , CPT1A , CYP2E1 , GAPDH , IL-1 β , IL-6 , NLRP3 , Occludin , Claudin-1 , ZO-1 The quantitative PCR primers for the gene were designed and synthesized by Beijing Qingke Biotechnology Co., Ltd.

[0097] 2. Experimental Methods (1) Determination of righting reflex recovery time in mice Male C57BL / 6 mice were acclimatized for one week and then randomly divided into four groups: a negative control group, a model group, a compound probiotic composition group, and a Haiwang Jinzun group (positive control), with six mice per group. All mice were fasted for 12 hours prior to the experiment, but allowed free access to water. The compound probiotic composition group was administered 100 mg / mouse of the corresponding compound probiotic composition suspension via gavage. The Haiwang Jinzun group was administered 100 mg / mouse of Haiwang Jinzun tablet solution via gavage. The control and model groups were administered the corresponding volume of physiological saline via gavage. Thirty minutes after administration, all groups except the control group were administered 15 mL / kg of 56-degree Hongxing Erguotou (a type of Chinese liquor) via gavage, while the control group was administered an equal volume of physiological saline. The righting reflex of the mice was observed every 5 minutes, and the alcohol tolerance time and sobering-up time were recorded.

[0098] (2) Protective effect of compound probiotic composition on alcohol intake-induced liver-intestinal injury in mice Establishment of a short-term, repeated alcoholic liver injury model in mice Male C57BL / 6 mice were acclimatized for one week and then randomly divided into three groups: a control group, a model group, and a compound probiotic composition group. Each group of mice was administered the corresponding intervention via gavage once daily for seven consecutive days. The compound probiotic composition group received 100 mg / mouse of the corresponding compound probiotic composition suspension via gavage, while the control and model groups received the corresponding volume of physiological saline. Two hours after the daily intervention, the model and compound probiotic composition groups were administered 10 mL / kg of 56-degree Erguotou (a type of Chinese liquor) via gavage, while the control group received the same volume of physiological saline. During the experiment, mice had free access to food and water. After the last alcohol gavage, all mice were fasted but allowed free access to water for 6 hours. Feces were collected, blood was drawn from the orbital rim, and the mice were euthanized by dislocation. Serum, intestinal tissue, and liver tissue were collected for subsequent biochemical index detection and histological analysis.

[0099] Measurement of serum LPS, D-Lactate, and TG levels, and ALT, AST, and DAO activities. The collected blood samples were centrifuged at 3500 r / min and 4℃ for 10 min, and the supernatant was collected to obtain serum. The levels of LPS, D-Lactate, and TG in the serum were measured using the corresponding indicator detection kits according to the instructions. The levels of ALT, AST, and DAO activities in the serum were measured using the corresponding indicator enzyme activity detection kits according to the instructions.

[0100] Measurement of GSH, MDA, and ATP levels, and the activities of ADH, ALDH, SOD, and CAT in liver tissue. The collected liver / intestinal tissues were added to an appropriate amount of physiological saline and then homogenized using a tissue homogenizer to prepare liver tissue homogenates. The levels of GSH, MDA, and ATP in the liver tissue were measured using the corresponding indicator detection kits according to the instructions. The activity levels of ADH, ALDH, SOD, and CAT were measured using the corresponding indicator enzyme activity detection kits according to the instructions.

[0101] Hematoxylin-eosin (H&E) staining of liver tissue Collected liver tissue samples from each group were fixed overnight at 4°C in 4% paraformaldehyde fixative. After fixation, the tissues were washed with PBS and then dehydrated, cleared, and embedded in paraffin according to standard procedures. Paraffin sections approximately 5 μm thick were cut using a microtome. After spreading and drying, the sections were dewaxed, rehydrated, stained with hematoxylin and eosin (H&E) according to standard procedures. The stained sections were then dehydrated, mounted, and observed and imaged under an optical microscope.

[0102] Real-time PCR Total RNA was extracted from the collected liver tissue using TRIzol reagent. An appropriate amount of RNA solution was reverse transcribed into total cDNA using an RT-PCR kit. The target gene was then quantitatively analyzed using an RT-qPCR kit and the Biorad CFX connect Real-Time System.

[0103] Mouse fecal 16S rRNA sequencing analysis Fresh mouse fecal samples were collected, rapidly frozen and hardened with liquid nitrogen, and total DNA was extracted. The variable region of the bacterial 16S rRNA gene was amplified and sequenced using high-throughput sequencing. The sequencing data were processed using standard biological procedures for analysis of the gut microbiota structure.

[0104] 3. Experimental Results (1) The compound probiotic composition significantly shortened the recovery time of righting reflex in mice after alcohol treatment. The recovery time of righting reflex after alcohol intake in mice in the negative control group, model group, compound probiotic combination group, and Haiwang Jinzun group, and the comparison results are as follows: Figure 3 As shown. By Figure 3 It was found that, compared with the model group, the righting reflex time of mice in the compound probiotic composition group was significantly shortened, and the righting reflex in some mice in this group did not disappear due to alcohol influence. The Haiwang Jinzun tablet group, as a positive control group, showed no significant difference in righting reflex time compared with the control group. The results indicate that the compound probiotic composition described in this invention can significantly shorten the righting reflex time of intoxicated mice, suggesting that the compound probiotic composition helps shorten the recovery time of bodily functions after alcohol intake and can be used for hangover relief.

[0105] (2) The compound probiotic composition can prevent liver dysfunction caused by alcohol intake. The effects of the control group, model group, and compound probiotic combination group on alcohol-induced liver morphology, liver function damage, oxidative stress, and related molecular indicators in mice, and their comparative results are as follows: Figure 4 As shown. By Figure 4 Compared with the control group, the model group mice showed significantly increased serum ALT and AST activities and TG levels, significantly decreased GSH and ATP levels, significantly increased MDA levels, and significantly decreased SOD and CAT activities in their liver tissue. However, no significant changes were observed in ADH and ALDH activities, suggesting that alcohol treatment induced liver damage accompanied by increased oxidative stress. H&E staining further indicated mild structural disorder and inflammatory cell infiltration in the liver tissue of the model group. In contrast, the compound probiotic combination group significantly prevented these abnormal changes. ADH and ALDH activities did not show adverse changes, and their levels were slightly higher than the control group. The liver tissue structure tended to be intact, and the overall related indicators recovered to control group levels.

[0106] (3) The compound probiotic composition can protect the intestinal barrier in the event of alcohol intake. Quantitative real-time PCR results showed that, compared with the control group, the model group had a higher concentration of lipid production-related genes. SREBP- 1c , FAS and SCD1 and inflammation-related genes IL-1β , IL-6 and NLRP3 The expression of these genes was significantly upregulated, along with genes related to fatty acid oxidation and alcohol metabolism. CPT1A and CYP2E1 Abnormal changes in gene expression were observed. However, the abnormal expression of the aforementioned related genes was corrected in the compound probiotic combination group, with their expression levels significantly decreasing compared to the model group, and some indicators approaching those of the control group.

[0107] The results showed that the intervention of the compound probiotic composition could alleviate the elevated level of alcohol-induced oxidative stress in the liver, reduce lipid production in the liver, stabilize the physiological indicators of liver function to be close to those of the control group, and help maintain liver metabolic homeostasis.

[0108] (4) The compound probiotic composition can stabilize the composition of the intestinal flora in the presence of alcohol. The effects of the control group, model group, and compound probiotic combination group on molecular indicators related to the intestinal barrier in alcohol-induced mice and their comparison results are as follows: Figure 5 As shown. By Figure 5 It was found that, compared with the control group, the serum DAO activity of mice in the model group was significantly increased, and the levels of D-Lactate and LPS were significantly increased; quantitative real-time PCR of intestinal tissue showed that intestinal barrier-related genes in the model group were significantly increased. ZO-1 , Occludin and Claudin-1 The expression of DAO, D-Lactate, and LPS was significantly downregulated compared to the control group, suggesting that alcohol treatment impairs intestinal barrier function. Compared to the model group, the levels of DAO, D-Lactate, and LPS in serum were significantly lower in the probiotic combination group, while the expression levels of intestinal barrier-related genes were higher in the probiotic combination group than in the control group. These results indicate that the probiotic combination can help protect intestinal barrier function, reduce serum levels of barrier-related indicators, and has a potential supporting role in alleviating gut-hepatic axis functional imbalances.

[0109] The effects of the control group, model group, and compound probiotic combination group on the level distribution of intestinal flora in alcohol-induced mouse feces are as follows: Figure 6 As shown. By Figure 6 It can be seen that after removing the exogenous flora from the compound probiotic combination group due to gavage intervention and restandardizing, the composition of endogenous intestinal flora in the feces of model group mice was significantly altered compared with the control group. At the genus level, this alteration is beneficial to the intestinal barrier homeostasis of mice. Faecalibaculum Genus Muribaculum Genus and Lachnospiraceae The relative abundances of bacteria such as family (genus not determined) decreased in the feces of the model group, while the relative abundances of Desulfovibrionaceae family (genus not determined) and Helicobacter genus related to intestinal barrier impairment and inflammation tended to increase in the feces of the model group, indicating that alcohol intake can disrupt the intestinal microbial homeostasis and be accompanied by an imbalance in the microbial community structure. In contrast, in the feces of the composite probiotic composition group, the overall distribution of the microbial community was close to that of the control group, and the structural characteristics were relatively balanced. The relative abundances of some of the above-mentioned genera related to intestinal homeostasis showed a recovery trend; at the same time, the relative abundances of the above-mentioned genera related to barrier impairment were also at a low level in the feces. The results showed that the composite probiotic composition could alleviate the alcohol-induced imbalance in the endogenous intestinal microbial composition and maintain the relative stability of the intestinal microbial community structure.

[0110] Test Example 5 Protective Effect of Composite Probiotic Composition on Hepato-Intestinal Injury in Sleep-Deprived Mice Taking the composite probiotic composition described in Example 1 of the present invention as an example, its protective effect on hepato-intestinal injury in sleep-deprived mice was tested.

[0111] 1. Experimental Materials (1) Experimental Animals Twenty-four male C57BL / 6 mice aged 7 - 8 weeks with a body weight of 20 - 25 g were purchased from the Experimental Animal Center of Sun Yat-sen University. The production license number of experimental animals is SCXK (Guangdong) 2021 - 0029. The mice were housed in the Experimental Animal Center of Sun Yat-sen University, and the license number for the use of experimental animals is SYXK (Guangdong) 2023 - 0112 of Sun Yat-sen University.

[0112] (2) Experimental Reagents Lipopolysaccharide (LPS), glutathione (GSH), malondialdehyde (MDA) detection kits (Wuhan Elite Biotechnology Co., Ltd.); alanine aminotransferase (ALT), aspartate aminotransferase (AST), superoxide dismutase (SOD) activity detection kits (Wuhan Elite Biotechnology Co., Ltd.); RT-PCR kit, RT-qPCR kit (Hunan Aikrui Bioengineering Co., Ltd.).

[0113] (3) Primer Sequences The primers used in the fluorescence quantitative PCR experiment included Occludin , Claudin-1 , ZO-1 Quantitative PCR primers for genes, which were designed and synthesized by Beijing Tsingke Biotechnology Co., Ltd.

[0114] 2. Experimental Methods (1) Establishment of a Subchronic Repeated Sleep Deprivation Model in Mice Male C57BL / 6 mice were acclimatized for one week and then randomly divided into four groups: control group, model group, model intervention group, and control intervention group, with six mice per group. Mice in each group were administered the corresponding intervention once daily by gavage for seven consecutive days before the start of the sleep deprivation experiment. Mice in the model intervention group and control intervention group were administered 100 mg / mouse of the corresponding compound probiotic suspension by gavage, while mice in the control group and model group were given the corresponding volume of physiological saline. The subchronic sleep deprivation experiment used a multi-platform pool method to establish the model, with several small, isolated platforms that could not be crossed within the pool. After the experiment began, mice in the model group and the model intervention group were placed in a multi-platform water tank for 20 hours each day for free movement. When they entered deep sleep or REM sleep, the decrease in muscle tone made the mice prone to slipping into the water and being passively awakened, thus achieving continuous sleep deprivation. Subsequently, the mice were transferred back to ordinary cages for 4 hours to recover, with free access to food and water. During this period, the mice were administered the corresponding intervention by gavage. Mice in the model intervention group were administered 100 mg / mouse of the corresponding compound probiotic suspension by gavage, while mice in the model group were given an appropriate volume of physiological saline. The sleep deprivation period was 7 days. During the sleep deprivation experiment, the control group and the control intervention group continued to be housed in ordinary cages and were also administered the corresponding intervention by gavage daily. Mice in the control intervention group were administered 100 mg / mouse of the corresponding compound probiotic suspension by gavage, while mice in the control group were given an appropriate volume of physiological saline. After the experiment, the mice in each group were fasted but allowed free access to water for 6 hours. Blood was collected from the orbital cavity and the mice were euthanized by dislocation. Serum, intestinal tissue, and liver tissue were collected for subsequent biochemical index detection.

[0115] (2) Determination of serum LPS levels and ALT and AST activities The collected blood samples were centrifuged at 3500 r / min and 4℃ for 10 min, and the supernatant was collected to obtain serum. The LPS level in the serum was measured using the corresponding indicator detection kits according to the instructions. The ALT and AST activity levels in the serum were measured using the corresponding indicator enzyme activity detection kits according to the instructions.

[0116] (3) Measurement of GSH, MDA levels and SOD activity in liver tissue The collected liver tissues were homogenized in a tissue homogenizer with an appropriate amount of physiological saline. The levels of GSH and MDA in the liver tissue were measured using the corresponding indicator detection kits according to the instructions. The SOD activity level was measured using the SOD enzyme activity detection kit according to the instructions.

[0117] (4) Real-time PCR Same as the real-time PCR experiment in Test Example 4.

[0118] 3. Experimental Results The effects of the control group, control intervention group, model group, and model intervention group on liver function and intestinal barrier-related molecular markers in sleep-deprived mice, and their comparative results are as follows: Figure 7 As shown. By Figure 7 Compared with the control group, the model group mice showed slightly elevated serum ALT and AST levels, slightly decreased GSH levels, slightly increased MDA levels, and slightly decreased SOD activity in liver tissue, indicating that sleep deprivation can induce oxidative stress and lipid peroxidation in the mouse liver, resulting in mild liver damage. Furthermore, the model group mice showed significantly elevated serum LPS levels, and quantitative real-time PCR in intestinal tissue showed... Occulin , Claudin-1 and ZO-1 The expression levels were significantly downregulated, indicating impaired intestinal barrier function in mice under sleep deprivation. Compared to the model group, the overall levels of serum biochemical indicators and liver oxidative stress indicators in the intervention group were close to those in the control group, while the expression levels of intestinal tight junction factors were higher. This suggests that the compound probiotic composition helps alleviate the increased liver oxidative stress burden under sleep deprivation and supports the maintenance of intestinal barrier function, thus contributing to liver metabolic homeostasis. Furthermore, the levels of GSH and SOD activity in the control intervention group showed an increasing trend, and the expression of SOD in intestinal tissue was significantly lower. Occludin , Claudin-1 and ZO-1 The expression level was slightly upregulated, while the other indicators remained similar to those of the control group, indicating that the compound probiotic composition of the present invention has the potential to regulate gut-liver axis homeostasis and maintain liver antioxidant capacity under non-sleep deprivation conditions.

[0119] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. A compound probiotic composition, characterized in that, It includes compound probiotics and compound compounds; the mass ratio of the compound probiotics and compound compounds is (3-9):(1-2); The compound probiotics consist of *Pediococcus pentosaceus* strain PP-01, *Pediococcus pentosaceus* strain PP-02, *Lactobacillus rhamnosus*, *Lactobacillus plantarum*, *Bifidobacterium bifidum*, *Saccharomyces boulardii*, and *Akermansia muciniphila* in a colony-forming unit ratio of (4–6):(4–6):(2–4):(1–3):(1–2):(1–2):(2–4), and the total number of live bacteria in the compound probiotics is at least 1 × 10⁻⁶. 10 CFU / g; The *Pediococcus pentosaceus* PP-01 strain was deposited at the China General Microbiological Culture Collection Center (CGMCC) on April 25, 2025, with accession number CGMCC No. 34357; The *Pediococcus pentosaceus* PP-02 strain was deposited at the China General Microbiological Culture Collection Center (CGMCC) on April 25, 2025, with accession number CGMCC No. 34358; The compound is composed of resveratrol, S-adenosylmethionine p-toluenesulfonic acid sulfate, curcumin, inulin, resistant starch, fructooligosaccharides, galactooligosaccharides, xylooligosaccharides and aloe polysaccharides in a mass ratio of (1-2):(4-6):(1-2):(3-5):(3-5):(3-5):(3-5):(1-2):(1-2).

2. The compound probiotic composition according to claim 1, characterized in that, The mass ratio of the compound probiotics to the compound compound is 3 to 9:

1.

3. The compound probiotic composition according to claim 2, characterized in that, The mass ratio of the compound probiotics to the compound compound is 3:

1.

4. A method for preparing the compound probiotic composition according to any one of claims 1 to 3, characterized in that, Includes the following steps: S1. Prepare freeze-dried bacterial powders of *Pediococcus pentosaceus* strain PP-01, *Pediococcus pentosaceus* strain PP-02, *Lactobacillus rhamnosus*, *Lactobacillus plantarum*, *Bifidobacterium bifidum*, *Saccharomyces boulardii*, and *Ackermania muscaria*, respectively. Mix the obtained freeze-dried bacterial powders of *Pediococcus pentosaceus* strain PP-01, *Pediococcus pentosaceus* strain PP-02, *Lactobacillus rhamnosaceus*, *Lactobacillus plantarum*, *Bifidobacterium bifidum*, *Saccharomyces boulardii*, and *Ackermania muscaria* uniformly according to the colony-forming unit ratio to obtain a product containing at least 1 × 10⁻⁶ viable bacteria. 10 CFU / g of compound probiotics; S2. The resveratrol, S-adenosylmethionine p-toluenesulfonate sulfate, curcumin, inulin, resistant starch, fructooligosaccharides, galactooligosaccharides, xylooligosaccharides and aloe polysaccharides are uniformly mixed according to the mass ratio to obtain a composite compound; Alternatively, resveratrol, S-adenosylmethionine p-toluenesulfonate sulfate, curcumin, inulin, resistant starch, fructooligosaccharides, galactooligosaccharides, xylooligosaccharides, and aloe polysaccharides may be weighed according to the aforementioned mass ratios and dissolved or dispersed in solvents to obtain concentrated solutions of each compound; wherein, resveratrol and curcumin are dissolved in propylene glycol, and S-adenosylmethionine p-toluenesulfonate sulfate, inulin, resistant starch, fructooligosaccharides, galactooligosaccharides, xylooligosaccharides, and aloe polysaccharides are dissolved or dispersed in sterile phosphate buffer; S3. The obtained compound probiotics and compound compound are mixed evenly according to the mass ratio to obtain the compound probiotic composition; Alternatively, the total mass of the compound probiotics and the concentrated solutions of each compound can be mixed according to the stated mass ratio, and sterile phosphate buffer can be added to prepare a homogeneous suspension. After freeze-drying, the compound probiotic composition can be obtained.

5. A formulation comprising the compound probiotic composition according to any one of claims 1 to 3.

6. The use of the compound probiotic composition according to any one of claims 1 to 3 or the preparation according to claim 5 in the preparation of a hangover relief and liver protection product.

7. The use of the compound probiotic composition according to any one of claims 1 to 3 or the formulation according to claim 5 in the preparation of a product for improving liver damage.

8. The application according to claim 7, characterized in that, The liver damage was caused by alcohol or sleep disorders.

9. The use of the compound probiotic composition of any one of claims 1 to 3 or the formulation of claim 5 in the preparation of a product that improves intestinal barrier damage.

10. The application according to claim 9, characterized in that, The intestinal barrier damage was caused by alcohol or sleep disorders.

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