A probiotic composition for alleviating acute oxidative stress and preparation and use thereof

Abstract

The application discloses a probiotic composition for relieving acute oxidative stress and preparation and application thereof, and belongs to the technical field of antioxidant strains. The probiotic composition for relieving acute oxidative stress has a synergistic effect, has a unique ability of removing free radicals and repairing damage of biological macromolecules such as DNA and protein caused by oxidative stress, can significantly up-regulate the level of key endogenous antioxidants and the activity of antioxidant enzymes in the liver tissue of an alcohol-induced acute oxidative stress mouse model, thereby strengthening the endogenous antioxidant defense system of the body, and has no toxic side effect, has a comprehensive effect of combining exogenous removal, damage repair and endogenous enhancement, is different from the existing single action mode technology, and provides a new biological way which is efficient and safe for precisely relieving acute oxidative stress, in particular, oxidative liver damage induced by drinking and the like.

Description

Technical Field

[0001] This invention relates to the field of antioxidant strain technology, and in particular to a probiotic composition for relieving acute oxidative stress, its preparation and application. Background Technology

[0002] Oxidative stress refers to an imbalance between the generation of free radicals and the body's antioxidant defense system, leading to an excessive accumulation of reactive oxygen species (ROS). ROS attack intracellular lipids, proteins, and DNA through oxidative reactions, causing physiological or pathological damage to cells, tissues, or organs. Oxidative stress is a significant contributing factor to many diseases, such as cardiovascular diseases, where free radicals and ROS damage vascular endothelial cells and promote the formation of atherosclerotic plaques; neurodegenerative diseases, such as Alzheimer's and Parkinson's, are closely related to neuronal death caused by oxidative damage in the brain; and cancer, where free radicals and ROS damage DNA and induce gene mutations, but high concentrations of free radicals and ROS can also inhibit tumor cell proliferation.

[0003] In recent years, a large number of products on the market have focused on synthetic drugs or plant extracts, mainly including antioxidants and anti-inflammatory drugs. However, these drugs have problems such as significant side effects and limited efficacy. Therefore, using probiotics to regulate the body's antioxidant balance has become an important research direction, and actively developing probiotics with stronger effects on oxidative stress relief is a goal that those in the field are relentlessly pursuing. Summary of the Invention

[0004] The purpose of this invention is to provide a probiotic composition for relieving acute oxidative stress, its preparation and application, so as to provide a probiotic composition and its lysate with a stronger effect on relieving oxidative stress, and to contribute to relieving oxidative stress and reducing the occurrence and development of diseases.

[0005] To achieve the above objectives, the present invention provides a probiotic composition for alleviating acute oxidative stress, wherein the probiotics are *Lactobacillus plantarum* STB9b and *Lactobacillus fermentum* Z-15; the Latin name of *Lactobacillus plantarum* STB9b is... Lactiplantibacillus plantarum The accession number is CCTCC NO.M 20241444; the Latin name of Lactobacillus fermentans Z-15 is... Limosilactobacillus fermentum The accession number is CCTCC NO.M 2024024.

[0006] Preferably, the ratio of viable Lactobacillus plantarum STB9b to Lactobacillus fermentum Z-15 in the probiotic composition is 1:1.

[0007] A method for preparing the probiotic composition for relieving acute oxidative stress as described above includes the following steps:

[0008] Lactobacillus plantarum STB9b and Lactobacillus fermentum Z-15 were inoculated into MRS liquid medium at an inoculation amount of 2-6% of the MRS liquid medium and fermented at 37°C for 8-16 hours. The precipitates were then centrifuged separately and resuspended in physiological saline to determine the bacterial concentration. Finally, the bacterial suspensions of the two strains were mixed.

[0009] Preferably, the bacterial concentration is determined to be 0.5 × 10⁻⁶. 9 -2×10 9 CFU / mL.

[0010] A probiotic lysate for relieving acute oxidative stress, comprising *Lactobacillus plantarum* STB9b and *Lactobacillus fermentum* Z-15; the Latin name for *Lactobacillus plantarum* STB9b is... Lactiplantibacillus plantarum The accession number is CCTCC NO.M 20241444; the Latin name of Lactobacillus fermentans Z-15 is... Limosilactobacillus leaven The accession number is CCTCC NO.M 2024024. The institution where it is deposited is Wuhan University, located in Wuhan, Hubei Province.

[0011] Preferably, the probiotic lysate is the supernatant obtained after the probiotics have been ultrasonically broken down and centrifuged.

[0012] The application of the probiotic composition for relieving acute oxidative stress as described above in the preparation of health food products, which are foods for preventing / repairing oxidative damage and / or improving the body's antioxidant level.

[0013] The use of a probiotic and / or composition for relieving acute oxidative stress as described above in the preparation of antioxidant pharmaceuticals.

[0014] The above-mentioned application of a probiotic lysate for relieving acute oxidative stress in the preparation of health food products, which are foods for preventing / repairing oxidative damage and / or improving the body's antioxidant level.

[0015] The application of a probiotic lysate for relieving acute oxidative stress, as described above, in the preparation of antioxidant drugs.

[0016] Therefore, the present invention provides a probiotic composition for alleviating acute oxidative stress, its preparation and application, and its specific technical effects are as follows:

[0017] (1) The probiotic composition for relieving acute oxidative stress provided by the present invention is composed of *Lactobacillus plantarum* (…). Lactiplantibacillus plantarum STB9b and Lactobacillus fermentum ( Limosilactobacillus leavenThe Z-15 strain has a synergistic effect with the two strains, and its ability to scavenge free radicals is better than that of Lactobacillus rhamnosus LGG, which is currently widely used for anti-oxidation.

[0018] (2) The probiotic composition and its lysate provided by the present invention for relieving acute oxidative stress have a unique ability to repair damage to biological macromolecules such as DNA and proteins caused by oxidative stress.

[0019] (3) The probiotic composition provided by the present invention, which relieves acute oxidative stress, can significantly upregulate the level of key endogenous antioxidants (such as glutathione GSH) and the activity of antioxidant enzymes (such as superoxide dismutase SOD) in the liver tissue of an alcohol-induced acute oxidative stress mouse model, thereby strengthening the body's endogenous antioxidant defense system without any toxic side effects.

[0020] (4) The probiotic composition provided by the present invention has a comprehensive effect of “exogenous scavenging, damage repair and endogenous enhancement”, which is different from the existing single mode of action technology. It provides a highly efficient and safe new biological approach for accurately relieving acute oxidative stress, especially oxidative liver damage induced by drinking.

[0021] The technical solution of the present invention will be further described in detail below with reference to the accompanying drawings and embodiments. Attached Figure Description

[0022] To more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings used in the description of the embodiments of the present invention will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0023] Figure 1 This is the result of the determination of the free radical scavenging ability of the probiotic / probiotic composition in Example 2 of the present invention; wherein A is the scavenging ability of DPPH; B is the scavenging ability of hydroxyl radicals; STB9b is the STB9b bacterial suspension treatment group; Z-15 is the Z-15 bacterial suspension treatment group; STB9b+Z-15 is the probiotic composition treatment group; LGG is the Lactobacillus rhamnosus LGG strain treatment group; STB1 is the Lactobacillus plantarum STB1 strain treatment group;

[0024] Figure 2This is the result of the determination of the ability of the probiotic / probiotic composition to repair DNA oxidative damage in vitro in Example 3 of the present invention; wherein lane 1 is a 5000bp marker; lane 2 is the undamaged plasmid group; lane 3 is the positive control group; lane 4 is the *Lactobacillus plantarum* STB9b suspension group; lane 5 is the *Lactobacillus fermentum* Z-15 suspension group; lane 6 is the combined *Lactobacillus plantarum* STB9b and *Lactobacillus fermentum* Z-15 suspension group; lane 7 is the *Lactobacillus plantarum* STB9b lysate group; lane 8 is the *Lactobacillus fermentum* Z-15 lysate group; and lane 9 is the combined *Lactobacillus plantarum* STB9b and *Lactobacillus fermentum* Z-15 lysate group.

[0025] Figure 3 This is the result of the determination of the ability of the probiotic / probiotic composition to repair oxidative damage of lactic acid bacteria proteins in vitro in Example 4 of the present invention; where A is the gray gel imaging image corresponding to the result; B is the color gel imaging image corresponding to the result; CK is the undamaged group; Con is the positive control group; NeCon is the negative control group with only damage; 9b is the *Lactobacillus plantarum* STB9b suspension group; Z-15 is the *Lactobacillus fermentum* Z-15 suspension group; 9b+Z-15 is the combined *Lactobacillus plantarum* STB9b and *Lactobacillus fermentum* Z-15 suspension group;

[0026] Figure 4 This is the result of the determination of the ability of the probiotic / probiotic composition in Example 5 of the present invention to alleviate the oxidative stress induced by alcohol consumption in the body; wherein A is reduced glutathione; B is glutathione peroxidase; C is the total antioxidant capacity of the liver; D is the total superoxide dismutase activity of the liver; E is the malondialdehyde content of the liver; and F is the catalase activity of the liver. Detailed Implementation

[0027] The technical solution of the present invention will be further described below with reference to the accompanying drawings and embodiments.

[0028] To make the objectives, technical solutions, and advantages of this application clearer, more thorough, and more complete, the technical solutions of the present invention will be clearly and completely described below with reference to the accompanying drawings and embodiments. The following detailed descriptions are all illustrations of embodiments, intended to provide further detailed explanation of the present invention. Unless otherwise specified, all technical terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains.

[0029] The instruments, equipment, reagents and materials used in the embodiments were all obtained through commercial means; the methods and steps not described in detail are all conventional techniques in the field.

[0030] The MRS liquid culture medium used in the examples had the following composition: 10.0 g peptone, 20.0 g glucose, 10.0 g beef extract, 5.0 g yeast extract, 5.0 g anhydrous sodium acetate, 2.0 g triammonium citrate, 2.0 g K2HPO4, 0.58 g MgSO4·7H2O, 0.25 g MnSO4·4H2O, 1.0 mL Tween-80, and 1000.0 mL distilled water. The pH was adjusted to 6.8, and the medium was sterilized at 121°C for 20 min.

[0031] Example 1

[0032] The specific steps for preparing the probiotic composition are as follows:

[0033] (1) Inoculate *Lactobacillus plantarum* STB9b into MRS liquid culture medium (inoculation amount: 4.0%) and ferment at 37℃ under normal atmospheric conditions for 12 h to obtain *Lactobacillus plantarum* fermentation broth. Centrifuge at 6000 rpm and 4℃ for 5 min to obtain *Lactobacillus plantarum* STB9b sludge. Then resuspend in physiological saline to make the concentration of *Lactobacillus plantarum* STB9b 1.0 × 10⁻⁶. 9 CFU / mL, to obtain a suspension of *Lactobacillus plantarum*.

[0034] (2) Inoculate *Lactobacillus fermentatus* Z-15 into MRS liquid medium (inoculation amount: 4.0%) and ferment at 37℃ under normal atmospheric conditions for 12 h to obtain *Lactobacillus fermentatus* fermentation broth. Centrifuge at 6000 rpm and 4℃ for 5 min to obtain *Lactobacillus fermentatus* Z-15 bacterial sludge. Then resuspend in physiological saline to make the concentration of *Lactobacillus fermentatus* Z-15 1.0 × 10⁻⁶. 9 CFU / mL yielded a suspension of fermenting *Lactobacillus mucinus*.

[0035] (3) Mix the plant lactobacillus suspension obtained in step (1) with the fermented mucus lactobacillus suspension obtained in step (2) at a mass ratio of 1:1 to obtain the probiotic composition.

[0036] Example 2

[0037] The ability of the probiotic composition prepared in Example 1 to scavenge free radicals was investigated, and the specific steps are as follows:

[0038] (1) Scavenging ability of DPPH free radicals. Centrifuge the probiotic composition prepared in Example 1 (3) at 4°C and 6000 rpm for 5 min, discard the supernatant, collect the bacterial cells, add 1×PBS buffer to resuspend, centrifuge again, discard the supernatant to collect the bacterial cells, weigh the fresh weight of the bacterial cells, and add 1×PBS buffer to resuspend the bacterial cells. Take 1 mL of bacterial suspension of different concentrations (10 7 10 8 10 9Add CFU / mL to a 2mL centrifuge tube, and add 0.2mL of DPPH dissolved in ethanol. Vortex until homogeneous, and incubate at 37°C in the dark for 30 minutes. Measure the absorbance at 517nm. Use 1×PBS buffer as a blank control. The experiment was repeated three times. This group was designated as the probiotic composition treatment group (STB9b+Z-15).

[0039] The group that used the *Lactobacillus plantarum* suspension prepared in Example 1 (1) and conducted experiments using the same method as described above is designated as the STB9b suspension treatment group (STB9b). The group that used the *Lactobacillus fermentans* suspension prepared in Example 1 (2) and conducted experiments using the same method as described above is designated as the Z-15 suspension treatment group (Z-15). The group that used the *Lactobacillus rhamnosus* LGG strain, which has a wide range of applications in antioxidants, and conducted experiments using the same method as described above is designated as the *Lactobacillus rhamnosus* LGG strain treatment group (LGG), serving as a positive control. The group that used the *Lactobacillus plantarum* STB1 strain, which had good antioxidant effects and was previously isolated in the inventor's laboratory, and conducted experiments using the same method as described above is designated as the *Lactobacillus plantarum* STB1 strain treatment group (STB1; for specific information on the *Lactobacillus plantarum* STB1 strain, please refer to Yang Rui's 2022 Master's thesis at Nanjing Agricultural University, entitled "Screening and Identification of Dominant Microorganisms in Guizhou Majiang White Sour Soup and Evaluation and Application of Their Probiotic Characteristics"), serving as a positive control.

[0040] The DPPH clearance rate is calculated using the following formula (I):

[0041] DPPH clearance rate (%) = [1-(A1-A2) / A0]×100 (Equation I).

[0042] In the formula, A1 is the absorbance of the DPPH solution and bacterial suspension after mixing; A2 is the absorbance of the 1×PBS buffer and bacterial suspension after mixing; and A0 is the absorbance of the DPPH solution and 1×PBS buffer after mixing.

[0043] (2) Scavenging ability of hydroxyl radicals.

[0044] The probiotic composition prepared in Example 1 (3) was centrifuged at 4°C and 6000 rpm for 5 min. The supernatant was discarded, and the bacterial cells were collected. The cells were resuspended in 1×PBS buffer, centrifuged again, and the supernatant was discarded. The fresh weight of the bacterial cells was weighed, and the cells were resuspended in 1×PBS buffer. The bacterial cell concentration was adjusted to 10. 7 10 8 10 9CFU / mL. Take 1 mL of bacterial suspension in a tube, add 1 mL of o-phenanthroline, 1 mL of 1×PBS buffer solution and 1 mL of distilled water, mix thoroughly, then add 1 mL of ferrous sulfate and 1 mL of H2O2. Incubate at 37℃ in the dark for 30 min, and measure the absorbance at 510 nm, recording it as A0. Use 1×PBS buffer as a blank control. Perform the experiment in triplicate.

[0045] The group that used the *Lactobacillus plantarum* suspension prepared in Example 1 (1) and conducted experiments using the same method as described above is designated as the STB9b suspension treatment group (STB9b). The group that used the *Lactobacillus fermentans* suspension prepared in Example 1 (2) and conducted experiments using the same method as described above is designated as the Z-15 suspension treatment group (Z-15). The group that used the *Lactobacillus rhamnosus* GG strain, which has a wide range of applications in antioxidants, and conducted experiments using the same method as described above is designated as the *Lactobacillus rhamnosus* GG strain treatment group (LGG), serving as a positive control. The group that used the *Lactobacillus plantarum* STB1 strain, which had good antioxidant effects and was previously isolated in the inventor's laboratory, and conducted experiments using the same method as described above is designated as the *Lactobacillus plantarum* STB1 strain treatment group (STB1; for specific information on the *Lactobacillus plantarum* STB1 strain, please refer to Yang Rui's 2022 Master's thesis at Nanjing Agricultural University, entitled "Screening and Identification of Dominant Microorganisms in Guizhou Majiang White Sour Soup and Evaluation and Application of Their Probiotic Characteristics"), serving as a positive control.

[0046] The hydroxyl radical scavenging rate is calculated using the following formula (II):

[0047] Hydroxyl radical scavenging rate (%) = [1-(A1-A0) / (A2-A0)]×100 (Formula II).

[0048] In the formula, A1 is the absorbance of the sample group where the test sample replaces 1×PBS buffer; A2 is the absorbance of the blank group where 1×PBS buffer replaces H2O2.

[0049] The results are as follows Figure 1 As shown, compared with *Lactobacillus plantarum* STB9b, *Lactobacillus fermentum* Z-15, and the positive control *Lactobacillus rhamnosus* GG and *Lactobacillus plantarum* STB1 suspension, the probiotic composition prepared in Example 1 has a higher free radical scavenging rate. The free radical scavenging effect it exerts is not significantly different from the recognized *Lactobacillus rhamnosus* GG, and is even slightly higher than the positive control.

[0050] Example 3

[0051] The ability of the probiotic composition prepared in Example 1 to repair oxidative damage to lactic acid bacteria DNA in vitro was investigated. The specific steps are as follows:

[0052] Centrifuge the fermentation broth at 4°C and 6000 rpm for 5 min, discard the supernatant, collect the bacterial cells, resuspend them in 1×PBS buffer, centrifuge again, discard the supernatant, collect the bacterial cells, weigh the fresh weight of the bacterial cells, resuspend the bacterial cells in 1×PBS buffer, sonicate at 400W for 30 min on ice, stopping for 2 seconds after each 1 second sonication, and then examine under a microscope to check for complete cell disruption. Obtain the bacterial lysate, centrifuge at 10000 rpm and 4°C for 5 min, and collect the supernatant. Supercoiled pUC19 plasmid DNA was used as the experimental material, and Fenton's reagent was used as the oxidative damage agent.

[0053] Mix 10 μL Tris-HCl buffer (100 Mm, pH 7.5), 10 μL pUC19 plasmid DNA (200 ng / μL), 20 μL of different samples, and 2 μL hydrogen peroxide (50 mM). Add 2 μL ferrous sulfate (5 mM) to the mixture to initiate the reaction. After 10 min, add 10 μL stop solution (8 M urea, 50% sucrose, 50 mM EDTA) to terminate the reaction. Electrophoresis of the reactants on a 1% agarose gel was performed. After electrophoresis, the agarose gel was stained with nucleic acid dyes and then imaged using a gel imaging system. 20 μL 1×PBS buffer was used as a negative control instead of the sample solution, and gallic acid samples of corresponding concentrations were used as positive controls.

[0054] The results are as follows Figure 2 As shown, *Lactobacillus plantarum* STB9b, *Lactobacillus fermentum* Z-15, the probiotic composition prepared in Example 1, and their corresponding bacterial lysates were tested. It was found that the lysate of the probiotic composition showed better protection against or repair of pUC19 plasmid DNA damage. Compared with the gallic acid positive control group, the band corresponding to the double-loop unopened band was brighter and accounted for a larger proportion, but it was far inferior to the blank control group (without any treatment). At the same time, the oxidative damage repair effect of the bacterial lysate was generally better, which may be due to the release of antioxidant enzymes in the bacteria by ultrasonic lysis, rather than the effect produced by the bacteria consuming free radicals themselves.

[0055] Example 4

[0056] The ability of the probiotic composition prepared in Example 1 to repair oxidative damage to lactic acid bacteria proteins in vitro was investigated. The specific steps are as follows:

[0057] Centrifuge the fermentation broth at 4℃ and 6000 rpm for 5 min, discard the supernatant, collect the bacterial cells, resuspend them in 1×PBS buffer, centrifuge again, discard the supernatant, collect the bacterial cells, weigh the fresh weight of the bacterial cells, and resuspend the bacterial cells in 1×PBS buffer.

[0058] First, preliminary experiments were conducted to determine the optimal working concentration of H2O2, finding a concentration that would cause significant but not excessive damage to bovine serum albumin (BSA). One BSA control tube and several experimental tubes with different H2O2 concentrations were set up. The system (total volume 100µL): Control tube: 40µL BSA (5mg / mL) + 60µL PBS. Experimental tubes: 40µL BSA (5mg / mL) + 20µL H2O2 of different concentrations + 40µL PBS, with final H2O2 concentrations of 5mM, 10mM, 20mM, and 50mM.

[0059] Both control and experimental tubes were incubated at 37°C for 2 hours. After incubation, both tubes were centrifuged at 10,000 rpm for 5 minutes at room temperature. Samples were then taken for SDS-PAGE electrophoresis to observe the bands. Selection criteria: The H2O2 concentration that reduced the brightness of the main BSA band (approximately 66 kDa) by about 50% and began to show slight tailing or polymerization / degradation bands was selected. Finally, an H2O2 concentration of 20 mM was determined to be used for subsequent formal experiments.

[0060] The total volume of the formal experimental system was 100 µL. The experimental treatment group consisted of 40 µL BSA (5 mg / mL) + 20 µL 20 mM H2O2 + 40 µL of 1.0 × 10⁻⁴ M H2O2. 9 CFU / mL bacterial suspension or the probiotic composition prepared in Example 1. Negative control group (completely undamaged): 40 µL BSA (5 mg / mL) and 60 µL PBS only. Damaged control group (negative control): 40 µL BSA (5 mg / mL) + 20 µL 20 mM H2O2 + 40 µL PBS. Positive control group: 40 µL BSA (5 mg / mL) + 20 µL 20 mM H2O2 + 40 µL 100 mM Vitamin C.

[0061] After gently mixing all samples, incubate in a 37°C water bath for 2 hours. After incubation, centrifuge the EP tubes at 10,000 rpm for 5 minutes at room temperature. Carefully aspirate 40 µL of the supernatant and add 10 µL of 5× SDS-PAGE loading buffer. Heat at 95°C for 5 minutes, then cool to room temperature before loading. Load 10 µL of sample into each electrophoresis lane. Electrophoresis conditions: 80V stacking gel, 100V separating gel. After electrophoresis, destain with rapid Coomassie blue staining solution and stain on a shaker. Then, perform imaging analysis using a gel imaging system.

[0062] The results are as follows Figure 3As shown, the probiotic composition prepared in Example 1 showed the best protective or repair effect on BSA protein, with the deepest protein band. Moreover, the protein band was deeper than that of the completely undamaged blank control group and the vitamin C positive control group, indicating that it had the best effect on repairing oxidative damage to BSA protein.

[0063] Example 5

[0064] This study investigated the ability of the probiotic composition prepared in Example 1 to alleviate alcohol-induced oxidative stress in vivo. Considering common daily life scenarios, the effects of *Lactobacillus plantarum* STB9b, *Lactobacillus fermentum* Z-15, and their composition on oxidative stress or antioxidant effects in mice were explored through alcohol-induced acute oxidative stress. The specific steps are as follows:

[0065] (1) Establish a safe and stable alcohol-induced oxidative stress model.

[0066] Nine-week-old male BALB / c mice weighing 25±2g were randomly divided into three groups (n=8 per group) and administered 4mg / g BW, 6mg / g BW, and 8mg / g BW, respectively (BW was purchased liquor with an alcohol content of 56% vol).

[0067] The righting reflex was used as the standard for determining the degree of intoxication: mice were placed on their backs with their abdomens and limbs facing upwards. If a mouse could not turn over within 30 seconds, it was considered to have lost its normal righting reflex. The point at which the righting reflex disappeared was defined as the intoxication point, and the time between the first drink and intoxication was defined as the alcohol tolerance time. The alcohol tolerance time of all three groups of mice was less than 20 minutes. To exclude the influence of individual differences, mice that did not lose the righting reflex within 1 hour were defined as not having acute intoxication. Considering the intoxication rate and safety, a dose of 4 mg / g BW was selected for subsequent experiments. The statistical results are shown in Table 1.

[0068] Table 1 Establishing an acute intoxication model

[0069] ;

[0070] (2) Animal experiment: Nine-week-old male BALB / c mice with a weight of 25±2g were randomly divided into five treatment groups (n=8 in each treatment group). The groups were randomly divided into blank control group, saline treatment negative control group, Lactobacillus plantarum STB9b bacterial solution group, Lactobacillus fermentum Z-15 bacterial solution group, and probiotic composition prepared in Example 1.

[0071] Mice were fasted for 3 hours before the experiment but allowed free access to water, followed by another 1 hour of fasting and water restriction. The blank control group received no treatment, while the negative control group received an equal volume (0.01 mL / g BW) of physiological saline followed by 2 hours of oral administration of baijiu (a type of Chinese liquor). Mice in the *Lactobacillus plantarum* STB9b culture group were administered 1×10⁶ bacteria via gavage. 9 cfu / mL (OD) 600 Two hours after administering a (0.01 mL / g BW) bacterial suspension (=1.0), the mice were then given baijiu (Chinese liquor); the mice in the fermented Lactobacillus mucinus Z-15 bacterial suspension group were given 1×10⁻⁶ bacteria via gavage each time. 9 cfu / mL (OD) 600 Two hours after administering a (0.01 mL / g BW) bacterial suspension (=1.0), the mice were then given baijiu (Chinese liquor); mice in the group receiving *Lactobacillus plantarum* STB9b bacterial suspension + *Lactobacillus fermentum* Z-15 bacterial suspension were given 1×10⁻⁶ bacteria per gavage. 9 cfu / mL (OD) 600 After 2 hours of adding (0.01 mL / g BW) bacterial suspension (=1.0), the liquor was then poured in.

[0072] The levels of superoxide dismutase (SOD), malondialdehyde (MDA), glutathione (GSH), and glutathione peroxidase (GSH-PX) in the livers of mice in each treatment group were detected using kits.

[0073] 3) Effects on the activities of superoxide dismutase (SOD), malondialdehyde (MDA), glutathione (GSH), and glutathione peroxidase (GSH-PX) in the liver of mice under alcohol-induced oxidative stress.

[0074] Blood was collected from the eyes of mice in each treatment group after enucleation. The mice were then euthanized by cervical dislocation, and dissected immediately. The livers were quickly removed, rinsed with pre-cooled physiological saline, and blotted dry with filter paper. A small amount of liver was cut off and a 10% liver homogenate was prepared using physiological saline as the homogenizing medium. The homogenate was centrifuged at 3000×g for 15 min and the supernatant was collected. The contents of superoxide dismutase (SOD), malondialdehyde (MDA), glutathione (GSH), glutathione peroxidase (GSH-PX), and total antioxidant capacity (T-AOC) in the mouse liver homogenate were determined according to the attached instructions using the kit.

[0075] The results are as follows Figure 4As shown, by measuring the activities of glutathione, glutathione peroxidase, superoxide dismutase, catalase, malondialdehyde, and total antioxidant enzymes in mouse liver, it was found that alcohol consumption induced strong oxidative stress in mice. These antioxidants also played a role in the process of consuming and converting ethanol and acetaldehyde. In the saline group and the blank control group, the antioxidant enzyme activities decreased or the antioxidant content increased significantly, indicating that the mice themselves resisted the oxidative stress caused by alcohol-induced ROS (reactive oxygen species). At the same time, the single strain and the strain combination treatment group showed that, compared with the blank treatment and the negative control group of saline, the strain treatment showed an effect of assisting the mice in coping with oxidative stress or antioxidation. The effect of the strain combination treatment group was significantly better than that of the single strain.

[0076] Therefore, the probiotic composition for alleviating acute oxidative stress provided by this invention has a synergistic effect, possessing a unique ability to scavenge free radicals and repair damage to biomolecules such as DNA and proteins caused by oxidative stress. It can significantly upregulate the levels of key endogenous antioxidants and the activity of antioxidant enzymes in the liver tissue of a mouse model of alcohol-induced acute oxidative stress, thereby strengthening the body's endogenous antioxidant defense system without any toxic side effects. It has a comprehensive effect combining "exogenous scavenging, damage repair, and endogenous enhancement," which is different from existing single-mode technologies. It provides a highly efficient and safe new biological approach for precisely alleviating acute oxidative stress, especially oxidative liver damage induced by alcohol consumption.

[0077] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and not to limit them. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can still be made to the technical solutions of the present invention, and these modifications or equivalent substitutions cannot cause the modified technical solutions to deviate from the spirit and scope of the technical solutions of the present invention.

Claims

1. A probiotic composition for alleviating acute oxidative stress, characterized in that: The probiotics are Lactiplantibacillus plantarum STB9b and Fermentum mucus lactobacillus Z-15; the Latin name of Lactiplantibacillus plantarum STB9b is Lactiplantibacillus plantarum , the accession number is CCTCC NO.M 20241444; the Latin name of Fermentum mucus lactobacillus Z-15 is Limosilactobacillus fermentum , the accession number is CCTCC NO.M 2024024; The ratio of viable counts of Lactiplantibacillus plantarum STB9b to Limosilactobacillus fermentum Z-15 in the probiotic composition is 1:

1. The method for preparing the probiotic composition comprises the following steps: Lactobacillus plantarum STB9b and Lactobacillus fermentum Z-15 were inoculated into MRS liquid medium at an inoculation rate of 2-6% of the medium volume. Fermentation was carried out at 37°C for 8-16 hours. The precipitates were then collected by centrifugation and resuspended in physiological saline to a confirmed bacterial concentration of 0.5 × 10⁻⁶. 9 -2×10 9 The concentration is CFU / mL, and then the bacterial suspensions of the two strains are mixed.

2. Use of a probiotic composition according to claim 1 for the preparation of a health food product for the alleviation of acute oxidative stress, characterized in that: The health food is used for relieving acute oxidative liver injury induced by alcohol.

3. Use of the probiotic composition for relieving acute oxidative stress according to claim 1 in the preparation of a drug for relieving acute oxidative liver injury induced by alcohol.

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