Lactobacillus paracasei for degrading acetaldehyde and application of lactobacillus paracasei

Abstract

The invention discloses casei paracasei capable of degrading acetaldehyde and application of the casei paracasei in ethanol or acetaldehyde related diseases. The invention provides a new lactobacillus paracasei strain A22032 screened and separated from long-life human intestinal tracts, the new lactobacillus paracasei strain A22032 has an excellent acetaldehyde degradation effect, the in-vivo acetaldehyde degradation rate reaches 69.23% and is obviously superior to that of the existing same strain, and a new scheme is provided for preventing and relieving ethanol or acetaldehyde related liver injury.

Description

Technical Field

[0001] This invention relates to the field of microbial technology, and in particular to a Lactobacillus paracasei that degrades acetaldehyde and its applications. Background Technology

[0002] Acetaldehyde (CH3CHO) is a highly reactive, toxic, and carcinogenic small molecule compound, classified as a Group 1 carcinogen by the International Agency for Research on Cancer (IARC) of the World Health Organization. The main sources of acetaldehyde in the human body include exogenous intake (such as alcoholic beverages, tobacco smoke, certain foods, and air pollution) and endogenous metabolism (with ethanol metabolism being the primary pathway). Ethanol metabolism mainly involves the oxidation of ethanol in the liver by alcohol dehydrogenase (ADH) to acetaldehyde, which is then metabolized into non-toxic acetic acid by aldehyde dehydrogenase (ALDH). The liver is the primary site of acetaldehyde metabolism and therefore the primary target organ for acetaldehyde toxicity.

[0003] The mechanisms by which acetaldehyde damages the liver involve multiple aspects: (1) Hepatotoxicity: Acetaldehyde has high chemical reactivity and can covalently bind with biomacromolecules such as proteins and DNA to form adducts, leading to the inactivation of key enzymes, damage to cell structure and DNA, directly causing hepatocyte inflammation, fatty degeneration and apoptosis. (2) Induction of oxidative stress: Acetaldehyde metabolism produces reactive oxygen species (ROS), which disrupt the balance of oxidation and antioxidant systems in the liver, attack the hepatocyte membrane, mitochondria and DNA double-strand structure, exacerbating inflammation and damage, and is a key link in the development of alcoholic liver disease (ALD). (3) Promotion of liver fibrosis: Long-term acetaldehyde exposure activates hepatic stellate cells (HSCs), which transform into myofibroblasts, synthesize and secrete large amounts of collagen fibers, leading to liver fibrosis, which may then develop into cirrhosis or even liver cancer. (4) Inhibition of mitochondrial function: Acetaldehyde damages the mitochondrial function of hepatocytes, affecting energy production and weakening the repair and regeneration capacity of hepatocytes. (5) ALDH2 gene defect: Approximately 30%-50% of East Asian populations carry loss-of-function mutations (ALDH2*2) in the aldehyde dehydrogenase 2 (ALDH2) gene, resulting in congenital insufficiency in acetaldehyde metabolism. After drinking alcohol, acetaldehyde accumulates, causing reactions such as facial flushing and palpitations (i.e., the "Asian red face" reaction), and significantly increasing the risk of liver damage and esophageal cancer.

[0004] Currently, the main preventive and interventional measures for acetaldehyde-related liver injury include: (1) abstinence from alcohol or alcohol restriction, but these are difficult to implement; (2) hepatoprotective drugs (such as silymarin and bicyclol), which are mainly used to repair damaged livers and have limited effect on blocking acetaldehyde absorption; and (3) supplementation with ALDH2 coenzyme (such as NAD+) or activators, which is still in the research stage, costly, and has uncertain in vivo effects. Therefore, it is crucial to develop new strategies to reduce acetaldehyde exposure from the source.

[0005] In recent years, microbial therapy has gained attention as an emerging health strategy. Certain probiotics can directly degrade exogenous or circulating acetaldehyde in the gut through their own enzyme systems (such as acetaldehyde dehydrogenase), reducing systemic acetaldehyde exposure levels and becoming a potential "enzyme replacement therapy." In existing technologies, probiotic strains have been reported to alleviate alcoholic liver injury. For example, *Lactobacillus rhamnosus* (CN116445360A) has antioxidant and intestinal epithelial cell barrier protective capabilities; *Lactobacillus plantarum* KLDS1.0344 combined with *Lactobacillus acidophilus* KLDS1.0901 (CN111437294A) can improve intestinal barrier and liver function and reduce inflammation levels; *Lactobacillus casei* grx12 (CN103893215A) can reduce blood lipids and the expression of liver inflammatory factors. However, these strains mainly target multiple aspects of alcoholic liver injury, and no existing technology has disclosed strains that can efficiently and specifically degrade acetaldehyde and directly prevent acetaldehyde-related liver injury.

[0006] Lactobacillus paracasei ( Lacticaseibacillus paracasei Lactobacillus paracasei is a common beneficial bacterium in the human gut, possessing probiotic properties such as immune regulation and gut health improvement; however, different strains exhibit significant functional differences. Currently, no Lactobacillus paracasei strains have been reported or applied to the efficient and specific degradation of acetaldehyde and the prevention of acetaldehyde-related liver injury. Summary of the Invention

[0007] This invention aims to at least solve one of the aforementioned technical problems existing in the prior art. Therefore, the object of this invention is to provide an acetaldehyde-degrading *Lactobacillus paracasei* strain and its application in the prevention of acetaldehyde-related diseases. This invention provides a novel *Lactobacillus paracasei* strain isolated from the intestinal flora of long-lived individuals, which exhibits excellent acetaldehyde degradation effects, achieving an in vivo acetaldehyde degradation rate of 69.23%, significantly superior to existing strains of the same species, providing a new approach for the prevention and mitigation of acetaldehyde-related liver injury.

[0008] In a first aspect, the present invention provides *Lactobacillus paracasei* having a 16S rDNA sequence as shown in SEQ ID NO: 1, or with at least 99.9% identity to the sequence shown in SEQ ID NO: 1.

[0009] In this invention, known computer algorithms can be used to determine the "identity" between two nucleic acid molecule sequences, such as the "FASTA" program, the GCG package, BLASTN, or FASTA. Commercially or publicly available programs can also include, for example, the DNAStar "MegAlign" program.

[0010] In this invention, the term "strain" should be understood as a single-celled microorganism capable of reproduction through cell division. Bacteria are classified by family, genus, and species. Each bacterial species contains multiple bacterial strains. The bacterial strain of this invention belongs to the species *Lactobacillus paracasei*. Therefore, "*Lactobacillus paracasei*" or "strain" in this invention refers not only to a specific bacterial strain, but also to all bacteria derived from or obtained from that strain, or corresponding to that bacterial strain and having the same metabolic function, such as at least one bacterium isolated from colonies derived from that strain.

[0011] In some embodiments of the present invention, the *Lactobacillus paracasei* is *Lactobacillus paracasei* (… Lacticaseibacillus paracasei Accession number A22032 is deposited at the Guangdong Provincial Microbial Culture Collection Center (GDMCC) on August 7, 2025, with accession number GDMCC No: 66822.

[0012] A second aspect of the invention provides a composition comprising the *Lactobacillus paracasei* described above, its culture, its metabolites, and / or products isolated from the *Lactobacillus paracasei*.

[0013] In some embodiments of the present invention, the *Lactobacillus paracasei* or its culture is selected from any of the following: (1) Live bacteria, attenuated bacteria, inactivated bacteria, or freeze-dried bacteria; (2) The fermentation broth of the Lactobacillus paracasei; (3) The supernatant of the fermentation broth of the Lactobacillus paracasei; (4) The inactivated fermentation broth of the aforementioned Lactobacillus paracasei; (5) The fermentation broth, fermentation broth supernatant and / or the concentrated or dried product of the inactivated fermentation broth in (2)-(4).

[0014] In this invention, "supernatant" or "supernatant" refers to the culture supernatant of a bacterial strain according to the invention, optionally containing compounds and / or cell debris of the strain, and / or metabolites and / or molecules secreted by the strain.

[0015] In some embodiments of the invention, the composition is provided in liquid or solid form.

[0016] In some embodiments of the present invention, the *Lactobacillus paracasei* in the composition is an attenuated bacterium, a killed bacterium, a freeze-dried bacterium, or an irradiated bacterium, for example, a heat-inactivated bacterium, such as pasteurized.

[0017] In some embodiments of the invention, the composition is in the form of a liquid, foam, cream, spray, powder (e.g., lyophilized powder), or gel.

[0018] In some embodiments of the present invention, the composition is in the form of powder, microencapsulated powder, capsule, tablet, lozenge, granule, oral liquid, suspension, emulsion, liquid formulation, sustained-release formulation, nanoformulation, or microencapsulated capsule.

[0019] In some embodiments of the invention, the composition further comprises one or more pharmaceutically acceptable carriers, excipients, or excipients. Such pharmaceutically acceptable excipients are well known to those skilled in the art.

[0020] In some embodiments of the present invention, the excipient may be at least one selected from carriers, excipients, diluents, lubricants, wetting agents, emulsifiers, suspension stabilizers, preservatives, sweeteners, and flavorings.

[0021] In some embodiments of the invention, the composition comprises one or more of the following: buffers (e.g., sodium bicarbonate, infant formula or sterile human milk or other agents that allow bacteria to survive and grow (e.g., to survive in the acidic environment of the stomach and to grow in the intestinal environment), lyophilization protectants, preservatives, stabilizers, binders, compactors, lubricants, dispersants, disintegrants, antioxidants, flavoring agents, sweeteners and colorants).

[0022] In some embodiments of the present invention, the composition further comprises one or more other active agents.

[0023] In some embodiments of the present invention, the other active agents may be one or more of probiotics and prebiotics, or a combination thereof.

[0024] In some embodiments of the present invention, the probiotics are selected from one or more of lactic acid bacteria, lactobacillus, lactococcus, butyric acid bacteria, bifidobacteria, thermophilic streptococcus, fecal streptococcus, and mesenteric streptococcus.

[0025] In some embodiments of the present invention, the probiotics are selected from at least one of the following: Bifidobacterium adolescentis, Bifidobacterium animalis, Bifidobacterium bifidum, Bifidobacterium breve, Bifidobacterium longum, Lactobacillus acidophilus, Lactobacillus curvatureii, Lactobacillus delbrueckii, Lactobacillus gasseri, Lactobacillus helveticus, Lactobacillus johnsonii, Lactobacillus mare's milk, Lactobacillus casei, Lactobacillus paracasei, Lactobacillus rhamnosus, Lactobacillus fermentum, Lactobacillus reuteri, Lactobacillus plantarum, Lactobacillus salivarius, Lactobacillus curvatureii, Lactobacillus sakei, Lactococcus lactis, and Lactococcus fatii.

[0026] In some embodiments of the present invention, the prebiotic is selected from inulin, mulberry leaf extract, berberine, Ganoderma lucidum, green coffee bean extract, oats, pectin, potato or its extract, citrus polyphenols, Ceylon cinnamon, chromium, ergothioneine, astaxanthin, quercetin, curcumin, proanthocyanidins, resistant dextrin, yeast β-glucan, ginseng or its extract, nutrient compounds, biotin, polydextrose, fructooligosaccharides (FOS), galactooligosaccharides (GOS), starch, cellulose, β-glucan, hemicellulose, lactulose, mannooligosaccharides, mannooligosaccharides (MOS), inulin rich in fructooligosaccharides, oligodextrose, tagatose, trans-galactooligosaccharides, pectin, resistant starch, xylooligosaccharides (XOS), and any combination thereof. In some embodiments, the other active agents may be antioxidants, such as vitamin C, vitamin E, vitamin A and their precursors (e.g., beta-carotene), tea polyphenols, anthocyanins, resveratrol, coenzyme Q10, ergothioneine, lipoic acid, selenium, zinc and any combination thereof.

[0027] In some embodiments of the invention, the composition may be formulated into a frozen composition, such as a frozen composition prepared by quick-freezing and drying, or freeze-drying, for storage and / or transportation.

[0028] In some embodiments of the invention, the composition may be used alone or in combination with a carrier such as a pharmaceutically acceptable carrier or a biocompatible scaffold.

[0029] In some embodiments of the invention, the composition is formulated for oral administration.

[0030] In some embodiments of the present invention, the composition is an enteric-coated formulation. In some embodiments of the present invention, The enteric-coated formulation is a dosage form with an enteric coating. For example, an enteric-coated formulation can be enteric granules, enteric tablets, or enteric capsules.

[0031] In some embodiments of the present invention, the composition is a capsule.

[0032] In some embodiments of the present invention, the capsule is a hard capsule or a soft capsule; or the capsule is a sustained-release capsule, a controlled-release capsule, or an enteric-coated capsule; or the capsule may be a microencapsulated capsule or a microcapsule.

[0033] In some embodiments of the present invention, the composition is any one of a drug, health product, or food.

[0034] In some embodiments of the present invention, the composition is an infant-suitable dosage form, a child-suitable dosage form, or an adult-suitable dosage form.

[0035] In some embodiments of the present invention, the composition is a gastrointestinal or non-gastrointestinal dosage form.

[0036] In some embodiments of the present invention, the health product or food is any one of a nutritional composition, candy, food bar, food additive, beverage additive, or dietary supplement.

[0037] A third aspect of the invention provides the use of the *Lactobacillus paracasei* and / or compositions described above in the preparation of food or health products.

[0038] In some embodiments of the present invention, the food or health product can help protect against chemically induced liver damage and / or provide antioxidant protection.

[0039] In some embodiments of the present invention, the chemically induced liver injury includes liver injury induced by ethanol or acetaldehyde.

[0040] In some embodiments of the present invention, the antioxidant includes preventing, improving or treating liver oxidative damage.

[0041] In some embodiments of the present invention, *Lactobacillus paracasei* is a legally permitted food ingredient. The Ministry of Health issued Announcement No. 20 of 2008, approving *Lactobacillus paracasei* as a new resource food. It has been on the "List of Microbial Strains that Can Be Used in Food" since 2010, and will be renamed *Lactobacillus paracasei* in 2025.

[0042] A fourth aspect of the invention provides the use of the *Lactobacillus paracasei* and / or compositions described above in the preparation of a pharmaceutical.

[0043] In some embodiments of the present invention, the drug is capable of: (1) Degrading ethanol and / or acetaldehyde in the body; (2) To prevent, improve or treat abnormal liver lipid metabolism; (3) To prevent, improve or treat liver injury induced by ethanol and / or acetaldehyde.

[0044] The application of the strains, inoculants, or their ferments or fermentation extracts of the present invention in the above-mentioned aspects also falls within the scope of protection of the present invention.

[0045] The beneficial effects of this invention are: This invention discovered a strain of Lactobacillus paracasei ( Lacticaseibacillus paracasei A22032 exhibits excellent acetaldehyde degradation, with an in vivo acetaldehyde degradation rate of 69.23%, significantly superior to existing strains of the same species. This provides a new approach for the prevention and relief of related diseases (such as ethanol or acetaldehyde-related liver injury). Attached Figure Description

[0046] Figure 1 This is a plate colony morphology diagram of strain A22032.

[0047] Figure 2 This is a Gram staining morphology diagram of strain A22032.

[0048] Figure 3 This is the result of the in vitro degradation rate of acetaldehyde by strain A22032.

[0049] Figure 4 This is the result of the survival ability test of strain A22032 in artificial gastrointestinal fluid.

[0050] Figure 5 The effect of strain A22032 on the ethanol content in the plasma of mice with ethanol or acetaldehyde-related liver injury.

[0051] Figure 6 The effect of strain A22032 on the acetaldehyde content in the plasma of mice with ethanol or acetaldehyde-related liver injury.

[0052] Figure 7 The effect of strain A22032 on TG content in the liver of mice with ethanol or acetaldehyde-related liver injury.

[0053] Figure 8 The effect of strain A22032 on TC content in the liver of mice with ethanol or acetaldehyde-related liver injury.

[0054] Figure 9 The study investigated the effect of strain A22032 on serum AST levels in mice with ethanol or acetaldehyde-related liver injury.

[0055] Figure 10 The effect of strain A22032 on serum ALT levels in mice with ethanol or acetaldehyde-related liver injury.

[0056] Figure 11 The effect of strain A22032 on serum LPS levels in mice with ethanol or acetaldehyde-related liver injury.

[0057] Figure 12 The effect of strain A22032 on the FITC-glucan content in the serum of mice with ethanol or acetaldehyde-related liver injury.

[0058] Figure 13 The study investigated the effect of strain A22032 on SOD levels in the livers of mice with ethanol or acetaldehyde-related liver injury.

[0059] Figure 14 The effect of strain A22032 on the 4-HNE content in the liver of mice with ethanol or acetaldehyde-related liver injury.

[0060] Figure 15The study investigated the effect of strain A22032 on MDA content in the livers of mice with ethanol or acetaldehyde-related liver injury. Detailed Implementation

[0061] The present invention will be further described in detail below through specific embodiments. Unless otherwise specified, the raw materials, reagents, or apparatus used in the embodiments and comparative examples are all available from conventional commercial sources or can be obtained by existing technical methods. Unless otherwise specified, the test or experimental methods are conventional methods in the art.

[0062] Experimental materials In the following examples, the MRS liquid culture medium was prepared as follows: Weigh 10 g glucose, 5 g beef extract, 5 g peptone, 2.5 g yeast extract, 1 g dipotassium hydrogen phosphate, 1 g triammonium citrate, 2.5 g anhydrous sodium acetate, 40 mL Tween-80, 0.29 g magnesium sulfate, 0.125 g manganese sulfate, and 500 mL distilled water. Adjust the pH to 6.2 ± 0.2, and autoclave at 121°C for 20 min. If a solid culture medium is required, add 6 g agar to the above liquid culture medium.

[0063] Example 1 This embodiment provides a method for the isolation, purification, and identification of strain A22032. The specific steps are as follows: Weigh 0.1 g of fecal sample from a healthy centenarian in Guangxi, add 0.9 mL of sterile saline, and vortex to mix. Dilute the sample suspension serially by 10-fold to obtain 10... -1 10 -2 10 -3 10 -4 10 -5 Sample suspensions at different dilution ratios. Take 0.1 mL of sample suspensions at different dilution ratios and spread them evenly on MRS solid medium. Incubate anaerobically at 37°C for 48 h.

[0064] Single colonies were picked, purified and cultured twice on MRS solid medium, and confirmed by microscopic examination after Gram staining.

[0065] The results are as follows Figure 1 and Figure 2 As shown.

[0066] It can be observed that strain A22032 is Gram-positive and presents as short rods. The bacteria are arranged singly, in pairs, or in short chains. The colonies are milky white, with a smooth and opaque surface, regular edges, and a diameter of approximately 1-2 mm, consistent with the morphological characteristics of *Lactobacillus paracasei*.

[0067] The obtained strain A22032 was further subjected to 16S rDNA amplification and sequencing by Sangon Biotech (Shanghai) Co., Ltd. The obtained 16S rDNA sequence is as follows:

[0068] The obtained 16S rDNA sequence was uploaded to the NCBI database for comparison. According to phylogenetic theory, strains with a similarity greater than 97% can be considered to be of the same species. The gene sequence of the target strain A22032 is similar to that of *Lactobacillus paracasei* (…). Lacticaseibacillus paracasei The homology was 99.86%. Therefore, strain A22032 was identified as Lactobacillus paracasei.

[0069] The obtained *Lactobacillus paracasei* A22032 was deposited at the Guangdong Provincial Microbial Culture Collection Center (GDMCC) on August 7, 2025, and its taxonomic name is... Lacticaseibacillus paracasei The address of the collection is 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, and the collection number is GDMCC No: 66822.

[0070] Example 2 In this embodiment, the acetaldehyde degradation capacity of Lactobacillus paracasei A22032 was determined by in vitro experiments.

[0071] The specific experimental method is as follows: After activating *Lactobacillus paracasei* A22032, centrifuge at 4°C and 10,000 rpm for 2 min, remove the supernatant, resuspend in PBS, and adjust the OD value to 0.6. Take 1 mL of the suspension, centrifuge again at 4°C and 10,000 rpm for 2 min, remove the supernatant, and obtain bacterial cells. Add 1.5 mL of 1 g / L MSM acetaldehyde medium (i.e., MSM medium containing 1 g / L acetaldehyde) to the bacterial cells and incubate for 24 h. Simultaneously, use 1.5 mL of sterile 1 g / L MSM acetaldehyde medium as a blank control.

[0072] Simultaneously, MSM standard solutions containing different concentrations of acetaldehyde (0, 100, 200, 400, 600, 800, 1000 mg / L) were prepared, and acetaldehyde standard curves were plotted. Specifically, 0.5 mL of the above-mentioned concentrations of acetaldehyde MSM medium, 1 mL of 4 mmol / L fructose aqueous solution, and 7 mL of 84 mmol / L resorcinol aqueous solution were respectively pipetted into test tubes, and MSM liquid medium was added to make up the total volume to 10 mL. The mixture was reacted in an 80°C water bath for 10 min, and then cooled in ice water. 200 μL of each solution was then pipetted into a 96 microplate, and the absorbance at OD555 nm was measured to plot the standard curve.

[0073] Centrifuge the culture (10,000 rpm, 10 min) to obtain the supernatant. Mix 20 μL of the supernatant with 20 μL of fructose, add 200 μL of resorcinol, incubate at 80°C for 10–30 min, cool in ice water, and then transfer 200 μL of each mixture to a 96 microplate. Measure the absorbance at OD555 nm, obtain the acetaldehyde content based on the acetaldehyde standard curve, and calculate the acetaldehyde degradation rate.

[0074] The results are as follows Figure 3 As shown.

[0075] The results showed that Lactobacillus paracasei A22032 achieved a 55% degradation rate of acetaldehyde in vitro, indicating that it has excellent in vitro degradation capabilities.

[0076] Example 3 In this embodiment, the resistance of Lactobacillus paracasei A22032 to gastric and intestinal fluids was tested.

[0077] The specific testing method is as follows: (1) Test for tolerance to gastrointestinal fluids: After activating *Lactobacillus paracasei* A22032 using MRS liquid medium, 1 mL of bacterial suspension was added to 9 mL of artificial gastric fluid (pH=3) and incubated at 37°C for 3 hours. 1 mL of supernatant was collected at 0 h, 1 h, and 3 h for plate counts. The viability was calculated using the following formula: Artificial gastric fluid survival rate (%) = ×100%.

[0078] Wherein, Nt represents the viable count of the strain after th hours of culture in artificial gastric fluid, and N0 represents the viable count of the strain at 0 hours.

[0079] (2) Test for tolerance to artificial intestinal fluid: Take 1 mL of the supernatant from the above steps, after culturing in artificial gastric fluid for 3 h, and add it to 9 mL of artificial intestinal fluid (pH=8.0). Incubate at 37℃ for 8 h. Collect the supernatant at 0 h, 2 h, 4 h, 6 h, and 8 h for plate count. Calculate the survival rate of the artificial intestinal fluid according to the above formula.

[0080] The results are as follows Figure 4 As shown.

[0081] It can be observed that Lactobacillus paracasei A22032 has a survival rate of 88% in artificial gastric fluid and 102% in artificial intestinal fluid, indicating that it has excellent intestinal colonization potential.

[0082] Example 4 In this embodiment, in vivo experiments were used to test the effects of Lactobacillus paracasei A22032 on in vivo ethanol or acetaldehyde metabolism and ethanol or acetaldehyde-related liver injury.

[0083] The specific steps are as follows: (1) Preparation of lyophilized Lactobacillus paracasei A22032 powder: Single colonies of *Lactobacillus paracasei* A22032 were picked up using a spreader, activated and transcultured twice in MRS solid medium, and then inoculated into 15 mL of MRS liquid medium. The culture was incubated anaerobically for 12 h to obtain seed culture 1. Seed culture 1 was then inoculated into fresh MRS liquid medium at an inoculation volume of 3% (150 mL) and incubated anaerobically for 20–24 h to obtain seed culture 2. Seed culture 2 was then inoculated into fresh MRS liquid medium at an inoculation volume of 3% (3 L) and incubated anaerobically for 20–24 h to obtain the scaled-up bacterial culture. The scaled-up bacterial culture was placed in an ice-water mixture (4°C) for 30 min, and centrifuged at 6500 rpm for 6 min at 4°C. The supernatant was discarded, and the bacterial cells were collected. The collected bacterial cells were washed twice with sterile physiological saline, and the saline was discarded to obtain A22032 bacterial cells. The obtained bacterial cells were added to 12% skim milk at a ratio of 1:1 (v / m) (12% skim milk: bacterial cell wet weight), stirred evenly, and then dispensed into 1.5 mL EP tubes. The tubes were then slowly frozen in a refrigerator (-20℃, 30 min; -80℃, 1 h). The frozen bacterial cells were then freeze-dried in a freeze dryer until they became a dry powder.

[0084] (2) Effects of Lactobacillus paracasei A22032 on acetaldehyde-related liver injury in animals: Thirty 6-week-old male C57BL / 6J mice were randomly selected and, after one week of acclimatization, were randomly divided into groups.

[0085] Thirty C57BL / 6J mice were stratified by body weight and randomly divided into three groups: normal group, model group and A22032 group, with 10 mice in each group. Except for the normal group, the other groups were modeled according to the following table.

[0086] Table 1 Treatment methods for each group of mice

[0087] On day 42, intestinal permeability of mice in each group was measured. After fasting for 6 hours, all mice were administered a single dose of 4 kDa FITC-glucan (concentration: 10 mg / 20 g) via gavage. Two hours after administration, blood was collected from the retroocular capillary plexus into heparinized tubes. The blood was centrifuged at 2000 × g for 5 minutes to obtain serum. The concentration of 4 kDa FITC-glucan in serum was measured using a fluorescence microplate reader (parameters Ex / Em = 490 / 520 nm), and the concentration of 4 kDa FITC-glucan in plasma samples was calculated based on the standard curve.

[0088] Standard curve: Set up 5 concentration gradients: 0, 0.1 μg / mL, 0.2 μg / mL, 0.3 μg / mL, 0.4 μg / mL, and 0.5 μg / mL, and perform measurements to create a standard curve.

[0089] On day 45, orbital blood was collected from mice in the model group and the A22032 group 5 hours after gavage with alcohol. Nine hours later, blood was collected from the heart after anesthesia, and the mice were euthanized by cervical dislocation. At the same time, the livers of mice in each group were collected.

[0090] Plasma was separated from blood samples collected 5 h later, and the ethanol content in the plasma was determined using a blood ethanol kit (Nanjing Jiancheng Bioengineering Institute, catalog number: E036-1-1).

[0091] The results are as follows Figure 5 As shown, the ethanol content in the plasma of the model group was significantly higher than that of the normal group. Compared with the model group, the ethanol content in the plasma of mice in the A22032 group was reduced by 20.61% (P<0.05).

[0092] Plasma was separated from blood samples collected 5 h ago, and the acetaldehyde content in the plasma was determined using an acetaldehyde kit (Megazyme, catalog number: K-ACHYD).

[0093] The results are as follows Figure 6 As shown, compared with the normal group, the acetaldehyde content in the plasma of the model group was significantly increased. Compared with the model group, the acetaldehyde content in the plasma of mice in the A22032 group was reduced by 69.23% (P<0.05).

[0094] This demonstrates that *Lactobacillus paracasei* A22032 has an excellent ability to metabolize ethanol and acetaldehyde in vivo.

[0095] The triglyceride (TG) content in the collected mouse liver samples was determined using the Tissue Cell Triglyceride (TG) Content Enzymatic Assay Kit (Pulley, Catalog No.: E1013).

[0096] The results are as follows Figure 7As shown, compared with the normal group, the TG content in the liver of the model group was significantly increased. Compared with the model group, the A22032 group significantly reduced the TG content in the liver of mice.

[0097] Collected mouse liver samples were analyzed using an enzymatic assay kit for total cholesterol (TC) content in tissue cells (Pulley, catalog number: E1015) to determine the total cholesterol (TC) content in the liver.

[0098] The results are as follows Figure 8 As shown, compared with the normal group, the TC content in the liver of the model group was significantly increased. Compared with the model group, the A22032 group significantly reduced the TC content in the liver of mice.

[0099] This demonstrates that Lactobacillus paracasei A22032 can significantly alleviate abnormal liver lipid metabolism caused by ethanol or acetaldehyde.

[0100] The serum samples collected from mice were tested for aspartate aminotransferase (AST) content using an aspartate aminotransferase (AST / GOT) test kit (Nanjing Jiancheng Bioengineering Institute, catalog number: C010-2-1).

[0101] The results are as follows Figure 9 As shown, compared with the normal group, the AST content in the model serum was slightly increased. Compared with the model group, the A22032 group reduced the AST content in mouse serum, although not significantly, but still close to that of the normal group overall.

[0102] Collected mouse serum samples were analyzed using an alanine aminotransferase (ALT / GPT) test kit (Nanjing Jiancheng Bioengineering Institute, catalog number: C009-2-1) to determine the alanine aminotransferase (ALT) content.

[0103] The results are as follows Figure 10 As shown in the figure, compared with the normal group, the serum ALT content in the model group was significantly increased. Compared with the model group, the A22032 group significantly reduced the serum ALT content in mice. This indicates that *Lactobacillus paracasei* A22032 can significantly improve liver injury associated with excessive accumulation of ethanol or acetaldehyde.

[0104] Collected mouse serum samples were used to determine the lipopolysaccharide (LPS) content in the serum using a mouse lipopolysaccharide (LPS) ELISA kit (Jianglai Biotechnology, catalog number: JL20691-96T).

[0105] The results are as follows Figure 11As shown in the figure, compared with the normal group, the LPS content in the serum of the model group was significantly increased. Compared with the model group, the A22032 group significantly reduced the LPS content in mouse serum. This indicates that *Lactobacillus paracasei* A22032 can reduce the endotoxin content in serum.

[0106] Mouse serum was collected from the orbital venous plexus, and the concentration of FITC-glucan in the serum was measured using a fluorescence microplate reader.

[0107] The results are as follows Figure 12 As shown, compared with the normal group, the FITC-glucan content in the serum of the model group was significantly increased. Compared with the model group, the A22032 group significantly decreased the FITC-glucan content in mouse serum. Figure 11 and Figure 12 This suggests that Lactobacillus paracasei A22032 can alleviate intestinal barrier damage caused by ethanol or acetaldehyde.

[0108] The superoxide dismutase (SOD) content in the collected mouse liver samples was determined using a superoxide dismutase (SOD) typing test kit (Nanjing Jiancheng Bioengineering Institute, catalog number: A001-2-2).

[0109] The results are as follows Figure 13 As shown, after A22032 intervention, the liver SOD activity in the intervention group was significantly lower than that in the model group (P<0.05), and then returned to a level that was not statistically different from that in the normal group (P>0.05). This indicates that A22032 intervention effectively alleviated the oxidative stress induced by the model, reduced the body's own antioxidant defense pressure, and thus restored the compensatory increase in SOD to normal physiological levels.

[0110] Collected mouse liver samples were used to determine the content of 4-hydroxynonenal (4-HNE) using a mouse 4-hydroxynonenal (4-HNE) ELISA kit (Jianglai Biotechnology, catalog number: JL20119-96T).

[0111] The results are as follows Figure 14 As shown, compared with the normal group, the 4-HNE content in the liver of the model group was significantly increased. Compared with the model group, the A22032 group significantly reduced the 4-HNE content in the liver of mice.

[0112] This demonstrates that the A22032 intervention not only alleviated the body's oxidative stress, eliminating the need for excessive compensation, but more importantly, it effectively reduced lipid peroxidation levels, thus providing direct protection for hepatocytes.

[0113] Collected mouse liver samples were used to determine the malondialdehyde (MDA) content in the liver using a malondialdehyde (MDA) assay kit (Nanjing Jiancheng Bioengineering Institute, catalog number: A003-1-2).

[0114] The results are as follows Figure 15 As shown, compared with the normal group, the MDA content in the liver of the model group was slightly increased. Compared with the model group, the A22032 group reduced the malondialdehyde (MDA) content in the liver of mice, although not significantly, but still close to that of the normal group overall.

[0115] In summary, A22032 intervention has a clear protective effect against liver injury induced by ethanol or acetaldehyde, and its mechanism may be related to improving oxidative stress.

[0116] 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. Paracaseicola casei, characterized in that, The Lactobacillus paracasei has a 16S rDNA sequence as set forth in SEQ ID NO: 1, or at least 99.9% identical to the sequence set forth in SEQ ID NO:

1.

2. The P. parapsilvii according to claim 1, characterized in that, The paracasei is Paracasei (Lactobacillus paracasei) Lactobacillus paracasei ) A22032, deposited in the Guangdong Microbial Culture Collection Center (GDMCC) on August 7, 2025, with the accession number GDMCC No: 66822.

3. A composition characterized in that, The composition contains the Lactobacillus paracasei, culture thereof, metabolite thereof, and / or product isolated from the Lactobacillus paracasei as set forth in claim 1 or 2.

4. The composition of claim 3, wherein, The Lactobacillus paracasei or culture thereof is selected from any one of the following: (1) live bacteria, attenuated bacteria, inactivated bacteria, or freeze-dried bacteria; (2) fermentation broth of the Lactobacillus paracasei; (3) supernatant of the fermentation broth of the Lactobacillus paracasei; (4) inactivated product of the fermentation broth of the Lactobacillus paracasei; (5) concentrated or dried product of the fermentation broth, the supernatant of the fermentation broth, and / or the inactivated product of the fermentation broth in (2)-(4).

5. The composition of claim 3, wherein The composition further comprises one or more pharmaceutically acceptable carriers, food carriers, excipients, and / or adjuvants.

6. The composition according to any one of claims 3-5, characterized in that, The composition is any one of a drug, a health product, or a food product; Preferably, the health product or the food product is any one of a nutritional composition, a candy, a food bar, a food additive, a drink additive, a dietary supplement.

7. Use of the Lactobacillus paracasei as set forth in any one of claims 1-2 and / or the composition as set forth in any one of claims 3-6 in the preparation of a food product or a health product.

8. Use according to claim 7, characterized in that, The food product or the health product is capable of assisting in the protection against chemical liver injury and / or antioxidation; Preferably, the chemical liver injury comprises ethanol or acetaldehyde-induced liver injury; Preferably, the antioxidation comprises prevention, improvement, or treatment of oxidative damage to the liver.

9. Use of the Lactobacillus paracasei as set forth in any one of claims 1-2 and / or the composition as set forth in any one of claims 3-6 in the preparation of a drug.

10. Use according to claim 9, characterized in that, The drug is capable of: (1) degrading ethanol and / or acetaldehyde in vivo; (2) preventing, improving, or treating abnormal lipid metabolism in the liver; (3) preventing, improving, or treating ethanol and / or acetaldehyde-induced liver injury.

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