Composition for protecting liver and reducing lipid and application thereof

Abstract

The invention provides a probiotic compound preparation with double effects of short-term hangover alleviating and long-term liver protection. The silybum marianum and hovenia dulcis thunb compound is fermented through the bifidobacterium animalis subsp. Animalis HX-BA21, and the concentration of the anti-inflammatory protein P75, the dihydromyricetin and the silybin which serve as core functional components is remarkably increased; and further compounding with corn oligopeptide, glutathione yeast and turmeric to construct a multi-channel synergistic system of anti-inflammation, metabolism acceleration, oxidation resistance, hepatocyte repair and lipid metabolism regulation. On one hand, alcohol metabolism is accelerated, the activity of alcohol dehydrogenase (ADH) and acetaldehyde dehydrogenase (ALDH) is improved, and rapid alcohol effect dispelling is achieved; on the other hand, the antioxidant capacity is enhanced (SOD and GSH-Px are remarkably improved, and MDA is reduced), intestinal tract-derived inflammation signals are synergistically inhibited (IL-6 and TNF-alpha are reduced), hepatocytes are synergistically repaired, and lipid metabolism is regulated (TC and TG of the liver and ALT and AST of serum are reduced) through P75 protein, so that long-term protection and hepatocyte repair of alcoholic liver injury are realized while the acute drunkenness symptom is relieved. The method has the advantages of high efficiency, safety and multi-target intervention, and is obviously superior to a single component or a simple physical mixture.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of bacterial fermentation, and particularly relates to a liver-protecting and lipid-lowering composition and application thereof. BACKGROUND

[0002] Alcoholic fatty liver (AFL) as an early stage of alcoholic liver disease is an important risk factor for developing hepatitis, liver fibrosis, liver cirrhosis and even liver cancer. Currently, AFL has become a major global public health problem. The pathogenesis of AFL is complex, mainly involving the direct toxic effects of ethanol and its metabolites (such as acetaldehyde) on hepatocytes, the impaired intestinal barrier function and endotoxin translocation caused by intestinal flora disorder, the dysregulated lipid metabolism (increased fatty acid intake / synthesis and decreased oxidation / output) in the liver, and the resulting oxidative stress and inflammatory response.

[0003] Currently, the primary and most effective method for the treatment of AFL in clinical practice is alcohol abstinence. However, due to the complexity of alcohol dependence, long-term abstinence compliance is poor, making this approach face great challenges in practical application. Drug therapy is bottlenecked, and existing drug treatments (such as liver-protecting drugs, antioxidants, etc.) mainly focus on relieving symptoms, with single target, and often have problems such as uncertain efficacy, obvious side effects, or questionable long-term safety, etc. There is a lack of ideal drugs that can intervene in the pathological process of AFL from multiple targets. There are some health products or functional foods on the market for assisting liver protection, such as products containing silymarin, curcumin, etc. However, these products generally have low bioavailability, relatively single target, slow onset, and other shortcomings, making it difficult to effectively reverse liver steatosis.

[0004] The invention patent with publication number CN103027231B “Compound probiotic fermented Chinese herbal medicine active health care liquid and preparation method thereof” utilizes compound probiotics Bacillus natto, Saccharomyces cerevisiae, Lactobacillus, Acidaminococcus, and Bifidobacterium to ferment Ginkgo biloba leaves, honeysuckle powder, etc. After fermentation, it has the functions of preventing and treating “three highs”, anti-tumor, anti-cancer, enhancing memory, protecting liver, improving immunity, preventing and treating coronary heart disease, etc. It also has the functions of regulating gastrointestinal balance, anti-aging, prolonging life, and aiding digestion, etc. It mainly combines the medical viewpoints of prevention, health care, treatment, and rehabilitation.

[0005] The invention patent with publication number CN104940240B “Probiotic preparation for alcoholism and liver protection, preparation method and application thereof” is made by mixing Lactobacillus bulgaricus, Bifidobacterium infantis, Bacillus, and Lactobacillus acidophilus, and fermenting the mixed bacteria at a constant temperature according to a certain proportion. The prepared mixed bacteria can improve the alcoholism speed, reduce the discomfort of “hangover” caused by excessive drinking; and long-term use can protect the liver, eliminate free radicals, reduce liver cell damage, enhance the body's immunity, regulate the balance of intestinal flora, and eliminate the gastrointestinal function disorder caused by alcoholism.

[0006] The invention patent with publication number CN 119950672 A discloses a formula for an alcoholism-relieving medicine and a production method thereof. The alcoholism-relieving medicine uses pure natural Chinese medicinal concentrated powder such as jujube fruit and radix puerariae, and is combined with nutritional ingredients such as corn peptides and gamma-aminobutyric acid. Through the synergistic effect of multiple components, the alcohol metabolism is effectively promoted, the symptoms of drunkenness are relieved, and the liver is protected. The formula is all-natural and does not contain chemical additives. It ensures the alcoholism-relieving effect while considering safety and taste, and does not cause additional burden to the body.

[0007] The invention patent with publication number CN 120459247 A provides a preparation method and application of a liver-protecting and alcoholism-relieving traditional Chinese medicine fermentation product. The fermentation product is prepared by fermenting traditional Chinese medicine compositions such as pueraria flower, jujube fruit, and amomum villosum using Monascus purpureus 7-3. The fermentation product strengthens the effects of traditional Chinese medicine on relieving alcoholism and protecting the liver through microbial transformation, can quickly relieve the symptoms of drunkenness and protect the liver, and has the characteristics of simple preparation process and significant economic benefits.

[0008] Many medicinal and edible traditional Chinese medicinal materials (such as hawthorn, radix puerariae, medlar, cassia seed, and gardenia) have been confirmed by modern pharmacology to have the activities of protecting the liver, reducing lipids, and resisting inflammation and oxidation. However, the effective components in these raw materials are often wrapped by cell walls or not easily absorbed directly due to large molecular weight, resulting in low bioavailability and limiting the full play of their efficacy. In recent years, the concept of gut-liver axis has been increasingly recognized, and the role of probiotics in the development of AFL has become increasingly clear. Supplementing probiotics can indirectly improve liver health through regulating intestinal microecological balance, strengthening intestinal barrier, reducing endotoxin into blood, and regulating bile acid metabolism. However, single probiotic preparations often have limited effect on complex AFL pathological networks.

[0009] Therefore, there is an urgent need in the art for an innovative solution that can integrate the regulation of intestinal flora and the dual advantages of direct liver protection and lipid reduction, and overcome the poor absorption of medicinal and edible components. Applying specific probiotic fermentation technology to carefully formulated medicinal and edible compositions is expected to have synergistic effects and produce new therapeutic effects, but there is currently no related report.

[0010] The present invention aims to pre-digest and release medicinal and edible effective substances through specific probiotic fermentation, thereby improving bioavailability; and to achieve liver protection and lipid reduction by synergistically exerting the effects of probiotic metabolites such as P75 protein and medicinal and edible components, thereby achieving short-term alcoholism relief and root intervention for AFL, which is significantly better than simple physical mixtures in terms of efficacy and safety. SUMMARY

[0011] The first object of the present application is to provide a multi-target fermentation complex with high efficiency of alcoholism relieving and systematic liver protection function, which comprises specific anti-inflammatory protein P75 and synergizes with active ingredients such as dihydro myricetin and silybin to construct a synergistic network of anti-inflammatory, antioxidant and lipid metabolism regulation.

[0012] The second object of the present application is to provide a functional composition comprising the fermentation complex and corn oligopeptide, glutathione yeast and curcuma, forming an integrated formula of "microbial fermentation-functional peptide-anti-inflammatory and antioxidant synergism".

[0013] The third object of the present application is to provide a preparation method of the fermentation complex.

[0014] The fourth object of the present application is to provide an application of the fermentation complex, which is used for preparing a food or drug for reducing ALT / AST, regulating TC / TG, inhibiting inflammatory factors such as IL-6 and TNF, and improving SOD and GSH-Px activities. The food or drug can achieve the effects of alcoholism relieving and liver protection and lipid reduction at the cellular and animal levels.

[0015] The present application is realized by the following technical solutions:

[0016] A fermentation complex with liver protection and lipid reduction function is obtained by fermenting silybum and jujube seed with animal bifidobacterium animalis subsp HX-BA21.

[0017] The weight ratio of the silybum and jujube seed is 1:1-1:2;

[0018] The viable count of animal bifidobacterium animalis subsp HX-BA21 in the fermentation complex is 1.0-3.5 x 10 8 CFU / mL.

[0019] The content of P75 protein in the fermentation complex is not less than 30 ng / mL, the content of dihydro myricetin is not less than 35 mg / L, and the content of silybin is not less than 35 mg / L.

[0020] The preparation method of the fermentation complex comprises the following steps:

[0021] The raw materials of jujube seed and silybum are crushed and sterilized, and then added to a fermentation medium;

[0022] Animal bifidobacterium animalis subsp HX-BA21 is inoculated into the fermentation medium and subjected to fermentation culture at 37 DEG C for 24 hours;

[0023] The fermentation broth is centrifuged, and the supernatant is taken to obtain the fermentation complex.

[0024] The fermentation medium consisted of: 10.0 g casein digest, 2.0 g triammonium citrate, 10.0 g beef extract, 0.2 g magnesium sulfate, 4.0 g yeast extract, 0.05 g manganese sulfate, 5.0 g sodium acetate, 2.0 g dipotassium hydrogen phosphate, 10.0 g glucose, 1.08 g Tween-80, and 8 g a combination of Hovenia dulcis and milk thistle.

[0025] The pH value of the fermentation medium is 5.8.

[0026] A liver-protecting and lipid-lowering composition, by weight, comprises 5-15 parts of the fermentation complex, 1-4 parts of corn oligopeptide, 1-4 parts of glutathione yeast, and 0.5-2.5 parts of turmeric.

[0027] A method for preparing the liver-protecting and lipid-lowering composition includes the step of mixing and drying the fermentation complex with corn oligopeptides, glutathione yeast, and turmeric.

[0028] The use of the composition in the preparation of food or medicine for the prevention and / or treatment of alcoholic liver injury.

[0029] The alcoholic liver injury mentioned above includes alcoholic fatty liver and alcoholic hepatitis.

[0030] The composition is used in the preparation of food or pharmaceuticals that enhance the activity of hepatic alcohol dehydrogenase and acetaldehyde dehydrogenase; or

[0031] Used in the preparation of food or pharmaceuticals that lower serum ALT and AST levels; or

[0032] It can be used in the preparation of foods or medicines that lower triglyceride and total cholesterol levels in the liver; or

[0033] It can be used to prepare food or medicines that enhance the activity of superoxide dismutase and glutathione peroxidase in the liver; or to prepare food or medicines that reduce the content of interleukin-6 and tumor necrosis factor-α in the liver. Detailed Implementation

[0034] The present invention will be further described in detail below with reference to specific embodiments. The following embodiments are not intended to limit the present invention, but only to illustrate the present invention. Unless otherwise specified, the experimental methods used in the following embodiments, and the experimental methods for which specific conditions are not specified in the embodiments, are generally performed under conventional conditions. The materials and reagents used in the embodiments are commercially available unless otherwise specified.

[0035] This invention relates to Bifidobacterium animalis subspecies HX-BA21, obtained from the feces of a two-month-old infant. This bacterium was deposited on December 6, 2024, at the China General Microbiological Culture Collection Center (CGMCC), with accession number CGMCC No. 32958. This bacterium is disclosed in Chinese invention patent application CN120682990A, entitled "Probiotic fermentation complex with cell repair and cell aging delaying effects and its application therein".

[0036] Example 1

[0037] For in vitro screening of strains, selected strains from the laboratory were activated on solid MRS medium and then enriched on MRS liquid medium. The strains were evaluated by measuring their free radical scavenging rate, SOD antioxidant enzyme activity, anti-lipid peroxidation capacity, and ability to produce alcohol dehydrogenase and acetaldehyde dehydrogenase. The selected strains included N63M6, P19M2, P19M3, BS686M3, 5M8, 24-55(HX-BA21), 24-58M13, 24-51, 24-53, 24-57, 24-33M1, and 24-33M2.

[0038] 1.1 Evaluation of Free Radical Scavenging Rate

[0039] DPPH, as a stable free radical, is often used to rapidly and quantitatively assess the total antioxidant capacity of samples, directly reflecting its effectiveness in scavenging free radicals. Therefore, the DPPH free radical scavenging rate of the strain was determined.

[0040] Table 1. DPPH free radical scavenging rate (%) of different strains

[0041] Strain Elimination rate (%) Strain Elimination rate (%) Strain Elimination rate (%) N63M6 62.54 5M8 93.21 24-53 97.21 P19M2 65.23 24-55(HX-BA21) 95.16 24-57 97.96 P19M3 73.21 24-58M13 77.85 24-33M1 71.48 BS686M3 98.62 24-51 61.29 24-33M2 59.88

[0042] As shown in Table 1, all 12 strains of bacteria have a certain ability to scavenge free radicals. Among them, strains 24-53, 24-57, 5M8, 24-55 (HX-BA21), and BS686M3 showed excellent performance and were selected for further testing.

[0043] 1.2 SOD Antioxidant Enzyme Activity

[0044] Superoxide dismutase (SOD) is a core endogenous enzyme in the body's fight against oxidative stress, responsible for catalyzing the dismutation reaction of superoxide anion free radicals. Measuring a specific activity can assess the product's key ability to enhance the cell's own defense system. Studies have shown that most probiotics possess SOD activity. Five initially screened probiotic strains were cultured in MRS liquid medium at 37°C for 24 hours, and the cell concentration was adjusted to 1.0 × 10⁻⁶ after resuspending. 9 The SOD activity of the bacteria was determined using the CFU / mL reference SOD activity assay kit (Beijing Solarbio Science & Technology Co., Ltd.), and the results are shown in Table 2.

[0045] Table 2 SOD activity of different strains

[0046] Strain Bacterial strain name SOD activity (U / mL) 24-53 Bifidobacterium longum subsp. longum 32.01 24-57 Bifidobacterium animalis subsp. lactis 52.87 5M8 Pediococcus pentosaceus 39.39 24-55(HX-BA21) Bifidobacterium animalis subsp. animalis 51.35 BS686M3 Lactobacillus salivarius 49.27

[0047] As shown in Table 2, all strains exhibited certain SOD antioxidant activity, with strains 24-57, 24-55 (HX-BA21) and BS686M3 showing excellent performance. These strains were selected for further experiments.

[0048] 1.3 Anti-lipid peroxidation capacity

[0049] The lipid peroxidation resistance capacity was assessed using linoleic acid as the unsaturated fatty acid, and the inhibition rate of lipid peroxidation by the strain against linoleic acid was determined. The end products of lipid peroxidation (such as malondialdehyde (MDA)) can damage proteins and nucleic acids in the body, leading to oxidative damage. This indicator is usually determined using the thiobarbituric acid method; the specific results are shown in Table 3.

[0050] Table 3. Anti-lipid peroxidation capacity of different strains

[0051] Strain Bacterial strain name Lipid peroxidation inhibition rate (%) 24-57 Bifidobacterium animalis subsp. lactis 58.21 24-55(HX-BA21) Bifidobacterium animalis subsp. animalis 63.73 BS686M3 Lactobacillus salivarius 59.66

[0052] By selecting indicators such as DPPH free radical scavenging, anti-lipid peroxidation, and SOD activity, we can indirectly verify that probiotics have a strong comprehensive antioxidant capacity, which can directly combat alcohol-induced oxidative stress and protect liver cell membranes and mitochondria from damage.

[0053] 1.4 Initial screening for P75 protein production

[0054] To screen for strains with anti-inflammatory potential, this invention quantitatively analyzed the P75 protein fermentation product of three candidate strains. The P75 protein was measured using an ELISA kit, with a known concentration of recombinant P75 protein as the standard curve. The concentration of P75 protein in the sample (ng / mL) was calculated based on the standard curve. The results are shown in Table 4.

[0055] Table 4. Concentration of P75 protein in fermentation supernatant of different strains

[0056] Strain Bacterial strain name P75 protein concentration ng / mL 24-57 Bifidobacterium animalis subsp. lactis 13.21 24-55(HX-BA21) Bifidobacterium animalis subsp. animalis 18.21 BS686M3 Lactobacillus salivarius 12.57

[0057] As shown in Table 4, strain HX-BA21 has a higher ability to produce P75 protein than the other two strains.

[0058] 1.5 Ability to produce alcohol dehydrogenase and acetaldehyde dehydrogenase

[0059] The determination of the enzyme production capacity of alcohol dehydrogenase (ADH) and aldehyde dehydrogenase (ALDH) aims to confirm that the strain can efficiently promote the breakdown of alcohol and its toxic metabolite acetaldehyde in the body, thereby reducing the metabolic burden on the liver from the source.

[0060] The three strains were tested according to the assay methods of the alcohol dehydrogenase kit (Soluble) and the acetaldehyde dehydrogenase kit (Soluble). Under the same conditions, the results are shown in Table 5.

[0061] Table 5. Activities of alcohol dehydrogenase and aldehyde dehydrogenase in different strains

[0062] Strain Bacterial strain name ADH (U / 106 cell) ALDH (U / 106 cell) 24-57 Bifidobacterium animalis subsp. lactis 14.21 29.21 24-55(HX-BA21) Bifidobacterium animalis subsp. animalis 18.31 37.69 BS686M3 Lactobacillus salivarius 10.96 9.01

[0063] As shown in Table 5, the activities of alcohol dehydrogenase and acetaldehyde dehydrogenase in Bifidobacterium animalis subspecies HX-BA21 are significantly higher than those of other strains, making it the optimal strain for hangover relief and liver protection products. Its high enzyme activity indicates that this strain can more effectively help the body metabolize alcohol and eliminate toxic acetaldehyde, thereby reducing alcohol damage to the liver at its source. Therefore, this strain was selected for subsequent experiments.

[0064] Example 2 Screening of Fermentation Raw Materials

[0065] To screen for food-medicine homologous materials that can alleviate alcoholic fatty liver through probiotic fermentation and synergistic effects, the initial screening of food-medicine homologous substances that have been proven to have activities such as alcohol detoxification, liver protection, lipid reduction, or antioxidant activity includes: kudzu root, Japanese raisin tree fruit, wolfberry fruit, licorice root, cassia seed, hawthorn, polygonatum rhizome, poria cocos, sea buckthorn, chrysanthemum, turmeric, jujube, and milk thistle.

[0066] To evaluate the synergistic growth effect of HX-BA21 with plant materials, 2% (w / v) of the material was added to MRS liquid medium and sterilized at 121℃ for 15 min. HX-BA21 was then inoculated into the medium containing the material, while an uninoculated medium containing the material served as a blank control group. Using the blank medium as a substrate, the OD difference (ΔOD600nm) after 24 hours of culture was calculated to compare the synergistic growth effect of different materials with HX-BA21.

[0067] Table 6. Synergistic effect of different raw materials on the growth of HX-BA21 (ΔOD600nm)

[0068]

[0069]

[0070] Table 6 shows that kudzu root, Japanese raisin tree fruit, hawthorn, turmeric, and milk thistle can all promote the growth of HX-BA21, providing a key basis for subsequent compounding or fermentation. Selecting Japanese raisin tree fruit and milk thistle, which have particularly prominent promoting effects, as fermentation substrates not only helps improve the growth of probiotics but may also enhance the synergistic potential of the final product in terms of hangover relief, liver protection, and lipid reduction through the metabolic transformation of active ingredients in the raw materials by the strains.

[0071] Example 3: Fermentation of Hovenia dulcis seeds and Milk thistle and preparation of probiotic formulations

[0072] To further enhance the bioavailability of the food-medicine homology components and the synergistic effect of probiotic metabolites, this embodiment uses Bifidobacterium animalis subspecies HX-BA21 to ferment a combination of Hovenia dulcis and Milk thistle to prepare a highly active fermented product. The specific steps are as follows:

[0073] 3.1 Culture medium preparation

[0074] Preparation of the combination of Hovenia dulcis and milk thistle: Weigh Hovenia dulcis powder and milk thistle powder in a mass ratio of 1:1 and mix them evenly.

[0075] According to the culture medium formula, add 10.0g casein digest, 2.0g triammonium citrate, 10.0g beef extract powder, 0.2g magnesium sulfate, 4.0g yeast extract powder, 0.05g manganese sulfate, 5.0g sodium acetate, 2.0g dipotassium hydrogen phosphate, 10.0g glucose, 1.08g Tween-80, and 5-20g of a combination of Japanese raisin tree seed and milk thistle. Add water to a final volume of 1L and adjust the pH to 5.8.

[0076] The composition was added at amounts of 5g, 8g, 11g, 14g, and 17g. After the bacterial suspension was cultured at 37℃ for 24h, the absorbance of the bacterial suspension at OD600 was measured. At the same time, a blank control group containing raw material culture medium without inoculation was set up. Using the blank culture medium as the base, the OD difference (ΔOD600nm) after 24 hours of culture was calculated and compared. The results are shown in Table 7. When the addition amount was 8g, the OD value was the highest and the number of viable bacteria in the bacterial suspension was the highest.

[0077] Table 7. Changes in fermentation results of different amounts of Hovenia dulcis and Milk thistle combinations.

[0078] Composition addition amount Delta OD 600 nm Bacterial liquid number of viable bacteria CFU / mL 5g 1.125 1.2 x 10 10 ]]> 8g 1.376 3.5 x 10 10 ]] 11g 1.065 2.9 x 10 9 ]] 14g 0.901 1.5 x 10 9 ]]> 17g 0.900 1.0 x 10 9 ]]>

[0079] 3.2 Preparation and Detection of Probiotic Formulations

[0080] After streaking the probiotics, single bacteria were picked and transferred to liquid MRS. After incubation at 37°C for 24 hours, an inoculum of 3% (v / v) of the activated probiotics was prepared, with an inoculum concentration of not less than 2 × 10⁻⁶. 8CFU / mL was added to a fermentation medium cooled to room temperature (10.0g casein digest, 2.0g triammonium citrate, 10.0g beef extract, 0.2g magnesium sulfate, 4.0g yeast extract, 0.05g manganese sulfate, 5.0g sodium acetate, 2.0g dipotassium hydrogen phosphate, 10.0g glucose, 1.08g Tween-80, 8g of a combination of Japanese raisin tree fruit and milk thistle, water was brought to a final volume of 1L, pH was adjusted to 5.8, and the mixture was sterilized at 121℃ for 20 minutes). Fermentation was carried out at 37℃ for 24 hours, followed by centrifugation at 3000 rpm and sterilization at 100℃ for 30 minutes to obtain the fermentation broth. Two parts corn oligopeptide, two parts glutathione yeast, and one part turmeric were added to 10 parts of the fermentation broth, and the mixture was dried to obtain the probiotic preparation.

[0081] Among them, the P75 protein can directly act on intestinal epithelial cells, powerfully inhibiting the production of pro-inflammatory factors at the source, thereby directly reducing the inflammatory storm suffered by the liver. By reducing intestinal inflammation, the P75 protein helps repair the intestinal barrier function and reduces the entry of enterogenic harmful substances such as endotoxins (LPS) into the blood, thereby indirectly blocking the continuous immune attack on the liver, which is crucial for controlling the progression of hepatitis.

[0082] Hovenia dulcis seeds, through components such as dihydromyricetin, significantly enhance the activity of hepatic alcohol dehydrogenase and aldehyde dehydrogenase, thereby powerfully promoting alcohol breakdown and reducing toxins at the source. Milk thistle, through components such as silymarin, can combat free radicals generated by alcohol metabolism, directly protecting liver cells, providing anti-inflammatory and antioxidant effects, and repairing damage. Besides their inherent hepatoprotective effects, their primary function in this system is as "inducers" of P75 protein, jointly creating an optimal fermentation environment to maximize P75 protein yield.

[0083] Corn oligopeptides are rich in branched-chain amino acids and antioxidant peptides, which can be directly absorbed and utilized to promote the synthesis of proteins in liver cells. Based on the systemic reduction of inflammation levels by P75 protein, they provide repair raw materials for liver cells, achieving a synergistic effect of "anti-inflammation" and "repair".

[0084] Glutathione yeast and turmeric form an "antioxidant / anti-inflammatory enhancement team." Glutathione yeast is one of the most important endogenous antioxidants in the human body, a core substance for scavenging free radicals and neutralizing acetaldehyde toxicity. Turmeric is a recognized natural antioxidant that can effectively inhibit alcohol-induced liver inflammation signaling pathways and reduce inflammatory damage to liver tissue. Together, they enhance the anti-inflammatory effect of the P75 protein, forming a second line of defense against inflammation in the liver. This system achieves a comprehensive approach, from rapid hangover relief to long-term liver protection, through the synergistic effect of multiple pathways including anti-inflammation, accelerated metabolism, antioxidation, and liver cell repair.

[0085] Comparative Example 1:

[0086] The difference from Example 3 is that a water extraction process is used. 4g each of Japanese raisin tree fruit and milk thistle are weighed, added to 1L of distilled water, boiled for 20 minutes, soaked for 24 hours, and then centrifuged.

[0087] Comparative Example 2:

[0088] The difference from Example 3 is that commercially available Lactobacillus rhamnosus GG of equal concentration was used for fermentation.

[0089] Comparative Example 3:

[0090] The difference from Example 3 is that fermentation was carried out using a commercially available, equal concentration of commercially available Bifidobacterium animalis subsp. ATCC 27536. Comparative Example 4:

[0091] The difference from Example 3 is that the combination of Hovenia dulcis seeds and milk thistle was replaced with 8g of Hovenia dulcis seeds during fermentation. Comparative Example 5:

[0092] The difference from Example 3 is that the combination of Japanese raisin tree seeds and milk thistle was replaced with 8g of milk thistle during fermentation. Comparative Example 6:

[0093] The difference from Example 3 is that corn oligopeptides are added to soybean protein peptides of equal mass during the preparation of the probiotic preparation.

[0094] Comparative Example 7:

[0095] The difference from Example 3 is that glutathione yeast was replaced with glutathione in the process of preparing the probiotic preparation.

[0096] Comparative Example 8

[0097] The difference from Example 3 is that turmeric was replaced with the natural anti-inflammatory agent epigallocatechin gallate (EGCG) during the preparation of the probiotic formulation.

[0098] Take the compounded probiotic preparation, add 5 times the volume of methanol, extract by ultrasonication at 400W for 20 min, centrifuge for 10 min (8000 r), take 0.1 mL of supernatant and dilute with methanol in a 5 mL volumetric flask, and use HPLC to quantitatively analyze and detect dihydromyricetin and silymarin. Detection method: flow rate 1.0 mL / min; injection volume 10 μL; column temperature 30℃; mobile phase acetonitrile (A)-0.1% phosphoric acid (B); gradient elution with 0 min 15% A, 10 min 12% A, 15 min 16% A, 16 min 90% A, 18 min 90% A, 19 min 15% A, and 30 min 15% A; detection at 290 nm.

[0099] Meanwhile, referring to the detection method in Example 1, the content of indicators DPPH, SOD, lipid peroxidation inhibition rate, ADH, ALDH, and P75 protein were evaluated.

[0100] Table 8. Changes in the content and function of active ingredients before and after fermentation.

[0101]

[0102]

[0103] Comparative analysis of HX-BA21 before and after fermentation of Hovenia dulcis and Milk thistle revealed that the efficient synthesis of the P75 protein strictly depends on a specific combination of "specific strain-specific substrate". In Comparative Example 1 (water extraction), the P75 protein was completely undetectable, confirming that it is not an inherent plant component but rather a product of microbial metabolism. When Comparative Examples 2 and 3 were replaced with other bacterial strains, the P75 protein yield decreased sharply compared to the examples, demonstrating the strain-level uniqueness of the Bifidobacterium animalis subspecies HX-BA21 in synthesizing this functional protein.

[0104] Comparative Examples 4 and 5 show that when the fermentation substrate was changed from the Hovenia dulcis-milk thistle complex to a single component, the P75 yield also decreased significantly. This strongly demonstrates that Hovenia dulcis and milk thistle, as a complex substrate, have a clear synergistic induction effect on the production of P75 protein, and neither can be omitted.

[0105] The key function of the compound components in this invention lies in forming a multi-pathway synergistic effect with the P75 protein. Data from Comparative Examples 6, 7, and 8 clearly show that, while maintaining a relatively constant P75 protein content, replacing any compound component leads to a significant decrease in key efficacy indicators.

[0106] In Comparative Example 6, replacing corn oligopeptides with soybean protein peptides led to a significant decrease in antioxidant indicators, indirectly demonstrating the unique advantages of corn oligopeptides in antioxidation and cell repair. In Comparative Example 7, replacing glutathione yeast with glutathione resulted in a significant decrease in ALDH activity compared to the control group. In Comparative Example 8, replacing turmeric with EGCG, while EGCG is a powerful antioxidant, curcumin may have a more prominent effect on specific anti-inflammatory pathways (such as NF-κB), which better complements the anti-inflammatory mechanism of P75 protein.

[0107] In summary, the production of P75 protein was enhanced by fermentation of Hovenia dulcis and Milk thistle with strain HX-BA21, and then a sophisticated multi-target functional network was constructed with three compound components.

[0108] Example 4 Cell Experiment

[0109] 4.1 Screening cell models by ethanol-induced concentration

[0110] L-02 cells that are growing normally were cultured in RPMI-1640 medium (containing 10% fetal bovine serum FBS) at 37°C in a 5% CO2 incubator, with the medium changed every 2-3 days. L-02 cells with confluence greater than 80% were digested with 0.25% EDTA-trypsin at 37°C for 3-5 minutes. Digestion was stopped when the cells detached in a flowing, sand-like manner. After counting, the cells were seeded into 96 empty culture plates at a density of 1×10⁶ cells / mL. 5 Cells were seeded at a density of 100 cells / mL and plated. After complete cell adhesion, a cell model was established. Groups were prepared with 0.1%, 0.5%, 0.9%, and 1.3% ethanol, 0.01% ferrous sulfate, and a blank control group containing only L-02 cells. After 48 hours of culture under these conditions, the cells were analyzed using the CCK-8 assay kit. Cell viability was calculated as [(experimental group - blank) / (blank control group - blank)] × 100%.

[0111] Table 9. Effects of different concentrations of ethanol on the growth of L-02 cells.

[0112]

[0113]

[0114] Table 9 shows that, compared with the blank control group (cell viability set at 100%), the 0.01% ferrous sulfate group and the 0.01% ethanol group had no significant effect on cell viability. The 1.3% ethanol group had a cell viability of 77.5, and observed cell volume increase, blurred edges, and significant cell detachment, indicating a significant decrease in cell viability. The 0.9% ethanol group had a cell viability of 87.5, which was lower than the previous group, but microscopic observation showed only a slight increase in cell volume, a relatively high number of viable cells, and fewer detached cells. Therefore, a 0.9% ethanol concentration was chosen to construct the alcoholic fatty liver (AFL) cell model.

[0115] 4.2 Establishment of AFL cell model

[0116] Take L-02 cells with 80% confluence from T25 culture flasks, digest them, count them, and then seed them to achieve a cell count of 6 × 10⁶ cells per well. 5 After cell adhesion, the experiment was divided into a blank control group, a negative control group, and a model group. The blank control group contained only L-02 cell suspension; the negative control group contained L-02 cell suspension with ferrous sulfate at a final concentration of 0.01%; the model group contained ethanol added to the negative control group to a final volume concentration of 0.9%, cultured to the 4th generation, and labeled AFL4. Oil Red O staining was used to observe lipid accumulation around the cell membrane, confirming the successful establishment of the model.

[0117] 4.3 Efficacy Testing

[0118] Following the procedure in 4.1, after cell adhesion, the experiment was divided into three groups: a blank control group (containing only L-02 cell suspension), a pre-treatment group (containing only AFL4 cell suspension), and drug-treated groups (AFL4 cell suspension supplemented with probiotic preparation, divided into high concentration (100ug / mL), medium concentration (70ug / mL), and low concentration (40ug / mL) groups, and control groups 2, 3, 6, 7, and 8 (70ug / mL). Cells were incubated at 37℃ in a 5% CO2 incubator, with medium changes every 2 days. Cell supernatants were collected after 16 days of observation. Biochemical indicators were measured in the collected cell supernatants according to the kit instructions, including TG (triglycerides), ALT (alanine transferase), AST (aspartate transferase), MDA (malondialdehyde), NEFA (free fatty acids), T-SOD (total superoxide dismutase), and GSH-Px (glutathione peroxidase). Meanwhile, ELISA was used to measure the levels of IL-6 (interleukin-6), IL-8 (interleukin-8), and TNF-α (tumor necrosis factor) in the cell supernatant of each group.

[0119] Table 10. Changes in TG, AST, ALT, MDA, NEFA, T-SOD, and GSH-Px in cell supernatants of each group.

[0120]

[0121]

[0122] As shown in Table 10, in an alcoholic fatty liver cell model, the probiotic preparation of this invention (high concentration group) exhibited comprehensive hepatoprotective effects. Compared with the model group, liver injury markers ALT and AST were significantly reduced by 69% and 63%, respectively, returning to normal levels; lipid metabolism indicators TG and NEFA decreased by 41% and 55%, respectively, effectively reversing fat accumulation; and the antioxidant system indicator T-SOD activity increased by 34%, especially glutathione GSH-Px, which increased significantly by 92%, significantly better than the blank control group. All key indicators showed clear dose-dependent improvement, and the overall efficacy of each concentration group of this invention comprehensively surpassed all comparative examples (e.g., the ALT value of comparative example 6 was only 52% of that of this invention), confirming the irreplaceable synergistic effect of specific strains and compound components.

[0123] Table 11 Changes in IL-6, IL-8, and INF-α in cell supernatants of each group

[0124]

[0125] As shown in Table 11, the probiotic formulation of this invention exhibits remarkable efficacy in inhibiting cellular inflammatory responses in alcoholic fatty liver disease. Compared to the model group, the high-concentration formulation significantly reduced key pro-inflammatory factors IL-6, IL-8, and TNF-α by 56%, 65%, and 26%, respectively, with IL-6 and TNF-α levels returning to normal ranges. Particularly noteworthy is the formulation's outstanding effect in inhibiting IL-8. This superior anti-inflammatory efficacy is directly related to the high production capacity of the P75 protein unique to the HX-BA21 strain. As a key microbial-derived anti-inflammatory effector molecule, the P75 protein can directly act on cellular inflammatory signaling pathways, effectively inhibiting the overexpression of pro-inflammatory factors. All test groups showed a clear dose-dependent improvement trend, and the overall anti-inflammatory effect of each concentration group of this invention was comprehensively superior to all comparative groups, fully demonstrating the unique and irreplaceable mechanism by which the HX-BA21 strain achieves precise anti-inflammatory effects through the production of P75 protein.

[0126] Example 5 Mouse Experiment

[0127] 5.1 Measurement of the effect of probiotic preparations on alcohol metabolism

[0128] Three-week-old healthy male Kunming mice, weighing 20±2g, were selected and randomly divided into 6 groups (n=10 per group) after one week of acclimatization. The groups were: a blank control group, an alcohol model control group, an experimental group (high concentration of the probiotic preparation composition from Example 3: 5mg / mL, medium concentration: 3mg / mL, low concentration: 1mg / mL), and a positive control group (Haiwang Brand Jinzun Granules aqueous solution). Mice in each group were fasted for 12 hours before being administered via gavage. Specific grouping and gavage methods are shown in Table 12. The intoxication and sobering states of the mice were monitored, and the intoxication time (from the administration of alcohol to the disappearance of the righting reflex) and sobering time (from the reappearance of the righting reflex to its return to normal) were recorded. Blood samples were collected from the orbital sinus of the mice at 0.5, 1.0, 2.0, and 3.0 hours after the second gavage, and serum ethanol concentration was measured using a kit.

[0129] Table 12 Animal grouping and gavage method

[0130]

[0131]

[0132] Table 13 Effects of probiotic preparations on alcohol intoxication in mice

[0133] Group name Drunk mice per group Drunk rate (%) Drunk time / min Sober time / min Blank group (C) 0 0 - - Alcohol model control group (E) 10 100％ 15.67 291.58 Positive control group (Y) 5 50 26.25 188.89 High concentration group (H) 5 50 29.71 167.21 Medium concentration group (Z) 6 60 23.65 189.05 Low concentration group (D) 8 80 20.71 212.36

[0134] The results showed that the probiotic preparation of the present invention exhibited a significant hangover-relieving effect. The results of different groups are shown in Table 13. Except for the blank group, all mice showed varying degrees of intoxication symptoms. Behavioralally, compared with the alcohol model group (intoxication rate 100%, sobering time 291.58 minutes), the high-concentration preparation group successfully reduced the intoxication rate to 50%, significantly prolonged the intoxication latency period to 29.71 minutes, and substantially shortened the sobering time to 167.21 minutes, demonstrating better effects than the positive control group. Furthermore, each dosage group showed a clear dose-dependent relationship.

[0135] In terms of alcohol metabolism kinetics, the probiotic preparation also showed significant effects. 0.5 hours after oral administration of alcohol, the serum ethanol concentration in the high-concentration group (19.27 mmol / L) was significantly lower than that in the model group (31.27 mmol / L), indicating that it effectively delayed ethanol absorption. By 3 hours after oral administration, the serum ethanol concentration in the high-concentration group had decreased to 9.99 mmol / L, with a significantly faster clearance rate than that in the model group (16.71 mmol / L). This indicates that the preparation not only delays alcohol absorption but also accelerates the clearance and metabolism of ethanol in the body, thereby comprehensively alleviating the symptoms of alcohol poisoning.

[0136] Table 14. Effects of probiotic preparations on serum ethanol concentration in mice (mmol / L)

[0137] Group name 0h 0.5h 1h 2h 3h Alcohol model control group (E) 0.01 31.27 35.71 28.42 16.71 Positive control group (Y) 0.02 23.29 27.61 20.11 10.28 High concentration group (H) 0.01 19.27 25.88 19.87 9.99 Medium concentration group (Z) 0.02 23.98 28.69 23.21 11.26 Low concentration group (D) 0.01 25.71 31.22 24.71 13.25

[0138] 5.2 Determination of the adjuvant protective effect of the formulation against alcoholic liver injury

[0139] Following the experiment described in 5.1, mice were randomly divided into 6 groups of 10 mice each, and tested for a total of 30 days. For the first 15 days, mice were administered the product via gavage according to their body weight. The control group and the alcohol model group were administered physiological saline via gavage, while the other groups were administered the corresponding product via gavage. The gavage dose for all groups was 12 mL / kg. One hour after the first gavage, a second gavage was administered. Except for the control group, all other groups were administered a 50% ethanol solution at a concentration of 8 mL / kg via gavage. For the next 15 days, the first gavage was the same as the first 15 days, and the second gavage used a 50% ethanol solution at a concentration of 14 mL / kg via gavage. Weight changes in the mice were recorded, and their fasting weight was measured before sacrifice.

[0140] Mice were fasted for 16 hours and then euthanized by an overdose of isoflurane. Blood samples were collected via the eyes and serum was prepared by centrifugation at 3000 rpm for 10 minutes at 4°C. The liver was completely dissected, rinsed with pre-cooled physiological saline, and dried before weighing. The left lobe of the liver was fixed in 4% paraformaldehyde, and the remaining liver tissue was flash-frozen in liquid nitrogen and then stored at -80°C for subsequent analysis. The liver index was calculated as (liver wet weight / final body weight) × 100%. Serum samples were collected, and liver function tests (AST and ALT) were measured using a kit. Liver tissue was homogenized, and oxidative stress markers (MDA, SOD, and GSH), fat content (TC and TG), and inflammatory factors (IL-6 and TNF-α) levels were measured using a kit. The results are shown in Tables 15-18.

[0141] Table 15 Mouse weight and liver index

[0142] Group name 0 days / g 15 days / g 30 days / g Liver index (%) Blank group 22.15 30.21 39.87 4.12 Alcohol model control group (E) 21.77 24.87 26.71 4.57 Positive control group (Y) 22.08 28.87 31.22 4.02 High concentration group (H) 21.37 29.11 32.11 4.03 Medium concentration group (Z) 22.95 27.21 30.98 4.11 Low concentration group (D) 23.21 27.01 30.11 4.25

[0143] Regarding overall growth and liver morphology, the control group of mice in the alcohol model showed slow weight gain and a significantly elevated liver index of 4.57%, indicating that alcohol intake led to developmental delay and pathological liver enlargement. In contrast, the high- and medium-concentration groups of this invention showed good weight recovery, with liver indices recovering to 4.03% and 4.11%, respectively, showing no significant difference from the normal group. This demonstrates that the formulation of this invention can effectively alleviate the negative effects of alcohol on growth and liver structure.

[0144] Table 16 Serum liver function indicators in mice

[0145] Group name ALT (U / L) AST (U / L) Blank group 42.31 37.62 Alcohol model control group (E) 79.21 59.88 Positive control group (Y) 58.73 48.71 High concentration group (H) 56.77 48.09 Medium concentration group (Z) 60.21 49.11 Low concentration group (D) 75.21 55.34

[0146] Regarding hepatocellular protection, serum ALT and AST activities in the alcohol model group increased to 79.21 U / L and 59.88 U / L, respectively, indicating significant liver damage. After intervention with the high-concentration formulation of this invention, these two indicators significantly decreased to 56.77 U / L and 48.09 U / L, with effects comparable to the positive control group and in a dose-dependent manner, confirming its excellent hepatocellular protective function.

[0147] Table 17 Liver-related Indicators Detection

[0148]

[0149]

[0150] Regarding antioxidant and lipid metabolism regulation, the liver MDA content in the alcohol model group increased to 7.99 mmol / mg, while SOD activity and GSH content significantly decreased. Simultaneously, TC and TG increased to 0.38 mmol / g and 0.36 mmol / mg, respectively, indicating severe oxidative stress and lipid accumulation. The high-concentration group of this invention successfully reduced MDA to 4.22 mmol / mg, significantly increased GSH to 598.76 μmol / g, and restored TC and TG to near-normal levels, demonstrating its effectiveness in alleviating alcoholic liver injury by enhancing the antioxidant defense system and regulating lipid metabolism.

[0151] Table 18 Inflammation markers in mouse liver

[0152] Group name IL-6 (pg / g) TNF-α Blank group 265.71 2127.38 Alcohol model control group (E) 517.22 4126.59 Positive control group (Y) 301.25 2875.21 High concentration group (H) 299.77 2217.65 Medium concentration group (Z) 381.56 2926.55 Low concentration group (D) 421.18 3019.89

[0153] Regarding inflammation suppression, the liver IL-6 and TNF-α levels in the alcohol model group were as high as 517.22 pg / g and 4126.59 pg / g, respectively, indicating a strong inflammatory response. The high-concentration group of this invention exhibited superior anti-inflammatory effects, reducing IL-6 and TNF-α levels to 299.77 pg / g and 2217.65 pg / g, respectively. The inhibitory effect on TNF-α was even better than that in the positive control group, highlighting the crucial anti-inflammatory effect produced by the synergistic effect of the P75 protein produced by the probiotic metabolism and the formulation.

[0154] This invention's probiotic formulation successfully constructs a dual-efficacy system of "short-term, highly effective hangover relief and long-term, fundamental liver protection." In an acute alcohol intoxication model, this formulation significantly prolongs the latency period of intoxication and simultaneously achieves rapid hangover relief by enhancing the breakdown of ethanol in the blood. In long-term intervention for alcoholic liver injury, the formulation, through a dual mechanism of action of "microbial functional proteins + plant active ingredients," achieves integrated intervention from intestinal-based anti-inflammatory measures to multi-pathway liver protection and lipid reduction. Its efficacy is significantly superior to simple physical mixtures, providing a novel, safe, and effective solution for the prevention and treatment of AFL.

Claims

1. A fermented complex with liver-protecting and lipid-lowering functions, wherein: the fermented complex is obtained by fermenting Silybum marianum and Hovenia dulcis with Bifidobacterium animalis subsp. animalis HX-BA21. 2.The fermented complex of claim 1, wherein: the weight ratio of Silybum marianum to Hovenia dulcis is 1:1-1:

2. 3.The fermented complex of claim 1, wherein: the content of P75 protein in the fermented complex is not less than 30 ng / mL, the content of dihydromyricetin is not less than 35 mg / L, and the content of silychristin is not less than 35 mg / L. 4.A method for preparing the fermented complex of claim 1, comprising the following steps: crushing and sterilizing raw materials of Silybum marianum and Hovenia dulcis, and adding them into a fermentation medium; inoculating Bifidobacterium animalis subsp. animalis HX-BA21 into the fermentation medium, and performing fermentation culture at 37℃ for 24 hours; centrifuging the fermentation liquid, and taking the supernatant to obtain the fermented complex. The viable cell number of Bifidobacterium animalis ssp. animalis HX-BA21 in the fermentation complex is 1.0-3.5 x 10 8 CFU / mL. 5.The method of claim 4, wherein: the fermentation medium comprises 10.0 g of casein enzymatic digest, 2.0 g of ammonium citrate tribasic, 10.0 g of beef extract powder, 0.2 g of magnesium sulfate, 4.0 g of yeast extract powder, 0.05 g of manganese sulfate, 5.0 g of sodium acetate, 2.0 g of potassium phosphate dibasic, 10.0 g of glucose, 1.08 g of Tween-80, 8 g of a combination of Hovenia dulcis and Silybum marianum, and the pH value of the fermentation medium is 5.

8. 6.A liver-protecting and lipid-lowering composition, comprising 5-15 parts by weight of the fermented complex of claim 1, 1-4 parts by weight of corn oligopeptide, 1-4 parts by weight of glutathione yeast, and 0.5-2.5 parts by weight of turmeric. 7.A method for preparing the liver-protecting and lipid-lowering composition of claim 6, comprising the step of mixing and drying the fermented complex with corn oligopeptide, glutathione yeast, and turmeric. 8.The use of the composition of claim 6 in the preparation of a food or a drug for preventing and / or treating alcoholic liver injury. 9.The use of the composition of claim 8, wherein: the alcoholic liver injury comprises alcoholic fatty liver and alcoholic hepatitis. 10.The use of the composition of claim 8, wherein: the composition is used in the preparation of a food or a drug for improving the activities of alcohol dehydrogenase and acetaldehyde dehydrogenase in the liver; or the composition is used in the preparation of a food or a drug for reducing the levels of ALT and AST in serum; or the composition is used in the preparation of a food or a drug for reducing the contents of triglyceride and total cholesterol in the liver; or the composition is used in the preparation of a food or a drug for improving the activities of superoxide dismutase and glutathione peroxidase in the liver; or the composition is used in the preparation of a food or a drug for reducing the contents of interleukin-6 and tumor necrosis factor-α in the liver. ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​

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