Probiotic fermented traditional Chinese medicine preparation and preparation method thereof

Abstract

The invention relates to the technical field of functional microorganism application, in particular to a probiotic fermented traditional Chinese medicine preparation and a preparation method thereof. The traditional Chinese medicine preparation is prepared by fermenting a radix puerariae extract and a hovenia dulcis thunb extract by utilizing Lodderomyces elongisporus, the contents of flavones and total phenols respectively reach 173.31 mu g / mL and 112.06 mu g / mL, and the traditional Chinese medicine preparation is high in oxidation resistance and can be used for efficiently removing various free radicals. The traditional Chinese medicine preparation is remarkable in hangover alleviating effect, capable of effectively relieving damage of alcohol to hepatocytes and improving the stability of the liver, and wide in application prospect.

Description

Technical Field

[0001] The present invention relates to the technical field of functional microorganism application, and in particular to a probiotic fermentation Chinese medicine preparation and a preparation method thereof. Background Art

[0002] Drinking alcohol can cause many adverse symptoms, such as blushing, nausea, headache, and confusion. It can not only easily lead to accidental injuries and cause social problems, but long-term drinking can also induce a variety of diseases, causing damage to tissues and organs such as the liver, cardiovascular system, and gastrointestinal tract, seriously endangering human health.

[0003] As research into the role of microbial preparations in human nutrition and health deepens, it has been discovered that yeast is rich in B vitamins and enzymes, which can deeply nourish and protect the liver, ensuring the proper functioning of its detoxification function. Yeast also contains numerous trace elements, which are important components of some enzymes.

[0004] Yeast fermentation, as a green processing method, offers numerous advantages. It can impart a unique flavor to products; it can also optimize the efficiency of the fermentation substrate and convert nutrients within it. Pueraria root and Hovenia dulcis fruit contain a variety of active ingredients, such as puerarin, daidzein, and other isoflavones. Yeast fermentation alters the molecular structure of these ingredients, making them more easily absorbed and utilized by the body, thereby increasing the bioavailability of kudzu root. Furthermore, enzymes produced by the yeast during fermentation can break down macromolecules in kudzu root, such as cellulose, into smaller molecules that are more easily digested and absorbed by the body, further promoting the absorption of nutrients. Therefore, the use of yeast fermentation to produce healthy hangover-relief products is of great significance. Summary of the Invention

[0005] The purpose of the present invention is to provide a probiotic fermentation Chinese medicine preparation and a preparation method thereof.

[0006] In order to achieve the purpose of the invention, the present invention adopts the following technical solutions:

[0007] One aspect of the present invention relates to a probiotic fermented Chinese medicine preparation, which is prepared by fermenting a Chinese medicine extract with Lodderomyces elongisporus.

[0008] The Lodea longispora yeast is Lodea longispora yeast W9, which was deposited in the General Microbiology Center of the China Culture Collection Administration (CGMCC) on August 12, 2024. The deposit address is: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, with the deposit number CGMCC No. 31572.

[0009] The traditional Chinese medicines are Pueraria root and Hovenia dulcis fruit.

[0010] The present invention also provides a method for preparing the probiotic fermentation Chinese medicine preparation, comprising the following steps:

[0011] (1) Raw material pretreatment: Grind the Pueraria root and Hovenia dulcis fruit raw materials through an 80-mesh sieve, mix them in a mass ratio of 3:2, and soak them in 10 times the volume of water for 120 min;

[0012] (2) Extraction of raw materials: the pretreated raw materials were extracted at 80°C for 60 minutes, filtered to obtain extract I; the same amount of water as in step (1) was added to the filtered residue, the mixture was extracted at 80°C for 60 minutes, filtered to obtain extract II; the same amount of water as in step (1) was added to the filtered residue, the mixture was extracted at 80°C for 60 minutes, filtered to obtain extract III; all the extracts were combined and concentrated in a water bath to obtain the Chinese medicine extract;

[0013] (3) Preparation of fermentation medium: Yeast powder and Lycium barbarum polysaccharide were added to the Chinese herbal medicine extract, sterilized at 121°C for 20 min, and cooled to room temperature to obtain fermentation medium;

[0014] (4) Probiotic fermentation: Loderma longisporum W9 powder was inoculated into the fermentation medium in proportion, and fermented at a constant temperature of 37°C for 48 hours. The solid matter was filtered to obtain the probiotic fermentation Chinese medicine preparation.

[0015] The concentration of the Chinese herbal medicine extract in step (2) is 0.02 g / mL.

[0016] The contents of yeast powder and wolfberry polysaccharide in the fermentation medium in step (3) are 16 g / L and 64 g / L, respectively.

[0017] The viable bacteria content of the Lodecanosa longispora W9 powder in step (4) is not less than 10 11 CFU / g.

[0018] The inoculation ratio of the Lodeella longispora W9 powder in step (4) is 40 g / L.

[0019] The probiotic fermentation Chinese medicine preparation is used in preparing a product with alcohol sobering effect.

[0020] Compared with the prior art, the present invention has the following beneficial effects:

[0021] The probiotic fermented traditional Chinese medicine preparation provided by the present invention has a soluble solid content of 5.21%, a total acid content of 7.35 g / kg, a pH value of 3.85, a viscosity of 1122.54 Pas, a logarithm value of the viable bacteria count of 8.32, a coliform count of ≤5, a mold count of ≤5, and no pathogenic bacteria detected, thus meeting the national standard requirements for fermented fruit and vegetable juice.

[0022] The flavonoid and total phenol contents in the probiotic fermented traditional Chinese medicine preparation reached 173.31 μg / mL and 112.06 μg / mL, respectively, which were increased by 51.84% and 31.20% compared with those before fermentation; the scavenging rates of DPPH, ABTS+, and hydroxyl free radicals reached 56.23%, 45.15%, and 42.52%, respectively, which were increased by 31.35%, 29.21%, and 35.93% compared with those before fermentation, respectively, and the antioxidant effect was very significant.

[0023] The probiotic fermented Chinese medicine preparation has a significant alcohol-relieving effect, and its effect is better than that of the alcohol-relieving medicine Jinzun tablets. It can effectively increase the levels of alcohol dehydrogenase (ADH) and acetaldehyde dehydrogenase (ALDH) in the liver of mice, promote the metabolism and clearance of alcohol, reduce the levels of aspartate aminotransferase (AST) and alanine aminotransferase (ALT) in the serum of mice, effectively reduce the damage of alcohol to liver cells, and increase the stability of the liver.

[0024] The results of animal experiments showed that compared with the model group, the drunkenness rate of the fermentation group mice that were gavaged with probiotic fermented Chinese medicine preparations was reduced by 42.86%, the drunkenness time was prolonged by 92.33%, and the sobering time was shortened by 41.70%. The sobering time of the fermentation group mice was shortened by 15.92% compared with the positive group that were gavaged with Jinzun tablets; the ethanol concentration in the serum of the fermentation group mice was reduced by 37.50% compared with the model group, and decreased by 21.05% compared with the positive group that were gavaged with Jinzun tablets; the levels of aspartate aminotransferase (AST) and alanine aminotransferase (ALT) in the serum of the fermentation group mice were significantly decreased compared with the model group (P < 0.01); the levels of alcohol-degrading enzymes ADH and ALDH in the liver of the fermentation group mice were significantly increased compared with the model group (P < 0.001); the liver damage of the fermentation group mice was the mildest, the liver cell structure was relatively complete, the shape of the hepatic cords was relatively clear, and no round vacuoles were seen in the cytoplasm.

[0025] The probiotic fermentation Chinese medicine preparation provided by the present invention can be widely used in the production of products with alcohol-relieving effects, and has broad prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 Statistical results of (A) alanine aminotransferase and (B) aspartate aminotransferase levels in the serum of mice in each group;

[0027] Figure 2 Statistical results of (A) superoxide dismutase activity and (B) reduced glutathione content in the liver of mice in each group;

[0028] Figure 3 Statistical results of (A) triglyceride and (B) cholesterol levels in the serum of mice in each group;

[0029] Figure 4Statistical results of (A) triglyceride and (B) cholesterol levels in the liver of mice in each group;

[0030] Figure 5 HE staining results of liver tissues of mice in each group; scale bar = 100 μm;

[0031] Figure 6 Figures show (A) serum lipopolysaccharide and (B) intestinal tight junction gene expression results for each group of mice;

[0032] Figure 7 H&E staining results of the livers of mice in each group; scale bar = 100 μm.

[0033] Figure 8 H&E staining of mouse liver tissue pathomorphology; A is the blank group, B is the model group, C is the positive group, D is the non-fermented group, and E is the fermented group;

[0034] Figure 9 Comparison of ALT (A) and AST (B) levels in the serum of mice in each group;

[0035] Figure 10 Comparison of ADH enzyme activity (A) and ALDH enzyme activity (B) in the liver of mice in each group. DETAILED DESCRIPTION

[0036] The following will describe in detail the implementation methods, product formulas, technical contents, achieved objectives and effects of this application with the help of accompanying drawings and examples.

[0037] Example 1 Screening and Identification of Alcohol-Degrading Enzyme-Producing Yeast

[0038] Alcohol detoxification enzymes are key biological enzymes that help the human body metabolize alcohol, mainly including alcohol dehydrogenase (ADH) and acetaldehyde dehydrogenase (ALDH). They help reduce the damage caused by alcohol to the body by breaking down ethanol and the intermediate product acetaldehyde in alcohol.

[0039] The present invention isolated and screened a yeast strain with high alcohol-degrading enzyme production from fermented milk lumps collected in Urumqi, Xinjiang. The yeast strain was identified as Lodderomyces elongisporus and named Lodderomyces elongisporus W9. The strain was deposited in the General Microbiology Center of the China Culture Collection of Microorganisms (CGMCC) on August 12, 2024. The deposit address is: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, with a deposit number of CGMCC No. 31572.

[0040] After the Lodwigia longispora W9 was cultured at a constant temperature of 28° C. for 48 hours in a YPD liquid culture medium, the enzyme activities of alcohol dehydrogenase (ADH) and acetaldehyde dehydrogenase (ALDH) in the bacterial lysis supernatant were as high as 38.28 U / mL and 180.71 U / mL, respectively, achieving unexpected technical results.

[0041] The degradation rate of the Lodzia longispora W9 to 10% volume fraction ethanol is as high as 36.06%, and the degradation rate to 1.0 g / L acetaldehyde is as high as 48.43%, with significant effects.

[0042] Example 2 Surface Adhesion Ability of Lodela longispora W9

[0043] 1. Surface hydrophobicity determination

[0044] The hydrophobicity of the bacterial surface is an important indicator for evaluating the adhesion of lactic acid bacteria, reflecting the strength of the interaction between bacteria and intestinal epithelial cells. Studies have found that the adhesion ability of bacteria to the host intestine is positively correlated with the surface hydrophobicity.

[0045] Lodzia longispora W9 and a commercially available control yeast strain ATCC9080, cultured to the stable growth phase, were centrifuged at 4°C and 10,000 rpm for 10 minutes. The cells were washed twice with PBS (pH 7.2) and resuspended in PBS until the OD value of the bacterial suspension at a wavelength of 600 nm was approximately 0.8, which was recorded as A0. 1 mL of a hydrophobic agent (xylene or chloroform) was added to 3 mL of the bacterial suspension, mixed thoroughly, and allowed to stand at room temperature for 20 minutes to allow the organic and aqueous phases to separate. The organic phase was removed, and the OD value of the aqueous phase was measured at 600 nm using PBS buffer as a control, which was recorded as A1. The surface hydrophobicity of the strain was calculated according to the following formula.

[0046] Surface hydrophobicity (%) = (1-A1 / A0) x 100%.

[0047] Table 1 Surface hydrophobicity of Lodrum longisporum W9

[0048] strain number Control bacteria ATCC9080 Loderma longisporum W9 Chloroform 21.17±1.51 68.72±1.28** Xylene 17.26±0.89 33.76±1.04**

[0049] Compared with the commercial control strain ATCC9080, **: P<0.01, the difference is extremely significant.

[0050] As shown in Table 1, regardless of whether xylene or chloroform was used for extraction, the surface hydrophobicity of the Lodena longispora W9 provided by the present invention was significantly higher than that of the control yeast ATCC9080 (p < 0.01). When chloroform was used as the organic solvent, the surface hydrophobicity of the Lodena longispora W9 reached 68.72%, an increase of 224.6% over the control yeast. This indicates that the Lodena longispora W9 has strong adhesion ability.

[0051] 2. Determination of self-agglutination ability

[0052] The self-aggregation ability of microorganisms makes an important contribution to the adhesion of intestinal cells and the avoidance of pathogen colonization.

[0053] Take 4 mL of the adjusted bacterial suspension, mix thoroughly, and incubate at room temperature for 6 hours. Take 1 mL of the bacterial suspension and use PBS buffer as a control to measure the OD value at 600 nm, which is recorded as A2. The calculation formula is as follows:

[0054] Self-aggregation rate (%) = (1-A2 / A0) x 100%.

[0055] Table 2 Self-aggregation effect of Lodeella longispora W9

[0056] strain number Control bacteria ATCC9080 Lodzia longispora W9 Self-agglutination rate (%) 18.68±1.12 67.21±1.07**

[0057] Compared with the commercial control strain ATCC9080, **: P<0.01, the difference is extremely significant.

[0058] From the results in Table 2, it can be seen that Lodzia longispora W9 has a strong self-aggregation ability, and its self-aggregation rate is as high as 67.21%, which is significantly higher than that of the control yeast ATCC9080, achieving an unexpected technical effect.

[0059] The above results show that the Lodzia longispora W9 provided by the present invention has a strong surface adhesion ability, can effectively enhance intestinal colonization, prolong the probiotic effect time, and at the same time help competitively inhibit the attachment of pathogens and maintain the balance of the bacterial flora.

[0060] Example 3 Ability of Lodzia longispora W9 to produce reduced glutathione and superoxide dismutase

[0061] Reduced glutathione (GSH) is a low molecular weight scavenger that can remove O 2- , H2O2, and LOOH, making GSH an important factor in measuring the body's antioxidant capacity. Superoxide dismutase (SOD) is a key antioxidant enzyme in organisms, its primary function being to catalyze the conversion of superoxide anions into hydrogen peroxide (H2O2) and oxygen (O2). SOD activity reflects the degree of intracellular oxidative stress; when its level is low, cells are susceptible to oxidative damage.

[0062] 1. Experimental methods

[0063] The strain, stored in glycerol at -80°C, was thawed and activated for the second generation. The bacterial suspension at the end of logarithmic growth was centrifuged at 4000 rpm for 10 minutes, and the supernatant discarded to obtain a bacterial slurry. The suspension was washed twice with PBS and resuspended. The bacterial suspension, with an OD value of approximately 1, was ultrasonically disrupted. Ultrasonication was performed at 300 W in an ice-water bath, with sonication every 3–5 seconds, followed by four intervals (each interval lasting approximately 30 seconds). The GSH and superoxide dismutase (SOD) production capacities of Lodovora longispora W9 were determined using the microbial reduced glutathione (GSH) assay kit and microbial superoxide dismutase (SOD) assay kit, respectively, provided by the Nanjing Jiancheng Bioengineering Research Institute.

[0064] 2. Experimental results

[0065] Table 3 Ability of Lodzia longispora W9 to produce reduced glutathione reductase and superoxide dismutase

[0066] strain number Control bacteria ATCC9080 Loderma longisporum W9 GSH content (umol / gprot) 8.31±1.42 21.84±0.78** SOD level (U / mgprot) 14.42±0.89 33.11±1.65**

[0067] Compared with the commercial control strain ATCC9080, **: P<0.01, the difference is extremely significant.

[0068] From the results in Table 3, it can be seen that compared with the control yeast, the contents of reduced glutathione (GSH) and superoxide dismutase (SOD) in the Lodwigia longispora W9 provided by the present invention were increased by 162.8% and 129.6%, respectively, and the yeast had extremely strong antioxidant capacity.

[0069] Example 4 In vitro antioxidant capacity detection of Lodela longispora W9

[0070] 1. Experimental methods

[0071] (1) DPPH free radical scavenging ability determination

[0072] 1 mL of bacterial suspension was mixed with 2 mL of DPPH free radical solution (0.1 mol / L) respectively. After incubation at room temperature in the dark for 30 min, the absorbance was measured at a wavelength of 517 nm. An equal volume of ethanol was used as a blank control. The DPPH free radical scavenging rate was calculated as follows:

[0073] DPPH free radical scavenging rate (%) = (1-bacterial suspension absorbance / blank group absorbance) × 100%.

[0074] (2) Determination of ABTS·+ free radical scavenging ability

[0075] Take 0.5 mL of bacterial suspension and 3.5 mL of ABTS·+ working solution and mix them evenly. After reacting for 6 minutes at room temperature, measure the absorbance at 734 nm. Use an equal volume of anhydrous ethanol as a blank control. The formula for calculating the ABTS+ free radical scavenging rate is as follows:

[0076] ABTS·+ free radical scavenging rate (%) = (1-absorbance of experimental group / absorbance of blank group) × 100%.

[0077] (3) Determination of hydroxyl radical scavenging ability

[0078] After the strain is activated for the second generation, take the bacterial suspension at the end of logarithmic growth, centrifuge at 4000rpm for 10 minutes, discard the supernatant, wash twice with PBS, and measure the OD of the bacterial suspension = 600. Add 1mL of bacterial suspension with an OD value of about 1 to the reaction system, then add 1mL of normal saline and 1mL of ferrous sulfate (3mmol / L). After mixing, add 1mL of hydrogen peroxide (3mmol / L), let it stand at room temperature for 10 minutes, then add 1mL of salicylic acid (3mmol / L, dissolved in ethanol), mix well, bathe in 37℃ water for 20 minutes, centrifuge, take the supernatant and measure the absorbance at 510nm. Replace the sample solution with an equal volume of normal saline as the control group, and use an equal volume of a mixture of normal saline and anhydrous ethanol as a blank to adjust to zero. Calculate the scavenging rate of hydroxyl radicals according to the following formula:

[0079] Hydroxyl radical scavenging rate = (As Ap) / As × 100%.

[0080] Wherein, Ap: OD510 value of Lodeella longispora W9, As: OD510 value of bacterial suspension after replacement with physiological saline.

[0081] 2. Experimental results

[0082] Table 4 Free radical scavenging ability of Lodeella longispora W9

[0083] strain number Control bacteria ATCC9080 Loderma longisporum W9 DPPH free radical scavenging rate (%) 30.32±0.76 60.65±1.26** ABTS·+ free radical scavenging rate (%) 31.33±1.17 45.68±0.76** Hydroxyl radical scavenging rate (%) 29.68±1.01 48.87±1.42**

[0084] Compared with the commercial control strain ATCC9080, **: P<0.01, the difference is extremely significant.

[0085] From the results in Table 4, it can be seen that compared with the control yeast, the scavenging rates of the Lodzia longispora W9 bacteria provided by the present invention on DPPH, ABTS·+ and hydroxyl free radicals were increased by 100.0%, 45.8% and 64.7%, respectively, and the bacteria had extremely strong in vitro antioxidant capacity.

[0086] Example 5 Application of Lodzia longispora W9 in Alleviating Chronic Alcohol-Induced Liver and Intestinal Damage in Mice

[0087] 1. Experimental Methods

[0088] 1.1 Preparation of Lodeyces longisporus W9 bacterial suspension

[0089] The strain at the end of logarithmic growth was selected and added to YPD liquid medium at a 2% inoculum and cultured in a 28°C incubator for 48 h. The bacteria were centrifuged at 4000 r / min for 10 min, and the bacteria were washed twice with sterile saline to adjust the bacterial solution concentration to 10 10 CFU / 200uL, store at 4℃ for later use.

[0090] 1.2 Animal grouping and establishment of a chronic alcohol-induced fatty liver mouse model

[0091] Male, five-week-old Kunming mice were randomly divided into a control group (NC), a group receiving oral administration of Lodosporin W9 (W9), a chronic alcohol model group (AC), a silymarin group (PC), and a Lodosporin W9 alcohol model group (AC+W9), with eight mice per group. The experiment began after one week of adaptive feeding. A 10% alcohol concentration, 10 mL / kg body weight, was administered via oral administration. The model was established for eight weeks, with free access to water. Specific experimental procedures are shown in Table 5.

[0092] Table 5 Animal experimental groups and operations

[0093]

[0094]

[0095] 1.3 Biochemical analysis

[0096] Total cholesterol (TC), triglycerides (TG), alanine aminotransferase (ALT), aspartate aminotransferase (AST), superoxide dismutase (SOD), and glutathione (GSH) were quantified using commercial kits (Nanjing Jiancheng, China). Tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), interleukin-10 (IL-10), and interleukin-1β (IL-1β) in the intestine were quantified using enzyme-linked immunosorbent assay kits (Shanghai ELISA, China).

[0097] 1.4 Histological analysis

[0098] Fresh liver and colon tissues were immediately fixed in 4% paraformaldehyde for 48 hours. Subsequently, tissue samples were dehydrated, paraffin-embedded, and cut into 4-micron-thick sections. After dewaxing, tissue samples were stained with hematoxylin and eosin (H&E) and analyzed using a light microscope.

[0099] 1.5 Real-time quantitative PCR (RT-qPCR)

[0100] Total RNA was extracted from liver tissue using Trizol reagent, and 1 μg of RNA was reverse transcribed into cDNA using the M5-SuperqPCR RT Kit with genomic DNA removal reagent. Liver mRNA expression was measured by SYBR green RT-qPCR using a 96-well instrument (Applied Biosystems, USA). Primer sequences are listed in Table 6. Target gene levels were quantified relative to Gapdh gene levels, and the results were analyzed using the 2-ΔΔCt analysis method.

[0101] Table 6 Primer sequences

[0102]

[0103] 1.6 Statistical analysis

[0104] Data are presented as mean ± standard deviation (SD). GraphPad Prism 9.5 was used to analyze significance using one-way or two-way analysis of variance (ANOVA) and post hoc Tukey-Kramer test. P values are as follows: *, 0.01 <P≤0.05;**,0.001<P≤0.01;***,P≤0.001。

[0105] 2. Experimental Results

[0106] (1) Effects of Lodwigia longispora W9 intervention on serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) activities in mice

[0107] Alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels in peripheral blood are important indicators for assessing liver damage. To assess liver damage, serum alanine aminotransferase and aspartate aminotransferase activities were measured using ALT and AST detection kits (Nanjing Jiancheng Bioengineering Institute), respectively.

[0108] The results are as follows Figure 1 As shown, long-term alcohol consumption led to significantly elevated serum AST and ALT levels in mice (93.56±4.074 U / L and 78.8±4.75 U / L) compared to normal control mice (42.59±5.59 U / L and 22.71±2.43 U / L, respectively) (p<0.05), indicating a successful establishment of a liver injury mouse model. Compared to the alcohol model group (AC group), serum AST and ALT activity levels in mice treated with Loderma longisporum W9 decreased by 48.15% and 47.08%, respectively, demonstrating a significant effect. This suggests that Loderma longisporum W9 can effectively alleviate liver damage in mice induced by long-term alcohol consumption.

[0109] (2) Effects of Lodwigia longispora W9 intervention on superoxide dismutase (SOD) activity and glutathione (GSH) content in mouse liver oxidative stress indicators

[0110] Excessive ethanol exposure can cause liver metabolism to produce a large amount of ROS, such as H2O2 and O2 - These free radicals quickly combine with ethanol or iron atoms within the cell to form reactive metabolites such as OH-, ferrous oxide (FeO), and hydroxyethyl (CH3CHOH-), ultimately causing oxidative stress. Intracellular antioxidants, such as SOD and GSH, can scavenge excess free radicals and play an important role in alleviating oxidative stress.

[0111] To evaluate liver oxidative stress damage, superoxide dismutase (SOD) activity and glutathione (GSH) in liver tissue were measured using SOD and GSH detection kits (Nanjing Jiancheng Bioengineering Institute), respectively.

[0112] The results are as follows Figure 2 As shown in Figure A, compared with the CTL group, SOD activity in the livers of mice in the alcohol model group (AC group) was significantly decreased, indicating that long-term alcohol exposure inhibits SOD enzyme activity. Compared with the alcohol model group (AC group), SOD activity and GSH content in the liver tissues of mice treated with Lodrum longisporum W9 were significantly increased. This suggests that Lodrum longisporum W9 effectively increases SOD and GSH levels in the livers of mice with alcoholic liver disease, potentially preventing or ameliorating chronic alcohol-induced liver oxidative stress damage.

[0113] (3) Effects of Lodzia longispora W9 intervention on lipid accumulation in the liver and serum of mice induced by chronic alcohol

[0114] Biochemical detection kits (Nanjing Jiancheng Bioengineering Institute) were used to detect the levels of triglyceride (TG) and cholesterol (TC) in mouse liver and serum, respectively.

[0115] The results are as follows Figure 3 As shown, compared with the CTL group, the levels of triglyceride (TG) and cholesterol (TC) in the liver and serum of mice in the alcohol model group (AC group) were significantly increased, indicating that long-term alcohol exposure leads to a large accumulation of lipids in the liver and serum. In contrast, the levels of triglyceride (TG) and cholesterol (TC) in the liver and serum of mice treated with Lodaria longispora W9 decreased by 55.77% and 37.71%, respectively, compared with the alcohol model group (AC group), demonstrating a highly significant effect.

[0116] (4) Effects of Lodzia longispora W9 intervention on chronic alcohol-induced liver tissue pathological damage in mice

[0117] Depend on Figure 4It can be seen that the liver tissue lobule structure of mice in the CTL group was intact, the cell boundaries were clear, and no fat vacuoles were seen. The liver tissue cell structure of mice in the alcohol model group (AC group) was destroyed, the liver fatty degeneration was obvious, fat vacuoles and inflammatory cell infiltration appeared, and the liver cells were swollen and deformed, indicating that the mice in the alcohol model group had developed alcoholic fatty liver and the liver tissue was severely damaged. However, the fatty degeneration of the liver tissue cells of mice in the group treated with Loderma longispora W9 was alleviated, the fat vacuoles were reduced, there was no obvious inflammatory infiltration, and the liver locks were radial. The pathological results were consistent with the results of liver and serum TC and TG indicators, indicating that Loderma longispora W9 can effectively improve liver fatty degeneration and inflammatory cell infiltration, and relieve alcoholic fatty liver and liver inflammation. (5) Effect of Loderma longispora W9 intervention on serum cytokine levels in mice induced by chronic alcohol

[0118] ELISA kits (Shanghai Enzyme-Linked Immunobiology Co., Ltd.) were used to detect the levels of IL-1β, TNF-a, IL-6, and IL-10 in serum.

[0119] Depend on Figure 5 As shown, long-term alcohol consumption significantly increased the levels of inflammatory factors IL-1β, TNF-α, and IL-6 in the mouse serum, while significantly decreasing IL-10 levels, indicating that long-term alcohol consumption leads to inflammation. However, serum cytokine levels were significantly restored in the mice treated with Loderma longisporum W9. This suggests that Loderma longisporum W9 can effectively alleviate alcohol-induced inflammation.

[0120] (6) Effects of Lodecanosa longispora W9 intervention on chronic alcohol-induced intestinal leakage and intestinal inflammation in mice

[0121] Long-term alcohol consumption not only causes irreversible damage to the liver but can also cause intestinal leakiness and intestinal inflammation. The toxic effects of alcohol continuously irritate the intestinal mucosa, gradually destroying the tight junctions between intestinal epithelial cells and increasing intestinal permeability. Various harmful substances and bacterial toxins can penetrate the intestinal barrier and enter the bloodstream, stimulating the body's immune response. Sustained immune system activation can trigger intestinal inflammation. Long-term intestinal inflammation can further impact overall health, interfere with nutrient absorption, weaken the body's immune system, and even lay the groundwork for other, more serious illnesses.

[0122] Depend on Figure 6 A shows that compared with the normal group, long-term drinking significantly increases the level of lipopolysaccharide (LPS) in serum, and Loderma longispora W9 can significantly inhibit the increase in LPS levels caused by alcohol. Moreover, compared with the normal group, the expression of intestinal tight junction genes in the alcohol model group (AC group) was significantly reduced; and compared with the AC group, after intervention with Loderma longispora W9, the expression level of intestinal tight junction genes in mice was significantly upregulated ( Figure 6 B).

[0123] The results of intestinal mucosal histological analysis were as follows Figure 7 As shown, chronic alcohol consumption causes the intestinal mucosal villi to partially break and detach, and the nuclei to become enlarged and rounded, mostly arranged in a single layer, with some stratified layers. The chromatin is largely vacuolated, with prominent nucleoli. The lamina propria is infiltrated with numerous chronic inflammatory cells. However, treatment with Lodzia longispora W9 significantly alleviated these symptoms, resulting in neatly arranged crypt cells, intact and smooth villi, and no obvious inflammatory infiltrates. These results demonstrate that Lodzia longispora W9 can significantly improve chronic alcohol-induced permeability and intestinal inflammation.

[0124] In summary, Lodrum longisporum W9 can significantly improve liver oxidative damage induced by chronic alcohol in mice, reduce liver lipid accumulation, and effectively alleviate liver and intestinal inflammation, achieving unexpected technical effects.

[0125] Example 6 Preparation Method of Loderma longisporum W9 Powder

[0126] Lodzia longispora W9 was inoculated at a 2% inoculum into 10 mL of liquid YPD medium and cultured in a constant temperature incubator at 25°C for 24 hours (first generation seed solution), thereby propagating to the second generation; the second generation seed was inoculated at a 2% inoculum into a 10 L fermenter containing liquid YPD medium, cultured for 48 hours, and the bacterial liquid was collected. The cells were centrifuged at 4000 rpm for 10 minutes, washed once with 0.9% physiological saline, and four times the amount of bacterial sludge was added to the protective agent and functional additive.

[0127] The bacteria were resuspended, freeze-dried, and vacuum-packed. The viable bacteria count in the obtained bacterial powder could reach 1×10 11 ~5×10 11 CFU / g.

[0128] Example 7 A probiotic fermented Chinese medicine preparation and its preparation method

[0129] A method for preparing a probiotic fermented Chinese medicine preparation comprises the following steps:

[0130] (1) Raw material pretreatment: Grind the Pueraria root and Hovenia dulcis fruit raw materials through an 80-mesh sieve, mix them in a mass ratio of 3:2, and soak them in 10 times the volume of water for 120 min;

[0131] (2) Extraction of raw materials: The pretreated raw materials were extracted at 80°C for 60 minutes, filtered to obtain extract I; the same amount of water as in step (1) was added to the filtered residue, the mixture was extracted at 80°C for 60 minutes, filtered to obtain extract II; the same amount of water as in step (1) was added to the filtered residue, the mixture was extracted at 80°C for 60 minutes, filtered to obtain extract III; the obtained extracts were combined and concentrated in a water bath to a concentration of 0.02 g / mL; and the Chinese medicine composite extract was obtained;

[0132] (3) Preparation of fermentation medium: Yeast powder and Lycium barbarum polysaccharide were added to the Chinese herbal medicine compound extract at a ratio of 16 g / L and 64 g / L, respectively, sterilized at 121°C for 20 min, and cooled to room temperature to obtain fermentation medium;

[0133] (4) Probiotic fermentation: Loderma longisporum W9 powder was inoculated into the fermentation medium at a ratio of 40 g / L, and fermented at a constant temperature of 37°C for 48 h. The solid matter was filtered to obtain the probiotic fermentation Chinese medicine preparation.

[0134] Example 8 Analysis of the physicochemical properties of probiotic fermented Chinese medicine preparations

[0135] 1. Soluble solids

[0136] Soluble solids content is an important indicator for evaluating food quality. It is also a parameter of food processing technology and can directly reflect the content of major nutrients in the product.

[0137] After testing, the soluble solid content of the probiotic fermented traditional Chinese medicine preparation provided by the present invention is 5.21%, which meets the product standards of beverages.

[0138] 2. Determination of viscosity

[0139] Viscosity is an important indicator for evaluating beverage quality and is tested using a viscometer.

[0140] The viscosity of the probiotic fermented traditional Chinese medicine preparation provided by the present invention is 1122.54 Pas, which is in line with the viscosity range of fermented fruit and vegetable juice.

[0141] 3. Determination of total acid and pH

[0142] Total acid and pH are important indicators for testing beverage quality. Testing the total acid and pH value of a beverage can determine whether its taste is suitable.

[0143] The probiotic fermented traditional Chinese medicine preparation provided by the present invention has a total acid content of 7.35 g / kg and a pH value of 3.85, and is sweet and sour with a good taste.

[0144] 4. Microbial content

[0145] The microbial indexes in the probiotic fermented Chinese medicine preparation provided by the present invention are measured as shown in Table 7 below. The content of the microbial indexes is reasonable and meets the product quality and safety standards.

[0146] Table 7 Microbial indicators in probiotic fermented Chinese medicine preparations

[0147]

[0148] Example 9 Effect of probiotic fermentation on active ingredients and antioxidant properties in traditional Chinese medicine preparations 1. Active ingredient content before and after fermentation

[0149] The contents of flavonoids and total phenols in the probiotic fermented Chinese medicine preparation prepared in Example 7 were respectively measured, and the fermentation medium prepared in step 3 of Example 7 was used as a control. The results are shown in Table 8.

[0150] Table 8 Comparison of active ingredient indicators before and after fermentation

[0151]

[0152] Note: The results are expressed as mean ± standard deviation, and different letters indicate significant differences (p < 0.05).

[0153] From the data in Table 8, it can be seen that the contents of flavonoids and total phenols in the probiotic fermented Chinese medicine preparation provided by the present invention reached 173.31 μg / mL and 112.06 μg / mL, respectively, which were increased by 51.84% and 31.20% compared with those before fermentation.

[0154] 2. Antioxidant function indicators before and after fermentation

[0155] The probiotic fermentation Chinese medicine preparation prepared in Example 7 was tested for DPPH and ABTS + , hydroxyl radical scavenging rate, and the fermentation medium prepared in step 3 of Example 7 was used as a control. The results are shown in Table 9.

[0156] Table 9 Comparison of antioxidant indicators before and after fermentation

[0157]

[0158] Note: The results are expressed as mean ± standard deviation, and different letters indicate significant differences (p < 0.05).

[0159] As shown in Table 9, the probiotic fermentation Chinese medicine preparation provided by the present invention has a significant effect on DPPH and ABTS. + The scavenging rates of hydroxyl free radicals reached 56.23%, 45.15% and 42.52% respectively, which were increased by 31.35%, 29.21% and 35.93% respectively compared with before fermentation, and the antioxidant effect was very significant.

[0160] Example 10 Analysis of the hangover-relieving efficacy of probiotic fermented Chinese medicine preparations

[0161] 1. Animal grouping and modeling

[0162] Forty-two healthy male KM mice weighing 22-25 g were purchased and randomly divided into six groups of seven mice each. After three days of adaptive feeding according to standard methods, they were fasted with or without water for 12 hours. The mice were weighed and marked with picric acid. The specific grouping and gavage method are shown in Table 10. The mice were observed, and the intoxication time (the time from gavage of alcohol to loss of righting reflex) and sobriety time (the time from loss of righting reflex to recovery) were recorded.

[0163] Table 10 Grouping and gavage of animals in different experimental groups

[0164]

[0165] 2. Experimental results

[0166] (1) Intoxication in mice

[0167] Table 11 Results of drunkenness of mice in each group

[0168]

[0169] Note: The results are expressed as mean ± standard deviation. Indicates significant difference (ρ<0.05), Indicates significant difference (ρ<0.01).

[0170] The results in Table 11 show that the model group mice had the shortest duration of intoxication and the longest sobering time. Compared with the model group, the fermentation group mice gavaged with the probiotic fermented Chinese medicine preparation had a 42.86% lower intoxication rate, a 92.33% longer duration of intoxication, and a 41.70% shorter sobering time. The sobering time of the fermentation group mice was 15.92% shorter than that of the positive group gavaged with the alcohol-detoxifying Jinzun tablet solution. This demonstrates that the probiotic fermented Chinese medicine preparation provided by the present invention has a highly significant alcohol-detoxifying effect, achieving unexpected technical benefits.

[0171] (2) Ethanol concentration in mouse serum

[0172] All mice were eyeballed and blood was collected. Serum was obtained by centrifugation at 3000 rpm for 10 min. Serum ethanol concentrations were measured in each group of mice. The results are shown in Table 12.

[0173] Table 12 Comparison of ethanol concentration in serum of mice in each group 2.5 hours after oral gavage

[0174]

[0175]

[0176] Note: The results are expressed as mean ± standard deviation, and different letters indicate significant differences (p < 0.05).

[0177] The results in Table 12 show that the model group mice had the highest ethanol concentration in their serum. The serum ethanol concentration in the fermentation group of mice given the probiotic fermented Chinese medicine preparation was 37.50% lower than that in the model group and 21.05% lower than that in the positive group given the alcohol-detoxifying Jinzun tablet solution. This demonstrates that the probiotic fermented Chinese medicine preparation provided by the present invention is highly effective in detoxifying alcohol.

[0178] 3. Aspartate aminotransferase and alanine aminotransferase levels in mouse serum

[0179] Alcohol administration can cause liver damage in mice, leading to elevated liver enzyme activity and the release of large amounts of aspartate aminotransferase (AST) and alanine aminotransferase (ALT) into the blood. Changes in serum ALT and AST activity can be used to assess the extent of liver damage in each experimental group.

[0180] Measurement method: The levels of aspartate aminotransferase (AST) and alanine aminotransferase (ALT) in serum samples of each group of mice were determined according to the instructions of the ALT and AST kits.

[0181] The results are as follows Figure 8 As shown, the ALT and AST levels in the serum of mice in the model group were the highest, indicating that when no intervention is given after oral administration of alcohol, there is a significant damage to the liver. Compared with the model group, the ALT and AST levels in the serum of the non-fermented group decreased after intervention, but the difference in ALT levels was not significant (P>0.05); the ALT and AST levels in the positive group decreased significantly compared with the model group (P<0.05), and the ALT and AST levels in the fermented group decreased extremely significantly compared with the model group (P<0.01). This shows that the probiotic fermented Chinese medicine preparation provided by the present invention can effectively alleviate alcohol-induced liver damage.

[0182] 4. Alcohol dehydrogenase (ADH) and acetaldehyde dehydrogenase (ALDH) levels in the liver

[0183] After the animals were sacrificed, the livers were removed by dissection and liver homogenates were prepared according to the method of Chen Yanyan (2022). The ADH and ALDH levels in the liver homogenates were determined according to the instructions of the ADH and ALDH kits.

[0184] The results are as follows Figure 9As shown, the ADH and ALDH levels in the livers of mice in the model group were the lowest, indicating that a large amount of alcohol-degrading enzymes were consumed in metabolism after oral administration of alcohol, resulting in a decrease in the levels of ADH and ALDH in the liver. Compared with the model group, the levels of ADH and ALDH in the liver of the non-fermented group increased significantly after intervention; the ADH and ALDH levels in the livers of mice in the positive group increased significantly compared with the model group (P < 0.05); and the ADH and ALDH levels in the livers of mice in the fermentation group increased extremely significantly compared with the model group (P < 0.001). This shows that the probiotic fermented Chinese medicine preparation provided by the present invention can significantly increase the level of alcohol-degrading enzymes in the liver.

[0185] 5. Liver tissue damage in mice

[0186] After fixation, the mouse liver was embedded in paraffin, sectioned, dewaxed, dried, and stained. The pathological changes of the liver tissue were observed under a 200× microscope and photographed.

[0187] The results are as follows Figure 10 As shown, the liver lobule structure of the blank group mice was clear, the hepatic cords were radial, the cell morphology was normal, and there was no dilation of the hepatic sinusoids and inflammatory infiltration. The cell boundaries of the model group disappeared, the cells were swollen, and inflammatory infiltration occurred, indicating that drinking alcohol can cause liver tissue damage in mice. Compared with the model group, the degree of liver cell damage in the positive group, the non-fermented group, and the fermented group mice was significantly reduced. Among them, the hepatic cords of the positive group were arranged neatly, and a small number of hepatocytes showed mild fatty degeneration; the liver tissue of the non-fermented group was rare with the portal area as the center, and a small number of hepatocytes around it showed mild fatty degeneration, and tiny round vacuoles were visible in the cytoplasm; while the liver cell structure of the fermented group was relatively complete, the shape of the hepatic cords was relatively clear, and no round vacuoles were seen in the cytoplasm. This shows that the probiotic fermented Chinese medicine preparation provided by the present invention can significantly reduce the damage of alcohol to liver tissue.

[0188] In summary, the probiotic fermentation Chinese medicine preparation provided by the present invention has greatly increased flavonoid and total phenol contents, enhanced antioxidant capacity, and significant alcohol-relieving effect. It can effectively reduce the damage of alcohol to liver tissue, and its effect is better than the alcohol-relieving medicine Jinzun tablets, and has broad application prospects.

Claims

1. A probiotic fermented Chinese medicine preparation, characterized in that: The Chinese medicine preparation is prepared by using Lodeyceps longisporus ( Lodderomyces elongisporus ) is prepared by fermenting Chinese herbal medicine extracts.

2. The Chinese medicine preparation according to claim 1, wherein The deposit number of the Lodeella longispora yeast is CGMCC No.31572.

3. The Chinese medicine preparation according to claim 2, wherein The traditional Chinese medicine extract is an extract of kudzu root and hovenia dulcis fruit.

4. The Chinese medicine preparation according to claim 3, wherein The preparation method of the Chinese medicine preparation comprises the following steps: (1) Pretreatment of Chinese medicinal materials: Grind the Pueraria root and Hovenia dulcis fruit through an 80-mesh sieve, mix them in a mass ratio of 3:2, and soak them in 10 times the volume of water for 120 min; (2) Extraction of Chinese herbal medicine: extract the pretreated Chinese herbal medicine raw material at 80°C for 60 min, filter, and obtain extract I; add the same amount of water as in step (1) to the filtered residue, extract at 80°C for 60 min, filter, and obtain extract II; add the same amount of water as in step (1) to the filtered residue, extract at 80°C for 60 min, filter, and obtain extract III; combine all the extracts, and concentrate in a water bath to obtain the Chinese herbal medicine extract; (3) Preparation of fermentation medium: Yeast powder and Lycium barbarum polysaccharide were added to the Chinese herbal medicine extract, sterilized at 121°C for 20 min, and cooled to room temperature to obtain the fermentation medium; (4) Probiotic fermentation: Lodzonella longisporum powder is inoculated into the fermentation medium in proportion, fermented at a constant temperature of 37°C for 48 hours, and the solid matter is removed by filtration to obtain the probiotic fermentation Chinese medicine preparation.

5. The Chinese medicine preparation according to claim 4, characterized in that The concentration of the Chinese herbal medicine extract in step (2) is 0.02 g / mL.

6. The Chinese medicine preparation according to claim 5, characterized in that The contents of yeast powder and wolfberry polysaccharide in the fermentation medium in step (3) are 16 g / L and 64 g / L, respectively.

7. The Chinese medicine preparation according to claim 6, characterized in that The amount of live bacteria in the Lodecanosa longispora powder in step (4) is not less than 10 11 CFU / g.

8. The Chinese medicine preparation according to claim 7, characterized in that The inoculation ratio of the Lodecanosa longispora powder in step (4) is 40 g / L.

9. Use of the Chinese medicine preparation according to any one of claims 1 to 8 in the preparation of a product having the effect of sobering up.

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