Milk tea, preparation method thereof, and application thereof in preventing alcoholic liver damage or sobering up

Abstract

The present invention relates to the field of food, and in particular to a milk tea and its preparation method and its application in preventing alcoholic liver damage or sobering up. The milk tea, by volume, comprises 35.6-40.6 parts of black tea extract, 60.9-64.9 parts of raw cow's milk and 1.0-1.2 parts of monk fruit concentrate; wherein, the preparation method of the black tea extract comprises the following steps: crushing the black tea, adding ultrapure water according to a material-liquid ratio of 1:25-30, extracting at 90-100°C, centrifuging to obtain a primary extraction supernatant and a precipitate; adding ethanol aqueous solution to the precipitate according to a material-liquid ratio of 1:25-30, extracting twice at 90-100°C, centrifuging to obtain a secondary extraction supernatant; combining the supernatants, concentrating to obtain the black tea extract. The milk tea of ​​the present invention prevents liver damage caused by AALI by promoting alcohol metabolism, reducing oxidative stress damage and alleviating liver inflammation.

Description

Technical Field

[0001] The present invention relates to the field of food, and in particular to milk tea, a preparation method thereof, and application of the milk tea in preventing alcoholic liver damage or sobering up. Background Art

[0002] Acute alcoholic liver injury (AALI) is a typical acute liver injury caused by a single large amount of alcohol. Its pathogenesis mainly includes three aspects: First, a large amount of alcohol is metabolized into acetic acid by alcohol dehydrogenase (ADH) and acetaldehyde dehydrogenase (ALDH) in the body, and is accompanied by the coenzyme NAD + The continuous consumption of intracellular NADH / NAD + An elevated ratio triggers the accumulation of reactive oxygen species (ROS). Furthermore, CYP2E1, a member of the cytochrome P450 enzyme family, is significantly induced under high alcohol consumption, further generating large amounts of ROS and initiating lipid peroxidation. Furthermore, ROS can activate the NF-κB signaling pathway, inducing the expression of pro-inflammatory cytokines such as TNF-α, IL-6, and IL-1β, exacerbating liver cell structural damage and dysfunction. Without timely intervention, alcoholic hepatitis can easily progress to liver fibrosis, or even liver failure.

[0003] Currently, the prevention and treatment strategies for ALD still primarily rely on pharmacological interventions, such as taurine and metadoxine mesylate. Although these drugs can alleviate alcohol-induced liver injury to a certain extent, they still have significant limitations in clinical application. Long-term use of taurine may cause side effects such as gastrointestinal discomfort, while metadoxine mesylate can improve lipid metabolism and oxidative stress in chronic ALD models, it is less effective in acute models and may cause toxic reactions due to increased metabolic burden on the liver.

[0004] Prior art CN108720013A discloses a health food composed of fructose and tea extract, which has the effect of sobering up and protecting the liver. The formula contains a high proportion of crystalline fructose of 85-95%, and tea extract of 3-15%. Fructose can accelerate the metabolism of alcohol in the body and promote its rapid excretion through the kidneys. Tea extract can enhance the activity of alcohol dehydrogenase and accelerate the metabolic conversion of alcohol. At the same time, tea extract can inhibit the reabsorption of alcohol by the renal tubules, promote the excretion of alcohol, and rapidly reduce the alcohol concentration in the body, thereby producing a rapid sobering effect. Tea extract also has a preventive effect on hyperuricemia that may be caused by fructose. Good results have been achieved in animal experiments and human applications. However, this existing technology is mainly based on a high proportion of crystalline fructose (85-95%), combined with tea extract to achieve the effects of sobering up and protecting the liver. Although it can accelerate the metabolism and excretion of alcohol to a certain extent, long-term or high-dose intake of fructose poses potential metabolic health risks, increases the lipid burden on the liver, and induces metabolic diseases such as fatty liver, obesity and hyperuricemia. It also has limited effect in regulating oxidative stress and inflammatory responses, and has limited effect on preventing alcoholic liver damage. Summary of the Invention

[0005] The present invention aims to provide a milk tea with black tea extract, raw milk and monk fruit concentrated juice as raw materials, a preparation method thereof and application of the milk tea in preventing alcoholic liver damage or sobering up.

[0006] In order to achieve the above object, the technical solution adopted by the present invention is:

[0007] A milk tea comprising, by volume, 35.6-40.6 parts of black tea extract, 60.9-64.9 parts of raw milk, and 1.0-1.2 parts of monk fruit concentrated juice;

[0008] The preparation method of the black tea extract comprises the following steps: crushing the black tea, adding ultrapure water at a material-liquid ratio of 1:25-30, extracting at 90-100°C, centrifuging to obtain a primary extraction supernatant and a precipitate; adding an ethanol aqueous solution to the precipitate at a material-liquid ratio of 1:25-30, extracting for the second time at 90-100°C, centrifuging to obtain a secondary extraction supernatant; combining the supernatants, concentrating, and obtaining the black tea extract.

[0009] This invention utilizes black tea, raw milk, and monk fruit concentrate to create a functional milk tea formula, aiming to balance the beverage's flavor structure and health benefits. Black tea is rich in bioactive ingredients such as tea polyphenols, theaflavins, and thearubigins, and possesses significant antioxidant and anti-inflammatory properties. Compared to industrially processed milk powder, raw milk retains a more complete natural nutritional structure, with a higher protein content and abundant whey protein and fat-soluble antioxidants, which help repair liver cell membranes, enhance antioxidant defenses, and strengthen tissue repair capacity. Mogroside V, a natural low-calorie sweetener, offers high sweetness and low calories. While satisfying the body's daily sweetness needs, it also scavenges free radicals and alleviates oxidative stress. Therefore, through experiments, the present invention optimized the preparation process for black tea extract. Combined with comprehensive sensory evaluation, the optimal ratio of black tea, raw milk, and monk fruit concentrate was determined, and the ratio of milk tea ingredients was systematically optimized to achieve an optimal formula with harmonious flavor, stable structure, and ease of commercialization. Based on the AALI mouse model, the preventive effect of formula milk tea on AALI was studied.

[0010] According to the embodiments of the present invention, the present invention can be further optimized. The following is the technical solution formed after optimization:

[0011] In one preferred embodiment, the milk tea comprises 35.6-40.6 parts of black tea extract, 60.9-64.9 parts of raw milk and 1.2 parts of monk fruit concentrated juice, by volume.

[0012] In one preferred embodiment, the milk tea comprises 40.6 parts of black tea extract, 64.9 parts of raw milk and 1.2 parts of monk fruit concentrated juice.

[0013] In one preferred embodiment, the volume concentration of mogroside V in the monk fruit concentrated juice is 1.8%-3.5%, the sugar content is 50-60%, and the sweetness is 1.5-1.8 times that of sucrose.

[0014] In one preferred embodiment, the centrifugal speed is 3500-4000 r / min and the time is 10-15 min.

[0015] In one preferred embodiment, the volume concentration of the ethanol aqueous solution is 55%-65%, preferably 60%.

[0016] In one preferred embodiment, the material-liquid ratio is 1:25-27; and the extraction temperature is 95-100°C.

[0017] In a preferred embodiment, the time for the first extraction and the second extraction is 30-40 minutes.

[0018] Based on the same inventive concept, the present invention also claims protection for a method for preparing the milk tea, comprising the following steps:

[0019] S1. Adding raw milk to the black tea extract and stirring evenly to obtain tea milk;

[0020] S2. Slowly adding the concentrated Momordica grosvenori juice into the tea milk, stirring evenly, and sterilizing at high temperature to obtain the milk tea.

[0021] Based on the same inventive concept, the present invention also claims protection for the use of the milk tea in preparing a reagent for preventing alcoholic liver damage.

[0022] Based on the same inventive concept, the present invention also claims protection for the use of the milk tea in preparing a hangover-relieving agent.

[0023] Therefore, the milk tea prepared by the present invention has a significant anti-intoxication and liver-protecting effect on mice with acute alcoholic liver injury. Experimental results show that the milk tea prepared by the present invention effectively alleviated the pathological damage of liver tissue in mice with acute alcoholic liver injury after 28 days of intervention with mice, enhanced the activities of key alcohol metabolism enzymes such as alcohol dehydrogenase (ADH) and acetaldehyde dehydrogenase (ALDH) in the liver, reduced the activities of aspartate aminotransferase (AST) and alanine aminotransferase (ALT) as well as the content of triglyceride (TG), accelerated the clearance of blood ethanol and acetaldehyde, and promoted alcohol metabolism; in addition, the milk tea prepared by the present invention enhanced the activities of catalase (CAT), superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) in the liver, and inhibited the production of malondialdehyde (MDA) and reactive oxygen species (ROS). It can also reduce the levels of proinflammatory cytokines such as interleukin-1β (IL-1β), interleukin-6 (IL-6), and tumor necrosis factor-α (TNF-α) to alleviate acute alcohol-induced liver inflammation.

[0024] In summary, the milk tea of ​​the present invention reduces liver damage caused by AALI by promoting alcohol metabolism, reducing oxidative stress damage, and alleviating liver inflammation, providing a theoretical basis for developing daily prevention strategies for alcoholic liver disease. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 This is the appearance of black tea soup obtained by different extraction processes.

[0026] Figure 2 This is a curve chart showing the effects of different tea-to-milk ratios on sensory evaluation.

[0027] Figure 3 This is a curve chart showing the effects of different amounts of monk fruit concentrate added on sensory evaluation.

[0028] Figure 4 is the mutual indicator result diagram of AALI mouse model; Figure 4 A is the H&E staining image of the AALI mouse model. Figure 4 B is a bar graph of serum AST levels in AALI mouse model. Figure 4 C is a bar graph of serum ALT levels in AALI mouse model. Figure 4 D is a bar graph of liver TG content in the AALI mouse model.

[0029] Figure 5 This is the result of milk tea alleviating liver pathological damage in AALI mice; Figure 5 A is the H&E staining image of mice in each group. Figure 5 B is a bar graph showing the liver tissue damage scores of mice in each group.

[0030] Figure 6 The effect of milk tea on the activity of enzymes related to alcohol metabolism and ethanol in the serum of AALI mice; Figure 6 A is a bar graph of serum TG levels in each group of mice. Figure 6 B is a bar graph of serum AST activity in each group of mice. Figure 6 C is a bar graph of the serum ALT levels of mice in each group. Figure 6 D is the bar graph of serum ADH activity of mice in each group. Figure 6 E is a bar graph of serum ALDH activity in each group of mice. Figure 6 F is a bar graph of the serum ethanol content of mice in each group. Figure 6 G is a bar graph of the serum acetaldehyde levels of mice in each group.

[0031] Figure 7 The effect of milk tea on oxidative stress and inflammatory response in the liver tissue of AALI mice; Figure 7 A is a bar graph of CAT content in each group of mice. Figure 7B is a bar graph of the GSH-Px content of mice in each group. Figure 7 C is a bar graph of the MDA content of mice in each group. Figure 7 D is the bar graph of the SOD levels of mice in each group. Figure 7 E is a bar graph of the ROS content in each group of mice. Figure 7 F is the bar graph of TNF-α content in each group of mice, Figure 7 G is a bar graph of IL-1β content in each group of mice. Figure 7 H is a bar graph of the IL-6 content in each group of mice.

[0032] Figure 8 The effect of milk tea on the expression of alcohol metabolism enzyme gene mRNA in the liver of AALI mice; Figure 8 A is a bar graph of the ADH6 gene levels in each group of mice. Figure 8 B is a bar graph of the ALDH1B1 gene levels in each group of mice. Figure 8 C is a bar graph of the ALDH2 gene levels in each group of mice. Figure 8 D is the bar graph of the CAT gene levels of mice in each group. Figure 8 E is a bar graph of the CYP2E1 gene levels in each group of mice. Figure 8 F is a bar graph showing the ACSS1 gene levels in each group of mice.

[0033] Figure 9 The effect of milk tea on the mRNA expression of TNF-α / NF-κB pathway related genes in the liver of AALI mice; Figure 9 A is a bar graph of the IL-1β gene levels in each group of mice. Figure 9 B is a bar graph of IL-6 gene levels in each group of mice. Figure 9 C is a bar graph of the NF-kBp65 gene levels in each group of mice. Figure 9 D is a bar graph showing the TNF-α gene levels in each group of mice.

[0034] Figure 10 The effect of milk tea on the expression of alcohol metabolism enzyme-related proteins in the liver of AALI mice; Figure 10 A is a bar graph of the ADH6 protein levels in each group of mice. Figure 10 B is a bar graph of the ALDH1B1 protein levels in each group of mice. Figure 10 C is a bar graph showing the ALDH2 protein levels in each group of mice. Figure 10 D is the bar graph of CAT protein levels in each group of mice. Figure 10 E is the bar graph of CYP2E1 protein levels in each group of mice, Figure 10 F is a bar graph showing the ACSS1 protein levels in each group of mice.

[0035] Figure 11 The effect of milk tea on the expression of TNF-α / NF-κB pathway related proteins in the liver of AALI mice; Figure 11 A is a bar graph of IL-1β-related protein levels in each group of mice. Figure 11 B is a bar graph of IL-6 related protein levels in each group of mice. Figure 11 C is a bar graph showing the levels of NF-kBp65 related proteins in each group of mice. Figure 11 D is a bar graph showing the levels of TNF-α-related proteins in each group of mice. DETAILED DESCRIPTION

[0036] The present invention is not limited to the following specific embodiments. Based on the disclosure of the present invention, a person skilled in the art may adopt a variety of other specific embodiments to implement the present invention. Any simple changes or modifications made to the design structure and concept of the present invention fall within the scope of protection of the present invention. It should be noted that the embodiments and features of the embodiments of the present invention may be combined with each other unless they conflict.

[0037] The black tea of ​​this invention was provided by Hunan Shimen Xiufeng Famous Tea Co., Ltd. (one bud, two leaves, autumn black tea). Raw cow milk was provided by Hunan Deren Animal Husbandry Co., Ltd. (freshly squeezed and unprocessed, based on Chinese Holstein cattle, with a total protein content of approximately 3.2%–3.5% and a whey protein content of 0.63%), Changsha, China. Monk fruit concentrate was provided by Hunan Huacheng Biological Resources Co., Ltd. (yellow concentrate, with a mogroside V content of 1.8%–3.5%, 1.5 times the sweetness of sucrose). Food-grade ethanol solution (95% ethanol) was purchased from Hunan Kelun Pharmaceutical Co., Ltd. (Hunan, China). This invention specifically emphasizes that to ensure the applicability and consistency of the optimized results, all sensory evaluations were performed on the finished product after the samples were sterilized at standard high temperature (121°C for 15 minutes) and cooled. The sensory evaluations reflect the true quality of the end product. And since this study uses beverage products as the target carrier, the raw milk is not treated with traditional dairy pasteurization (72–85°C), but is instead treated with overall high-temperature sterilization (121°C) to ensure the safety and stability of the finished formula.

[0038] Experimental design and data analysis were performed using Design Expert 13.0 software. To mitigate the influence of extreme values, the highest and lowest scores were removed and the average calculated. The scoring criteria were based on the national standard GB / T 21733-2008, "Tea Beverages," with appropriate adjustments based on the characteristics of the present invention.

[0039] The experimental data were fitted with a quadratic polynomial regression model to describe the effects of various factors and their interactions on sensory scores. The model expression is as follows:

[0040]

[0041] Where Y represents the predicted sensory score, A, B, and C represent the coded values ​​of the three independent variables. β0 is the constant coefficient; β i is the linear coefficient; β ii is the quadratic coefficient, β ij is the interaction term coefficient.

[0042] All data are expressed as mean ± standard deviation (mean ± SD). Statistical analysis was performed using SPSS Statistics software (version 19.0, SPSS Inc., Chicago, USA). For data that passed the homogeneity of variance test, one-way analysis of variance (ANOVA) combined with Duncan's test and the least significant difference (LSD) multiple comparison method was used to analyze differences between groups. Different letters indicate statistically significant differences between groups (P < 0.05); identical letters indicate no statistically significant differences (P > 0.05). Tissue sections were viewed and analyzed using CaseViewer software (3DHISTECH, Budapest, Hungary), and graphs were drawn using GraphPad Prism software (version 8.0.2; GraphPad Software, La Jolla, USA).

[0043] Example 1

[0044] The tea leaves (autumn black tea) were crushed to obtain tea powder, ultrapure water was added according to the ratio of tea to water (mass ratio), the mixture was stirred evenly, and then placed in a preheated water bath for extraction with continuous stirring. The mixture was centrifuged (4000 r / min, 15 min) to obtain a primary extraction supernatant and a precipitate. An ethanol-water solution was added to the precipitate according to the ratio of tea to water (mass ratio), the mixture was extracted again, and the mixture was centrifuged (4000 r / min, 15 min) to obtain a secondary extraction supernatant and a precipitate. The above two supernatants (the primary extraction supernatant and the secondary extraction supernatant) were combined and filtered, concentrated by rotary evaporation, and filtered to obtain a black tea soup, which was stored at -20°C.

[0045] The effects of material-to-water ratio, extraction temperature, ethanol concentration, and extraction time on the quality of black tea liquor were investigated. Sensory scores (aroma, liquor color, and flavor) were used as evaluation indicators. The evaluation indicators are shown in Table 1.

[0046] The following extraction parameter combinations (Table 2) were set to determine the optimal extraction process for black tea.

[0047] The sensory evaluation results of black tea extract by 30 normal people with normal sense of smell and taste and of different ages are shown in Table 3.

[0048] The sensory evaluation results of black tea soup obtained by different extraction parameters were verified experimentally, as shown in Table 3.

[0049] To further validate the functional scientificity of the optimized black tea extraction process, we used CCK-8 (Novizen, A311) to examine its protective effects against ethanol-induced injury in mouse hepatocytes (AML-12) using the mouse hepatocyte model. Black tea extracts were obtained according to the extraction conditions in Table 3, followed by freeze-drying. Extracts were uniformly prepared at a concentration of 100 μg / mL and sterilized through a 0.22 μm filter. After pre-treating cells (co-cultured with cells) for 2 hours, 100 mM ethanol was added and incubated for 24 hours. Subsequently, 10 μL of CCK-8 reagent was added to each well, shaken, and the cells were incubated in a dark incubator for 2 hours. Absorbance was read at 450 nm. The absorbance of the blank control (cultured with culture medium only) was subtracted from the absorbance of all treatment groups. The final cell viability test results are shown in Table 4.

[0050] The results showed that compared with the normal group, cell viability was significantly reduced after ethanol treatment, while cell viability was significantly improved after intervention with black tea using different extraction processes. Among them, the extraction method with a material-water ratio of 1:25, an extraction time of 30 minutes, an ethanol concentration of 60%, and an extraction temperature of 100°C was the most superior in improving cell viability.

[0051] The optimal extraction process combination is: material-water ratio of 1:25, extraction temperature of 100℃, ethanol concentration of 60% (v / v), and extraction time of 30min. The experimental results show that the material-water ratio is a key factor affecting the quality of tea soup. A too low material-water ratio will result in a light color and a light tea flavor, affecting the overall flavor performance; while a too high material-water ratio can enhance the color, aroma and flavor, but at the same time cause the astringency to increase, reducing the overall sensory score. Temperature, ethanol concentration and time also have an important influence on the extraction effect: within a certain range, as the temperature increases, the extraction time prolongs and the ethanol concentration increases, the color of the tea soup gradually deepens and the tea flavor enhances, but at the same time it also enhances the bitterness ( Figure 1 ).

[0052] The composition of the extract obtained by the optimal extraction process was analyzed, and the contents of each component in the water extract were as follows: tea polyphenols, theaflavins, soluble sugars and free amino acids reached 43.05%, 14.26%, 1.5%, 5.8% and 2%, respectively.

[0053] We also compared the sensory properties of black tea extracted by the following two extraction methods:

[0054] Method B (referring to prior art CN117442670A): Tea leaves (black tea) are ground to obtain tea powder. 10 volumes of ultrapure water (dry weight) are added, stirred, and then placed in a preheated 90°C waterbath for 2 hours with constant stirring. The mixture is centrifuged (5000 rpm, 10 minutes) to obtain a primary extraction supernatant and a precipitate. Eight volumes of ultrapure water are added to the precipitate, and the mixture is again extracted for 2 hours. The mixture is centrifuged (5000 rpm, 10 minutes) to obtain a secondary extraction supernatant and a precipitate. The two supernatants (primary and secondary extraction supernatants) are combined, filtered, concentrated by rotary evaporation, and filtered to obtain a tea extract. The contents of tea polyphenols, theaflavins, soluble sugars, and free amino acids in the water extract are 41.56%, 12.56%, 1.3%, 6.02%, and 3.62%, respectively.

[0055] Method C (referring to prior art CN108714164A): Take an appropriate amount of tea leaves (autumn black tea) and add a 60% ethanol-water solution (solid-to-liquid ratio of 1:25). Reflux extraction is performed for 2 hours at 80-100°C. Concentration and filtration are then performed to obtain a black tea extract. The contents of the components in the aqueous extract are: tea polyphenols, theaflavins, soluble sugars, and free amino acids are 37.54%, 11.67%, 0.17%, 3.49%, and 3.53%, respectively.

[0056] The sensory score of the black tea soup obtained by method B was 82.2, and the sensory score of the black tea soup obtained by method C was 83.8.

[0057] Example 2

[0058] The black tea soup prepared under the optimal experimental conditions of Example 1 and a certain proportion of raw cow's milk and monk fruit concentrated juice are mixed in sequence. First, a certain amount of black tea soup is placed in a clean mixing container; then a certain amount of raw cow's milk is slowly added, while continuously stirring at a uniform speed to fully blend it to avoid protein coagulation or stratification; after the mixed liquid is uniform, a certain amount of monk fruit concentrated juice is added dropwise, and stirring is continued until the system is stable and consistent without stratification or precipitation, thereby obtaining a functional milk tea product. During the entire mixing process, stirring is maintained at about 300 rpm for 2-3 minutes to ensure that each component is fully homogenized and the taste is coordinated and consistent, and milk tea is prepared. The preparation process is as follows:

[0059] Tea soup → add raw milk → add monk fruit concentrated juice → high temperature sterilization → milk tea is finished.

[0060] Key points: (1) Blending tea and milk: Pour the prepared black tea into a flask, add fresh raw milk of the day, and stir evenly with a glass rod. (2) Adding sugar substitute to adjust flavor: Use a pipette to draw an appropriate amount of monk fruit concentrate juice, slowly add it to the mixed tea and milk, and stir evenly with a glass rod. (3) High-temperature sterilization: Place the prepared milk tea into a high-temperature sterilizer and sterilize at 121℃ for 15 minutes to obtain the finished milk tea.

[0061] Based on this, we studied the effects of varying the volume ratio of black tea extract to raw milk (1:3, 1:2, 2:3, 1:1, and 2:1) and the amount of monk fruit concentrate added (0.5ml, 1ml, 1.5ml, 2ml, and 2.5ml) on the sensory evaluation of milk tea (aroma, soup color, and flavor; Table 5). This approach helped determine the optimal addition range for the main ingredients. Subsequently, we established a formula based on the amount of black tea, raw milk, and monk fruit concentrate added (Table 6). The sensory evaluation of milk tea was based on a comprehensive assessment of color, aroma, flavor, texture, and mouthfeel, reflecting the overall product acceptance.

[0062] The results are as follows Figure 2 and Figure 3 As shown in the figure, with the total solution volume held constant at 100 mL, the sensory score of milk tea initially increased and then decreased with increasing ratios of black tea extract to raw milk. The highest sensory score was achieved at a ratio of 2:3 (black tea extract:raw milk). Below 2:3, the milk flavor was overpowering and the tea flavor was insufficient. Above 2:1, the milk flavor was less pronounced and the astringency increased, affecting taste harmony. Overall, the 2:3 ratio demonstrated the best flavor harmony and sensory acceptance, and was therefore selected for subsequent optimization experiments on the addition level of monk fruit juice concentrate. Further single-factor experiments explored the effect of monk fruit juice concentrate addition on the sensory quality of milk tea. As the addition level increased, the sweetness of the milk tea gradually increased, and the sensory score also showed an initial upward trend followed by a downward trend. The highest sensory score was achieved at an addition level of 1.5 mL / 100 mL. When the added amount is less than 1.2mL / 100mL, the sweetness is insufficient and the flavor of the milk tea is not harmonious; when it is higher than 1.6mL / 100mL, the sweetness is too strong and accompanied by a bitter aftertaste, affecting the overall taste.

[0063] Based on this, a total of 15 combination schemes were set up to explore the effects of the amount of tea soup added, the amount of raw milk added, and the amount of monk fruit concentrated juice added on the sensory score of milk tea (Table 7).

[0064] To further validate the differences in the effects of different milk tea ratios in an ethanol-induced liver injury model, evaluate their potential for preventing alcoholic liver injury, and support the selection of optimal sensory formulations, the present invention established an ethanol-induced cell injury model using mouse hepatocyte AML-12 cells based on 14 optimized milk tea ratios. The CCK-8 assay was used to evaluate the effects of different milk tea ratios on cell viability, identifying the optimal formulation that combines both palatability and liver-protective properties. Each milk tea group was prepared according to the 15 combinations listed in Table 8. 100 ml of milk tea was extracted from each group, sterilized through a 0.22 μm filter, and diluted to a uniform concentration of 100 μg / mL (calculated based on tea polyphenol content) as the experimental concentration. Cells were seeded in 96-well plates and pretreated with the milk tea extracts from each group for 2 hours after adherence. The cells were then incubated with 100 mM ethanol for 24 hours. The untreated group was used as the normal control and the ethanol group was used as the model group. Finally, the cell viability was detected by CCK-8 reagent, the absorbance value was read at a wavelength of 450nm, and the synergistic index method was used to determine whether the three raw material components had a synergistic effect in intervening in cell viability.

[0065] .

[0066] in, CI <10.9 indicates synergistic effect, 0.9≤ CI ≤1 has an additive effect, CI >1 indicates antagonistic effects, and D1, D2, and D3 represent the amounts of the three ingredients (black tea, raw milk, and monk fruit concentrate) in the actual mixture. 1、 Dx 2、 Dx3 is the dose required to achieve the same effect as when the ingredient is used alone. The results are shown in the table below.

[0067] The results showed that cell viability in the ethanol group decreased significantly. Among the 15 milk tea ratios, most combinations had varying degrees of protective effects on cells. A significant synergistic effect was observed when the black tea soup was added at a ratio of 35.6-40.6, the raw milk was added at a ratio of 60.9-64.9, and the monk fruit concentrate was added at a ratio of 1.0-1.2. The most effective group, group 12 (black tea soup: raw milk: monk fruit concentrate = 40.6:64.9:1.2), showed the most significant protective effect and exhibited a strong synergistic effect. However, some formulas, such as groups 14 and 15, although composed of the same three components, did not exhibit a synergistic effect compared to the individual components. Therefore, based on the sensory scores and cell viability, the optimal formula volume ratio was obtained as follows: black tea soup: raw milk: monk fruit concentrate juice = 40.6:64.9:1.2. This ratio was used to conduct three parallel experiments to verify the production of milk tea. The test was repeated three times, and the product sensory score was 89.7-89.9 points.

[0068] The milk tea prepared above was concentrated using a rotary evaporator set at 40°C and 110 rpm. The evaporator was stopped when the volume was reduced to 1 / 12.3 of its original volume. The resulting concentrate was used as the sample for subsequent experiments. This concentration factor was based on the human-animal dose conversion formula recommended by the US Food and Drug Administration (FDA). According to this formula, when converting the human equivalent dose to the mouse equivalent dose, a conversion factor of approximately 12.3 must be multiplied.

[0069] Key points:

[0070] The initial water temperature of the water bath should be below 10°C, the initial temperature of the rotary evaporator should be set to 20°C, and the water bath temperature should be gradually increased with a temperature gradient of 5°C to an end point of 40°C.

[0071] When the water temperature in the rotary evaporator rises to a certain temperature, there will be obvious boiling and bubbling. At this time, immediately turn off the instrument and loosen the air valve to release the air to prevent the instrument from sucking back. Continue rotary evaporation until the sample no longer boils and temporarily maintain the device temperature at the current setting. Repeat this 2-3 times until the sample no longer boils and bubbles, then continue to increase the device temperature setting.

[0072] Example 3

[0073] In this example, the enzyme activity levels of AST (C010-2-1) and ALT (C009-2-1) in mouse serum were determined according to the instructions of the kit provided by Nanjing Jiancheng Bioengineering Institute (Nanjing, China).

[0074] The activities of ADH (ADH-1-W) and ALDH (ALDH-1-G) in mouse serum were determined according to the kit instructions of Suzhou Keming Biotechnology Co., Ltd. (Jiangsu, China).

[0075] The ethanol (EtOH, ADS-W-FM030-48) content in mouse serum was determined according to the kit instructions of Jiangsu Addison Biotechnology Co., Ltd. (Jiangsu, China).

[0076] The acetaldehyde (K-ACHYD) content in mouse serum was determined according to the kit instructions of Megazyme Biotechnology Co., Ltd., Ireland.

[0077] The levels of TG, SOD, CAT, GSH-Px, MDA, and ROS in mouse liver tissue were determined according to the kit instructions of Nanjing Jiancheng Bioengineering Institute (Nanjing, China).

[0078] The levels of TNF-α, IL-6, and IL-1β in mouse liver tissues were determined using ELISA kits purchased from Wuhan Huamei Bioengineering Co., Ltd. (Hubei, China).

[0079] Data were initially processed using Excel. Analysis was performed using IBM Statistics SPSS (version 19.0, SPSS, Inc., Chicago, IL, USA). Results are presented as mean ± standard deviation (mean ± SD), and bar graphs were created using GraphPad Prism (version 9.5.1; La Jolla, CA, USA). Western blot analysis was performed using ImageJ software. Statistical significance was defined as P < 0.05 and P < 0.01 as extremely significant.

[0080] The experimental steps are as follows:

[0081] 1. Construction of a mouse model of acute alcoholic liver injury

[0082] Five-week-old SPF-free C57BL / 6J male mice were purchased from Hunan Slake Jingda Laboratory Animal Co., Ltd. (Hunan, China; License No. SCXK (Xiang) 2019-0004). All animal experiments were performed in accordance with the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health (NIH Publication No. 8023, revised 1978). Mice were housed in an environment with a temperature of 25°C ± 1°C, a humidity of 55% ± 5%, and a 12-h light / dark cycle.

[0083] After a one-week acclimation period, 20 mice were randomly divided into a blank control group (CK group) and a model group (MOD group), with 10 mice in each group. Mice in the MOD group were gavaged once with 50% edible alcohol (12 mL / kg body weight) at a dose of 0.01 mL per gram of body weight; mice in the CK group were gavaged with an equal volume of normal saline. Six hours after gavage, mice were anesthetized with an intraperitoneal injection of 2% pentobarbital. Eyeballs were removed and blood was collected. Blood was centrifuged at 3000 rpm and 4°C for 10 minutes. The supernatant serum was collected and stored at -80°C for subsequent analysis of serum markers. Serum AST (C010-2-1) and ALT (C009-2-1) enzyme activities were determined according to the kit instructions provided by Nanjing Jiancheng Bioengineering Institute (Nanjing, China). Serum ADH (ADH-1-W) and ALDH (ALDH-1-G) activities were determined according to the kit instructions provided by Suzhou Keming Biotechnology Co., Ltd. (Jiangsu, China). Liver tissue was obtained from both groups of mice. After removal, the largest lobe was fixed in 4% paraformaldehyde for observation of liver pathological changes. Liver histopathological examination allows direct observation of hepatocyte morphology, histological structure, inflammatory activity, degree of fibrosis, and abnormal material deposition, visualizing the extent of liver pathological damage in each experimental group. The left lobe was fixed in 4% (v / v) paraformaldehyde for 48 hours and then embedded in paraffin blocks. Liver tissue sections were examined under a light microscope using H&E staining (Eclipse Ci-L, Nikon, Japan). The remaining liver was washed with 0.9 wt% NaCl, wrapped in tin foil, fixed in liquid nitrogen, and stored at -80°C for subsequent preparation of liver homogenates. Triglyceride levels in mouse liver tissue were measured according to the kit instructions provided by Nanjing Jiancheng Bioengineering Institute (Nanjing, China). If the serum transaminase (ALT, AST) activities of mice in the MOD group are significantly increased, and the liver tissue shows pathological characteristics such as color change, softening and swelling, it is confirmed that the acute alcoholic liver injury model has been successfully established.

[0084] After the alcohol infusion was completed, the mice were dissected and the livers were taken for hematoxylin-eosin (H&E) staining. Figure 4 As shown in the figure, the liver tissue sections of mice showed that the liver cells of CK group were uniform in size, neatly arranged, and intact in structure, without fatty degeneration and inflammatory cell infiltration. However, MOD liver tissue showed more ballooning of hepatocytes, cytoplasmic vacuoles (yellow arrows), fatty degeneration of hepatocytes, and tiny round vacuoles in the cytoplasm (green arrows). Hepatocytes also showed watery degeneration, cell swelling, and loose and lightly stained cytoplasm (black arrows). Occasionally, small focal infiltration of neutrophils was seen (red arrows). The liver plates were irregularly arranged, and the hepatic sinusoids were obviously squeezed ( Figure 4A). In addition, compared with the CK group, the serum ALT and AST activities and the TG content in the liver of the mice in the MOD group were significantly increased (P<0.05; Figure 4 B, 4C, and 4D), indicating that the AALI model was successfully constructed.

[0085] 2. Animal Experiment Design

[0086] For this experiment, 100 five-week-old C57BL / 6J male mice weighing 18-22 g were purchased from Hunan Slake Jingda Experimental Animal Co., Ltd. (Hunan, China). All mice were acclimated under the same environmental conditions for one week and then divided into 10 groups (10 mice per group) according to their body weight: blank control group (CK group), model group (MOD group), black tea group (BT group), raw milk group (FM group), monk fruit concentrated juice group (MOG group), low-dose milk tea group (LMT group), medium-dose milk tea group (MMT group), high-dose milk tea group (HMT group), milk tea group B (BMT group), and milk tea group C (CMT group).

[0087] The black tea used in the high-dose milk tea B group was concentrated from the black tea soup extracted by method B in Example 1, and the remaining components of the milk tea were the same as the optimal formula.

[0088] The black tea used in the high-dose milk tea C group was concentrated from the black tea soup extracted by method C in Example 1, and the remaining components of the milk tea were the same as the optimal formula.

[0089] Except for normal saline, the gavage materials used in the other treatment groups (black tea, raw milk, and monk fruit concentrated juice) were consistent with the dosage of each component in the high-dose milk tea group (HMT group), and all were concentrated 12.3 times based on the original volume and gavage-fed continuously for 28 days at a dose of 0.01 mL / g body weight.

[0090] During the gavage treatment period, mice in the CK and MOD groups were gavage-fed with an equal volume of normal saline daily; mice in the BT group were gavage-fed with black tea extract; mice in the FM group were gavage-fed with fresh, untreated raw cow's milk; and mice in the MOG group were gavage-fed with monk fruit concentrated juice. The LMT, MMT, and HMT groups were gavage-fed with different doses of the optimal formula milk tea of ​​Example 1 of the present invention. The MMT and HMT groups were gavage-fed with milk tea prepared by concentrating the LMT group's milk tea by 1 and 2 times, respectively, at the same ingredient ratio. The BMT and CMT groups were gavage-fed with milk tea prepared by concentrating the black tea extracts extracted by methods B and C by 12.3 times, respectively.

[0091] This study used the conversion formula recommended by the US Food and Drug Administration (FDA) to convert the human equivalent dose to the mouse dose, with a conversion ratio of approximately 12.3 times. Therefore, except for normal saline, all gavages used in the remaining treatment groups were concentrated 12.3 times above the original human dose recommendation and administered continuously for 28 days at a dose of 0.01 mL / g body weight. On day 29, mice in all groups except the CK group were gavage-administered 12 mL / kg body weight of 50% edible alcohol to establish the AALI model; the CK group was gavage-administered with an equal volume of normal saline.

[0092] Each mouse was weighed regularly once a week. Six hours after gavage on the 29th day, the mice were anesthetized with an intraperitoneal injection of 2% sodium pentobarbital solution, and their eyes were removed for blood collection. The blood samples were allowed to rest at room temperature (25 ± 2°C) for 2 hours, then centrifuged at 3000 rpm for 10 minutes at 4°C to separate the serum, which was then stored at -80°C for subsequent analysis of serum alcohol metabolism-related enzyme activity indicators. Subsequently, liver samples were removed from the mice, washed with 0.9% NaCl, wrapped in tin foil, fixed in liquid nitrogen, and transferred to a -80°C freezer for subsequent storage.

[0093] 3. Liver function and liver histopathological analysis

[0094] Three mice were randomly selected from each group. The left lobe of the liver was fixed with 4% paraformaldehyde for 48 hours, then embedded in paraffin. Tissue sections were prepared and stained using the H&E method. An optical microscope (Eclipse Ci-L, Nikon, Japan) was used to observe liver histological changes and evaluate cell morphology, structural integrity, fatty degeneration, and inflammatory cell infiltration (Table 9) to visually reflect the degree of pathological damage in the liver of each group of mice. Figure 5 shown.

[0095] At the same time, 0.1 g of liver tissue was taken and homogenized according to the mass volume ratio of tissue to PBS buffer of 1:9. The supernatant was collected after centrifugation at 4°C and 16,000g for 20 minutes. The liver antioxidant enzyme activity, lipid peroxidation products, and liver inflammatory factors were tested. The results were as follows: Figure 6 and Figure 7 shown.

[0096] The histopathological analysis of liver tissues by H&E staining revealed that ( Figure 5A) In the CK group, hepatocytes showed normal structural arrangement and no obvious pathological changes. However, in the MOD group, liver tissue showed significant damage, manifested by extensive hepatocyte fatty degeneration, the appearance of round vacuoles of varying sizes within the cytoplasm (green arrows), accompanied by extensive hydropic degeneration (black arrows), a small amount of ballooning (yellow arrows), and small focal neutrophil infiltration (red arrows), demonstrating severe pathological changes. Following different treatments, liver tissue damage was alleviated to varying degrees. In the HMT group, hepatocytes showed essentially intact structural integrity, with only minor fatty vacuoles and no obvious hydropic degeneration, suggesting that high-dose milk tea has a potent preventive effect. In the LMT group, punctate necrosis, characterized by nuclear fragmentation and chromatin loss, was still observed in the liver tissue of mice. Hepatocyte fatty degeneration and minor inflammatory infiltration were still prevalent in the BT, FM, and MOG groups treated with each of the ingredients alone. Punctate necrosis was also observed in the BT and FM groups, indicating that the protective effect of each ingredient alone was less pronounced than that of the combined formula. Comprehensive histological scoring results showed that liver pathological changes in the MOD group were significantly aggravated, while liver damage was significantly alleviated after functional milk tea treatment. The liver tissue damage in the BMT and CMT groups was significantly less severe than that in the MOD group, with smaller lesions, more orderly hepatocyte arrangement, and decreased inflammatory cell infiltration, suggesting a protective effect on liver tissue. However, compared with the HMT group, the improvement in the BMT and CMT groups was relatively weak (comprehensive histological scores of 1.51 and 1.36, respectively), and the degree of tissue structural recovery had not yet reached near-normal levels. This suggests that the HMT group had the most significant intervention effect among the three milk tea groups (comprehensive histological scores of each group were: MOD: 3.33, BT: 2.33, FM: 2.33, MOG: 2.67, LMT: 1.67, MMT: 1.33, HMT: 1.00, BMT: 1.34, CMT: 1.55). Figure 5 B).

[0097] Compared with the CK group (TG: 0.10, ALT: 11.08, AST: 15.07), the levels of TG (0.63), ALT (24.47), and AST (41.21) in the serum of mice in the MOD group were significantly increased (P < 0.05; Figure 6A, 6B, 6C). After intervention with milk tea and its single raw material, the levels of TG (BT: 0.36, FM: 0.37, MOG: 0.48, LMT: 0.2, MMT:0.15, HMT: 0.13, BMT: 0.17, CMT: 0.22), ALT (BT: 16.85, FM: 16.82, MOG: 19.7, LMT:11.90, MMT: 11.41, HMT: 10.40, BMT: 12.30, CMT: 13.23) and AST (BT: 30.64, FM: 27.78, MOG: 32.9, LMT: 22.30, MMT: 19.83, HMT: 19.04, BMT: 23.05, CMT: The level of HBeAg (24.11) in the 34-48 group was significantly lower than that in the MOD group (P < 0.05), indicating that the intervention had a good hepatoprotective effect.

[0098] Further detection of key enzyme activities of alcohol metabolism and ethanol concentration revealed that compared with the CK group (ADH: 17.12, ALDH: 0.81, ethanol: 0.60, acetaldehyde: 0.11), the activities of ADH (7.95) and ALDH (0.61) in the serum of mice in the MOD group were significantly decreased (P < 0.05; Figure 6 D, 6E), and the concentrations of ethanol (6.15) and acetaldehyde (6.78) in serum were significantly increased (P < 0.05; Figure 6 F, 6G), indicating that the ability to metabolize alcohol is impaired. Compared with the MOD group, the intervention groups, BMT and CMT groups could significantly increase the activities of ADH (BT: 13.41, FM: 12.92, MOG: 10.97, LMT: 20.15, MMT: 20.11, HMT: 21.71, BMT: 20.22, CMT: 19.16) and ALDH (BT: 0.86, FM: 0.89, MOG: 0.75, LMT: 1.14, MMT: 1.21, HMT: 1.35, BMT: 1.15, CMT: 1.04) (P < 0.05), and significantly reduce the levels of serum ethanol (BT: 3.77, FM: 3.58, MOG: 4.39, LMT: 2.57, MMT: 2.42, HMT: 2.28, BMT: 2.44, CMT: 2.55) and acetaldehyde (BT: 5.35, FM: 5.42, MOG: 5.83, LMT: 4.64, MMT: 4.23, HMT: 4.15, BMT: 4.56, CMT:4.78) contents, showing a good metabolic effect.

[0099] Compared with the CK group (SOD: 120.04, CAT: 51.96, GSH-Px: 43.99, MDA: 0.46, ROS: 43.21), the activities of SOD (70.60), CAT (17.79), and GSH-Px (21.67) in the liver tissue of mice in the MOD group were significantly decreased (P < 0.05; Figure 7 D, 7A, 7B), while the levels of MDA (2.50) and ROS (147.87) were significantly increased (P < 0.05; Figure 7 C, 7E). In contrast, the levels of SOD (BT: 90.52, FM: 90.12, MOG: 85.51, LMT: 106.62, MMT: 108.03, HMT:109.71, BMT: 100.28, CMT: 102.34), CAT (BT: 26.94, FM: 32.40, MOG:24.44, LMT: 40.59, MMT: 41.65, HMT:46.90, BMT: 40.72, CMT: 38.56) and GSH-Px (BT:33.41, FM: 32.60, MOG: 28.12, LMT: 38.93, MMT: 40.20, HMT: 40.60, BMT: The activities of MDA (BT: 1.71, FM: 1.54, MOG: 2.32, LMT: 1.28, MMT: 0.99, HMT: 0.79, BMT: 1.27, CMT: 1.32) and ROS (BT: 110.59, FM: 112.55, MOG: 125.68, LMT: 91.52, MMT: 88.45, HMT: 84.34, BMT: 91.30, CMT: 88.56) were significantly decreased in the treatment group (P< 0.05). Among them, both the BMT group and the CMT group showed good effects in improving antioxidant enzyme activities and reducing oxidative stress products, which were significantly better than the MOD group (P < 0.05), but the degree of improvement was slightly lower than that of the HMT group.

[0100] In terms of inflammatory factor levels, compared with the CK group (TNF-α: 160.65, IL-6: 59.60, IL-1β: 65.56), the contents of TNF-α (816.42), IL-6 (193.77), and IL-1β (248.12) in the liver tissue of mice in the MOD group were significantly increased (P<0.05; Figure 7Milk tea and single raw material groups could significantly reduce TNF-α (BT: 613.36, FM: 654.70, MOG: 717.75, LMT: 528.12, MMT: 497.93, HMT: 483.19, BMT: 515.62, CMT: 555.26), IL-6 (BT: 127.48, FM: 136.67, MOG: 147.52, LMT: 104.05, MMT: 90.83, HMT: 72.65, BMT: 105.24, CMT: 112.18) and IL-1β (BT: 176.75, FM: 186.58, MOG: 192.83, LMT: The levels of the three inflammatory factors in the BMT and CMT groups were significantly lower than those in the MOD group (P < 0.05), but the decrease was less than that in the HMT group, indicating that they had a certain effect in inhibiting the inflammatory response, but still inferior to the intervention advantage of the HMT group.

[0101] 4. Real-time quantitative PCR analysis of alcohol metabolism enzyme genes

[0102] Total RNA was extracted from mouse liver samples using the RNA Easy Fast Animal Tissue Kit (TIANGEN, Beijing, China). The extracted total RNA was reverse transcribed into cDNA using the FastKinggDNAdiscreasing RT SuperMix Kit (TIANGEN, Beijing, China). Gene expression levels were detected using the Talent qPCR Pre Mix (SYBR Green) Kit (TIANGEN, Beijing, China). Each sample was replicated three times. After the reaction, the average Ct value of the three replicates was calculated, and the relative expression of the target gene mRNA was calculated using β-actin as the internal reference. The relative expression of the target gene mRNA in each treatment group was 2 -△△ct .

[0103] The calculation formula for ∆∆CT is:

[0104] △△ct=(Ct value of target gene - Ct value of internal reference) 处理组 - (Ct value of target gene - Ct value of reference gene) CK组 .

[0105] Primers were synthesized by Beijing Qingke Bioengineering Co., Ltd. (Beijing, China). The primer sequences are shown in Table 10.

[0106] β-actin was used as an internal reference to calculate the relative mRNA expression of the target gene.

[0107] The results are as follows Figure 8 and Figure 9 The results showed that in terms of mRNA expression levels, compared with the CK group, the expression of alcohol metabolism-related genes such as ADH6 (0.71), ALDH2 (0.95), ALDH1B1 (0.69), ACSS1 (0.37) and CAT (0.64) in the liver of mice in the MOD group were significantly downregulated (P < 0.05; Figure 8 A. Figure 8 B. Figure 8 C. Figure 8 E. Figure 8 F), while the expression of genes such as CYP2E1 (6.32), NF-κBp65 (1.88), TNF-α (1.85), IL-1β (1.89) and IL-6 (2.75) were significantly upregulated (P < 0.05; Figure 8 E. Figure 9 A. Figure 9 B. Figure 9 C. Figure 9D). After milk tea intervention, compared with the MOD group, the livers of mice in all doses of milk tea groups had significantly increased ADH6 (BT: 1.29, FM: 1.31, MOG: 0.99, LMT: 1.54, MMT: 1.90, HMT: 1.91, BMT: 1.35, CMT:1.25), ALDH2 (BT: 0.95, FM: 0.98, MOG: 0.91, LMT: 1.26, MMT: 1.33, HMT: 1.44, BMT:1.27, CMT: 1.15), ALDH1B1 (BT: 1.01, FM: 1.15, MOG: 0.79, LMT: 1.38, MMT: 1.49, HMT:1.54, BMT: 1.45, CMT: The expressions of BT: 2.07, FM: 2.45, MOG: 0.78, LMT: 2.54, MMT: 3.18, HMT: 3.33, BMT: 2.46, CMT: 2.34), and CAT (BT: 0.79, FM: 1.17, MOG: 1.08, LMT:1.50, MMT: 1.73, HMT: 1.70, BMT: 1.62, CMT: 1.54) were significantly up-regulated in the model group (P<0.05). Meanwhile, the expressions of CYP2E1 (BT: 3.04, FM: 3.46, MOG: 3.47, LMT: 2.43, MMT: 2.34, HMT: 1.64, BMT: 2.87, CMT: 2.98) and NF-κBp65 (BT: 1.50, FM: 1.56, MOG: 1.70, LMT: 1.22, MMT: 1.12, HMT: 1.07, BMT: 1.25, CMT: 1.32), TNF-α (BT: 1.60, FM: 1.61, MOG: 1.59, LMT: 1.36, MMT: 1.10, HMT: 1.22, BMT: 1.34, CMT: 1.49), IL-1β (BT: 1.60, FM: 1.59, MOG: 1.72, LMT: 1.28, MMT: 1.27, HMT: 1.22, BMT: 1.34, CMT: 1.40) and IL-6 (BT: The relative expression levels of 1137 NMT: 1.54, 1.71, 1.97, 2.19, FM: 2.05, MOG: 2.28, LMT: 1.39, MMT: 1.30, HMT: 1.16, BMT: 1.65, and CMT: 1.69 were also significantly downregulated (P < 0.05).The BMT and CMT groups significantly improved the expression of metabolism-related genes such as ADH6, ALDH2, ALDH1B1, ACSS1, and CAT. Gene expression levels were significantly higher than those in the MOD group (P < 0.05), but significantly lower than those in the MMT group, suggesting a facilitative effect on the recovery of liver alcohol metabolism. Furthermore, the BMT and CMT groups significantly downregulated the mRNA expression levels of CYP2E1, NF-κBp65, TNF-α, IL-1β, and IL-6 (P < 0.05), demonstrating a positive effect in inhibiting the activation of inflammatory signaling pathways. However, the magnitude of downregulation was still lower than that in the MMT group, suggesting that while BMT and CMT have some anti-inflammatory effects, they are not as significant as those in the MMT and HMT groups. In addition, in the intervention of milk tea raw materials, the regulatory trends of ADH6, ALDH2, ALDH1B1, ACSS1, CYP2E1, NF-κBp65, TNF-α, IL-1β and IL-6 in the BT group and the FM group were also consistent with those of the milk tea dosage groups.

[0108] 5. Western blot analysis of alcohol metabolism enzyme genes. Protein extraction and detection were performed using 50 mg of liver tissue from three mice in each group. Sterile medical scissors were used to cut 50 mg of liver tissue, washed with ice-cold PBS, and then mixed with 500 µL of RRIPA lysis buffer in a biological sample homogenizer. The tissue was repeatedly ground into a liver tissue homogenate. Lysis was continued on ice for 15 minutes. Centrifugation was performed at 12,000 rpm for 15 minutes at 4°C. The supernatant was transferred to a 1.5 mL microcentrifuge tube and protein quantified using a BCA kit. The expression of the target protein was analyzed by Western blot according to the following table:

[0109] The primary antibodies used are as follows: ADH6 (67709-1-Ig), ALDH1B1 (15560-1-AP), ALDH2 (68237-1-Ig), CAT (66765-1-Ig), ACSS1 (17138-1-AP), CYP2E1 (67263-1-Ig), GAPDH (60004-1-Ig), TNF-α (17590-1-AP), IL-1β (16806-1-AP), IL-6 (21865-1-AP), and NF-κB p65 (10745-1-AP) were all purchased from Proteintech (Chicago, USA). Secondary antibodies used included Goat anti-Rabbit IgG (H&L, 511203) and Rabbit anti-Mouse IgG (H&L, 701051), purchased from Chengdu Zhengneng Biotechnology Co., Ltd. (Sichuan, China). The dilution ratios of the universal antibody diluent and the antibodies used for primary and secondary antibody incubation are shown in (Table 12).

[0110] The grayscale values ​​of Western blot bands were analyzed using Image J software (version 1.53c, Bethesda, MD, USA). Figure 10 shown.

[0111] The results showed that in terms of protein expression levels, compared with the CK group, the protein expression levels of ALDH1B1 (0.66) and CAT (0.68) in the MOD group were significantly downregulated, while the protein expression level of CYP2E1 (1.14) was significantly increased (P < 0.05; Figure 10 B. Figure 10D). After milk tea intervention, compared with the MOD group, the expressions of ADH6 (BT: 0.74, FM: 0.89, MOG: 0.99, MT: 1.18, BMT: 1.15, CMT: 1.05), ALDH2 (BT: 0.97, FM: 0.97, MOG: 1.03, MT: 1.20, BMT: 1.10, CMT: 1.06), ALDH1B1 (BT: 0.85, FM: 0.88, MOG: 0.83, MT: 1.16, BMT: 1.05, CMT: 1.01), ACSS1 (BT: 0.86, FM: 0.99, MOG: 1.01, MT: 1.07, BMT: 1.06, CMT: The protein expression levels of BT: 1.01, FM: 0.83, MOG: 0.63, MT: 0.50, BMT: 0.55, CMT: 0.58) were significantly up-regulated (P < 0.05; Figure 10 A. Figure 10 C. Figure 10 B. Figure 10 D. Figure 10 F). In the milk tea formula, the BMT group and the CMT group also showed a good regulatory effect on the expression of alcohol metabolism-related proteins. Specifically, both the BMT group and the CMT group significantly upregulated the protein expression of ALDH1B1, ACSS1 and CAT (P < 0.05), and the CYP2E1 protein expression was also significantly decreased compared with the MOD group (P < 0.05), but the magnitude of these adjustments was lower than that of the HMT group. In the single raw material intervention groups, none of the groups significantly increased the expression of ADH6 protein, but the protein levels of ALDH1B1, ACSS1 and CAT were significantly upregulated, and only the MOG group significantly downregulated the expression of CYP2E1 at the protein level (P < 0.05; Figure 10 E).

[0112] In terms of inflammatory signaling pathway-related proteins, compared with the CK group, the expression levels of NF-κBp65 (1.29), TNF-α (1.16), IL-1β (1.13), and IL-6 (1.39) proteins in the liver of mice in the MOD group were significantly upregulated (P < 0.05; Figure 11 A. Figure 11 B. Figure 11 C. Figure 11D). Compared with the MOD group, milk tea and its single ingredient intervention groups reduced the expression of the above inflammatory factors to varying degrees. The expressions of TNF-α (BT: 0.93, FM: 0.77, MOG: 0.64, MT: 0.58, BMT: 0.60, CMT:0.61), IL-1β (BT: 0.83, FM: 0.82, MOG: 0.82, MT: 0.55, BMT: 0.62, CMT: 0.69), IL-6 (BT: 0.83, FM: 0.72, MOG: 0.78, MT: 0.41, BMT: 0.49, CMT: 0.55) and NF-κBp65 (BT:1.15, FM: 0.66, MOG: 0.51, MT: 0.59, BMT: 0.53, CMT: 0.52) showed downregulation in expression, with the HMT group being the most significant (P < 0.05; Figure 11 A. Figure 11 B. Figure 11 C. Figure 11 D). In the milk tea intervention group, the BMT and CMT groups also significantly reduced the expression levels of the above-mentioned inflammation-related proteins (P < 0.05), especially in the inhibition of TNF-α, IL-6, and NF-κBp65, but the regulation amplitudes were lower than those in the HMT group. In the single raw material treatment groups, the BT group only significantly reduced the IL-6 protein level (P < 0.05; Figure 11 B), FM and MOG groups significantly downregulated the expression of TNF-α, IL-6 and NF-κBp65 proteins (P<0.05; Figure 11 B. Figure 11 C. Figure 11 D).

[0113] In summary, the present invention successfully constructs a functional milk tea with both excellent sensory quality and AALI liver protection function. This formula can significantly improve the efficiency of alcohol metabolism by regulating the expression of key alcohol metabolism enzymes such as ADH6, ALDH1B1, ALDH2, ACSS1, and inhibit the generation of ROS induced by CYP2E1, activating antioxidant defense systems such as SOD, CAT, and GSH-Px, thereby effectively alleviating the oxidative stress and tissue damage caused by AALI. At the same time, by inhibiting the TNF-α / NF-κB signaling pathway and the expression of its downstream inflammatory factors IL-6 and IL-1β, the regulation of the inflammatory response is achieved. The study also found that there is a synergistic effect between the components of milk tea, among which the HMT group has the most significant protective effect, which is better than each single raw material, indicating that scientific formula optimization is the key to achieving functional superposition.

[0114] It should be noted that the above embodiments are merely examples for the purpose of clearly illustrating the present invention and are not intended to limit the embodiments of the present invention. A person skilled in the art would be able to make other variations or modifications based on the above description. It is not possible to enumerate all embodiments here. Any obvious variations or modifications arising from the technical solution of the present invention remain within the scope of protection of the present invention.

Claims

1. A milk tea, characterized in that: By volume, it comprises 35.6-40.6 parts of black tea extract, 60.9-64.9 parts of raw milk and 1.0-1.2 parts of monk fruit concentrated juice; The preparation method of the black tea extract comprises the following steps: crushing the black tea, adding ultrapure water at a material-liquid ratio of 1:25-30, extracting once at 90-100°C, centrifuging to obtain a primary extraction supernatant and a precipitate; adding an ethanol aqueous solution to the precipitate at a material-liquid ratio of 1:25-30, extracting twice at 90-100°C, centrifuging to obtain a secondary extraction supernatant; combining the supernatants, concentrating, and obtaining the black tea extract.

2. The milk tea according to claim 1, characterized in that The milk tea comprises 35.6-40.6 parts of black tea extract, 60.9-64.9 parts of raw milk and 1.2 parts of monk fruit concentrated juice by volume.

3. The milk tea according to claim 1, characterized in that In the monk fruit concentrated juice, the volume concentration of mogroside V is 1.8%-3.5%, and the sugar content is 50-60%.

4. The milk tea according to claim 1, characterized in that The centrifugal speed is 3500-4000 r / min, and the time is 10-15 min.

5. The milk tea according to claim 1, characterized in that The volume concentration of the ethanol aqueous solution is 55%-65%.

6. The milk tea according to claim 1, characterized in that The solid-liquid ratio is 1:25-27; the extraction temperature is 95-100℃.

7. The milk tea according to claim 1, characterized in that The time for the first extraction and the second extraction is 30-40 minutes.

8. A method for preparing milk tea according to any one of claims 1 to 7, characterized in that: The following steps are involved: S1. Adding raw milk to the black tea extract and stirring evenly to obtain tea milk; S2. Slowly adding the Momordica grosvenori concentrated juice into the tea milk, stirring evenly, and sterilizing to obtain the milk tea.

9. Use of the milk tea according to any one of claims 1 to 7 in preparing an agent for preventing alcoholic liver damage.

10. Use of the milk tea according to any one of claims 1 to 7 in preparing a hangover sobering agent.

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