Exocarpium citri grandis composite extract for dispelling effects of alcohol and protecting liver and preparation method thereof

Abstract

The invention belongs to the field of development of medicinal and edible plants, and particularly relates to an exocarpium citri grandis composite extract for dispelling effects of alcohol and protecting liver and a preparation method thereof. The compound extract comprises exocarpium polysaccharide, exocarpium oligosaccharide, exocarpium flavone and exocarpium essential oil. Essential oil is treated by adopting a beta-cyclodextrin inclusion technology, polysaccharide and oligosaccharide are compounded into a culture medium, and the culture medium is fermented by complex flora such as lactobacillus plantarum and streptococcus thermophilus and then is mixed with inclusion essential oil and flavone to play a synergistic effect. Experimental results show that the exocarpium citri grandis composite extract has a remarkable hangover alleviating effect and lipid metabolism capability in an acute alcoholism mouse model, and the effectiveness of the exocarpium citri grandis composite extract in the aspects of hangover alleviating and liver protection is proved.

Description

Technical Field

[0001] The invention belongs to the field of development of medicinal and edible plants, and particularly relates to a tangerine peel composite extract for sobering up and protecting the liver and a preparation method thereof. Background Art

[0002] Huajuhong (Chinese orange peel) is a specialty of Huazhou City, Maoming City, Guangdong Province. It holds a National Geographical Indication designation and is a valuable Chinese medicinal material. With an aromatic aroma and a bitter, slightly pungent flavor, it enters the lung and spleen meridians and is considered one of China's "Four Great Southern Medicinal Herbs" and one of the "Ten Great Guangdong Medicinal Herbs." It has the effects of regulating qi, relieving fullness, and dispelling dampness and phlegm. It is used to treat coughs with excessive phlegm, food stagnation, alcohol-induced vomiting, and abdominal distension. Modern research has revealed that Huajuhong contains active ingredients such as flavonoids, polysaccharides, essential oils, and coumarins, resulting in antioxidant benefits, blood sugar regulation, lipid metabolism, and immune enhancement.

[0003] Alcohol is metabolized in the body primarily through three pathways: the alcohol dehydrogenase system, the CYP2E1 metabolic system, and the catalase system. Long-term excessive drinking can lead to over 200 diseases affecting multiple organs, including the brain, heart, gastrointestinal tract, and liver. The liver is the primary site of alcohol metabolism in the body. The initial stage of alcoholic liver disease is alcoholic fatty liver disease, characterized by hepatic steatosis (ALD). The accumulation of triglycerides in hepatocytes can progress to hepatitis and / or fibrosis, ultimately leading to cirrhosis, liver necrosis, or liver cancer. Alcohol abuse has become a global public health concern, with chronic drinkers susceptible to gastric ulcers, alcoholic hepatitis, cirrhosis, and other diseases.

[0004] Polyphenols, polysaccharides, and oligosaccharides, as natural bioactive ingredients, can prevent or alleviate alcoholic fatty liver disease through antioxidant, anti-inflammatory, anti-apoptotic, and lipid metabolism and intestinal microbial regulation. Therefore, the present invention extracts multiple active ingredients from the medicinal and edible plant, Citrus aurantium, and formulates them into a functional food with alcohol-relief and liver-protecting properties, which has promising market prospects. Summary of the Invention

[0005] The present invention overcomes the defects of the prior art and provides a composite extract of Citrus aurantium dulcis for sobering up and protecting the liver, and a preparation method thereof. The composite extract comprises Citrus aurantium dulcis polysaccharides, Citrus aurantium dulcis oligosaccharides, Citrus aurantium dulcis flavonoids and Citrus aurantium dulcis essential oil. The essential oil is treated by β-cyclodextrin inclusion technology, and the polysaccharides and oligosaccharides are compounded into a culture medium. After fermentation by a composite bacterial community such as Lactobacillus plantarum and Streptococcus thermophilus, the culture medium is mixed with the inclusion essential oil and flavonoids to exert a synergistic and synergistic effect. The experimental results show that the composite extract of Citrus aurantium dulcis exhibits significant sobering efficacy and lipid metabolism ability in an acute alcohol poisoning mouse model, confirming its effectiveness in sobering up and protecting the liver. The composite extract of Citrus aurantium dulcis provided by the present invention can increase the activation rate of alcohol dehydrogenase and acetaldehyde dehydrogenase, the key enzymes of alcohol metabolism, thereby achieving the effect of accelerating alcohol metabolism, and accelerating the restoration of liver metabolic homeostasis through antioxidant, anti-inflammatory and other pathways.

[0006] The purpose of the present invention is achieved by at least one of the following technical solutions.

[0007] The present invention provides a method for preparing a composite extract of Citrus aurantium dulcis for sobering up and protecting the liver, comprising the following steps:

[0008] (1) Slice the whole fruit of the dried tangerine peel, crush it, sieve it, defat it with anhydrous ethanol, and evaporate the ethanol;

[0009] (2) placing the tangerine peel powder obtained in step (1) in an essential oil extractor, adding zeolite and water, and maintaining a slight boiling to obtain tangerine peel essential oil and filtrate residue;

[0010] (3) Mixing tangerine peel essential oil with an equal amount of anhydrous ethanol, slowly dropping it into a saturated aqueous solution of β-cyclodextrin and stirring to include it, taking it out and letting it cool to room temperature and then refrigerating it, filtering it and drying it to obtain an essential oil inclusion complex (hereinafter referred to as inclusion essential oil);

[0011] (4) inoculating the filter residue obtained in step (2) with Aspergillus niger and Trichoderma spp., extracting with ethanol after fermentation, centrifuging the filtrate and filter residue, concentrating the filtrate, and freeze-drying to obtain citrus red flavonoids;

[0012] (5) placing the filter residue obtained in step (4) in clean water, performing ultrasonic-assisted extraction for two hours, and then filtering. Repeating the extraction process once for the filter residue, combining the filtrates of step (2) and step (5) to obtain a polysaccharide extract, half of which is used in step (6), and the other half is concentrated under reduced pressure to obtain a polysaccharide concentrate, adding ethanol to the concentrate and mixing thoroughly, refrigerating and standing, separating the supernatant and the precipitate, and freeze-drying the precipitate to obtain the tangerine peel polysaccharide;

[0013] (6) adding a composite enzyme to the polysaccharide extract obtained in step (5), performing enzymatic hydrolysis for a period of time, heating to terminate the enzyme reaction, centrifuging to obtain the supernatant, adding ethanol to fully mix, refrigerating and standing, taking the precipitate, and freeze-drying to obtain tangerine peel oligosaccharides;

[0014] (7) crushing the polysaccharide and oligosaccharide obtained above, sieving them, adding purified water to the fixed volume to form a culture medium, and mixing them with Lactobacillus delbrueckii culture medium to form a new culture medium;

[0015] (8) inoculating the activated mixed strains into the culture medium described in step (7), and obtaining a fermentation liquid after fermentation on a shaking table;

[0016] (9) The inclusion essential oil obtained in step (3), the flavonoids of Citrus grandis obtained in step (4), and the fermentation liquid obtained in step (8) are uniformly mixed to obtain a composite extract of Citrus grandis with the effects of sobering up and protecting the liver.

[0017] Furthermore, the mesh size of the sieve after pulverization in step (1) is 40 to 60 meshes.

[0018] Furthermore, the temperature of the stirring inclusion process in step (3) is 40° C. to 60° C., and the stirring inclusion process is carried out for 2 to 3 hours.

[0019] Furthermore, in step (4), the inoculation ratios of Aspergillus niger and Trichoderma are 2% to 3% and 1% to 2% respectively (based on the mass of the fermented product), the pH of the fermentation process is 6.0 to 7.0, the time is 12 to 24 hours, and the fermentation temperature is 25 to 35° C.; the ethanol concentration is 60% to 80%, the material-liquid ratio is 1:20 to 25 (g / ml), the extraction temperature is 45 to 55° C., and the extraction is carried out by centrifugation at 2000 to 3000 r / min for 10 to 15 minutes.

[0020] Furthermore, in step (5), the material-liquid ratio is 1:25-35 (g / ml), the ultrasonic power is 300-400w, the temperature is 85-90°C, the added ethanol is 4-4.5 times the volume of anhydrous ethanol (relative to the volume of the concentrate), and the refrigerated standing time is 12-24h.

[0021] Furthermore, the complex enzyme in step (6) is α-glucosidase and α-galactosidase, and the added amounts are 0.5% to 1.5% (w / w) and 0.2% to 0.5% (w / w), respectively. The enzymatic hydrolysis temperature is 40 to 60°C, the pH is 4.5 to 6.5, the ultrasonic (200 to 300w) assisted enzymatic hydrolysis time is 2 to 3h, the added ethanol is 4 to 4.5 times the volume of anhydrous ethanol (relative to the volume of the supernatant), and the refrigerated standing time is 12 to 24h.

[0022] Furthermore, in step (7), the amounts of polysaccharide, oligosaccharide and pure water (in mass percentage) are 10% to 40%, 10% to 40% and 10% to 60% respectively, and the mesh size of the sieve is 20 to 60 meshes.

[0023] Furthermore, the culture medium in step (7) is composed of a mixture of polysaccharides, oligosaccharides and pure water and a Lactobacillus delbrueckii culture medium, and the volume ratio of the Lactobacillus delbrueckii culture medium to the mixture is 1-2:1-2.

[0024] Furthermore, the mixed strains described in step (8) are Lactobacillus plantarum, Streptococcus thermophilus, Lactobacillus casei, and yeast. Based on the quality of the fermentation system, the strain inoculation ratios are 1.5% to 2.5%, 0.5% to 1.5%, 1.5% to 2.5%, and 0.5% to 1.0%, respectively. The fermentation temperature is 30-40° C., the shaking table speed is 180 to 200 r / min, and the time is 36 to 48 h.

[0025] Furthermore, the mass percentages of the components in the composition in step (9) are 10% to 30% of the inclusion essential oil, 30% to 60% of the flavonoids, 30% to 60% of the fermentation liquid, and the balance is water.

[0026] Compared with the prior art, the advantages and beneficial effects of the present invention are:

[0027] 1. Different technical methods are used to extract essential oils, flavonoids, polysaccharides and oligosaccharides from the raw material of Conghua Tangerine Peel in sequence, which not only fully utilizes the raw materials but also obtains a variety of active ingredients for subsequent compounding.

[0028] 2. The formula ingredients are tangerine peel polysaccharides, flavonoids, oligosaccharides and essential oils to form a four-component synergistic enhancement system. Among them, polysaccharides and oligosaccharides are regulated by the gut-liver axis, flavonoids work together by activating key enzymes, and essential oils work together through anti-oxidation to comprehensively achieve the effect of sobering up and protecting the liver.

[0029] 3. Find suitable extraction methods for the four active ingredients: polysaccharides, flavonoids, oligosaccharides and essential oils, to improve their yields.

[0030] 4. The obtained tangerine peel polysaccharide extract is treated with α-glucosidase and α-galactosidase to degrade a portion of the polysaccharide into oligosaccharides, while retaining a portion of the polysaccharide. The polysaccharide and oligosaccharide mixture is then used to prepare a fermentation medium, thereby obtaining two active ingredients, polysaccharides and oligosaccharides, and utilizing their physiological functions to play a role in the alcohol detoxification and liver protection complex.

[0031] 5. The fermentation method is used to extract flavonoids from tangerine peel. Compared with the ordinary ethanol extraction method, the flavonoid content in the fermentation liquid after fermentation is increased by about 1 times.

[0032] 6. Using a mixture of polysaccharides and oligosaccharides as a culture medium, the activity of polysaccharides and oligosaccharides was further improved by fermentation with a composite strain of Lactobacillus plantarum, Streptococcus thermophilus, Lactobacillus casei and yeast. Compared with the unfermented polysaccharide and oligosaccharide mixture, the activation rates of the fermented polysaccharide and oligosaccharide mixed fermentation liquid on alcohol dehydrogenase and acetaldehyde dehydrogenase increased by 1.0 times and 1.3 times, respectively. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 The effects of different fermentation times on enzymes related to alcohol metabolism ((a) is the ADH activation rate, (b) is the change in ALDH activation rate).

[0034] Figure 2 The effect on serum lipid metabolism (changes in total cholesterol TC content) in mice with acute alcohol poisoning model; NC and MC in the figure are the blank group and model group, respectively. Compared with the NC group (#), p<0.05; compared with the MC group (*), p<0.05.

[0035] Figure 3 The effect on serum lipid metabolism in mice with acute alcohol poisoning model (changes in total triglyceride TG content);

[0036] In the figure, NC and MC are the blank group and model group, respectively. Compared with the NC group (#), p<0.05; compared with the MC group (*), p<0.05.

[0037] Figure 4 The effect on lipid metabolism in the liver of mice with acute alcohol poisoning model (changes in aspartate aminotransferase (AST) content);

[0038] In the figure, NC and MC are the blank group and model group, respectively. Compared with the NC group (#), p<0.05; compared with the MC group (*), p<0.05.

[0039] Figure 5 The effect on lipid metabolism in the liver of mice with acute alcohol poisoning model (changes in alanine aminotransferase ALT content);

[0040] In the figure, NC and MC are the blank group and model group, respectively. Compared with the NC group (#), p<0.05; compared with the MC group (*), p<0.05.

[0041] Figure 6 This is a comparison of the liver status of mice in each scheme.

[0042] Figure 7 The figure is a bar graph of the liver index of mice in each regimen; NC and MC are the blank group and model group, respectively. Compared with the NC group (#), p<0.05; compared with the MC group (*), p<0.05.

[0043] Figure 8The study is about the effect of composite extracts of Lactobacillus plantarum, Streptococcus thermophilus, Lactobacillus casei and yeast at different inoculation ratios on enzymes related to alcohol metabolism.

[0044] Figure 9 It is the effect of composite extracts with different contents of fermentation broth, flavonoids and essential oils on enzymes related to alcohol metabolism. DETAILED DESCRIPTION

[0045] The present invention is further described in detail below with reference to specific examples. The examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. The test methods used in the following examples are all conventional methods unless otherwise specified.

[0046] Unless otherwise specified, the materials and reagents used are commercially available.

[0047] Example 1

[0048] (1) Slice the white inner peel of fresh tangerine peel, crush it, pass it through a 40-mesh sieve, and defatted it by reflux with anhydrous ethanol. Then dry it with hot air at a temperature of 40°C for 24 hours.

[0049] (2) The tangerine peel powder obtained in step (1) is placed in an essential oil analyzer, zeolite and water are added at a material-liquid ratio of 1:40 (g / ml), and the mixture is kept slightly boiling to obtain tangerine peel essential oil and filtrate residue. The essential oil is mixed with an equal amount of anhydrous ethanol, and the mixture is slowly dripped into a saturated aqueous solution of β-cyclodextrin (35.0 mg / ml). The mixture is stirred for inclusion at 60° C. for 2 h, taken out and allowed to cool to room temperature and refrigerated for 24 h, and the filter residue is filtered and dried in vacuo at 50° C. to obtain the inclusion essential oil.

[0050] (3) Inoculating the activated Aspergillus niger (2%) and Trichoderma spp. (1%) into the filter residue obtained in step (2), and fermenting the mixture on a shaker at a temperature of 25° C. and a pH of 6.0 for 24 hours to obtain a fermented product. The fermented product was placed in 80% ethanol at a solid-liquid ratio of 1:25 (g / ml) at a temperature of 45° C., extracted for 40 minutes, and centrifuged at 2000 r / min for 10 minutes to separate the filtrate and filter residue. The supernatant was concentrated under reduced pressure and freeze-dried to obtain citrus flavonoids.

[0051] (4) The filter residue in step (3) is placed in clean water with a material-liquid ratio of 1:25 (g / ml), the temperature is 85°C, and the extraction is carried out under 400W ultrasonic assisted extraction for two hours, followed by filtration. The filter residue is subjected to the extraction process once, and the filtrates of step (2) and step (4) are combined to obtain a polysaccharide extract, half of which is used in step (5), and the other half is concentrated under reduced pressure to obtain a polysaccharide concentrate; 4 times the volume of anhydrous ethanol is added to the concentrate and mixed thoroughly, and the concentrate is allowed to stand in a refrigerator for 12 hours, and the filtrate and filter residue are separated by centrifugation at a speed of 3000 r / min. The filter residue is rinsed with anhydrous ethanol to evaporate the water, and then freeze-dried to obtain the tangerine peel polysaccharide.

[0052] (5) Add α-glucosidase and α-galactosidase to the polysaccharide extract obtained in step (4) in an amount of 0.5% (w / w) respectively.

[0053] and 0.2% (w / w), temperature 40°C, pH 4.5, 200w ultrasonic assisted enzymatic hydrolysis for 3 hours, heating at 90°C for 10 minutes to terminate the enzyme reaction, centrifugation to obtain the supernatant, add 4 times the volume of anhydrous ethanol to fully mix, let it stand in the refrigerator for 12 hours, and precipitate and freeze-dry to obtain tangerine peel oligosaccharides.

[0054] (6) The polysaccharide and oligosaccharide prepared above were crushed and passed through a 60-mesh sieve, and the following raw materials were mixed with Lactobacillus delbrueckii culture medium in a volume ratio of 1:1 to form a culture medium. Activated Lactobacillus plantarum, Streptococcus thermophilus, Lactobacillus casei, and yeast (with inoculation amounts of 1.5%, 1.5%, 2%, and 0.5%, respectively) were inoculated into the above culture medium and fermented in a shaking incubator at 30° C. and 180 rpm for 48 hours to obtain a fermentation liquid.

[0055] (7) A composite extract of Citrus aurantium wilfordii with the effects of sobering up and protecting the liver, comprising the following raw materials in percentage by weight: 15% of inclusion essential oil, 40% of flavonoids, 40% of fermentation liquid, and 5% of water.

[0056] Example 2

[0057] (1) Slice the white inner peel of fresh tangerine peel, crush it, pass it through a 45-mesh sieve, and defatted it by reflux in anhydrous ethanol. Then dry it with hot air at a temperature of 45°C for 24 hours.

[0058] (2) The tangerine peel powder obtained in step (1) is placed in an essential oil analyzer, zeolite and water are added, the material-liquid ratio is 1:40 (g / ml), and the tangerine peel essential oil and the filtrate residue are obtained by keeping the mixture slightly boiling. The essential oil is mixed with an equal amount of anhydrous ethanol, and the mixture is slowly dripped into a saturated aqueous solution of β-cyclodextrin (35.0 mg / ml). The mixture is stirred and included at 45° C. for 2.2 h, taken out and placed at room temperature and refrigerated for 24 h, and the residue is filtered.

[0059] The inclusion essential oil was obtained by vacuum drying at 50°C.

[0060] (3) The activated Aspergillus niger (2.6%) and Trichoderma (1.3%) were inoculated into the filter residue obtained in step (2) at a temperature of 28° C. and a pH of 6.4, and the mixture was fermented on a shaker for 20 hours to obtain a fermentation product. The fermentation product was placed in 65% ethanol at a solid-liquid ratio of 1:21 (g / ml).

[0061] The temperature was 50°C, the extraction was carried out for 40 minutes, the filtrate and residue were separated by centrifugation at 2800 r / min for 10 minutes, and the supernatant was concentrated under reduced pressure.

[0062] Lyophilization yields tangerine peel flavonoids.

[0063] (4) The filter residue in step (3) is placed in clean water with a material-liquid ratio of 1:27 (g / ml), the temperature is 86°C, and 300W ultrasonic-assisted extraction is performed for two hours, followed by filtration. The filter residue is subjected to the extraction process once, and the filtrates of step (2) and step (4) are combined to obtain a polysaccharide extract, half of which is used in step (5), and the other half is concentrated under reduced pressure to obtain a polysaccharide concentrate; 4.1 times the volume of anhydrous ethanol is added to the concentrate and mixed thoroughly, and the concentrate is allowed to stand in a refrigerator for 14 hours, and the filtrate and filter residue are separated by centrifugation at a speed of 2500 r / min. The filter residue is rinsed with anhydrous ethanol to evaporate the water, and then freeze-dried to obtain the tangerine peel polysaccharide.

[0064] (5) Add α-glucosidase and α-galactosidase to the polysaccharide extract obtained in step (4) in an amount of 0.7% (w / w)

[0065] and 0.3% (w / w), temperature 45°C, pH 5.0, 220w ultrasonic assisted enzymatic hydrolysis for 2.2 hours, heating at 90°C for 10 minutes to terminate the enzyme reaction, centrifugation to obtain the supernatant, adding 4.1 times the volume of anhydrous ethanol to fully mix, and standing in the refrigerator for 14 hours. The precipitate is freeze-dried to obtain tangerine peel oligosaccharides.

[0066] (6) The polysaccharide and oligosaccharide prepared above were crushed and passed through a 60-mesh sieve, and the following raw materials were mixed with Lactobacillus delbrueckii culture medium in a volume ratio of 1:1.5 to form a culture medium. Activated Lactobacillus plantarum, Streptococcus thermophilus, Lactobacillus casei, and yeast (with inoculation amounts of 1.5%, 0.5%, 2%, and 1%, respectively) were inoculated into the above culture medium and fermented in a shaking incubator at 32° C. and 185 rpm for 40 hours to obtain a fermentation liquid.

[0067] (7) A composite extract of Citrus aurantium wilfordii with the effects of sobering up and protecting the liver, comprising the following raw materials in percentage by weight: 10% of inclusion essential oil, 30% of flavonoids, and 60% of fermentation liquid.

[0068] Example 3

[0069] (1) Slice the white inner peel of fresh tangerine peel, crush it, pass it through a 55-mesh sieve, and defatted it by reflux with anhydrous ethanol. Then dry it with hot air at a temperature of 50°C for 24 hours.

[0070] (2) The tangerine peel powder obtained in step (1) is placed in an essential oil analyzer, zeolite and water are added, the material-liquid ratio is 1:40 (g / ml), and the tangerine peel essential oil and the filtrate residue are obtained by keeping the mixture slightly boiling. The essential oil is mixed with an equal amount of anhydrous ethanol, and the mixture is slowly dripped into a saturated aqueous solution of β-cyclodextrin (35.0 mg / ml). The mixture is stirred and included at 55° C. for 2.4 h, taken out and placed at room temperature and refrigerated for 24 h, and the residue is filtered.

[0071] The inclusion essential oil was obtained by vacuum drying at 50°C.

[0072] (3) The activated Aspergillus niger (2.5%) and Trichoderma (1.5%) were inoculated into the filter residue obtained in step (2) at a temperature of 32° C. and a pH of 6.5, and the mixture was fermented on a shaker for 18 hours to obtain a fermentation product. The fermentation product was placed in 75% ethanol at a solid-liquid ratio of 1:23 (g / ml).

[0073] The temperature was 50°C, the extraction was carried out for 40 minutes, the filtrate and residue were separated by centrifugation at 2900 r / min for 10 minutes, and the supernatant was concentrated under reduced pressure.

[0074] Lyophilization yields tangerine peel flavonoids.

[0075] (4) The filter residue in step (3) is placed in clean water with a material-liquid ratio of 1:32 (g / ml), the temperature is 88°C, and 300W ultrasonic-assisted extraction is performed for two hours, followed by filtration. The filter residue is subjected to the extraction process once, and the filtrates of step (2) and step (4) are combined to obtain a polysaccharide extract, half of which is used in step (5), and the other half is concentrated under reduced pressure to obtain a polysaccharide concentrate; 4.4 times the volume of anhydrous ethanol is added to the concentrate and mixed thoroughly, and the concentrate is allowed to stand in a refrigerator for 20 hours, and the filtrate and filter residue are separated by centrifugation at a speed of 2800 r / min. The filter residue is rinsed with anhydrous ethanol to evaporate the water, and then freeze-dried to obtain the tangerine peel polysaccharide.

[0076] (5) Add α-glucosidase and α-galactosidase to the polysaccharide extract obtained in step (4) in an amount of 1.2% (w / w)

[0077] and 0.4% (w / w), temperature 55°C, pH 6.0, 270w ultrasonic assisted enzymatic hydrolysis for 2.5 hours, heating at 90°C for 10 minutes to terminate the enzyme reaction, centrifugation to obtain the supernatant, adding 4.4 times the volume of anhydrous ethanol to fully mix, standing in the refrigerator for 20 hours, and precipitating and freeze-drying to obtain tangerine peel oligosaccharides.

[0078] (6) The polysaccharide and oligosaccharide prepared above were crushed and passed through a 60-mesh sieve, and the following raw materials were mixed with Lactobacillus delbrueckii culture medium in a volume ratio of 1:0.5 to form a culture medium. Activated Lactobacillus plantarum, Streptococcus thermophilus, Lactobacillus casei, and yeast (with inoculation amounts of 1.5%, 0.5%, 1.5%, and 0.5%, respectively) were inoculated into the above culture medium and fermented in a shaking incubator at 37° C. and 190 r / min for 42 hours to obtain a fermentation liquid.

[0079] (7) A composite extract of Citrus aurantium wilfordii with the effects of sobering up and protecting the liver, comprising the following raw materials in percentage by weight: 10% of inclusion essential oil, 60% of flavonoids, and 30% of fermentation liquid.

[0080] Example 4

[0081] (1) Slice the white inner peel of fresh tangerine peel, crush it, pass it through a 60-mesh sieve, and defatted it by reflux with anhydrous ethanol. Then dry it with hot air at a temperature of 60°C for 24 hours.

[0082] (2) The tangerine peel powder obtained in step (1) is placed in an essential oil analyzer, zeolite and water are added at a material-liquid ratio of 1:40 (g / ml), and the mixture is kept slightly boiling to obtain tangerine peel essential oil and filtrate residue. The essential oil is mixed with an equal amount of anhydrous ethanol, and the mixture is slowly dripped into a saturated aqueous solution of β-cyclodextrin (35.0 mg / ml). The mixture is stirred for inclusion at 40° C. for 3 h, taken out and allowed to cool to room temperature and refrigerated for 24 h, and the filter residue is filtered and dried in vacuo at 50° C. to obtain the inclusion essential oil.

[0083] (3) Inoculating the activated Aspergillus niger (3%) and Trichoderma (2%) into the filter residue obtained in step (2), fermenting on a shaker at a temperature of 35° C. and a pH of 7.0 for 12 hours to obtain a fermented product. The fermented product was placed in 60% ethanol at a solid-liquid ratio of 1:20 (g / ml) at a temperature of 55° C., extracted for 40 minutes, centrifuged at a speed of 3000 r / min for 10 minutes to separate the filtrate and filter residue, and the supernatant was concentrated under reduced pressure and freeze-dried to obtain citrus flavonoids.

[0084] (4) The filter residue in step (3) is placed in clean water with a material-liquid ratio of 1:35 (g / ml), the temperature is 90°C, and 300W ultrasonic-assisted extraction is performed for two hours, followed by filtration. The filter residue is subjected to the extraction process once, and the filtrates of step (2) and step (4) are combined to obtain a polysaccharide extract, half of which is used in step (5), and the other half is concentrated under reduced pressure to obtain a polysaccharide concentrate; 4.5 times the volume of anhydrous ethanol is added to the concentrate and mixed thoroughly, and the concentrate is allowed to stand in a refrigerator for 24 hours, and the filtrate and filter residue are separated by centrifugation at a speed of 2000 r / min. The filter residue is rinsed with anhydrous ethanol to evaporate the water, and then freeze-dried to obtain the tangerine peel polysaccharide.

[0085] (5) Add α-glucosidase and α-galactosidase to the polysaccharide extract obtained in step (4) at an addition amount of 1.5% (w / w)

[0086] and 0.5% (w / w), temperature is 60°C, pH is 6.5, 300W ultrasound assisted enzymatic hydrolysis for 2 hours, heating at 90°C for 10 minutes to terminate the enzyme reaction, centrifugation to obtain the supernatant, add 4.5 times the volume of anhydrous ethanol to fully mix, let it stand in the refrigerator for 24 hours, and precipitate and freeze-dry to obtain tangerine peel oligosaccharides.

[0087] (6) The polysaccharide and oligosaccharide prepared above were crushed and passed through a 60-mesh sieve, and the following raw materials were mixed with Lactobacillus delbrueckii culture medium in a volume ratio of 1:2 to form a culture medium. Activated Lactobacillus plantarum, Streptococcus thermophilus, Lactobacillus casei, and yeast (with inoculation amounts of 2.5%, 1%, 2.5%, and 0.5%, respectively) were inoculated into the above culture medium and fermented in a shaking incubator at 40° C. and 200 rpm for 36 hours to obtain a fermentation liquid.

[0088] (7) A composite extract of Citrus aurantium wilfordii with the effects of sobering up and protecting the liver, comprising the following raw materials in percentage by weight: 10% of inclusion essential oil, 30% of flavonoids, 30% of fermentation liquid, and 30% of water.

[0089] Comparative Example 1

[0090] (1) Slice the fresh whole fruit of Citrus aurantium, crush it, pass it through a 60-mesh sieve, and defatted it by reflux with anhydrous ethanol. Then dry it with hot air at a drying temperature of 50°C for 24 hours.

[0091] (2) The tangerine peel powder obtained in step (1) was added to tap water and extracted for two hours with a solid-liquid ratio of 1:25 (g / ml). The filtrate and residue were filtered with four layers of filter cloth, and the extraction process was repeated once for the residue. The two filtrates were combined to obtain the tangerine peel water extract.

[0092] Comparative Example 2

[0093] (1) is similar to Example 1, except that the step in step (4) is replaced by: the filter residue obtained in step (3) is mixed with the filter residue at a material-liquid ratio of 1:

[0094] 25 is mixed with distilled water, the extraction temperature is 85℃, and the extraction is carried out in a water bath with shaking for 2h. After centrifugation, the filter is suctioned and the extraction process is repeated once for the residue. The filtrates of step (2) and step (4) are combined to obtain a polysaccharide extract, half of which is used in step (5), and the other half is concentrated under reduced pressure to obtain a polysaccharide concentrate; 4 times the volume of anhydrous ethanol is added to the concentrate and mixed thoroughly, and the concentrate is allowed to stand in the refrigerator for 12h. The filtrate and residue are separated by centrifugation at a speed of 3000r / min, and the residue is rinsed with anhydrous ethanol to evaporate the water and then freeze-dried to obtain the tangerine peel polysaccharide.

[0095] Comparative Example 3

[0096] (1) Similar to Example 1, except that the step in step (3) is replaced by: placing the filter residue obtained in step (2) in 80% ethanol, with a solid-liquid ratio of 1:25 (g / ml), and a temperature of 45°C. After extraction for 40 minutes, filtering is performed, the filtrate and filter residue are separated, and the supernatant is concentrated under reduced pressure and freeze-dried to obtain citrus red flavonoids.

[0097] Comparative Example 4

[0098] (1) Similar to Example 1, except that the steps in step (5) are replaced by: the polysaccharide extract obtained in step (4) is subjected to ultrasonic-assisted enzymatic hydrolysis at 40°C for 45 minutes at 200W, heated at 90°C for 10 minutes to terminate the enzyme reaction, centrifuged to obtain the supernatant, added with 4 times the volume of anhydrous ethanol to fully mix, and allowed to stand in the refrigerator for 12 hours. The precipitate is freeze-dried to obtain tangerine peel oligosaccharides.

[0099] Comparative Example 5

[0100] (1) Similar to Example 1, except that the inoculum ratio in step (6) is replaced by: activated Lactobacillus plantarum, Streptococcus thermophilus, Lactobacillus casei, and yeast (their inoculum amounts are 1%, 1%, 1%, and 1%, respectively).

[0101] Comparative Example 6

[0102] (1) Similar to Example 1, except that the inoculum ratio in step (6) is replaced by: activated Lactobacillus plantarum, Streptococcus thermophilus, Lactobacillus casei, and yeast (their inoculum amounts are 3%, 0.1%, 0.5%, and 0.2%, respectively).

[0103] Comparative Example 7

[0104] (1) Similar to Example 1, except that the inoculum ratio in step (6) is replaced by: activated Lactobacillus plantarum, Streptococcus thermophilus, Lactobacillus casei, and yeast (their inoculum amounts are 0.5%, 0.3%, 2%, and 3%, respectively).

[0105] Comparative Example 8

[0106] (1) Similar to Example 1, except that the ratio in step (7) is replaced by: inclusion essential oil 60%, flavonoids 20%, fermentation broth 10%, and water 10%.

[0107] Comparative Example 9

[0108] (1) Similar to Example 1, except that the ratio in step (7) is replaced by: inclusion essential oil 10%, flavonoids 10%, fermentation broth 70%, water 10%.

[0109] Comparative Example 10

[0110] (1) Similar to Example 1, except that the ratios in step (7) are replaced by: inclusion essential oil 5%, flavonoids 70%, fermentation broth 20%, and water 5%.

[0111] Test results

[0112] 1. Comparison of active ingredient extraction methods

[0113] Table 1 Comparison of whether essential oils are included or not

[0114] sample Advantages state Encapsulated essential oils High water dispersion stability, difficult to oxidize and deteriorate, low volatility at room temperature solid powder essential oils Low water dispersion stability, easy to oxidize and deteriorate, high volatility at room temperature liquid

[0115] Table 2 Comparison of yields of tangerine peel polysaccharide obtained by different methods

[0116] method Polysaccharide yield / % Example 1 (Ultrasound-assisted water extraction and alcohol precipitation) <![CDATA[20.01±0.24 a ]]> Example 2 (Ultrasound-assisted water extraction and alcohol precipitation) <![CDATA[19.64±0.54 a ]]> Example 3 (Ultrasound-assisted water extraction and alcohol precipitation) <![CDATA[20.23±0.17 a ]]> Example 4 (Ultrasound-assisted water extraction and alcohol precipitation) <![CDATA[20.71±0.88 a ]]> Comparative Example 2 (Ordinary Water Extraction and Alcohol Precipitation) <![CDATA[15.67±0.31 b ]]>

[0117] Table 3 Comparison of the yield of flavonoids obtained by different methods

[0118]

[0119]

[0120] Table 4 Comparison of oligosaccharide yields obtained by different methods

[0121] method Oligosaccharide yield / % Example 1 (enzymatic hydrolysis) <![CDATA[10.84±1.03 a ]]> Example 2 (enzymatic hydrolysis) <![CDATA[10.37±0.73 a ]]> Example 3 (enzymatic hydrolysis) <![CDATA[10.81±0.31 a ]]> Example 4 (enzymatic hydrolysis) <![CDATA[10.61±0.69 a ]]> Comparative Example 4 (ultrasound-assisted extraction method) <![CDATA[7.67±0.45 b ]]>

[0122] As shown in Tables 1-4, the yields of polysaccharides, flavonoids, and oligosaccharides under conventional extraction methods are 15.67%, 7.37%, and 7.67%, respectively. The yields of polysaccharides, flavonoids, and oligosaccharides under the optimized extraction method of the present invention are 19.64% to 20.71%, 12.94% to 14.12%, and 10.37% to 10.84%, respectively. The stability of the inclusion essential oil is much higher than that of ordinary essential oil. After 28 days, the release rate of ordinary essential oil reaches 81%, while the release rate of the inclusion essential oil after 28 days is only 31%. Ordinary essential oils are almost insoluble in aqueous solution, while inclusion compounds can form a stable aqueous solution system. In comparison, the preferred extraction method of the present invention can greatly improve the yield and bioavailability of the active ingredients of Citrus aurantium.

[0123] 2. In vitro alcohol metabolic activity

[0124] The activities of alcohol metabolic enzymes alcohol dehydrogenase (ADH) and acetaldehyde dehydrogenase (ALDH) as well as the in vitro antioxidant capacity of each active ingredient were examined using a multifunctional microplate reader.

[0125] Table 5 Comparison of activation rates of key enzymes in alcohol metabolism by unfermented, single strain and composite strain fermentation broths

[0126]

[0127]

[0128] Note: The inoculation amounts of Lactobacillus plantarum, Streptococcus thermophilus, Lactobacillus casei and yeast are 2.0%, 1.0%, 2.0% and 0.5% respectively.

[0129] Fermentation with different bacterial species affects the content of active ingredients in the fermentation broth. As shown in Table 5, the activation rate of the fermented oligosaccharide-polysaccharide mixture is significantly higher than that of the unfermented mixture, and the activation effect of the alcohol metabolic enzymes in the fermentation of the four mixed strains is significantly better than that of the fermentation of single strains, two mixed strains, and three mixed strains. The four-strain mixed fermentation broth has the highest activation rates of alcohol dehydrogenase and acetaldehyde dehydrogenase, at 41.9% and 32.5%, respectively. The increase in the activation rates of alcohol dehydrogenase and acetaldehyde dehydrogenase reduces the accumulation of ethanol in the blood (significant inhibition of the central nervous system, resulting in a state of drunkenness such as drowsiness or coma) and the accumulation of acetaldehyde in the body (producing a state of drunkenness such as nausea, vomiting, coma, and discomfort, which can damage the liver). This shows that the fermentation broth obtained by the preferred four mixed strain fermentation methods of the present invention has a good alcohol sobering effect.

[0130] Depend on Figure 1 It can be seen that when the fermentation time is short, the activation rate of both enzymes increases slowly as the fermentation time increases. However, if the fermentation time is too long, the activation rate of both enzymes will drop rapidly. The fermentation time of the present invention (36-48h) can maximize the activation rate of both enzymes in the fermentation broth, indicating that this time period is the optimal fermentation time for mixed fermentation.

[0131] Table 6 Comparison of activation rate and antioxidant activity of key enzymes in alcohol metabolism by single active ingredient and different combinations

[0132]

[0133]

[0134] Note: The polysaccharides, oligosaccharides, flavonoids, essential oils and fermentation broth used were obtained by extraction from the same mass of whole tangerine peel and the same parameters as in Example 1.

[0135] As shown in Table 6, the composite extract (fermentation broth + flavonoids + essential oil) of the preferred combination of the present invention has better alcohol dehydrogenase activation rate, acetaldehyde dehydrogenase activation rate, DPPH free radical scavenging rate, and ABTS free radical scavenging rate than any single or two-by-two combination, showing better alcohol metabolism and antioxidant capacity.

[0136] 3. Data on the effect of animal experiments on alleviating alcoholic fatty liver disease

[0137] Four-week-old male C57BL / 6J mice were used as experimental animals. An acute alcohol poisoning mouse model was established by oral administration of liquor to study the regulatory effects of Examples 1 and 2 and Comparative Example 1 on the alcohol metabolism function of mice. Each group of mice was orally administered with 8, 10, 12, and 14 mL / kg of liquor according to body weight. The health status, food intake, water intake, intoxication state, and number of sleep episodes of each group of mice were observed to determine the optimal liquor dosage for this experiment.

[0138] Four experimental groups were set up, namely a blank control group (NC), a model control group (MC), Example 1 and 2 groups, and a comparative example 1 group, with 12 mice in each group. The raw materials were all administered to mice by oral gavage. Except for the normal group and the model group, which were gavaged with physiological 0.9% saline (10 mL / kg), the other groups (based on body weight) were dosed with Example 1 and Comparative Example 1. 30 minutes after each gavage, the blank control group was gavaged with 10 mL / kg (based on body weight) of physiological saline, and the other groups were gavaged with 10 mL / kg of white wine. The gavage was continued for 10 days, and the body weight was measured. During this period, feed and water were available to the mice freely.

[0139] After the final oral gavage, mice were fasted for 12 hours but not water. The mice were weighed, and eye blood, heart, liver, spleen, kidney, and lungs were collected and rinsed in pre-chilled saline. The organs were dried with filter paper, weighed, and photographed. Organ index (organ mass as a percentage of body mass) was calculated. The left lobe of the mouse liver was removed, and 0.5 g of liver tissue was added to 4.5 mL of saline. The mixture was homogenized in an ice-water bath to prepare a 10% (mass-to-volume) tissue homogenate. The mixture was centrifuged at 2500 rpm for 10 minutes, and the supernatant was collected for analysis.

[0140] (1) Biochemical index detection

[0141] Serum biochemical profiles, including total cholesterol (TC) and total triglycerides (TG), were estimated using an automated blood chemistry analyzer.

[0142] (2) Basic indicator testing

[0143] The intoxication state and number of sleep episodes in each group of mice were observed to determine the optimal alcohol dosage. During the intervention period, the mice's weight changes were recorded weekly. Following oral administration of white wine, the mice's intoxication latency (the time from alcohol consumption to ataxia) and intoxication duration (the time from ataxia to recovery of autonomous activity) were recorded. Ataxia was defined as loss of the righting reflex.

[0144] (3) Liver index detection

[0145] Take 0.5 g of liver tissue and add 4.5 mL of normal saline. Grind and homogenize in an ice-water bath to make a 10% (mass-to-volume ratio) tissue homogenate. Centrifuge at 2500 r / min for 10 min. Take the supernatant and detect the aspartate aminotransferase (AST) and alanine aminotransferase (ALT) levels according to the kit instructions.

[0146] Table 7 Basic indicators of drunkenness in each scheme

[0147] Group Drunkenness latency time / min Drunk time / min MC <![CDATA[33.3±1.7 c ]]> <![CDATA[133.3±4.2 a ]]> Comparative Example 1 <![CDATA[45.4±2.5 b ]]> <![CDATA[113.3±4.1 b ]]> Example 1 <![CDATA[56.1±2.1 a ]]> <![CDATA[89.4±4.5 c ]]> Example 2 <![CDATA[55.3±1.9 a ]]> <![CDATA[92.8±3.7 c ]]>

[0148] Compared with the alcohol model group, Example 1, Example 2 and Comparative Example 1 can reduce mortality, prolong the incubation time of drunkenness and reduce the duration of drunkenness. Compared with Comparative Example 1, Examples 1 and 2 of the present invention can significantly prolong the incubation time of drunkenness and significantly reduce the duration of drunkenness, showing a better hangover effect.

[0149] Drinking a lot of alcohol can lead to fat accumulation and dyslipidemia, which is manifested by increased total cholesterol (TC) levels. Figure 2 As can be seen, compared with the blank group, the total cholesterol content in the alcohol model group was significantly increased, indicating that the acute alcohol intoxication model was successfully established. Example 1, Example 2, and Comparative Example 1 all reduced total cholesterol content. Among them, Examples 1 and 2 significantly reduced total cholesterol content and were lower than the blank group, showing a significant effect in improving lipid deposition.

[0150] Drinking a lot of alcohol can lead to fat accumulation and dyslipidemia, which is manifested by increased total triglyceride (TG) content. Figure 3 As can be seen, compared with the blank group, the total triglyceride in the model group was significantly increased, indicating that the acute alcohol intoxication model was successfully established. Example 1, Example 2, and Comparative Example 1 all reduced total triglyceride, with Examples 1 and 2 being superior to Comparative Example 1, demonstrating a significant effect in regulating lipolysis.

[0151] When liver cells are damaged, the cell membrane permeability increases, and the concentration of aspartate aminotransferase (AST) can be used as an indicator of liver cell damage. Figure 4 As shown, compared with the blank group, the AST concentration in the model group increased significantly. The AST concentrations in Examples 1 and 2 were significantly reduced, and compared with Comparative Example 1, the AST concentrations were lower, indicating a stronger ability to repair liver damage.

[0152] When liver cells are damaged, the cell membrane permeability increases, and the concentration of alanine aminotransferase (ALT) can be used as an indicator of liver cell damage. Figure 5 It can be seen that compared with the blank group, the alanine aminotransferase concentration in the model group increased significantly. The alanine aminotransferase concentrations in Examples 1 and 2 were significantly reduced, and compared with Comparative Example 1, the alanine aminotransferase concentrations were lower, showing a stronger liver damage repair ability.

[0153] Depend on Figure 6 It can be seen that compared with the blank group (NC), hepatic fatty degeneration, inflammation and swelling were observed in the alcohol model group (MC), while they were alleviated to a certain extent in Comparative Example 1, Example 1 and Example 2, among which Examples 1 and 2 showed better intervention ability.

[0154] Depend on Figure 7 It can be seen that compared with the blank group, the liver accounted for a higher proportion of body weight in the alcohol model group. Comparative Example 1, Example 1 and Example 2 achieved a certain reduction, among which Examples 1 and 2 had better relief effects than Comparative Example 1, indicating that the composite extract of the present invention has a good effect on repairing liver damage.

[0155] Depend on Figure 8 It can be seen that the alcohol dehydrogenase activation rate and acetaldehyde dehydrogenase activation rate in Example 1 group were significantly higher than those in Comparative Examples 5, 6, and 7. This indicates that the preferred inoculation ratio of the present invention enables the composite extract to have a good alcohol sobering and liver protecting effect.

[0156] Depend on Figure 9 It can be seen that the alcohol dehydrogenase activation rate and acetaldehyde dehydrogenase activation rate in Example 1 group were significantly higher than those in Comparative Examples 8, 9, and 10. This indicates that the preferred addition ratio of fermentation broth, flavonoids, and essential oils of the present invention can enable the composite extract to achieve a synergistic effect.

[0157] The above embodiments of the present invention are merely examples for the purpose of clearly illustrating the present invention and are not intended to limit the embodiments of the present invention. Those skilled in the art will appreciate that other variations or modifications may be made based on the above description. It is not necessary and impossible to enumerate all embodiments here. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention are intended to be included within the scope of protection of the claims of the present invention.

[0158] In the description of this specification, the reference terms "one embodiment", "some embodiments", "example", "specific example", or "some examples" mean that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic expressions of the above terms should not be understood as necessarily referring to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described can be combined in any one or more embodiments or examples in a suitable manner. In addition, those skilled in the art can combine and combine different embodiments or examples described in this specification.

Claims

1. A method for preparing a composite extract of Citrus aurantium dulcis for detoxifying alcohol and protecting the liver, characterized in that: The following steps are involved: (1) Slice the whole fruit of the dried tangerine peel, crush it, sieve it, defat it with anhydrous ethanol, and evaporate the ethanol; (2) placing the tangerine peel powder obtained in step (1) in an essential oil extractor, adding zeolite and water, and maintaining a slight boiling to obtain tangerine peel essential oil and filtrate residue; (3) Mix the tangerine peel essential oil with an equal amount of anhydrous ethanol, slowly drip it into a saturated aqueous solution of β-cyclodextrin, stir and include it, take it out, cool it to room temperature and refrigerate it, filter it and dry it to obtain the included essential oil; (4) inoculating the filter residue obtained in step (2) with Aspergillus niger and Trichoderma spp., extracting with ethanol after fermentation, centrifuging the filtrate and filter residue, concentrating the filtrate, and freeze-drying to obtain citrus red flavonoids; (5) placing the filter residue obtained in step (4) in clean water, performing ultrasonic-assisted extraction and filtering, repeating the extraction process on the filter residue, combining the filtrates of step (2) and step (5) to obtain a polysaccharide extract, half of which is used in step (6), and the other half is concentrated under reduced pressure to obtain a polysaccharide concentrate, adding ethanol to the concentrate and mixing thoroughly, refrigerating and standing, separating the supernatant and the precipitate, and freeze-drying the precipitate to obtain the tangerine peel polysaccharide; (6) adding a complex enzyme to the polysaccharide extract obtained in step (5), performing enzymatic hydrolysis, then heating to terminate the enzyme reaction, centrifuging to obtain the supernatant, adding ethanol to mix thoroughly, refrigerating and standing, taking the precipitate, and freeze-drying to obtain tangerine peel oligosaccharides; (7) crushing the polysaccharide and oligosaccharide obtained above, sieving them, adding purified water to the fixed volume to form a culture medium, and mixing them with Lactobacillus delbrueckii culture medium to form a new culture medium; (8) inoculating the activated mixed strains into the culture medium described in step (7), and obtaining a fermentation liquid after fermentation on a shaking table; (9) The inclusion essential oil obtained in step (3), the Citrus aurantium flavonoids obtained in step (4), and the fermentation liquid obtained in step (8) are uniformly mixed to obtain a Citrus aurantium composite extract.

2. The method for preparing a compound extract of Citrus aurantium dulcis for detoxifying alcohol and protecting liver according to claim 1, characterized in that: The mesh size of the sieve after pulverization in step (1) is 40 to 60 meshes.

3. The method for preparing a compound extract of Citrus aurantium dulcis for detoxifying alcohol and protecting liver according to claim 1, characterized in that: The temperature of the stirring inclusion process in step (3) is 40° C. to 60° C., and the stirring inclusion process is carried out for 2 to 3 hours.

4. The method for preparing a compound extract of Citrus aurantium dulcis for detoxifying alcohol and protecting liver according to claim 1, characterized in that: In step (4), the inoculation ratios of Aspergillus niger and Trichoderma are 2% to 3% and 1% to 2% respectively, the pH of the fermentation process is 6.0 to 7.0, the fermentation time is 12 to 24 hours, the fermentation temperature is 25 to 35° C., the ethanol concentration is 60% to 80%, the material-liquid ratio is 1:20 to 25 (g / ml), the extraction temperature is 45 to 55° C., and the extraction is carried out by centrifugation at 2000 to 3000 r / min for 10 to 15 minutes.

5. The method for preparing a compound extract of Citrus aurantium dulcis for detoxifying alcohol and protecting liver according to claim 1, characterized in that: In step (5), the material-liquid ratio is 1:25-35 g / ml, the ultrasonic power is 300-400 w, the temperature is 85-90 ° C, the added ethanol is 4-4.5 times the volume of the concentrated solution, and the refrigerated standing time is 12-24 h.

6. The method for preparing a compound extract of Citrus aurantium dulcis for detoxifying alcohol and protecting liver according to claim 1, characterized in that: The complex enzyme in step (6) is α-glucosidase and α-galactosidase, and the addition amounts are 0.5% to 1.5% (w / w) and 0.2% to 0.5% (w / w), respectively. The enzymatic hydrolysis temperature is 40 to 60° C., the pH is 4.5 to 6.5, the ultrasonic (200 to 300w) assisted enzymatic hydrolysis time is 2 to 3 hours, the added ethanol is 4 to 4.5 times the volume of anhydrous ethanol relative to the volume of the supernatant, and the refrigerated standing time is 12 to 24 hours.

7. The method for preparing a compound extract of Citrus aurantium dulcis for detoxifying alcohol and protecting liver according to claim 1, characterized in that: In step (7), the amounts of polysaccharide, oligosaccharide and pure water are 10% to 40%, 10% to 40% and 10% to 60% respectively in terms of mass percentage, and the mesh size of the sieve is 20 to 60 meshes; the culture medium in step (7) is composed of a mixture of polysaccharide, oligosaccharide and pure water and a Lactobacillus delbrueckii culture medium, and the volume ratio of the Lactobacillus delbrueckii culture medium to the mixture is 1 to 2:1 to 2.

8. The method for preparing a compound extract of Citrus aurantium dulcis for detoxifying alcohol and protecting liver according to claim 1, characterized in that: The mixed strains described in step (8) are Lactobacillus plantarum, Streptococcus thermophilus, Lactobacillus casei and yeast. Based on the quality of the fermentation system, the strain inoculation ratios are respectively 1.5% to 2.5%, 0.5% to 1.5%, 1.5% to 2.5% and 0.5% to 1.0%. The fermentation temperature is 30-40° C., the shaking table speed is 180 to 200 r / min and the time is 36 to 48 h.

9. The method for preparing a compound extract of Citrus aurantium dulcis for detoxifying alcohol and protecting liver according to claim 1, characterized in that: The mass percentages of the components in the composition in step (9) are 10% to 30% of the inclusion essential oil, 30% to 60% of the flavonoids, 30% to 60% of the fermentation liquid, and the balance is water.

10. The compound extract of Citrus aurantium obtained by the preparation method according to any one of claims 1 to 9, characterized in that: The composite extract has an activation rate of more than 40% for alcohol dehydrogenase and an activation rate of more than 30% for acetaldehyde dehydrogenase.

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