CN116496859A

CN116496859A - Ginseng millet wine and preparation method and application thereof

Info

Publication number: CN116496859A
Application number: CN202310735654.8A
Authority: CN
Inventors: 郑飞; 吴泳锡; 皮子凤; 越皓; 戴雨霖
Original assignee: Changchun University of Chinese Medicine
Current assignee: Changchun University of Chinese Medicine
Priority date: 2023-06-21
Filing date: 2023-06-21
Publication date: 2023-07-28

Abstract

The invention provides ginseng millet wine and a preparation method and application thereof, and relates to the technical field of health care wine. The invention saccharifies the pulping and saccharifying enzyme and the millet of the ginseng, then ferments and decocts the wine, and finally prepares the ginseng millet wine which is clear and transparent and has moderate sweet and sour; 303 aroma components are detected in the ginseng millet wine, so that the ginseng millet wine has special taste of ginseng, is mellow in wine quality and has no obvious bitter taste of ginseng. The ginseng millet wine obtained by the invention is rich in rare ginsenoside, activates alcohol dehydrogenase, accelerates alcohol metabolism rate, and can play a role in protecting liver by inhibiting oxidative stress reaction.

Description

Ginseng millet wine and preparation method and application thereof

Technical Field

The invention belongs to the technical field of health care wine, and particularly relates to ginseng millet wine and a preparation method and application thereof.

Background

Millet is used as characteristic food in northeast China, wherein the content of crude protein and starch is higher than that of rice and wheat, and is a high-quality raw material for brewing wine. In addition, the nutrient elements such as vitamins, minerals, trace elements and the like are also rich. However, the efficacy of the traditional millet wine is not outstanding.

The ginseng is the dried root and rhizome of ginseng of Araliaceae, and is widely applied to the fields of health-care food and food because of remarkable pharmacodynamic activity since the ginseng is taken as a new resource food. Ginsenoside is the main active ingredient, and has abundant bioactivity, such as anti-tumor, anti-fatigue, immune system regulating, anti-inflammatory, anti-aging, etc. Ginsenoside is a glycoside compound formed by connecting ginsenoside aglycone and glycosyl through glycosidic bond, and triterpene saponin is the main component. At present, more than one hundred ginsenosides have been separated, wherein the common ginsenosides are mainly protopanaxadiol type ginsenosides and protopanaxatriol type ginsenosides, and the common ginsenosides are mainly distinguished by different 6-position substituents of parent nuclei. Research shows that the pharmacological activity and medicinal value of deglycosylated ginsenoside are superior to those of main ginsenoside, and compared with the main ginsenoside, the deglycosylated rare ginsenoside is easier to enter blood circulation and exert action, so that the deglycosylated rare ginsenoside is a candidate drug molecule with great potential. Factors such as the type of ginsenoside, the number of substituents, the number of saccharides, and the configuration will affect the pharmacological activity.

The ginseng product in the Changbai mountain area is lack of characteristics and has low added value. The ginseng wine circulated in the market is ginseng soaked wine, the efficacy is not obvious, the mechanism is not clear, the flavor of the wine is poor, and no relevant report on anti-alcoholic liver injury exists. In order to meet the requirements of industrial production, along with the progress of scientific research, ginseng high-added-value products are continuously developed and converted and are applied to industrial production.

Disclosure of Invention

In view of the above, the invention aims to provide a preparation method of the ginseng millet wine, which has good vinosity and high wine yield, and the ginsenoside forms various rare saponins after fermentation and conversion, so that the ginseng millet wine has the health care functions of higher alcohol metabolism rate and strong anti-alcoholic liver injury.

In order to achieve the above object, the present invention provides the following technical solutions:

the invention provides a preparation method of ginseng millet wine, which comprises the following steps:

pulping Ginseng radix, adding saccharifying enzyme into semen Panici Miliacei, and saccharifying to obtain sweet mash; adding 0.5-0.7wt% of yeast into the mash for fermentation, and filtering residues in the fermentation liquor after the fermentation is finished for ageing; and (3) after the ageing is finished, obtaining fermented mash, and decocting the fermented mash to obtain the ginseng millet wine.

Preferably, the millet processing mode is as follows: soaking the millet for 6-10 hours, steaming for 40-60 minutes at normal pressure, and then spreading for cooling to 25-30 ℃.

Preferably, the saccharification time is 15-30 h, and the saccharification temperature is 45-55 ℃.

Preferably, the fermentation temperature is 23-33 ℃, and the fermentation time is 6-10 d.

Preferably, the ageing temperature is 10-20 ℃, and the ageing time is 15-20 d.

Preferably, the temperature of the wine decocting is 90-120 ℃, and the time of wine decocting is 8-15 min.

Preferably, the addition amount of the saccharifying enzyme is 0.4-0.6wt%.

Preferably, the mass ratio of the ginseng to the millet is 1:1-2.

The invention also provides the ginseng millet wine obtained by the preparation method.

The invention also provides an application of the ginseng millet wine in preparing an anti-alcoholic liver injury product.

Compared with the prior art, the invention has the following beneficial effects:

(1) According to the process for preparing the ginseng millet wine, the fresh ginseng is added to provide a proper acidic environment for fermentation liquor, so that more active ingredients are obtained; under the condition of microbial fermentation, the conversion of polysaccharide-based ginsenoside to rare ginsenoside is facilitated. Through detection, the prepared ginseng millet wine is rich in 92 ginsenosides, and the bioavailability of the ginsenosides is improved.

(2) The ginseng millet wine prepared by the invention can accelerate the alcohol metabolism rate of a human body and reduce the discomfort of hangover or drunk people; the rare ginsenoside obtained by the preparation process of the invention also has the health-care function of resisting alcoholic liver injury for the ginseng millet wine, is suitable for various sub-health people, and provides a reference for developing and utilizing high-value health products of the ginseng millet wine.

Drawings

FIG. 1 is a BPI chart of aroma components of ginseng millet wine in example 1 of the present invention;

FIG. 2 shows the results of chromatographic analysis of ginsenoside in the case of example 1 of the present invention and commercial ginseng wine;

FIG. 3 shows the results of AO/EB fluorescent staining of LO 2-intervening cells according to example 1 of the present invention;

FIG. 4 shows the result of SDS-PAGE gel of LO2 cellular proteins according to example 1 of the present invention;

FIG. 5 shows the effect of example 1 of the invention on LO2 cell protein expression;

FIG. 6 shows the biochemical index of LO2 cells measured in example 1 of the present invention;

FIG. 7 is a test of the relative levels of induced ROS in zebra fish in example 1 of the present invention;

FIG. 8 is a graph showing the measurement of the biochemical markers of induced zebra fish according to example 1 of the present invention;

FIG. 9 shows the measurement of alcohol dehydrogenase content in example 1 of the present invention.

Detailed Description

pulping Ginseng radix, adding saccharifying enzyme into semen Panici Miliacei, and saccharifying to obtain sweet mash; preferably adding 0.5-0.7wt% of yeast into the mash for fermentation, and filtering residues in the fermentation liquor for ageing after the fermentation is finished; after ageing, obtaining fermented mash, and decocting the fermented mash to obtain ginseng millet wine; more preferably 0.6wt% yeast is added for fermentation. In a specific embodiment of the present invention, the method for pulping ginseng comprises the steps of: and (3) taking the cleaned fresh ginseng, cutting the fresh ginseng into slices with the thickness of 2-3mm, and adding water with the mass of 3-5 times for pulping. In a specific embodiment of the invention, the yeast is Saccharomyces cerevisiae, a high activity yeast available from Dandelion, inc. of Lesfu group, france. The amount of yeast added directly affects the properties of the fermented wine. The addition amount of the yeast is controlled to be 0.5-0.7wt%, for example, when the addition amount of the yeast is less than 0.5wt%, the transformation efficiency reduction trend becomes slow, and when the addition amount of the yeast is more than 0.7wt%, the yeast reproduction rate is too high, unpleasant taste is easily generated, and the sensory score is reduced.

In the present invention, the millet processing mode is preferably: soaking millet for 6-10 hours, steaming for 40-60 minutes at normal pressure, and then spreading for cooling to 25-30 ℃; the millet processing mode is more preferably as follows: soaking the millet for 5-8 hours, steaming for 45-55 minutes at normal pressure, and then spreading for cooling to 28 ℃. In a specific embodiment of the present invention, ginseng pulp and saccharifying enzyme are added to spread and cooled steamed millet to carry out saccharification reaction.

In the invention, the saccharification time is preferably 15-30 hours, and the saccharification temperature is preferably 45-55 ℃; the saccharification time is more preferably 18-24 hours, and the saccharification temperature is more preferably 48-50 ℃.

In the invention, the fermentation temperature is preferably 23-33 ℃, and the fermentation time is preferably 6-10 d; the fermentation temperature is more preferably 25-30 ℃, and the fermentation time is more preferably 7-9 d. The low fermentation temperature can lead to low yeast metabolism activity and low wine production rate, and people participate in millet and cannot ferment completely under the same fermentation time, so that the alcohol degree is lower, and the alcohol degree is stable due to the rising of the temperature. When the temperature is too high, the growth and the reproduction of yeast are not suitable, the growth and the metabolism rate of yeast are fast, a large amount of alcohol can be locally generated in a short time, the growth of microorganisms is inhibited, the premature senility phenomenon is caused, various bad flavor substances are accumulated, the wine is insufficient in aftertaste, spicy, heavy in bitter taste and not mature and mellow enough, the wine is easy to rancidity in the later fermentation stage due to the too high temperature, the whole sensory quality of the wine body is damaged, and the content of active ingredients is reduced. According to the invention, the fermentation temperature and the fermentation time are adjusted to enable the obtained ginseng millet wine to be fermented more fully, and the fermentation is ended when the alcoholic strength reaches 7-9% vol, so that the obtained ginseng millet wine is mellow and smooth, and has no obvious bitter taste of ginseng.

In the invention, the ageing temperature is preferably 10-20 ℃, and the ageing time is preferably 15-20 d; the ageing temperature is more preferably 15-18 ℃, and the ageing time is more preferably 16-18 d.

In the invention, the temperature of the wine decoction is preferably 90-120 ℃, and the wine decoction time is preferably 8-15 min; the temperature of the wine decocting is more preferably 100-110 ℃, and the time of wine decocting is more preferably 10-12 min.

In the invention, the addition amount of the saccharifying enzyme is preferably 0.4-0.6wt%; more preferably 0.5wt%. The added amount of the saccharifying enzyme is too high, and the content of the initial saccharifying enzyme is higher, so that the generated alcohol, aroma and other components are more, but the autolysis phenomenon occurs along with the early aging of the later saccharomycetes, so that the fermentation is stopped in advance, the generated amount of the alcohol is reduced, and the sensory score is lowered; when the addition amount of the saccharifying enzyme is low, insufficient nutrient substances required for growth in the mash can cause metabolism to stop, and the alcoholic strength of the mash is also reduced. The addition amount of the saccharifying enzyme can better promote the saccharification reaction of ginseng and millet. The saccharifying enzymes described in the present invention are purchased from Angel Yeast Co.

In the invention, the mass ratio of the ginseng to the millet is preferably 1:1-2; more preferably 1:1.5. The invention satisfies the pH value of the fermentation liquid system by controlling the adding amount of ginseng, provides a proper acidic environment, further obtains more active ingredients, and is favorable for converting polysaccharide-based ginsenoside into rare ginsenoside under the condition of microbial fermentation.

The invention also provides the ginseng millet wine obtained by the preparation method. The total saponin content in the prepared ginseng millet wine is more than or equal to 90mg/100mL, and the rare ginsenoside is more than or equal to 18mg/100mL, wherein the rare ginsenoside is Rh1+ Rg2+ F2+ Rg3+ CK + Rh2+ Rg4+ Rg6+ Gypenoside XVII.

The invention also provides an application of the ginseng millet wine in preparing an anti-alcoholic liver injury product. In the present invention, the preparation of anti-alcoholic liver injury products includes, but is not limited to, anti-alcoholic liver injury health care products.

The technical solutions provided by the present invention are described in detail below with reference to examples, but they should not be construed as limiting the scope of the present invention.

Example 1

A preparation method of ginseng millet wine comprises the following steps: firstly, taking cleaned fresh ginseng according to the mass ratio of the fresh ginseng to the millet of 1:1, cutting the fresh ginseng into slices with the mass of about 2-3mm, adding 3 times of water for pulping to obtain fresh ginseng pulp, simultaneously cleaning and soaking the millet for 12 hours, steaming the soaked millet for 60 minutes at normal pressure, and then spreading for cooling to 30 ℃. Fresh ginseng pulp and 0.5wt% of saccharifying enzyme are added to cooled millet for saccharification for 24 hours to obtain the mash. Adding 0.7wt% of high-activity yeast for brewing wine into the mash after activating, uniformly stirring, adding 400wt% of Changbai mountain mineral water, fermenting, ending the fermentation when the alcoholic strength reaches 7-9% vol, filtering residues in the fermentation liquid, ageing, and after ageing, performing pasteurization on the aged fermentation mash for 10min at 100 ℃ to obtain ginseng millet wine; wherein the fermentation temperature is 28 ℃, the fermentation time is 8 days, the ageing temperature is 15 ℃, and the ageing time is 15 days.

The HPLC-MS/MS combined technology is used for detecting the ginsenoside which is the effective component in the ginseng millet wine, and 92 ginsenosides are detected. Wherein, the total saponin content is more than or equal to 90mg/100mL, and the rare ginsenoside content is more than or equal to 18mg/100mL.

Example 2

Firstly taking the cleaned fresh ginseng according to the mass ratio of the fresh ginseng to the millet of 1:2, cutting the fresh ginseng into slices with the mass of 2-3mm, adding 3 times of water for pulping to obtain fresh ginseng pulp, simultaneously cleaning and soaking the millet for 8 hours, steaming the soaked millet for 50 minutes at normal pressure, and then spreading for cooling to 25 ℃. Fresh ginseng pulp and 0.6wt% of saccharifying enzyme are added to cooled millet for saccharification for 30 hours to obtain the mash. Adding 0.5wt% of high-activity yeast for brewing according to the amount of fermentation liquid to be fermented, activating, adding the activated high-activity yeast into the mash, uniformly stirring, adding 400wt% of Changbai mountain mineral water, fermenting, ending the fermentation when the alcoholic strength reaches 7-9% vol, filtering residues in the fermentation liquid, ageing, and after the ageing is ended, performing pasteurization on the aged fermentation mash for 8min under the condition of 120 ℃ to obtain ginseng millet wine; wherein the fermentation temperature is 23 ℃, the fermentation time is 10 days, the ageing temperature is 14 ℃, and the ageing time is 20 days.

The effective ginsenosides in the ginseng millet wine are detected by an HPLC-MS/MS combined technology, and 92 ginsenosides are detected. Wherein, the content of the total ginsenoside is more than or equal to 90mg/100mL, and the rare ginsenoside is more than or equal to 18mg/100mL.

Example 3

Firstly taking fresh ginseng according to the mass ratio of fresh ginseng to millet of 1:1.5, cutting the fresh ginseng into slices with the mass of about 2-3mm, adding 3 times of water for pulping to obtain fresh ginseng pulp, simultaneously cleaning and soaking the millet for 12 hours, steaming the soaked millet for 60 minutes at normal pressure, and then spreading for cooling to 30 ℃. Fresh ginseng pulp and 0.5wt% of saccharifying enzyme are added to cooled millet for saccharification for 24 hours to obtain the mash. Adding 0.3-0.7wt% of high-activity yeast for brewing into the mash after activating, stirring uniformly, adding 400wt% of Changbai mountain mineral water, fermenting, ending fermentation when the alcoholic strength reaches 7-9 vol%, filtering residues in the fermentation broth, ageing, and performing pasteurization after ageing, and decocting the aged fermentation broth at 100deg.C for 10min to obtain Ginseng semen Panici Miliacei wine; wherein the fermentation temperature is 33 ℃, the fermentation time is 6 days, the later fermentation temperature is 15 ℃, and the fermentation time is 15 days.

Comparative example 1

In example 1, the mass ratio of fresh people to millet is replaced by 1.5:1, and the other conditions are the same as in example 1.

The ginsenoside as an active ingredient in the fermented wine was detected by HPLC-MS/MS combination technique, and 90 kinds of ginsenoside were detected in the fermented liquid, and no ginsenoside such as PPD, PPT, 20-O-Glc-Rf was detected as compared with the case 1.

Comparative example 2

The mass ratio of millet to ginseng in example 1 was replaced with 0.5:2.5, and the other conditions were the same as in example 1.

Comparative example 3

The amount of the high-activity yeast for brewing in example 1 was replaced with 0.4 wt% and the other conditions were the same as those in example 1.

Compared with the example 1, the yellow wine has low alcoholic strength, bitter and astringent taste and clear and low flavor.

Comparative example 4

The amount of the high-activity yeast for brewing in example 1 was replaced with 1.0 wt% and the other conditions were the same as those in example 1.

Compared with example 1, the fermented wine has insufficient aroma and light color.

Comparative example 5

The fermentation temperature was changed to 35℃and the fermentation time was changed to 10 days in example 1, and the other conditions were the same as in example 1.

Compared with example 1, the content of effective components is reduced, the amount of mixed bacteria is increased, the acidity of the wine body is increased, and the taste is too sour.

Test example 1

Aroma components of the ginseng millet wine obtained in examples 1 to 3 were analyzed. The analytical chromatographic conditions were: chromatographic column: DB-WAX (30.00 m.times.0.25 mm.times.0.25 μm); the temperature of the sample inlet is 250 ℃; the carrier gas is high-purity helium (He) with the purity of 99.9 percent; the flow rate is 1mL/min; sample injection mode: not split; the temperature programming conditions are as follows: the initial temperature is 40 ℃; hold for 5min, raise to 220℃at 5℃per min, raise to 250℃at 20℃per min, hold for 2.5min. Mass spectrometry conditions: using electron bombardment source anion scan patterns (EI ^- ) The method comprises the steps of carrying out a first treatment on the surface of the Interface temperature 260 ℃; the ion source temperature is 230 ℃; the temperature of the quadrupole rods is 150 ℃; the scanning range m/z is 20-500. Headspace solid phase microextraction conditions: extraction head (50/30. Mu. MDVB/CAR/PDMS SPMEfiber); 1mL of ginseng fermentation broth is taken to be placed in a 20mL headspace bottle, balanced for 15min at 50 ℃, extracted for 30min and desorbed for 5min. FIG. 1 is an embodiment1, a BPI chart of aroma components of the ginseng millet wine; the 303 aroma components of the ginseng millet wine of example 1 are shown in table 1. The analysis results of example 2 and example 3 were similar to those of example 1.

Table 1 data on 303 aroma components in ginseng millet wine (n=3)

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The alcohol degrees of the ginseng millet wine of examples 1 to 3 and the ginseng millet wine obtained in comparative examples 1 to 5 were detected, and the products were subjected to sensory evaluation. The experimental method of sensory evaluation is that the nose smell, mouth taste and eye view are combined; a total of 100 professionally trained personnel participated in the evaluation. The evaluation criteria are shown in Table 2, and the evaluation results are shown in Table 3.

Table 2 sensory evaluation criteria for ginseng millet wine

Table 3 sensory evaluation of Ginseng millet wine of examples 1 to 3 and comparative examples 1 to 5

The ginseng millet wine obtained in the embodiments 1-3 is pale yellow, clear and transparent, moderate in sweetness and sourness, has the special taste of ginseng, mellow and sweet in wine quality, and has no obvious bitter taste of ginseng.

Test example 2

Chromatographic analysis was performed on ginsenoside in example 1 and commercial ginseng wine (produced from Shenyang eastern spring wine works, preparation method: ginseng was soaked in 42% white spirit and prepared to 8% concentration for identification). The analytical chromatographic conditions were: chromatographic column: agilentZORBAXeclipse CalusC 18 (2.1X105 mm,3.5 μm); gradient elution, mobile phase: (a) 0.1% formic acid in water- (B) acetonitrile; mobile phase gradient: 0-5min, B15-17%; 5-10min, B17-19%; 10-20min, B19-21%; 20-26min, B21-28%; 26-37min, B28% -30%;37-45min, B30-36%; 45-50min, B36-45%; 50-58min, B45-65%; 58-60min, B65-80%; column temperature is 35 ℃, flow rate is 0.4mL/min, and sample injection quantity is 5 mu L. Mass spectrometry conditions: using electrospray negative ion scanning mode (ESI) ^- ) The method comprises the steps of carrying out a first treatment on the surface of the Dryer flow rate (N) ₂ ) The dryer temperature is 9L/min, the atomization voltage is 3500kV, the capillary voltage is 3500kV, the fragmentation voltage is 175V, the cone hole voltage is 65V, and the mass scanning range is m/z100-2000. Experimental data were analyzed using masshunterqualitive analysis software.

As can be seen from FIG. 2, the ginseng millet wine (FIG. 2B) has significantly reduced contents of ginsenoside mRg, re, mRb1 and Rc, relatively increased contents of ginsenoside Rb1 and Rd, and the rare ginsenoside formed by conversion mainly has PPD, PPT, 20 (R) Rh3, 20 (S) Rh3 and the like, compared with the commercial ginseng wine (FIG. 2A). The analysis results of example 2 and example 3 were similar to those of example 1.

Test example 3

The experiment adopts a cell experiment, and LO2 cells of normal liver epithelial cells of human beings are selected for modeling, and the LO2 normal liver epithelial cell line is derived from the institute of biophysics of China academy of sciences. The reagent is a Total Cholesterol (TC) determination kit, a Triglyceride (TG) determination kit, a total protein determination kit, a Malondialdehyde (MDA) determination kit, a superoxide dismutase (T-SOD) determination kit and a glutathione peroxidase (GSH-Px) determination kit which are purchased from Nanjing institute of biological engineering.

The sample feeding method comprises the following steps: and taking LO2 cells of normal liver epithelial cells of human beings, and randomly dividing the LO2 cells into a normal group, a model group, a positive control group and a high-medium-low dose group of ginseng millet wine after subculturing. PBS was added to the normal group (CON), 8% ethanol was added to the model group (AL), and 8% yellow wine (available from Zhejiang Guyue Longshan Shaoxing wine Co., ltd.) was added to the positive control group (H). A low dose group of ginseng millet wine (GL, example 1 diluted to 2% alcoholicity), a medium dose group of ginseng millet wine (GM, example 1 diluted to 4% alcoholicity), and a high dose group of ginseng millet wine (GH, example 1). The measuring method comprises the following steps: AO/EB staining was used to detect apoptosis of LO2, and by double staining, the status of living cells (nuclear chromatin with green uniform fluorescence), early apoptotic cells (nuclear chromatin with green granule fluorescence), late apoptotic cells (nuclear chromatin with orange red granule fluorescence) and non-apoptotic dead cells (nuclear chromatin with orange red uniform fluorescence) could be observed. Apoptosis was detected under a fluorescence microscope after staining, and the staining results are shown in fig. 3. BCA assay kit was used to determine protein concentration. Depending on the protein concentration and the desired volume, 5 XSDS was added to the lysate to give a loading volume of 20. Mu.L. Protein samples were quantified at 15 μg per well. Proteins were separated by 10% SDS-PAGE gel electrophoresis and transferred to PVDF membrane. And (5) observing whether the protein is successfully transferred or not by using a ponceau staining method. Then blocked with 5% skimmed milk at room temperature for 3h. The corresponding primary antibody (1:1000) was added and incubated overnight at 4℃and the corresponding secondary antibody was added and incubated at room temperature for 2h. Imaging was performed using a high sensitivity ECL solution development chemiluminescent instrument, and the experimental results are shown in fig. 4. Total Cholesterol (TC), triglyceride (TG), malondialdehyde (MDA), superoxide dismutase (T-SOD) and glutathione peroxidase (GSH-Px) were all measured according to the procedure of the instruction on the kit, and the experimental results are shown in FIG. 6.

As can be seen from fig. 3, AO/EB staining was used to detect the apoptosis cycle of LO2 cells. The cells of the CON group exhibited uniform green fluorescence with clear nucleoli and approximately normal cell morphology (fig. 3A). While the green and orange fluorescence of the green particles of groups AL (fig. 3B) and H (fig. 3C) are evident, the total cell volume is also significantly reduced. While the relative intensities of the orange fluorescence of the three groups GL (figure 3D), GM (figure 3E) and GH (figure 3F) gradually subside, and the cell volume is also enlarged, which shows that the ginseng millet yellow wine improves the early apoptosis and the late apoptosis of LO2 cells induced by alcohol compared with other alcohol type products, and the higher the concentration, the more obvious the activity.

As can be seen from fig. 4 and 5, the expression of the AL group PI3K proteins was down-regulated (p < 0.05) compared to the CON group. Up-regulation of PI3K protein expression was observed following treatment with ginseng millet wine. After alcohol induction, protein expression of AL and H AKT and Bcl-2 was reduced and significantly up-regulated (p < 0.05) following treatment with ginseng millet wine. Meanwhile, the expression level of the CON group Bax protein is lower, but the AL group is obviously up-regulated, and the expression level of the protein is obviously down-regulated after the treatment of the ginseng millet wine.

As can be seen from fig. 6, the high, medium and low dose groups GSH-PX (fig. 6B) and SOD (fig. 6A) had increased activity (p < 0.05), TC (fig. 6D), TG (fig. 6E) and MDA (fig. 6C) levels decreased (p < 0.05) compared to the AL group. In conclusion, the protection of the LO2 cells in example 1 is realized by recovering the mitochondrial apoptosis pathway, can raise the ratio of Bcl-2 to Bax, reduce the occurrence of apoptosis, and has a certain protection effect on resisting alcoholic liver injury. In FIG. 6, T-SOD has an ordinate unit of nmol/mgprot, GSH-PX has an ordinate unit of nmol/mgprot, MDA has an ordinate unit of nmol/mgprot, TC has a unit of mmol/g, and TC has a unit of mmol/g.

Test example 4

Pharmacodynamic tests were performed on the ginseng millet wine of example 1. The experiment adopts an animal experiment, and the animal is zebra fish, an AB wild strain and Nanjing Yishuli peanut science and technology company. The reagent is a Total Cholesterol (TC) determination kit, a Triglyceride (TG) determination kit, a total protein determination kit, a Malondialdehyde (MDA) determination kit, a superoxide dismutase (T-SOD) determination kit and a glutathione peroxidase (GSH-Px) determination kit which are purchased from Nanjing institute of biological engineering.

The sample feeding method comprises the following steps: healthy zebra fish larvae were randomly selected, and after adaptive breeding, they were randomly divided into 50 animals each, namely, normal group, model group, positive control group, ginseng millet wine (example 1), medium (example 1 diluted to 4% alcohol), and low dose group (example 1 diluted to 2% alcohol). PBS was added to the normal group (CON), 8% ethanol was added to the model group (AL), and yellow wine was added to the positive control group (H). A low dose group of ginseng millet wine (GL, 2% alcoholicity), a medium dose group of ginseng millet wine (GM, 4% alcoholicity) and a high dose group of ginseng millet wine (GH, 8% alcoholicity). After 128h of dosing, staining was performed by adding DCFH-DA stain (20. Mu.g/mL) followed by incubation in the dark for 2h. The staining solution was then washed out and examined by photographic observation under a fluorescence microscope against a blue fluorescent background for the change in ROS content in each group of zebra fish, as shown in fig. 7. The zebra fish liver tissue homogenate was prepared by a high-speed homogenizer, centrifuged (4000 r/min,10 min) for biochemical index determination, total Cholesterol (TC), triglyceride (TG), malondialdehyde (MDA), superoxide dismutase (T-SOD) and glutathione peroxidase (GSH-Px) were all determined according to the instruction method steps on the kit, and the experimental results are shown in FIG. 8.

ROS are markers of oxidative stress, as can be seen from fig. 7, the green fluorescence of the blank group is the weakest (fig. 7A), the green fluorescence intensity of the model group juvenile fish is the strongest (fig. 7B), the green fluorescence of the positive control group juvenile fish is slightly lower than that of the model group (fig. 7C), and the green fluorescence intensity of the ginseng millet wine low dose group (fig. 7D), the medium dose group (fig. 7E) and the high dose group (fig. 7F) is gradually reduced, indicating that ROS in zebra fish are gradually reduced. As can be seen from fig. 8, the enzyme activities of MDA (fig. 8C), T-SOD (fig. 8A), TC (fig. 8D), TG (fig. 8E), GSH-Px (fig. 8B) all had significant differences (p < 0.05) in the liver homogenate of the zebra fish in the model group, compared with the blank group, wherein the enzyme activities of MDA and T-SOD had very significant differences (p < 0.01), indicating that the model was established. Compared with the model group, the activity of T-SOD and GSH-Px enzymes in the high-dose group, the medium-dose group and the low-dose group of the ginseng millet wine have significant differences (p < 0.05). In FIG. 8, T-SOD has an ordinate unit of nmol/mgprot, GSH-PX has an ordinate unit of nmol/mgprot, MDA has an unit of nmol/mgprot, TC has an unit of mmol/g, and TC has an unit of mmol/g.

Combining fig. 7 and 8, the ginseng millet wine of the invention has strong health care function of resisting alcoholic liver injury. In the whole experimental process, the activity state of the zebra fish is not abnormal, biochemical indexes in the detection process are not abnormal, the health of the tested animals is not obviously influenced, and the safety is high.

Test example 5

Pharmacokinetic experiments were performed on the ginseng millet wine of example 1. The 20 rats were randomly divided into 5 groups after one week of adaptive feeding, which were ginseng millet wine high dose group (example 1), commercial ginseng wine group a, commercial ginseng wine group B, commercial yellow wine group a, commercial yellow wine group B, labeled and weighed. Blood is taken out 0h, 0.5h, 1h, 1.5h, 2h, 3h, 4h, 6h, 12h, 24h and 48h after the stomach is irrigated, a blood sample is centrifuged for 30min by a high-speed refrigerated centrifuge 3000r/min, and supernatant is taken out for standby after centrifugation. The results show that the half-life period and the maximum time to reach the serum concentration (Tmax) of the ethanol in the ginseng millet wine group rats are lower than those of other commercial wine group rats, the ethanol content in the serum reaches the peak value at the time of 0.74h of the ginseng millet wine group rats, the rapid elimination process is shown, and no ethanol is detected in the serum at the sampling point of the third hour. Compared with other commercial alcohol group rats, the metabolism reaction of the ginseng millet wine group rats to the ethanol is most rapid. The relevant pharmacokinetic parameters are shown in table 4.

TABLE 4 metabolism kinetic parameters of ethanol in serum of rats in the Ginseng Pan Rice wine group and other commercially available wines group

Test example 6

The alcohol dehydrogenase was measured on the ginseng millet wine of example 1. The absorbance change was measured at a detection wavelength of 340 nm. The viability of ALT, AST, AKP was also observed by in vitro LO2 cell experiments, as shown in fig. 9. Alcohol Dehydrogenase (ADH) is a zinc-containing metalloprotease which mainly exists in the liver and is the most important participating enzyme and metabolizing enzyme for alcohol metabolism, and the activation rate of the ginseng millet wine on the ADH is ordered from large to small: GSLP-III > GSLP-I > GSLP-II (FIG. 9A), it is shown that the ginseng millet wine obtained by the invention has the activity of activating ADH and ALDH to a certain extent, and can accelerate alcohol metabolism and reduce blood alcohol content. The research shows that the ginseng millet wine has an activating effect on alcohol metabolism related enzymes, particularly has a stronger activating effect on GSLP-III, thus being beneficial to reducing the harm of acetaldehyde generated by alcohol metabolism to human bodies. Also, as can be seen in fig. 9B, to some extent, ginseng millet wine was able to reduce ALT, AST and AKP activity in the LO2 model.

The foregoing is merely a preferred embodiment of the present invention and it should be noted that modifications and adaptations to those skilled in the art may be made without departing from the principles of the present invention, which are intended to be comprehended within the scope of the present invention.

Claims

1. A preparation method of ginseng millet wine is characterized by comprising the following steps:

2. The method according to claim 1, wherein the millet processing method is as follows: soaking the millet for 6-10 hours, steaming for 40-60 minutes, and then spreading for cooling to 25-30 ℃.

3. The method according to claim 1, wherein the saccharification time is 15-30 hours and the saccharification temperature is 45-55 ℃.

4. The preparation method of claim 1, wherein the fermentation temperature is 23-33 ℃ and the fermentation time is 6-10 d.

5. The method according to claim 1, wherein the aging temperature is 10-20 ℃ and the aging time is 15-20 d.

6. The preparation method according to claim 1, wherein the temperature of the decoction is 90-120 ℃, and the time of the decoction is 8-15 min.

7. The method according to claim 1, wherein the saccharifying enzyme is added in an amount of 0.4 to 0.6wt%.

8. The preparation method of claim 1, wherein the mass ratio of the people to the millet is 1:1-2.

9. The ginseng millet wine obtained by the preparation method of any one of claims 1 to 8.

10. Use of a ginseng millet wine of claim 9 in the preparation of an anti-alcoholic liver injury product.