Lactobacillus plantarum and application thereof in fermentation of ginsenoside

Abstract

The invention belongs to the technical field of medicinal and edible Chinese herbal medicine alcoholic drink, and particularly relates to lactobacillus plantarum and application thereof in fermentation of ginsenoside. The lactobacillus plantarum is specifically lactobacillus plantarum L100, and the preservation number of the lactobacillus plantarum L100 is CGMCC (China General Microbiological Culture Collection Center) No. 34425. The lactobacillus plantarum L100 has the function of converting and preparing the rare ginsenoside, basic components of ginseng can be directionally converted into high-value rare ginsenoside (such as Rg3, Rg5 and Rh1), and particularly, the lactobacillus plantarum L100 has extremely high specificity on generation of F5 and Rh10. Ginseng fermentation liquor obtained by fermenting ginseng through lactobacillus plantarum L100 and extracting solutions of medicinal and edible components such as codonopsis pilosula, astragalus membranaceus, liquorice and cistanche deserticola are used for preparing the liqueur, and a liqueur product with potential effects in the aspects of oxidation resistance, inflammation resistance, immunoregulation and the like is obtained.

Description

Technical Field

[0001] This invention belongs to the field of medicinal and edible herbal liqueur technology, and specifically relates to a herbal liqueur with antioxidant and free radical scavenging functions and its preparation method. Background Technology

[0002] Liqueurs are a type of reconstituted alcoholic beverage made by blending the active ingredients of traditional Chinese medicine with the base spirit through extraction or co-fermentation processes, using distilled or fermented spirits as the base. They possess both complex flavors and health benefits. In recent years, with the upgrading of consumer demand, probiotic dynamic fermentation technology has gradually replaced the traditional static extraction process, driving the liqueur industry towards functionalization and high quality. Traditional static extraction methods generally suffer from low material contact efficiency and limited mass transfer kinetics, resulting in low dissolution rates of active ingredients and a limited range of flavor profiles. In contrast, probiotic dynamic fermentation technology, by constructing a multi-phase synergistic system of microorganisms, traditional Chinese medicine, and base spirit, can significantly improve the biotransformation efficiency of active ingredients.

[0003] Against the backdrop of the rapid development of the health industry, the market demand for functional liqueurs that combine the characteristics of traditional alcoholic beverages with targeted health benefits is growing. Taking ginseng liqueur as an example, its core active ingredient, ginsenosides, has antioxidant, anti-fatigue, and immunomodulatory effects. However, traditional processes often employ a single ethanol extraction method, resulting in low dissolution rates and poor bioavailability of the original ginsenosides (such as Rb1 and Rg1) due to their large molecular weight and high polarity. Studies have shown that rare ginsenosides modified by deglycosylation (such as Rg3, Rh2, and CK) exhibit significantly improved bioactivity and absorption efficiency due to optimized molecular structure. However, the content of rare ginsenosides in natural ginseng is extremely low, and conventional processing techniques struggle to achieve effective enrichment, severely hindering the functional breakthroughs of ginseng liqueurs.

[0004] Currently, the preparation of rare ginsenosides mainly relies on chemical hydrolysis or in vitro enzymatic hydrolysis processes, which suffer from problems such as product structural damage and insufficient transformation specificity. In contrast, microbial transformation technology exhibits significant advantages due to its green safety and reaction specificity. Among them, Lactobacillus (Lactobacillus) Lactobacillus Due to its β-glucosidase activity, it can precisely cleave the glycosidic bonds of ginsenosides, and has been proven to be a highly efficient biotransformation medium. Most importantly, this technology has unique application value in liqueur systems: through microbial transformation, the content of rare ginsenosides in liqueurs can be significantly increased; simultaneously, using liqueurs as a carrier can effectively enhance the bioactivity and human absorption efficiency of ginsenosides and other active ingredients, thereby overcoming the technical shortcomings of low active ingredient content and poor absorption rate in traditional liqueurs.

[0005] Based on the above analysis, and considering the existing processes for preparing rare ginsenosides, Lactobacillus (Lactobacillus spp.) is used. LactobacillusThe strain is used as a biological transformation medium, and specific beta-glucosidase activity thereof is used to efficiently cut specific glycoside bonds of ginsenosides to form rare saponins, and the rare saponins are further applied to liquor systems, so that the content of rare ginsenosides in liquor is significantly improved, and the bioactivity and absorption efficiency of ginsenosides and other active ingredients are effectively enhanced, thereby overcoming the core problems of low content of active ingredients and poor absorption rate in traditional liquor. SUMMARY

[0006] Therefore, the application provides a method for preparing rare ginsenosides and compound liquor based on lactic acid bacteria transformation and application thereof.

[0007] One of the technical solutions provided by the application is a lactobacillus plantarum, specifically lactobacillus plantarum (L100). Lactiplantibacillus plantarum The strain has been preserved in the China General Microbiological Culture Collection Center (CGMCC) on May 6, 2025, and the address is No. 1, Beichen West Road, Haidian District, Beijing, and the preservation number is CGMCC No. 34425.

[0008] The lactobacillus plantarum L100 has the function of transforming and preparing rare ginsenosides, and can directionally transform ginseng basic ingredients into high-value rare saponins (such as Rg3, Rg5, Rh1, etc.), especially has strong specificity for the generation of F5 and Rh10.

[0009] The second technical solution provided by the application is the application of the lactobacillus plantarum L100 in the first technical solution, especially in the preparation of ginsenosides.

[0010] The third technical solution provided by the application is a ginsenoside-enriched liquor, which is mainly prepared by mixing and processing ginseng, radix codonopsis, liquorice, cistanche and other medicinal and edible plants and white wine. Further, the liquor is prepared by compounding the following components in the weight fraction ratio: 50-60 parts of radix codonopsis extract, 80-90 parts of ginseng fermentation liquor, 200-300 parts of cistanche flavone extract, 50-60 parts of cistanche phenethyl alcohol glycoside extract, and 5-15 parts of ginseng powder in 500-600 parts of base liquor soaking solution. Further, the preparation method of the liquor is as follows: (1) Wash ginseng, radix codonopsis, astragalus, liquorice and cistanche, dry and then grind into fine powder, and pass through a 40 or 50 mesh sieve for standby; (2) Codonopsis extract: the above fine powder of Codonopsis and 75%-85% vol edible alcohol are mixed at 1:20-30 (m:v) to carry out ethanol reflux extraction, the extraction time is 90-110 min, the extraction temperature is 75-80 ℃, and the Codonopsis extract is obtained after filtration (30-50 microns) and is prepared; (3) Cistanche flavone extract: the above crushed Cistanche and 65%-75% vol edible alcohol are mixed at 1:25-30 (m:v) to obtain a raw liquid at room temperature for 110-120 min, the raw liquid is centrifuged (7000 rpm, 10 min), the supernatant is evaporated with ethanol (40 mbar vacuum, temperature 40 ℃), and the Cistanche flavone extract is obtained by adding water to 50%-55% of the volume of the supernatant; the precipitate after centrifugation is prepared; (4) Cistanche phenethyl alcohol glycoside extract: the precipitate after centrifugation of the raw liquid in the preparation of the Cistanche flavone extract is mixed with pure water at 1:15-18 (m:v) to extract at 70-75 ℃ for 55-65 min, the supernatant is obtained by centrifugation, and the Cistanche phenethyl alcohol glycoside extract is obtained; (5) Base liquor soaking liquid: 10-12 parts by weight of Codonopsis, 10-12 parts by weight of Astragalus and 1.4-1.6 parts by weight of licorice are added to 400-420 parts by weight of base liquor, and the mixture is soaked at a temperature of 25-28 ℃ for 34-38 h, and the base liquor soaking liquid is obtained by filtration (30-50 microns) and is prepared; (6) Ginseng fermentation liquid: ginseng powder and sterile water are prepared at a ratio of 1:28-32 (m:v), and the mixture is subjected to water bath at 78-82 ℃ for 18-22 min, and then cooled to room temperature to serve as a fermentation substrate; The fermentation substrate is 10-12% (v:v) of Lactobacillus plantarum L100 culture solution, the culture solution is washed with sterile water until no culture medium is present, and then added to the above fermentation substrate, and then subjected to fermentation at 37-38 ℃ for 190-192 h; after the fermentation is completed, the mixture is subjected to water bath at 80-85 ℃ for 20-25 min, and then centrifuged at 7000-7500 rpm for 10-12 min to obtain the ginseng fermentation liquid; Further, the Lactobacillus plantarum L100 culture solution is inoculated into a fermentation medium and cultured until the OD600 is 8-10; Further, the fermentation medium comprises: peptone 1 g, beef powder 0.8 g, yeast powder 0.4 g, glucose 2 g, potassium phosphate dibasic 0.2 g, diammonium hydrogen citrate 0.2 g, sodium acetate 0.5 g, magnesium sulfate 0.02 g, manganese sulfate 0.004 g, Tween 80 0.1 g, and water is added to 100 mL, and then sterilized at 118 ℃ for 15 min; Further, the culture conditions are as follows: after inoculation, the mixture is placed at 37-38 ℃ for 24-26 h. (7) 50-60 parts of the extract of radix codonopsis, 80-90 parts of the fermentation liquor of radix ginseng, 200-300 parts of the extract of flavonoids of cistanche, 50-60 parts of the extract of phenylethanoid glycoside of cistanche, 5-15 parts of radix ginseng powder are added in 500-600 parts of base liquor; (8) The prepared liquor is subjected to impurity removal treatment, and the supernatant is obtained after centrifugation at 7000 rpm for 10 min; Further filtration is performed using a filter with a pore size of less than 0.5 μm.

[0011] Beneficial effects: 1. The present application first provides a plant lactobacillus L100 capable of producing rare ginsenosides. The fermentation treatment of the ginseng powder by the strain significantly improves the relative content of rare ginsenosides. The content of ginsenoside F5 increases by more than 11 times (+1155.71%), indicating that the precursor substances are efficiently converted into target ginsenosides. The increase of ginsenoside Rh10 reaches 354.97%. The increase of the remaining ginsenosides shows a gradient distribution: the increase of ginsenoside RT5 is 36.97%, the increase of ginsenosides Rh8, Rg3 and Rh1 is between 60.40% and 92.56%, and the increase of Rg5, Rg6, isoginsenoside Rh3 and Rg2 is more than 110%. It is fully confirmed that the plant lactobacillus L100 can convert the basic components of ginseng into high-value rare ginsenosides (such as Rg3, Rg5, Rh1, etc.) through biological transformation, especially for the generation of F5 and Rh10. The strain provides an effective strategy for the synergistic development of functional components of ginseng, and significantly improves the value of active ingredients of the fermentation product.

[0012] 2. The present application uses the ginseng fermentation liquor obtained by fermenting ginseng with the plant lactobacillus L100, and the extract of radix codonopsis, astragalus, licorice, cistanche and other medicinal and edible ingredients for the preparation of liquor. On the one hand, the herbal liquor shows strong scavenging ability, indicating that it has good antioxidant capacity. On the other hand, the liquor is rich in various bioactive ingredients, especially the content of scopolin is significantly higher than that of general commercially available liquor, showing its unique health value and pharmacological potential. The contents of total ginsenosides and flavonoids are also at a high level, further verifying the potential efficacy of the liquor in antioxidant, anti-inflammatory, immune regulation and other aspects. BRIEF DESCRIPTION OF DRAWINGS

[0013] Figure 1 It is the single colony morphology of the plant lactobacillus L100.

[0014] Figure 2 It is the cell morphology of the plant lactobacillus L100.

[0015] Figure 3 It is the phylogenetic tree of the plant lactobacillus L100.

[0016] Figure 4 Growth curve of Lactobacillus plantarum L100.

[0017] Figure 5 Results of Lactobacillus plantarum L100 tolerance determination.

[0018] Figure 6 Acid production capacity determination of Lactobacillus plantarum L100. DETAILED DESCRIPTION

[0019] The present application is further described in the following specific embodiments. Unless otherwise specified, the technical means, materials and the like involved in the following embodiments can be known to those skilled in the art, and appropriate ones can be selected from the known means and materials capable of solving the corresponding technical problems. In addition, the embodiments should be understood as illustrative rather than limiting the scope of the present application, and the essence and scope of the present application are only limited by the claims. For those skilled in the art, various changes or modifications to the material components and amounts in these embodiments without departing from the essence and scope of the present application also fall within the protection scope of the present application.

[0020] Example 1 Isolation and identification of strains 1. Isolation of strains (1) Take 10 g of fermented grains sample in 90 mL of sterile water, shake at 220 r / min for 20 min at 37 ℃, mix well and perform gradient dilution, take 100 μL of 10 -3 , 10 -4 , 10 -5 of the bacterial liquid to MRS plate, and incubate in a 37 ℃ incubator for 2-3 days. The colonies with yellow rings on the plate are picked and streaked on MRS plates, and incubated in a 37 ℃ incubator for 2-3 days. The single colony morphology is observed, and the morphology is observed under a microscope. A total of 13 strains are screened, which are GT2, GT102, GT49, L30, GT196, GT24, L68, GT444, GT131, GT478, L32, GT141, and L100.

[0021] Lactic acid bacteria can achieve partial biological transformation of ginsenosides through directional enzymatic mechanism. Further, 0.05% ferric citrate and 0.3% esculin are added to the MRS medium, and the 13 strains screened above are respectively cultured at 37 ℃ for 48 h, and the β-glucosidase production capacity of the strains is determined, and the results are shown in Table 1.

[0022] Table 1 β-glucosidase primary screening results

[0023] The β-glucosidase specifically removes one or more glucose groups by hydrolyzing the β-glycosidic bond at the end of the glucose group of ginsenoside (such as Rb1, Rg3, etc.); this enzymatic reaction converts the prototype saponin with large molecular weight and high polarity into rare saponin (such as C-K, Rh2, etc.) with small molecular weight, low polarity and higher biological activity. The β-glucosidase generates escinogen by enzymatic hydrolysis of escin, and Fe 3+ The chelate shows brown-black color, so the MRS medium with added ferric citrate and escin can be used to screen strains with good β-glucosidase production capacity.

[0024] As shown in Table 1, the strains L30, L68 and L100 have good β-glucosidase activity and have potential for saponin conversion applications.

[0025] (2) 2 g of ginseng powder was prepared with 60 mL of sterile water prepared in advance, and was placed in a water bath at 80 ℃ for 20 min. After cooling to room temperature, 6 mL of Lactobacillus plantarum culture solution (L30, L68, L100) was added to the above mixture of ginseng powder and sterile water, and was placed at 37 ℃ for 8 d of fermentation. After centrifugation at 7000 rpm for 10 min, the supernatant was obtained to obtain ginseng fermentation liquor.

[0026] The preparation method of the Lactobacillus plantarum culture solution is to inoculate Lactobacillus plantarum into a fermentation medium and place it at 37 ℃ for 24 h of culture until the OD600 is about 8; The fermentation medium consists of 1 g of proteose peptone, 0.8 g of beef powder, 0.4 g of yeast powder, 2 g of glucose, 0.2 g of potassium phosphate, 0.2 g of diammonium hydrogen citrate, 0.5 g of sodium acetate, 0.02 g of magnesium sulfate, 0.004 g of manganese sulfate, 0.1 g of Tween 80, and water to make up to 100 mL. Sterilization at 118 ℃ for 15 min; The contents of ginsenoside Re and Rg1 in the ginseng fermentation liquor were detected by HPLC to further verify the saponin conversion capacity of each strain, as follows: Agilent C18 column (250 mm x 4.6 mm, 5 μm) with acetonitrile as mobile phase A and water as mobile phase B at a flow rate of 1 mL / min, injection volume of 10 μL, gradient elution program: 0-35 min, 19% A; 35-55 min, 19-29% A; 55-70 min, 29% A; 70-100 min, 29-40% A. The peak areas of ginsenoside Re and Rg1 standard solutions with concentrations of 100 μg / mL, 200 μg / mL, 300 μg / mL, 400 μg / mL, 500 μg / mL, 800 μg / mL and 1000 μg / mL were determined under the same conditions, and a standard curve was drawn. The detection wavelength was 203 nm, and the linear regression equations of ginsenoside Re and Rg1 were: y = 2.8876x - 11.97, R 2 = 0.9993; y = 3.5069x - 18.73, R 2 = 0.9992. The results are shown in Table 2.

[0027] Table 2 Changes in ginsenoside Re and Rg1 before and after fermentation

[0028] As shown in Table 2, the contents of ginsenoside Re and Rg1 in the strain L100 transformation system decreased most significantly, with a decrease of 43.14% for ginsenoside Rg1 and 53.88% for ginsenoside Re, indicating that the strain L100 showed good ginsenoside conversion rate. Therefore, the strain was determined to be used for subsequent studies.

[0029] 2. Strain identification (1) The single colony morphology of strain L100 is shown in Figure 1 , and the cell morphology is shown in Figure 2 .

[0030]

[0031] Phylogenetic tree constructed by NCBI Blast sequence alignment ( Figure 3 ), and strain L100 was identified as Lactiplantibacillus plantarum It was named Lactobacillus plantarum ( Lactiplantibacillus plantarum L100. It was deposited at the China General Microbiological Culture Collection Center on May 6, 2025, with accession number CGMCC No. 34425.

[0032] 3. Determination of physicochemical properties (1) Growth curve Strain L100 was inoculated into 20 mL of MRS liquid medium at a 2% inoculum. Starting at 0 h, the OD of the bacterial culture was measured every 2 h. 600 .

[0033] The results are as follows Figure 4 As shown, the strain has a lag phase of 0-4 h, a logarithmic growth phase of 4-12 h, and a stationary phase of 12-24 h.

[0034] (2) Tolerance test Strain L100 was inoculated into MRS liquid medium and incubated statically at 37 °C for 16 hours. After the bacterial growth stabilized, the concentration of the culture medium was measured using a spectrophotometer, and the suspensions of each strain were uniformly adjusted to a standardized concentration of OD600 = 1.0. Subsequently, serial dilutions were performed using sterile water to prepare 10⁻⁻⁶ saturated solutions. 1 Up to 10⁻ 5 The continuous dilution system, the spare bacterial suspension contains the original solution and the above dilution gradient.

[0035] Organic acid tolerance test: Lactic acid and acetic acid at final concentrations of 0, 3, 6, 9, and 12 g / L were added to MRS liquid medium, respectively. After the plates solidified, 10 μL of each medium was spread onto the plates. -1 Up to 10 -5 The diluted bacterial culture was incubated at 37 °C for 48 h.

[0036] Ethanol tolerance test: The tolerance test followed the organic acid test procedure, but the inhibitor was replaced with anhydrous ethanol, and the volume concentration gradient was set to 0%, 4%, 8%, 12%, and 16% (v / v).

[0037] Temperature tolerance experiment: Five treatment groups were set up with culture temperatures of 37, 39, 41, 43, and 45 ℃. Standard MRS solid culture medium was used, and the sampling method was the same as before.

[0038] The results are as follows Figure 5As shown, L100 still maintained a certain growth at 12 g / L lactic acid concentration, grew better in 2 g / L and 4 g / L acetic acid concentration, L100 still maintained growth activity at 12% ethanol, and showed a certain tolerance in high temperature environment.

[0039] (3) Acid production capacity determination Sorghum hydrolysate preparation: high-quality sorghum was ground into powder, mixed according to the ratio of 1:3 (w / v) of sorghum powder and water, and gelatinized in a 60-70 ℃ water bath for 30 min. Then it was heated to 90 ℃, and then liquefying enzyme (1 μL / g of raw material) was added, and it was liquefied at 90 ℃ for 20 min. Then it was boiled at high temperature for 20 min, and after the end, the original volume was supplemented with water, and immediately cooled to 60 ℃. According to the proportion of 2 μL / g of raw material, glucoamylase was added, and the water bath was continuously incubated at 60 ℃ for 4 h. After the completion of saccharification, it was cooled to 40 ℃, and acid protease (1 μL / g of raw material) was added, and the water bath was acted at 40 ℃ for 2 h. After cooling to room temperature, it was filtered with 4 layers of gauze and prepared for use.

[0040] Strain L100 was inoculated into 100 mL sorghum hydrolysate at an inoculation amount of 2%, and incubated in a 37 ℃ incubator for 7 d. The lactic acid and acetic acid contents were determined by high performance liquid chromatography.

[0041] As shown in Figure 6 After 7 d of culture, L100 produced 8.02 g / L of lactic acid and 0.44 g / L of acetic acid, indicating that it had good acid production performance.

[0042] (4) Saponin conversion capacity determination 2 g of ginseng powder was prepared with 60 mL of sterile water in advance, and was placed in a 80 ℃ water bath for 20 min. After cooling to room temperature, 6 mL of Lactobacillus plantarum culture solution (the preparation method of the culture solution was the same as step 1 of embodiment 1) was washed with sterile water until there was no culture medium, and was added to the above mixture of ginseng powder and sterile water. After 8 d of fermentation at 37 ℃, centrifugation was performed at 7000 rpm for 10 min to obtain ginseng fermentation broth; Based on UHPLC-QTOF-MS / MS analysis of the relative contents of some rare saponins in the samples before and after the addition of Lactobacillus plantarum fermentation in the above steps, the changes in the contents of different saponins were calculated by peak area. The results are shown in Table 3.

[0043] Table 3 Changes in rare saponins before and after fermentation

[0044] As shown in Table 3, the fermentation of Lactobacillus plantarum on ginseng powder significantly increased the relative content of all 10 rare saponins detected, with an increase ranging from 36.97% to 1155.71%. Among them, ginsenoside F5 had the most prominent increase, with a peak area growth of more than 11 times (+1155.71%), indicating that its precursor material was efficiently converted into the target saponin; ginsenoside Rh10 was second, with an increase of 354.97%. The remaining saponins showed a gradient distribution in the increase: pseudoginsenoside RT5 had the smallest increase (+36.97%), ginsenosides Rh8, Rg3, and Rh1 had an increase of 60.40%-92.56%, and Rg5, Rg6, isoginsenoside Rh3, and Rg2 had an increase of more than 120% (111.13%-123.86%).

[0045] The results fully confirmed that Lactobacillus plantarum L100 can convert the basic components of ginseng into high-value rare saponins (such as Rg3, Rg5, Rh1, etc.) through biological transformation, especially for the generation of F5 and Rh10. This strain provides an effective strategy for the synergistic development of ginseng functional components, significantly improving the value of active ingredients in the fermentation product.

[0046] Example 2: A liquor and a preparation method thereof Wash and dry ginseng, codonopsis, astragalus, licorice, and cistanche, and then grind them into fine powder, pass through a 40-mesh sieve, and reserve for use.

[0047] (1) Mix 3 g of finely ground codonopsis and 75 mL of 80% Vol edible alcohol, and extract under reflux at 80°C for 90 min. Filter the extract liquid through a 50-micron membrane, and reserve for use as the codonopsis extract liquid; (2) Mix 5 g of finely ground cistanche and 150 mL of 70% Vol edible alcohol, and extract at room temperature (25°C) for 2 h to obtain a raw liquid. Centrifuge the raw liquid at 7000 rpm for 10 min, evaporate 100 mL of supernatant ethanol (40 mbar vacuum, temperature 40°C), and then add water to 50 mL to obtain a cistanche flavone extract liquid. Reserve the precipitate after centrifugation of the raw liquid; (3) Mix 5 g of the precipitate from step (2) with 80 mL of pure water, and extract at 70°C for 1 h. Centrifuge at 7000 rpm for 10 min, and take the supernatant to obtain a cistanche phenethyl alcohol glycoside extract liquid, and reserve for use; (4) Add 10 g of codonopsis, 10 g of astragalus, and 1.4 g of licorice to 410 g of a base liquor with a clear aroma, and soak at a temperature of 25°C for 36 h. Filter (50 microns) to obtain a base liquor soaking liquid, and reserve for use; (5) 2 g of ginseng powder was prepared with 60 mL of sterile water prepared in advance, and it was placed in a water bath at 80°C for 20 min. After cooling to room temperature, it was used as a fermentation substrate. 6 mL of Lactobacillus plantarum L100 culture solution was washed with sterile water until there was no culture medium, and then added to the above-mentioned ginseng fermentation substrate. It was placed at 37°C for 8 days of fermentation. After the fermentation was completed, it was placed in a water bath at 80°C for 20 min. After centrifugation at 7000 rpm for 10 min, ginseng fermentation liquor was obtained; The preparation method of the Lactobacillus plantarum culture solution is as follows: after inoculation, it is placed at 37°C for 24 h of culture. The fermentation medium consists of: peptone 1 g, beef powder 0.8 g, yeast powder 0.4 g, glucose 2 g, potassium phosphate dibasic 0.2 g, diammonium citrate 0.2 g, sodium acetate 0.5 g, magnesium sulfate 0.02 g, manganese sulfate 0.004 g, Tween 80 0.1 g, and water to make up to 100 mL. Sterilization at 118°C for 15 min. (6) 11 g of radix codonopsis extract, 16 g of ginseng fermentation liquor, 54 g of cistanche flavone extract, 11 g of cistanche phenethyl alcohol glycoside extract, and 1.2 g of ginseng powder were added to 108 g of base liquor soaking solution, and mixed to obtain liquor.

[0048] (7) The prepared liquor was subjected to impurity removal treatment. The supernatant was obtained after centrifugation at 7000 rpm for 10 min, and filtered using a filter with a pore size of 0.2 μm to obtain the final herbal liquor product.

[0049] Example 3: Determination of antioxidant capacity of liquor The free radical scavenging capacity and total antioxidant capacity of the ginseng powder before and after fermentation and the herbal liquor prepared in Example 2 were detected by DPPH method and ABTS method, respectively.

[0050] Principle of detection: DPPH method utilizes the single electron transfer reaction between 2,2-diphenyl-1-picrylhydrazyl radical and antioxidants, and the free radical scavenging efficiency is characterized by the absorbance attenuation at 517 nm. ABTS method generates blue-green ABTS cation radical (ABTS) by means of potassium persulfate pre-oxidation. Antioxidants cause the fading of ABTS by hydrogen donor mechanism, and the absorbance change reflects the broad-spectrum scavenging capacity. The detection wavelength is 734 nm. The experiment analyzes the antioxidant mechanism from the perspectives of free radical targeting scavenging, oxidation substrate neutralization, etc., providing theoretical support for the identification of functional ingredients of liquor.

[0051] Detection of free radical scavenging capacity (DPPH) and determination of ABTS cation radical scavenging capacity DPPH, ABTS (ready-to-use solution): Shanghai Maikelin Biochemical Technology Co., Ltd. 0.05 g of DPPH was accurately weighed and dissolved in a 1000 mL volumetric flask with anhydrous ethanol. The solution was prepared immediately before use.

[0052] The calculation method is as follows: The DPPH free radical scavenging rate of the sample = [A0-(A1-A2)] / A0*100% In the formula: A0 is the absorbance of 1 mL of DPPH free radical solution + 1 mL of double distilled water; A1 is the absorbance of 1 mL of DPPH free radical solution + 1 mL of sample; and A2 is the absorbance of 1 mL of anhydrous ethanol + 1 mL of sample.

[0053] The ABTS cation free radical scavenging rate of the sample = [A0-(A1-A2)] / A0*100% In the formula: A0 is the absorbance of 4 mL of ABTS cation radical ethanol solution + 1 mL of double distilled water; A1 is the absorbance of 4 mL of ABTS cation radical ethanol solution + 1 mL of sample; and A2 is the absorbance of 4 mL of double distilled water + 1 mL of sample.

[0054] Table 4: Results of antioxidant activity detection

[0055] As can be seen from Table 4, after fermentation treatment by strain L100, the DPPH free radical and ABTS cation free radical scavenging capacity of ginseng was significantly improved, In addition, in terms of free radical scavenging, the herbal liqueur also showed strong scavenging ability. The above results show that the active ingredients of Chinese herbal medicine in the herbal liqueur can significantly scavenge DPPH free radicals and ABTS cation free radicals, indicating its good antioxidant capacity.

[0056] Example 4: Determination of total saponins, flavones and scabiosioside in the liqueur The total saponins, flavones and scabiosioside contents in the final herbal liqueur obtained in Example 2 were detected. The specific operations are as follows: (1) The absorbance of ginsenoside Re standard solution with mass concentrations of 100 μg / mL, 200 μg / mL, 300 μg / mL, 400 μg / mL, 500 μg / mL and 600 μg / mL was determined. The specific operation steps are as follows: First, 40 μL of each solution was placed in a 80 ℃ constant temperature water bath to evaporate the solvent, then 20 μL of 5 wt% vanillin-glacial acetic acid solution and 80 μL of perchloric acid were added in turn, and reacted at 60 ℃ water bath for 15 minutes. After the reaction was completed, the sample was placed in an ice bath for 5 minutes, and then 200 μL of glacial acetic acid was added to terminate the reaction. Finally, the absorbance was measured at a wavelength of 540 nm. According to the measured absorbance value and the corresponding saponin solution concentration, a standard curve was drawn, and the standard equation y=2.2094x-0.0809 (R 2= 0.9996).

[0057] The total saponin content in the herbal liqueur was detected by the above method.

[0058] (2) Take rutin standard control solution 0 mL, 1 mL, 2 mL, 3 mL, 4 mL, 5 mL, and place it in a 25 mL volumetric flask. Add 0.3 mL of 5% NaNO2, 6 min later, add 0.3 mL of 10% Al(NO3)3, 6 min later, add 4 mL of 1 mol / L NaOH solution, and dilute to the mark with 70% ethanol solution. After 15 min, measure the absorbance value at 510 nm. With the absorbance value as the abscissa and the rutin mass concentration as the ordinate, a standard curve is drawn: y = 0.009x + 0.0011, R 2 = 0.9999.

[0059] The flavonoid content in the herbal liqueur was detected by the above method.

[0060] (3) Take methanol as mobile phase A and 0.1% formic acid solution as mobile phase B, flow rate: 1 mL / min, injection volume 10 μL, Agilent C18 chromatographic column (250 mm x 4.6 mm, 5 μm), detection wavelength 330 nm, gradient elution program: 0-17 min, 26.5% A; 17-20 min, 26.9-29.5% A; 20-27 min, 29.5% A; Under the same conditions, the peak area of the echinacoside standard solution with mass concentration of 100 μg / mL, 200 μg / mL, 300 μg / mL, 400 μg / mL, 500 μg / mL, 800 μg / mL, 1000 μg / mL was determined, and a standard curve was drawn, and the standard equation y = 13540x + 49.292, R 2 = 0.9999.

[0061] The echinacoside content in the herbal liqueur was detected by the above method.

[0062] Table 5 Liqueur active substance detection results

[0063] Table 5 shows that the liqueur is rich in various bioactive ingredients, especially the content of echinacoside is significantly higher than that of general commercially available liqueur, showing its unique health value and pharmacological potential. The contents of total saponins and flavonoids are also at a high level, further verifying the potential efficacy of the liqueur in antioxidant, anti-inflammatory, and immune regulation.

[0064] Example 5 A liqueur and a preparation method thereof Wash and dry ginseng, radix codonopsitis, radix astragali, licorice and cistanche, then grind them into fine powder, pass through a 50-mesh sieve, and reserve for use.

[0065] (1) Mix 5 g of the fine powder of radix codonopsitis and 150 mL of 80% Vol edible alcohol, and extract at 75°C for 110 min. Filter the extract liquid through a 50-micron membrane, and reserve for use as the radix codonopsitis extract liquid; (2) Mix 5 g of the fine powder of cistanche and 125 mL of 70% Vol edible alcohol, and extract at room temperature (25°C) for 2 h. Centrifuge the original liquid at 7000 rpm for 10 min, and then evaporate 100 mL of supernatant ethanol (40 mbar vacuum, temperature 40°C), and then add water to 50 mL to obtain the cistanche flavone extract liquid. Reserve the precipitate after centrifugation of the original liquid; (3) Mix 5 g of the precipitate of step (2) and 90 mL of pure water, and extract at 75°C for 65 min. Centrifuge the supernatant at 7000 rpm for 10 min to obtain the cistanche phenethyl alcohol glycoside extract liquid, and reserve for use; (4) Add 12 g of radix codonopsitis, 12 g of radix astragali, and 1.6 g of licorice to 420 g of base liquor of the light-flavor type, and soak at a temperature of 28°C for 38 h. Filter (50 microns) to obtain the base liquor soaking liquid, and reserve for use; (5) Prepare 2 g of ginseng powder with 60 mL of sterile water prepared in advance, and then place it in a water bath at 80°C for 20 min. After cooling to room temperature, use it as a fermentation substrate. Wash 7 mL of Lactobacillus plantarum culture liquid with sterile water until no culture medium is present, and then add it to the above ginseng fermentation substrate. Place it at 37°C for fermentation for 190 h. After fermentation, place it in a water bath at 80°C for 20 min. Centrifuge at 7000 rpm for 10 min to obtain the ginseng fermentation liquid; The Lactobacillus plantarum culture liquid is prepared as follows: inoculate and then place it at 37°C for 24 h of static culture. The fermentation medium consists of: peptone 1 g, beef powder 0.8 g, yeast powder 0.4 g, glucose 2 g, potassium phosphate dibasic 0.2 g, diammonium hydrogen citrate 0.2 g, sodium acetate 0.5 g, magnesium sulfate 0.02 g, manganese sulfate 0.004 g, Tween 80 0.1 g, and water to make up to 100 mL. Sterilize at 118°C for 15 min. (6) Add 60 g of the radix codonopsitis extract liquid, 90 g of the ginseng fermentation liquid, 300 g of the cistanche flavone extract liquid, 60 g of the cistanche phenethyl alcohol glycoside extract liquid, and 15 g of ginseng powder to 600 g of the base liquor soaking liquid, and mix well to obtain the liquor.

[0066] (7) Perform impurity removal treatment on the prepared liquor, centrifuge at 7000 rpm for 10 min, and then take the supernatant. Use a filter with a pore size of 0.2 μm to obtain the final herbal liquor product.

[0067] While the present application has been disclosed in terms of preferred embodiments thereof, it will be apparent to those skilled in the art that various changes, modifications, substitutions and alterations can be made thereto without departing from the spirit and scope of the application as defined by the appended claims and their equivalents.

Claims

1. A strain of *Lactobacillus plantarum*, characterized in that, Specifically, Lactobacillus plantarum ( Lactiplantibacillus plantarum L100, accession number: CGMCC No.34425.

2. The application of Lactobacillus plantarum L100 as described in claim 1 in the preparation of ginsenosides.

3. The application as described in claim 2, characterized in that, The ginsenosides include, but are not limited to: RT5, Rh8, Rg3, Rh1, Rg5, Rg6, Rh3, Rg2, F5, and Rh10.

4. A method for preparing a liqueur, characterized in that, The liqueur is composed of the following components in the following weight fraction ratios: 50-60 parts of Codonopsis pilosula extract, 80-90 parts of ginseng fermentation liquid, 200-300 parts of Cistanche deserticola flavonoid extract, 50-60 parts of Cistanche deserticola phenylethanol glycoside extract, and 5-15 parts of ginseng powder are added to 500-600 parts of base liquor soaking liquid. The ginseng fermentation liquid was obtained by fermenting ginseng with Lactobacillus plantarum L100.

5. The preparation method according to claim 4, characterized in that, The preparation method of ginseng fermentation liquid is as follows: Ginseng powder and sterile water are mixed at a mass-volume ratio of 1:28-32, and the mixture is placed in a water bath at 78-82 ℃ for 18-22 min. After cooling to room temperature, it is used as the fermentation substrate. Add Lactobacillus plantarum L100 to the above fermentation substrate and ferment at 37-38 ℃ for 190-192 h; sterilize after fermentation and centrifuge to obtain ginseng fermentation broth.

6. The preparation method according to claim 4, characterized in that, Includes the following steps: (1) Wash the ginseng, codonopsis, astragalus, licorice and cistanche, dry them, grind them into fine powder, and pass them through a 40 or 50 mesh sieve for later use; (2) Codonopsis pilosula extract: The finely powdered Codonopsis pilosula and 75%-85% vol edible alcohol were mixed at a mass-volume ratio of 1:20-30 and then subjected to ethanol reflux extraction for 90-110 min at an extraction temperature of 75-80 ℃. After filtration, the Codonopsis pilosula extract was obtained and set aside for later use. (3) Cistanche deserticola flavonoid extract: The above-mentioned pulverized Cistanche deserticola and 65%-75% vol edible alcohol were mixed at a mass-volume ratio of 1:25-30 and extracted at room temperature for 110-120 min to obtain the stock solution. After centrifuging the above stock solution, the supernatant was evaporated and water was added to 50%-55% of the supernatant volume to obtain Cistanche deserticola flavonoid extract; the precipitate after centrifugation of the stock solution was reserved. (4) Extract of phenylethanoid glycosides of Cistanche deserticola: The precipitate after centrifugation of the original solution in the preparation of Cistanche deserticola flavonoid extract is mixed with purified water at a mass-volume ratio of 1:15-18 and extracted at 70-75℃ for 55-65 min. The supernatant is then collected by centrifugation to obtain the extract of phenylethanoid glycosides of Cistanche deserticola. (5) Base liquor soaking solution: Add 10-12 parts of Codonopsis pilosula, 10-12 parts of Astragalus membranaceus, and 1.4-1.6 parts of Glycyrrhiza uralensis to 400-420 parts of light-aroma base liquor, soak at 25-28 ℃ for 34-38 h, filter to obtain base liquor soaking solution, and set aside for later use; (6) Ginseng fermentation liquid: Ginseng powder and sterile water are mixed at a mass-volume ratio of 1:28-32, and the mixture is placed in a water bath at 78-82 ℃ for 18-22 min. After cooling to room temperature, it is used as a fermentation substrate. Take Lactobacillus plantarum L100 culture medium at a volume ratio of 10-12% of the fermentation substrate. Wash the culture medium with sterile water until it is free of culture medium, add it to the above fermentation substrate, and ferment at 37-38 ℃ for 190-192 h. After fermentation, incubate in a water bath at 80-85 ℃ for 20-25 min, and centrifuge to obtain ginseng fermentation broth. (7) Add 50-60 parts of Codonopsis pilosula extract, 80-90 parts of ginseng fermentation liquid, 200-300 parts of Cistanche deserticola flavonoid extract, 50-60 parts of Cistanche deserticola phenylethanol glycoside extract, and 5-15 parts of ginseng powder to 500-600 parts of base liquor soaking liquid, and mix to obtain liquor.

7. The preparation method according to claim 6, characterized in that, The Lactobacillus plantarum L100 culture medium is prepared by inoculating Lactobacillus plantarum L100 into a fermentation medium and incubating it statically at 37-38 ℃ for 24-26 h. The fermentation medium consisted of: 1 g peptone, 0.8 g beef meal, 0.4 g yeast powder, 2 g glucose, 0.2 g dipotassium hydrogen phosphate, 0.2 g diammonium hydrogen citrate, 0.5 g sodium acetate, 0.02 g magnesium sulfate, 0.004 g manganese sulfate, and 0.1 g Tween 80. Water was added to bring the volume to 100 mL, and the mixture was sterilized at 118 °C for 15 min.

8. The preparation method according to claim 6, characterized in that, The prepared liqueur was purified by centrifugation at 7000 rpm for 10 min, and the supernatant was collected. Then it was filtered using a filter with a pore size of less than 0.5 μm.

9. A liqueur prepared by the method according to any one of claims 4-8.

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